# Patent

## Title
Screening assay for insecticides

## Abstract

The present invention relates to polypeptides, preferably from Drosophila melanogaster (DmShaI) as target for insecticides.

## Background
This application is a National Stage application of International Application No. PCT/EP2009/064700, filed Nov. 5, 2009, which claims the benefit of U.S. Provisional Application No. 61/111,870, filed Nov. 6, 2008; U.S. Provisional Application No. 61/139,667, filed Dec. 22, 2008; U.S. Provisional Application No. 61/139,676, filed Dec. 22, 2008; and U.S. Provisional Application No. 61/139,686, filed Dec. 22, 2008; the entire contents of which are hereby incorporated herein by reference.


The present invention relates to a potassium channel with the activity of a voltage-gated potassium channel ShaI (Shaker cognate I or Shaker-like), preferably from Drosophila melanogaster (DmShaI) as target for insecticides. In one embodiment the voltage-gated potassium channel ShaI is co-expressed with its accessory protein KChIP (potassium channel-interacting protein), preferably its putative accessory protein CG5890, the Drosophila KChIP (potassium channel-interacting protein) ortholog. The present invention provides polypeptides with the activity of an insect voltage-gated potassium channel ShaI (Shaker cognate I or Shaker-like), preferably from Drosophila melanogaster (DmShaI) and its accessory protein KChIP (potassium channel-interacting protein), preferably its putative accessory protein CG5890, the Drosophila KChIP (potassium channel-interacting protein) ortholog as targets for insecticides, provides novel nucleic acid sequences encoding a polypeptide with the activity of an insect voltage-gated potassium channel ShaI (Shaker cognate I or Shaker-like) and its accessory protein KChIP (potassium channel-interacting protein) and functional equivalents of the aforementioned nucleic acid sequences.
The present invention relates further to the use of a polypeptide with the activity of an insect voltage-gated potassium channel ShaI and/or its accessory protein KChIP (potassium channel-interacting protein) in a method and in an assay for identifying insecticidally active compound that reduces the activity of an insect voltage-gated potassium channel ShaI (Shaker cognate I or Shaker-like) and/or its accessory protein KChIP (potassium channel-interacting protein). Furthermore, the invention relates to insecticidal compounds identified by the above mentioned method and the use of these compounds as insecticides.
In another embodiment, the present invention relates to a potassium channel with the activity of a Shaker potassium channel, preferably from Drosophila melanogaster as target for insecticides. In one embodiment the Shaker channel is co-expressed with a Hyperkinetic beta subunit, preferably H-kv beta subunit A or C subtype, preferably from Drosophila melanogaster. 
The present invention further provides polypeptides with the activity of an insect Shaker channel, preferably from Drosophila melanogaster and a Hyperkinetic beta subunit, preferably H-kv beta subunit A or C subtype in an screening assay for insecticides, provides novel nucleic acid sequences encoding a polypeptide with the activity of an insect Shaker channel and a Hyperkinetic beta subunit, preferably H-kv beta subunit A or C subtype and functional equivalents of the aforementioned nucleic acid sequences.
The present invention furthermore relates further to the use of a polypeptide with the activity of an insect Shaker channel and/or a Hyperkinetic beta subunit, preferably H-kv beta subunit A or C subtype respectively in a method and in an assay for identifying insecticidally active compound that reduces the activity of an insect Shaker channel and a Hyperkinetic beta subunit, preferably H-kv beta subunit A or C subtype respectively. Furthermore, the invention relates to insecticidal compounds identified by the above mentioned method and the use of these compounds as insecticides.
In another embodiment, the present invention relates to a G-protein coupled receptor (GPCR) with the activity of an octopamine receptor, preferably from Drosophila melanogaster as target for insecticides.
The present invention further provides polypeptides with the activity of an an octopamine receptor, preferably from Drosophila melanogaster in an screening assay for insecticides, provides novel nucleic acid sequences encoding a polypeptide with the activity of an octopamine receptor, preferably from Drosophila melanogaster and functional equivalents of the aforementioned nucleic acid sequences.
The present invention relates further to the use of a polypeptide with the activity of an octopamine receptor selected from the group consisting of oa2, preferably from Drosophila melanogaster, Oamb, Oct-beta-2R and Oct-beta-3R in a method and in an assay for identifying insecticidally active compound that reduces the activity of an octopamine receptor selected from the group consisting of oa2, preferably from Drosophila melanogaster, Oamb, Oct-beta-2R and Oct-beta-3R. Furthermore, the invention relates to insecticidal compounds identified by the above mentioned method and the use of these compounds as insecticides.
In another embodiment, the present invention relates to a SK-channel as target for insecticides.
The present invention furthermore provides polypeptides with the activity of an insect small-conductance Ca2+-activated potassium channel as targets for insecticides, provides novel nucleic acid sequences encoding a polypeptide with the activity of an insect small-conductance Ca2+-activated potassium channel and functional equivalents of the aforementioned nucleic acid sequences.
The present invention relates further to the use of a polypeptide with the activity of an insect small-conductance Ca2+-activated potassium channel in a method and in an assay for identifying insecticidally active compound that reduces the activity of an insect small-conductance Ca2+-activated potassium channel. Furthermore, the invention relates to insecticidal compounds identified by the above mentioned method and the use of these compounds as insecticides.
Insects cause many human and animal diseases. Insects are also responsible for substantial agricultural and property damage resulting in economic loss. In spite of all the pesticide poison's used but also misused, insects still destroy over 30% of the world's food crops each year.
Many approaches have been developed in order to limit the damages caused by insects.
One approach is the use of chemicals for insect control. The problem of many insecticides, for example like DDT, is the fact that they require a application in high concentrations and they have a unspecific, broad spectrum of activity. Chemical pesticides generally affect beneficial as well as nonbeneficial species. Many of them are persistent in the environment and accumulate therefore in the food chain.
Another approach are transgenic crops that express insecticidal toxins, such as protein toxins from the bacterium Bacillus thuringiensis. 
Insect pests tend to acquire resistance to all kinds of insecticides because they have an exceptional ability to adapt to their environment, due to a mechanisms which leads to the rapid development of resistance in an insect population, such as short life cycles, a high reproductive rate and the ability to travel long distances. At the moment pests exhibiting insecticide resistance is increasing.
Therefore, there is still a need to find effective and economic insecticides with a very specific effect against insect pests. The more effective an insecticide is the less environmental hazard it creates.
This can be achieved by identification and isolation of a gene that codes for a protein which will control insect development and/or surviving.



BRIEF DESCRIPTION OF THE DRAWINGS
FIG. 1: Vector NTI-generated map of the primary Sha1 clone showing the major features of the construct.
FIG. 2: Vector NTI-generated map of the primary KChIP clone showing the major features of the construct.
FIG. 3: Vector maps of pcDNA3/AcGFP-Sha1 and pcDNA3/AcGFP-Sha1delN2-40aa.
FIG. 4: Patch clamp data for full length and truncated constructs Sha1 and Sha1_delN when coexpressed with AmCyan as well as AcGFP-tagged versions (top); schematic representation of GFP-Sha1 pore mutant and patch clamp data for wild type and pore mutant.
FIG. 5: Sha1_DelN clones tested on FLIPR with KCl depolarization.
FIG. 6: K channel blocker inhibition curves of known K-channel blockers tested on FLIPR.
FIG. 7: Screening Sha1_delN Clone10 subclones by KCl depolarization on FLIPR.
FIG. 8: Patch clamp data for clones 10-2, 10-3 and 10-6. IV curves showing the relationship of applied voltage to current.
FIG. 9: Sha1_DelN C10-3 cells at passage 12. Left panel—fluorescence excitation, right panel—normal light.
FIG. 10: Effect of BaCl2 on Sha1 channel activity on FLIPR.
FIG. 11: Sha1_delN C10-3 function on FLIPR at passage 20.
FIG. 12: CHO-K1 cells stably expressing Sha1_delN were either mock transfected (lower line) or cotransfected with KChIP (upper line).
FIG. 13: Sha1/KChIP inhibition by arachidonic acid measured by whole cell patch-clamp; Right panel: Peak current amplitudes; Left panel: Integrated area.
FIG. 14: I/V curves for clones 18-13-6 and 18-13-20.
FIG. 15: Sha1/KChIP clone screening and Neo/Zeo control line. ScreenWorks screenshots showing primary FLIPR data and a reduced data table for the 19 clones and the Neo/Zeo pool (middle panel, wells H4-6).
FIG. 16: Sha1/KChIP final subclone screening. Reduced FLIPR data from subclone screening resulting in the final cell line.
FIG. 17: Sha1/KChIP assay FLIPR response profiles.
FIG. 18: Effect of dye in activation buffer measured on FLIPR Tetra.
FIG. 19: Comparison of BD and Greiner 384-well assay plates.
FIG. 20: Cell density optimization measured on FLIPR.
FIG. 21: Dye concentration evaluation for Sha1/KChIP clones 18-13 and 18-28.
FIG. 22: Effect of DMSO on Sha1/KChIP assay window size, standard deviations and Z′ statistics.
FIG. 23: Effect of dye loading time on Sha1/KChIP assay window size, standard deviation and resulting Z′ statistic.
FIG. 24: The effects of pre-cooling cells to room temperature on the Sha1/KChIP assay prior to dye-loading.
FIG. 25: Group-averaged primary FLIPR data and preliminary EC50 for Sha1/KChIP18-13 cell line.
FIG. 26: Comparison of GraphPad Prism scatter plots showing maximum and minimum responses with resultant statistics of the Sha1/KChIP assay for MDC and Axygen FLIPR 384 tips.
FIG. 27: Determination of stability of reagents used in the Sha1/KChIP 18-13 assay over time.
FIG. 28: Three-day minimum, midpoint and maximum statistics for Sha1/KChIP 20 assay cells.
FIG. 29: Three-day KCl dose response curves and EC50s of Sha1/KChIP 20 assay cells.
FIG. 30: Three-day amiloride dose response curves in Sha1/KChIP 20 cells.
FIG. 31: Sha1/KChIP 20 direct-to-plate assay results.
FIG. 32: ScreenWorks screenshots showing primary FLIPR data for BIOMOL compound screening in Sha1/KChIP 20 cells.
FIG. 33: Assay plate views showing percent inhibition of Sha1/KChIP response by BIOMOL compounds.
FIG. 34: Compound preparation protocol for BioFocus screening.
FIG. 35: Sha1/KChIP FLIPR BioFocus 1° screening—activation and distribution of actives on Sha1/KChIP.
FIG. 35A: PCR products from the reactions described in Table A1.
FIG. 36: VectorNTi map of the final pExSelect_Shaker construct.
FIG. 37: Assay data for mock clones upon KCl injection.
FIG. 38: Assay data for Shaker and Hkvβ clones upon KCl injection.
FIG. 39: Re-test assay data for two mock clones and six clones each from both Shaker plus A or C subtype.
FIG. 40: A-3A10 signal stability at different cell passages.
FIG. 41: A-3A10 signal stability after freezing and thawing.
FIG. 42: TEA effect on A-3A10 clone.
FIG. 43: Hypertonic and isotonic solutions analysis.
FIG. 44: A-3A10 II limiting dilution clone selection at FLIPR384.
FIG. 45: n° 1 and n° 9 II limiting dilution clones analyzed at FLIPRTETRA.
FIG. 46: Cell density dependency and KCl dose-response.
FIG. 47: DMSO sensitivity: 10000 c/w-24 h.
FIG. 48: DMSO sensitivity: 7500 c/w-24 h.
FIG. 49: DMSO sensitivity: 5000 c/w-24 h.
FIG. 50: Clone stability in culture and after freezing/thawing: 10000, 7500, 5000 c/w-24 h.
FIG. 51: EC50 reproducibility on three different days.
FIG. 52a: Patch clamp analysis of clone 3a10 subtype A.
FIG. 52b: Patch clamp analysis of clone 7d1 subtype C.
FIG. 53: Activation of Dm-Shaker in cells: a) Activation of Dm-Shaker in cells cultured for 10-11 passages; b) Activation of Dm-Shaker in cells cultured for 17-20 passages; c) Activation of Dm-Shaker in cells after thawing.
FIG. 54: The I-V relationship of voltage dependency of the activity of DM-Shaker.
FIG. 55a: Schematic of calcium release from activation of G-alpha-16-protein.
FIG. 55b: Vector maps of pcDNA3.1-OcrR for and pcDNA3.1-OcrR-GFP.
FIG. 55c: Fluorescence change in response to 1 μM and 100 nM octopamine.
FIG. 55d: Calcium-sensing Fluo-4 fluorescent dye response to octopamine or tyramine stimulation in CHO cells stably expressing G-alpha-16 and transiently expressed tagged or untagged OctR-pcDNA3.1.
FIG. 56: Stable pool of zeocin-selected CHO-G-alpha-16 cells identified by green fluorescent protein expression.
FIG. 57: Screening of all GFP-positive monoclonal cell lines for response to octopamine.
FIG. 58: Response of clones #13 and #30 to octopamine on FLIPR Tetra.
FIG. 59: Single-cell response to octopamine for clones #13 and #30.
FIG. 60: OCTR clone 55 selection.
FIG. 61: Homogeneity assay of OCTR clone 55.
FIG. 62: Antagonist dose response curves and agonist dose response curves in transfected and parent cell lines analysed on FLIPR.
FIG. 63: Octopamine EC50 curves in different DMSO concentrations in 1st and 2nd additions.
FIG. 64: Graphical representation of effect of cell density measured on FLIP.
FIG. 65: Graphical representation of effect of dye concentration on assay window size.
FIG. 66: Graphical results of time course experiment of effect of dye loading time.
FIG. 67: Graphical results of effect of different tip washings measured on FLIPR.
FIG. 68: Octopamine and mianserin dose response curves run on three separate days in OCTR cells.
FIG. 69: A scatter plot of plate #7, showing the controls and test wells for the agonist portion of the screen (left) and antagonist portion of the screen (right).
FIG. 70: Vector map of pCRII-SK2+4D
FIG. 71: Vector map of pTriEx3 Neo SK
FIG. 72: Activation curves for CHO-K1 cells transfected with pTriEx3 Neo SK and activated with ionomycin.
FIG. 73: Activation curves comparing normal assay buffer and Ca2+ free buffer.
FIG. 74: EC50 curves for SK/CHO-K1 and SK/pTx-CHO cells activated with increasing amounts of ionomycin.
FIG. 75: Graphical representation of assay window size variability in relation to dye loading time.
FIG. 76: Graphical representation of DMSO concentration effect on ionomycin activation.
FIG. 77: Graphical representation of data used for statistically testing the probability of a differential response occurring by chance in the assay.
FIG. 78: Graphical result for BAY K-8644 L-type Ca2+ channel agonist.
FIG. 79: Graphical result for Propafenone.
FIG. 80: Graphical result for tetraethylammonium (SK3) as percent baseline.
FIG. 81: Graphical EC50 result for 4-aminopyridine (SK3-4) as a percent baseline.
FIG. 82: Graphical EC50 results for propafenone (SK3).
FIG. 83: Graphical representation of characterization of DmSK expression in CHO cells by functional expression assay.
FIG. 84: Graphical representation of the effect of Propafenone on DmSK expressing CHO cells
FIG. 85: Graphical results of whole cell patch-clamp assay for DmSK expressing CHO cells subjected to 70 μm propafenone.
FIG. 86: Schematic of an automated scheduled process for a single assay experiment.
FIG. 87: Schematic of Tripos compound dilution file.
FIG. 88: Schematic of Divpick compound dilution control file.
FIG. 89: Schematic of Read 1 plate map.
FIG. 90: Schematic of Read 2 plate map.
FIG. 91: Screenshot of Batch Export statistics interface.
FIG. 92: Reference graph of activity of rat SK channel in oocyte expression system.



The present invention provides a insect voltage-gated potassium channel ShaI (Shaker cognate I or Shaker-like) and/or its accessory protein KChIP (potassium channel-interacting protein) as new target for insecticides. The present invention puts further a method and an assay at disposal for identifying insecticidally active compounds that reduce the activity of a insect voltage-gated potassium channel ShaI (Shaker cognate I or Shaker-like) and/or its accessory protein KChIP (potassium channel-interacting protein).
The outward voltage-dependent K+ currents found in many Drosophila embryonic and larval neurons arise from a mix of currents generated from the Shaker family members Shaw, Shab and ShaI (Tsunoda, S. & L. Salkoll (1995) J Neuroscience. March; 15(3):1741-1754). ShaI generates the transient component (the “A-type” current) of these macroscopic K+ currents and modulates these currents by, for example, causing a delayed current spike in certain neurons (Yu, D., Feng, C. & A. Guo (1999) J Neurobiology. August; 40(2):158-70). Alternative splicing, post-translational modifications and other ShaI-associated processes are likely responsible in part for the large repertoire of modulations seen in these neurons (Choi, J. C., Park, D. & L. C. Griffith (2004) J Neurophysiology. 91:2353-2365).
KChIP, a Kv4.x (ShaI) accessory protein, has been shown to dramatically increase the trafficking of ShaI to the cell membrane, probably by promoting tetrameric channel assembly, and to cause distinct changes in ShaI channel gating properties (Kunjilwar, K., Strang, C., DeRubeis, D. & P. J. Pfaffinger (2004) J Biological Chemistry. December; 279(52): 54542-54551).
Despite ShaI's role in neuronal transmission, the validation case for this target remains to be established. Our in situ data indicate low-levels of expression in the embryonic ventral nerve cord and visceral musculature. In situs at the Berkeley Drosophila Genome Project show staining in embryonic/larval visceral muscle, longitudinal visceral muscle fibers, ventral midline, and embryonic central nervous system including the ventral nerve cord. DmShaI mRNA is a rare transcript with an expression peak at mid-embryonic stages (in-house Lynx data). There are two intronic transposon insertion lines at the Harvard Medical School Drosophila Stock Collection, but no viability data is associated with these, and no other ShaI mutants are available. There is no microarray or northern data in the public domain.
No insecticide on the market has been identified as having modulation of ShaI potassium channels as its primary mode of action, meaning that a compound does not merely contribute by a “side effect” to a insecticidal activity, but its activity is the key lethal effect.
In another embodiment, the present invention provides a insect Shaker channel and/or a Hyperkinetic beta subunit, preferably H-kv beta subunit A or C subtype respectively as new target for insecticides. The present invention puts further a method and an assay at disposal for identifying insecticidally active compounds that reduce the activity of a insect Shaker channel and/or a Hyperkinetic beta subunit, preferably H-kv beta subunit A or C subtype respectively.
The Drosophila Hyperkinetic (Hk) mutations alter a gene encoding a homolog of the mammalian K+ channel P subunit. Wang et al. (Biophysical Journal Volume 71, December 1996, 3167-3176) have shown that the Hk P subunit modulates a wide range of the Shaker (Sh) K+ current properties. Coexpression of Hk with Sh in Xenopus oocytes is also known from CHOUINARD et al. (Proc. Natl. Acad. Sci. USA, Vol. 92, pp. 6763-6767, July 1995 Neurobiology) which demonstrated that this coexpression increases current amplitudes and changes the voltage dependence and kinetics of activation and inactivation.
Surprisingly it was found, that the coexpression Shaker channel and a Hyperkinetic beta subunit, preferably H-kv beta subunit A or C subtype respectively leads to a new screening assay for insecticides.
No insecticide on the market has been identified as having modulation of Shaker channel and/or a Hyperkinetic beta subunit, preferably H-kv beta subunit A or C subtype respectively as its primary mode of action, meaning that a compound does not merely contribute by a “side effect” to a insecticidal activity, but its activity is the key lethal effect.
In another embodiment, the present invention provides a insect octopamine receptor selected from the group consisting of oa2, preferably from Drosophila melanogaster, Oamb, Oct-beta-2R and Oct-beta-3R as new target for insecticides. The present invention puts further a method and an assay at disposal for identifying insecticidally active compounds that reduce the activity of a insect octopamine receptor selected from the group consisting of oa2, preferably from Drosophila melanogaster, Oamb, Oct-beta-2R and Oct-beta-3R.
Octopamine, a biogenic monoamine structurally related to noradrenaline, is a major neurotransmitter, neuromodulator and neurohormone, mediating diverse physiological processes in peripheral and central nervous system of invertebrates. Together with tyramine, octopamine is the only neuroactive non-peptide transmitter whose physiological role is restricted to invertebrates.
The action of octopamine is mediated through various receptor classes like oa2, Oamb, Oct-beta-2R or Oct-beta-3R.
The fact that octopamine receptors can only/mainly be found in invertebrates makes them a significant target for insecticides. Compounds that specifically modulate octopamine receptors should therefore have low vertebrate toxicity. In addition to selectivity, octopamine receptors are a good target for insecticides due to its neuronal expression. Together with tyramine, octopamine is the only neuroactive non-peptide transmitter whose physiological role is restricted to invertebrates.
Although octopamine is a principal neuromediator in insects, its receptors have proven to be difficult to clone. To date, only a few octopamine receptors have been cloned. The present invention puts a method at disposal for cloning octopamine receptors and for introducing them in membranes, preferably of cells. Preferably additionally linked proteins are introduced in the membrane.
Octopamine receptors can modulate their action through cyclic AMP production or intracellular calcium release, dependent on the receptor isoform. Octopamine receptors, preferably oa2 endogenously signals though cAMP. A detection of the activity of the receptor is difficult.
Therefore the present invention puts a method at disposal to force coupling to calcium, which leads to calcium release upon its activation. The calcium release is measurable by fluorescent calcium sensing dyes.
Surprisingly it was found, that the expression of octopamine receptor selected from the group consisting of oa2, preferably from Drosophila melanogaster, Oamb, Oct-beta-2R and Oct-beta-3R and expression of a promiscuous G-alpha protein leads to a new screening assay for insecticides.
GPCRs mediate signal transduction across a cell membrane upon the binding of a ligand to an extracellular portion of a GPCR. The intracellular portion of a GPCR interacts with a G-protein to modulate signal transduction from outside to inside a cell. A GPCR is therefore said to be “coupled” to a G-protein. G-proteins are composed of three polypeptide subunits: an a subunit, which binds and hydolyzes GTP, and a dimeric βγ subunit. Certain G-proteins are considered “promiscuous” G-proteins because their G subunits allow them to couple with GPCRs that normally couple with G-proteins of other families.
No insecticide on the market has been identified as having modulation of octopamine receptor selected from the group consisting of oa2, preferably from Drosophila melanogaster, Oamb, Oct-beta-2R and Oct-beta-3R as its primary mode of action, meaning that a compound does not merely contribute by a “side effect” to a insecticidal activity, but its activity is the key lethal effect.
In another embodiment, the present invention provides a small-conductance calcium-activated potassium channel as new target for insecticides. The present invention puts further a method and an assay at disposal for identifying insecticidally active compounds that reduce the activity of an insect small-conductance Ca2+-activated potassium channel.
Calcium-activated potassium channels are a functionally diverse group of ion channels activated by an increase in intracellular calcium. In mammals they are found in a majority of nerve cells where they contribute to the shaping of action potentials and regulate neuronal excitability. More specifically, their currents underlie the after hyper-polarization that follows an action potential; they also appear to be involved in neuronal firing frequency precision (for review see: Stocker et al., Nature Reviews Neuroscience 5, 2004).
This class of potassium channel exists in three general types based on single channel conductances. The large-, intermediate- and small-conductance channels are termed BK, IK and SK, respectively. In Drosophila, the genes are termed slo, slack and SK, respectively.
Each type of potassium ion channel shows a distinct pharmacological profile. Potassium ion channels have been associated with a number of physiological processes, including regulation of heartbeat, dilation of arteries, release of insulin, excitability of nerve cells, and regulation of renal electrolyte transport. Therefore potassium ion channels are already known as a therapeutic target in the treatment of a number of diseases as disclosed in WO 2005 099711 and US 2005 0239800.
Specifically, SK channels have been shown to have distinct pharmacological profiles. As disclosed in WO 2005 100349, different compounds were found with clinically relevant psycho activity using patch clamp techniques. The evaluated compounds are structurally related to tricyclic antidepressants and include amitriptyline, carbamazepine, chlorpromazine, cyproheptadine, imipramine, tacrine and trifluperazine. Each of the compounds tested was found to block SK2 channel currents with micro-molar affinity. A number of neuromuscular inhibiting agents exist that affect SK channels, e. g. apamin, atracurium, pancuronium and tubocurarine (Shah et al., Br J Pharmacol 129: 627-30 (2000)).
Assays which use SK channels as target for pharmacological active compounds for treatment of diseases are also described:
Recombinant rat brain SK2 channels were expressed in HEK293 mammalian cells to study by patch clamp technique the effect of centrally acting muscle relaxant compounds, like chlorzoxazone (Cao et al., J. Pharmacol. Exp. Ther. 296: 683-689, 2001). The effect of metal ions on the activation of recombinant human SK4 channels has also been studied by patch clamp technique with transformed HEK293 cells (Cao et al., FEBS, 446: 137-141, 1999).
A method of identifying a compound which increases or decreases the potassium ion flux through a calcium-activated potassium SK channel is described in WO 98/11139. Until now, classical molecular targets in insects were acetylcholinesterase, voltage-dependent sodium channels, ionotropic receptors such as nicotinic acetylcholine and GABA receptors. Gautier et al. (J. Pharm. Exp. Therapeutics, jpet.107.128694, 2007) have brought evidence for the participation of calcium-activated potassium channel as an indirect target in insecticide neurotoxicity. They demonstrated by knockdown of DUM (dorsal unpaired median) neuron BK channels by antisense oligonucleotides treatment in DUM neurons from cockroaches (Periplaneta americana) that DMDS (di-methyl disulfide) inhibits calcium-activated potassium currents.
Further specific toxins, which inhibit the activity Ca2+-activated potassium channels, have been identified from several organisms, the most well-known being apamin from bee venom as disclosed for example in U.S. Pat. No. 5,607,843.
Nevertheless, at the moment no insecticide on the market has been identified that has modulation of Ca2+-activated potassium channels, preferably of an SK channel, as its primary mode of action, meaning that a compound does not merely contribute by a “side effect” to a insecticidal activity, but its activity is the key lethal effect.
In general, there is a great demand for the detection of polypeptides which might constitute novel targets for insecticides. The reasons are the above mentioned resistance problems and the ongoing endeavor to identify novel insecticidal active ingredients which are distinguished by a wide as possible spectrum of action, ecological and toxicological acceptability and/or low application rates.
The present invention now provides an insect voltage-gated potassium channel ShaI (Shaker cognate I or Shaker-like) and/or its accessory protein KChIP (potassium channel-interacting protein) as targets for insecticides and a method and an assay for identifying insecticidally active compound that reduces the activity of an insect voltage-gated potassium channel ShaI (Shaker cognate I or Shaker-like) and/or its accessory protein KChIP (potassium channel-interacting protein).
The present invention also provides an Shaker channel and/or a Hyperkinetic beta subunit, preferably H-kv beta subunit A or C subtype respectively as targets for insecticides and a method and an assay for identifying insecticidally active compound that reduces the activity of Shaker channel and/or a Hyperkinetic beta subunit, preferably H-kv beta subunit A or C subtype respectively.
The present invention further provides an octopamine receptor selected from the group consisting of oa2, preferably from Drosophila melanogaster, Oamb, Oct-beta-2R and Oct-beta-3R as targets for insecticides and a method and an assay for identifying insecticidally active compound that reduces the activity of octopamine receptor selected from the group consisting of oa2, preferably from Drosophila melanogaster, Oamb, Oct-beta-2R and Oct-beta-3R.
The present invention furthermore provides an insect small-conductance Ca2+-activated potassium channel as targets for insecticides and a method and an assay for identifying insecticidally active compound that reduces the activity of an insect small-conductance Ca2+-activated potassium channel.
In practice, the detection of novel targets entails great difficulties since the inhibition of the activity of a polypeptide frequently has no further effect on the survival of insects. This may be attributed to the fact that insects may switches to alternative activities, hence the number of protein-encoding genes is at least 3 fold higher than that of microorganisms.
Furthermore, in the case of the SK-channel, with regard to the research results in human medicine, e.g. WO 2005 100349, which teaches that even a inhibition of the potassium ion flow through the potassium ion channels is useful in the treatment of diseases, it was surprisingly that the SK channels of the invention are targets for insecticides as thy influence their survival.
It is an object of the present invention to identify novel targets which are essential for the development or survival of insects, and to provide methods which are suitable for identifying insecticidal active compounds.
We have found that this object is achieved by the use of a polypeptide with the activity of an insect voltage-gated potassium channel ShaI (Shaker cognate I or Shaker-like) and/or its accessory protein KChIP (potassium channel-interacting protein).
One embodiment of the present invention is directed to a method for identifying an insecticidal active compound that reduces the activity of a polypeptide with the activity of an insect voltage-gated potassium channel ShaI (Shaker cognate I or Shaker-like) and/or its accessory protein KChIP (potassium channel-interacting protein) which method comprises:

    
    
        a) assembling in a membrane a polypeptide with the activity of an insect voltage-gated potassium channel ShaI (Shaker cognate I or Shaker-like) and/or its accessory protein KChIP (potassium channel-interacting protein), which is originally not present in said membrane,
        b) applying at one side of the membrane the compound suspected of having the ability to inhibit the activity of said polypeptide which is originally not present said membrane,
        c) determining the activity of said polypeptide and
        d) identifying a compound applied in (b) that reduces the activity of said polypeptide.
    
    


In another embodiment, we have found that this object is achieved by the use of a polypeptide with the activity of an insect Shaker channel and/or a Hyperkinetic beta subunit, preferably H-kv beta subunit A or C subtype respectively.
One embodiment of the present invention is directed to a method for identifying an insecticidal active compound that reduces the activity of a polypeptide with the activity of an insect Shaker channel and/or a Hyperkinetic beta subunit, preferably H-kv beta subunit A or C subtype respectively which method comprises:

    
    
        a) assembling in a membrane a polypeptide with the activity of an insect Shaker channel and/or a Hyperkinetic beta subunit, preferably H-kv beta subunit A or C subtype respectively, which is originally not present in said membrane,
        b) applying at one side of the membrane the compound suspected of having the ability to inhibit the activity of said polypeptide which is originally not present said membrane,
        c) determining the activity of said polypeptide and
        d) identifying a compound applied in (b) that reduces the activity of said polypeptide.
    
    


In another embodiment, we have found that this object is achieved by the use of a polypeptide with the activity of an insect octopamine receptor selected from the group consisting of oa2, preferably from Drosophila melanogaster, Oamb, Oct-beta-2R and Oct-beta-3R.
One embodiment of the present invention is directed to a method for identifying an insecticidal active compound that reduces the activity of a polypeptide with the activity of an insect octopamine receptor selected from the group consisting of oa2, preferably from Drosophila melanogaster, Oamb, Oct-beta-2R and Oct-beta-3R which method comprises:

    
    
        a) assembling in a membrane a polypeptide with the activity of an insect octopamine receptor selected from the group consisting of oa2, preferably from Drosophila melanogaster, Oamb, Oct-beta-2R and Oct-beta-3R, which is originally not present in said membrane,
        b) applying at one side of the membrane the compound suspected of having the ability to inhibit the activity of said polypeptide which is originally not present said membrane,
        c) determining the activity of said polypeptide and
        d) identifying a compound applied in (b) that reduces the activity of said polypeptide.
    
    


In another embodiment, we have found that this object is achieved by the use of a polypeptide with the activity of an insect small-conductance Ca2+-activated potassium channel.
One embodiment of the present invention is directed to a method for identifying a insecticidally active compound that reduces the activity of an insect small-conductance Ca2+-activated potassium channel which method comprises:

    
    
        a) assembling in a membrane a polypeptide with the activity of an insect small-conductance Ca2+-activated potassium channel, which is originally not present in said membrane,
        b) applying at one side of the membrane the compound suspected of having the ability to inhibit the activity of said polypeptide which is originally not present said membrane,
        c) determining the activity of said polypeptide and
        d) identifying a compound applied in (b) that reduces the activity of said polypeptide.
    
    


According to the present invention, a membrane is a structure like a semipermeable sheet or layer which acts as a barrier between two phases or solutions, whereby the membrane is solvent permeable, preferably water permeable. In one embodiment the membrane is a biological membrane, biomembrane or a lipid layer or lipid bilayer. The membrane is preferably composed of a fluid lipid bilayer. In one embodiment the membrane is the outer surface of a cell, cell compartment, vesicle, liposomes (vesicles made of phospholipids which are amphiphilic molecules), polymer vesicles or synthosomes.
Polymer vesicles are often referred as “polymersomes”, which have been studied in detail and progress has been summarized in reviews [Discher et al., Science 2002, 297; 967 973]. Polymersomes or polymer vesicles consist of self-assembled di- or triblock copolymers.
The Synthosome, which is a functionalized nanocompartment system, has been developed for putative biotechnological applications [Nardin et al., Chem. Commun. 2000, 1433 1434]. A Synthosome is a hollow sphere consisting of a mechanically stable vesicle with a block copolymer membrane and an engineered transmembrane protein acting as the selective gate.
The, preferably insect, voltage-gated potassium channel ShaI (Shaker cognate I or Shaker-like) and/or its accessory protein KChIP (potassium channel-interacting protein) of the invention is assembled or intercalated, embedded or integrated, terms which are synonymously and interchangeable, in the membrane.
The, preferably insect, Shaker channel and/or a Hyperkinetic beta subunit, preferably H-kv beta subunit A or C subtype respectively of the invention is assembled or intercalated, embedded or integrated, terms which are synonymously and interchangeable, in the membrane.
The, preferably insect, octopamine receptor selected from the group consisting of oa2, preferably from Drosophila melanogaster, Oamb, Oct-beta-2R and Oct-beta-3R of the invention is assembled or intercalated, embedded or integrated, terms which are synonymously and interchangeable, in the membrane.
The insect small-conductance Ca2+-activated potassium channel is assembled or intercalated, embedded or integrated, terms which are synonymously and interchangeable, in the membrane.
For the purposes of the invention, as a rule the plural is intended to encompass the singular and vice versa.
Unless otherwise specified, the terms “polynucleotides”, “nucleic acid” and “nucleic acid molecule” are interchangeably in the present context. Unless otherwise specified, the terms “peptide”, “polypeptide” and “protein” are interchangeably in the present context. The term “sequence” may relate to polynucleotides, nucleic acids, nucleic acid molecules, peptides, polypeptides and proteins, depending on the context in which the term “sequence” is used. The terms “gene(s)”, “polynucleotide”, “nucleic acid sequence”, “nucleotide sequence”, or “nucleic acid molecule(s)” as used herein refers to a polymeric form of nucleotides of any length, either ribonucleotides or de-oxyribonucleotides. The terms refer only to the primary structure of the molecule.
Thus, the terms “gene(s)”, “polynucleotide”, “nucleic acid sequence”, “nucleotide sequence”, or “nucleic acid molecule(s)” as used herein include double- and single-stranded DNA and RNA. They also include known types of modifications, for example, methylation, “caps”, substitutions of one or more of the naturally occurring nucleotides with an analog. Preferably, the DNA or RNA sequence comprises a coding sequence encoding the herein defined polypeptide.
A “coding sequence” is a nucleotide sequence, which is transcribed into a RNA, e.g. a regulatory RNA, such as a miRNA, a ta-siRNA, cosuppression molecule, a RNAi, a ribozyme, etc. or into a mRNA which is translated into a polypeptide when placed under the control of appropriate regulatory sequences. The boundaries of the coding sequence are determined by a translation start codon at the 5′-terminus and a translation stop codon at the 3′-terminus. A coding sequence can include, but is not limited to mRNA, cDNA, recombinant nucleotide sequences or genomic DNA, while introns may be present as well under certain circumstances.
As used in the present context a nucleic acid molecule may also encompass the untranslated sequence located at the 3′ and at the 5′ end of the coding gene region, for example at least 500, preferably 200, especially preferably 100, nucleotides of the sequence upstream of the 5′ end of the coding region and at least 100, preferably 50, especially preferably 20, nucleotides of the sequence downstream of the 3′ end of the coding gene region. In the event for example the antisense, RNAi, snRNA, dsRNA, siRNA, miRNA, ta-siRNA, cosuppression molecule, ribozyme etc. technology is used coding regions as well as the 5′- and/or 3′-regions can advantageously be used.
However, it is often advantageous only to choose the coding region for cloning and expression purposes.
“Polypeptide” refers to a polymer of amino acid (amino acid sequence) and does not refer to a specific length of the molecule. Thus peptides and oligopeptides are included within the definition of polypeptide. This term does also refer to or include post-translational modifications of the polypeptide, for example, glycosylations, acetylations, phosphorylations and the like. Included within the definition are, for example, polypeptides containing one or more analogs of an amino acid (including, for example, unnatural amino acids, etc.), polypeptides with substituted linkages, as well as other modifications known in the art, both naturally occurring and non-naturally occurring.
The terms “comprise” or “comprising” and grammatical variations thereof when used in this specification are to be taken to specify the presence of stated features, integers, steps or components or groups thereof, but not to preclude the presence or addition of one or more other features, integers, steps, components or groups thereof.
The terms “reduction”, “repression”, “decrease” or “inhibition” relate to a corresponding change of a property in an organism, a part of an organism such as a tissue, or in a cell. Under “change of a property” it is understood that the activity is changed in a specific volume or in a specific amount of protein relative to a corresponding volume or amount of protein of a control, reference or wild type.
The terms “reduction”, “repression”, “decrease” or “inhibition” include the change of said property in only parts of the subject of the present invention, for example, the modification can be found in compartment of a cell, like an organelle, or in a part of a organism, like tissue, wing, leg, trunk etc. Preferably, the “reduction”, “repression”, “decrease” or “inhibition” is found cellular, thus the term “reduction, decrease or inhibition of an activity” relates to the cellular reduction, decrease or inhibition compared to the wild type cell or to the control cell.
Accordingly, the term “reduction”, “repression”, “decrease” or “inhibition” means that the specific activity of a gene product, a protein or a regulatory RNA as well as the amount of a compound or metabolite, e.g. of a polypeptide, a nucleic acid molecule, a ion or an encoding mRNA or DNA, can be reduced, decreased or inhibited in a specific volume. The terms “reduction”, “repression”, “decrease” or “inhibition” include that the reason for said “reduction”, “repression”, “decrease” or “inhibition” can be a chemical compound that is administered to the organism or part thereof.
The terms “reduction”, “repression”, or “decrease” are exchangeable. The term “reduction” shall include the terms “repression”, “decrease” or “inhibition” if not otherwise specified.
Reduction is also understood as meaning the modification of the activity. In this context, the function or activity, e.g. the “functional activity” or the “biological activity”, is reduced by at least 10%, advantageously 20%, preferably 30%, especially preferably 40%, 50% or 60%, very especially preferably 70%, 80%, 85% or 90% or more, very especially preferably are 95%, more preferably are 99% or more in comparison to the control, reference or wild type. Most preferably the reduction, decrease or deletion in activity amounts to essentially 100%. Thus, a particularly advantageous embodiment is the inactivation, e.g. inhibition of the function of a compound, e.g. a polypeptide or a nucleic acid molecule.
The terms “wild type”, “control” or “reference” are exchangeable and can be a cell or a part of organisms such as an organelle or a tissue, or an organism, in particular an insect, which was not modified or treated according to the herein described process according to the invention. Accordingly, the cell or a part of organisms such as an organelle or a tissue, or an organism, in particular an insect used as wild type, control or reference corresponds to the cell, organism or part thereof as much as possible and is in any other property but in the result of the process of the invention as identical to the subject matter of the invention as possible. Thus, the wild type, control, or reference is treated identically or as identical as possible, saying that only conditions or properties might be different which do not influence the quality of the tested property.
Preferably, any comparison is carried out under analogous conditions. The term “analogous conditions” means that all conditions such as, for example, culture or growing conditions, assay conditions (such as buffer composition, temperature, substrates, pathogen strain, concentrations and the like) are kept identical between the experiments to be compared.
The “reference”, “control”, or “wild type” is preferably a subject, e.g. an organelle, a cell, a tissue, an organism, in particular an insect, which was not modified or treated according to the herein described process of the invention and is in any other property as similar to the subject matter of the invention as possible. The reference, control, or wild type is in its genome, transcriptome, proteome or metabolome as similar as possible to the subject of the present invention. Preferably, the term “reference-” “control-” or “wild type-”-organelle, -cell, -tissue or -organism, relates to an organelle, cell, tissue or organism, which is nearly genetically identical to the organelle, cell, tissue or organism, of the present invention or a part thereof preferably 95%, more preferred are 98%, even more preferred are 99.00%, in particular 99.10%, 99.30%, 99.50%, 99.70%, 99.90%, 99.99%, 99.999% or more. Most preferable the “reference”, “control”, or “wild type” is a subject, e.g. an organelle, a cell, a tissue, an organism, which is genetically identical to the organism, cell or organelle used according to the process of the invention except that the responsible or activity conferring nucleic acid molecules or the gene product encoded by them are amended, manipulated, exchanged or introduced according to the inventive process.
Preferably, the reference, control or wild type differs form the subject of the present invention only in the activity of the polypeptide of the invention or the polypeptide used in the method of the invention.
“Significant decrease”: referring to the activity of the polypeptide encoded by a nucleic acid sequence according to the invention, this is understood as meaning a decrease in activity of the polypeptide treated with a test compound in comparison with the control. e.g. in comparison with the activity of the polypeptide which has not been incubated with the test compound, with a magnitude outside a measurement error.
Reference to the “functional activity” of an ion channel should be understood as a reference to any one or more of the functions and/or traits which an ion channel performs or is involved in.
Reference to the “functional activity” of an octopamine receptor should be understood as a reference to any one or more of the functions and/or traits which an octopamine receptor performs or is involved in.
In one embodiment the term “functional activity” or “biological activity” of a polypeptide with the activity of an insect voltage-gated potassium channel ShaI (Shaker cognate I or Shaker-like) and/or its accessory protein KChIP (potassium channel-interacting protein) is defined by the transport of potassium ions across membranes.
In another embodiment the term “functional activity” or “biological activity” of a polypeptide with the activity of an insect Shaker channel and/or a Hyperkinetic beta subunit, preferably H-kv beta subunit A or C subtype respectively is defined by the transport of potassium ions across membranes.
In another embodiment the term “functional activity” or “biological activity” of a polypeptide with the activity of an octopamine receptor selected from the group consisting of oa2, preferably from Drosophila melanogaster, Oamb, Oct-beta-2R and Oct-beta-3R is defined by the fact, that octopamine receptors are selectively blocked by α-adrenergic antagonists and activated by α-adrenergic agonists.
In another embodiment the term “functional activity” or “biological activity” of a polypeptide with the activity of an insect small-conductance Ca2+-activated potassium channel is defined by the transport of potassium ions across membranes. This transport is activated by calcium ions, with a half maximal activation or Ca2+ sensitivity K0.5 selected from the group of intervals 200-1000 nM, 300-900 nM, 300-800 nM and 400-800 nM. The SK channels of the invention have a single-channel conductance selected from the group of intervals 2-20 pS, 3-20 pS, 4-15 pS, 5-12 pS and 5-10 pS. The biological activity of a polypeptide of the invention is apamine-insensitive.
The term “activity” of a compound refers to the function of a compound in a biological system such as a cell, an organ or an organism. For example, the term “activity” of a compound refers to the enzymatic function, regulatory function or its function as binding partner, transporter, regulator, or carrier, etc of a compound.
Insecticidal activity of a compound refers to the ability of said compound to kill or paralyze insects, or to inhibit the insect development or growth in such a manner that the insects provide less damage. Compounds having insecticidal activity are also referred to as toxic to insects. Insecticidal activity of a compound induce not just to death of insects, but also include other detrimental effects on insects such as sickness, anti-feedant activity, growth retardation, reduced reproductive ability and reduced fecundity.
A compound with a insecticidal activity as used herein is a “insecticide”. The term “insecticide” generally refers to chemicals, biological agents, and other compounds that adversely affect insect viability, e.g., that kill, paralyze, sterilize or otherwise disable insect species in the areas of agricultural crop protection, human and animal health.
In one embodiment the method of the invention is implemented with a membrane which comprises at least one polypeptide encoded by a nucleic acid molecule selected from the group consisting of:

    
    
        a) a nucleic acid molecule encoding the polypeptide shown in SEQ ID NO: 2, 6, 10, 14, 18, 22, 26 and/or 30;
        b) a nucleic acid molecule shown in SEQ ID NO: 1, 5, 9, 13, 17, 21, 25 and/or 29;
        c) a nucleic acid molecule, which, as a result of the degeneracy of the genetic code, can be derived from a polypeptide sequence according to SEQ ID NO: 2, 6, 10, 14, 18, 22, 26 and/or 30;
        d) a nucleic acid molecule having at least 50% identity with the nucleic acid molecule sequence of a polynucleotide comprising the nucleic acid molecule shown in SEQ ID NO: 1, 5, 9, 13, 17, 21, 25 and/or 29;
        e) a nucleic acid molecule encoding a polypeptide having at least 50% identity with the amino acid sequence of the polypeptide encoded by the nucleic acid molecule of (a) to (c) and having the activity of an insect voltage-gated potassium channel ShaI (Shaker cognate I or Shaker-like) and/or its accessory protein KChIP (potassium channel-interacting protein) respectively;
        f) nucleic acid molecule which hybridizes with a nucleic acid molecule of (a) to (c) under stringent hybridization conditions;
        g) a nucleic acid molecule encoding a polypeptide which can be isolated with the aid of monoclonal or polyclonal antibodies made against a polypeptide encoded by one of the nucleic acid molecules of (a) to (e) and having the activity of a an insect voltage-gated potassium channel ShaI (Shaker cognate I or Shaker-like) and/or its accessory protein KChIP (potassium channel-interacting protein) respectively;
        h) a nucleic acid molecule encoding a polypeptide comprising the consensus sequence as depicted in SEQ ID NO: 33 and/or 34 respectively or one or more motifs as depicted in SEQ ID NO: 35, 36, 37, 38, 39, 40, 41, 42, 43, 44, 45, 46, 47, 48, 49, 50, 51, 52, 53, 54 and/or 55, and/or 56, 57, 58, 59, 60, 61, 62, 63, 64, 65, 66, 67, 68, 69, 70 and/or 71 respectively;
        i) nucleic acid molecule which comprises a polynucleotide, which is obtained by amplifying a cDNA library or a genomic library using the primers in SEQ ID NO: 3, 4; 7, 8; 11, 12; 15, 16; 19, 20; 23, 24; 27, 28 and/or 31, 32 respectively;
        and
        j) a nucleic acid molecule which is obtainable by screening a suitable nucleic acid library under stringent hybridization conditions with a probe comprising a complementary sequence of a nucleic acid molecule of (a) or (b) or with a fragment thereof, having at least 15 nt, preferably 20 nt, 30 nt, 50 nt, 100 nt, 200 nt or 500 nt of a nucleic acid molecule complementary to a nucleic acid molecule sequence characterized in (a) to (e) and encoding a polypeptide and having the activity of a an insect voltage-gated potassium channel ShaI (Shaker cognate I or Shaker-like) and/or its accessory protein KChIP (potassium channel-interacting protein) respectively.
    
    


In one embodiment the method of the invention is implemented with a membrane which comprises at least a functional equivalent or homologue of a polypeptide encoded by a nucleic acid molecule selected from the group as depicted above under item a), b), c), d), e), f), g), h), i) or j), for example a ShaI_delN mutant, which has an N-terminal deletion for the 2-40 amino acid coding region.
In one embodiment the method of the invention is implemented with a membrane which comprises at least a functional equivalent or homologue of a polypeptide with the activity of a voltage-gated potassium channel ShaI as depicted in SEQ ID NO: 2, 6, 10, 14, 18 and/or 22 or homolog thereof and/or a ShaI_delN mutant, which has an N-terminal deletion for the 2-40 amino acid coding region linked to a polypeptide with the activity of its accessory protein KChIP as depicted in SEQ ID NO: 26 and/or 30 or homolog thereof.
In another embodiment the method of the invention is implemented with a membrane which comprises at least one polypeptide encoded by a nucleic acid molecule selected from the group consisting of:
a) a nucleic acid molecule encoding a polypeptide comprising the polypeptide shown in SEQ ID NO: 73, 75, 77, 79, 81, 83, 85 and/or 87;
b) a nucleic acid molecule comprising the nucleic acid molecule shown in SEQ ID NO: 72, 74, 76, 78, 80, 82, 84 and/or 86;
c) a nucleic acid molecule, which, as a result of the degeneracy of the genetic code, can be derived from a polypeptide comprising a polypeptide sequence according to SEQ ID NO: 73, 75, 77, 79, 81, 83, 85 and/or 87;
d) a nucleic acid molecule having at least 50% identity with the nucleic acid molecule sequence of a polynucleotide comprising the nucleic acid molecule shown in SEQ ID NO: 72, 74, 76, 78, 80, 82, 84 and/or 86;
e) a nucleic acid molecule encoding a polypeptide having at least 50% identity with the amino acid sequence of the polypeptide encoded by the nucleic acid molecule of (a) to (c) and having the activity of an insect Shaker channel and/or a Hyperkinetic beta subunit, preferably H-kv beta subunit A or C subtype respectively;
f) nucleic acid molecule which hybridizes with a nucleic acid molecule of (a) to (c) under stringent hybridization conditions;
g) a nucleic acid molecule encoding a polypeptide which can be isolated with the aid of monoclonal or polyclonal antibodies made against a polypeptide encoded by one of the nucleic acid molecules of (a) to (e) and having the activity of a an insect Shaker channel and/or a Hyperkinetic beta subunit, preferably H-kv beta subunit A or C subtype respectively;
h) a nucleic acid molecule encoding a polypeptide comprising the consensus sequence as depicted in SEQ ID NO: 102 and/or 103 respectively or one or more motifs as depicted in SEQ ID NO: 104, 105, 106, 107, 108, 109, 110, 111, 112, 113, 114, 115, 116, 117, 118, 119, 120, 121, 122, 123, 124 and/or 125 and/or 126, 127 and/or 128 respectively;
i) nucleic acid molecule which comprises a polynucleotide, which is obtained by amplifying a cDNA library or a genomic library using the primers in SEQ ID NO: 88, 89; 90, 91; 92, 93; 94, 95; 96, 97; 98, 99 and/or 100, 101 respectively;
and
j a nucleic acid molecule which is obtainable by screening a suitable nucleic acid library under stringent hybridization conditions with a probe comprising a complementary sequence of a nucleic acid molecule of (a) or (b) or with a fragment thereof, having at least 15 nt, preferably 20 nt, 30 nt, 50 nt, 100 nt, 200 nt or 500 nt of a nucleic acid molecule complementary to a nucleic acid molecule sequence characterized in (a) to (e) and encoding a polypeptide and having the activity of a an insect Shaker channel and/or a Hyperkinetic beta subunit, preferably H-kv beta subunit A or C subtype respectively.
In one embodiment the method of the invention is implemented with a membrane which comprises at least a functional equivalent or homologue of a polypeptide encoded by a nucleic acid molecule selected from the group as depicted above under item a), b), c), d), e), f), g), h), i) orj), whereby the nucleic acid molecule comprises a Kozak sequence (e.g. ACCATG).
In one embodiment the method of the invention is implemented with a membrane which comprises at least a functional equivalent or homologue of a polypeptide encoded by a nucleic acid molecule selected from the group as depicted above under item a), b), c), d), e), f), g), h), i) orj), whereby the nucleic acid molecule comprises a sequence coding for a 400 bp or 500 bp 5′-fragment of the Shaker channel, preferably comprising the Shaker ATG codon, preferably together with a proper Kozak, and/or 1770 bp fragment of the Shaker channel and/or for a Hyperkinetic beta subunit, preferably H-kv beta subunit A or C subtype.
In one embodiment the method of the invention is implemented with a membrane which comprises any combination of at least a polypeptide selected from the group consisting of SEQ ID NO: 73, 75, 77, 79, 81 and 83 or a functional equivalent or homologue thereof and a polypeptide selected from the group consisting of SEQ ID NO: 85 and 87 or a functional equivalent or homologue thereof.
In another embodiment the method of the invention is implemented with a membrane which comprises at least one polypeptide encoded by a nucleic acid molecule selected from the group consisting of:
a) a nucleic acid molecule encoding the polypeptide shown in SEQ ID NO: 130, 134, 138, 142, 146, 150, 154, 158, 162, 166, 170 and/or 174;
b) a nucleic acid molecule shown in SEQ ID NO: 129, 133, 137, 141, 145, 149, 153, 157, 161, 165, 169 and/or 173;
a nucleic acid molecule, which, as a result of the degeneracy of the genetic code, can be derived from a polypeptide sequence according to SEQ ID NO: 130, 134, 138, 142, 146, 150, 154, 158, 162, 166, 170 and/or 174;
d) a nucleic acid molecule having at least 50% identity with the nucleic acid molecule sequence of a polynucleotide comprising the nucleic acid molecule shown in SEQ ID NO: 129, 133, 137, 141, 145, 149, 153, 157, 161, 165, 169 and/or 173;
e) a nucleic acid molecule encoding a polypeptide having at least 50% identity with the amino acid sequence of the polypeptide encoded by the nucleic acid molecule of (a) to (c) and having the activity of an insect octopamine receptor selected from the group consisting of oa2, preferably from Drosophila melanogaster, Oamb, Oct-beta-2R and Oct-beta-3R;
f) nucleic acid molecule which hybridizes with a nucleic acid molecule of (a) to (c) under stringent hybridization conditions;
g) a nucleic acid molecule encoding a polypeptide which can be isolated with the aid of monoclonal or polyclonal antibodies made against a polypeptide encoded by one of the nucleic acid molecules of (a) to (e) and having the activity of a an insect octopamine receptor selected from the group consisting of oa2, preferably from Drosophila melanogaster, Oamb, Oct-beta-2R and Oct-beta-3R;
h) a nucleic acid molecule encoding a polypeptide comprising the consensus sequence as depicted in SEQ ID NO: 177, 178 and/or 179 respectively or one or more motifs as depicted in SEQ ID NO: 180, 181, 182, 183, 184, 185, 186, 187, 188, 189 and/or 190, and/or 191, 192, 193, 194, 195, 196, 197, 198, 199, 200, 201, 202, 203, 204, 205, 206, 207 and/or 208, and/or 209, 210, 211, 212, 213, 214, 215, 216, 217, 218, 219, 220, 221, 222, 223, 224, 225 and/or 226 respectively;
i) nucleic acid molecule which comprises a polynucleotide, which is obtained by amplifying a cDNA library or a genomic library using the primers in SEQ ID NO: 131, 132; 135, 136; 139, 140; 143, 144; 147, 148; 151, 152; 155, 156, 159, 160; 163, 164; 167, 168; 171, 172 and/or 175, 176;
and
j) a nucleic acid molecule which is obtainable by screening a suitable nucleic acid library under stringent hybridization conditions with a probe comprising a complementary sequence of a nucleic acid molecule of (a) or (b) or with a fragment thereof, having at least 15 nt, preferably 20 nt, 30 nt, 50 nt, 100 nt, 200 nt or 500 nt of a nucleic acid molecule complementary to a nucleic acid molecule sequence characterized in (a) to (e) and encoding a polypeptide and having the activity of a an insect octopamine receptor selected from the group consisting of oa2, preferably from Drosophila melanogaster, Oamb, Oct-beta-2R and Oct-beta-3R.
In one embodiment the method of the invention is implemented with a membrane which comprises at least a functional equivalent or homologue of a polypeptide encoded by a nucleic acid molecule selected from the group as depicted above under item a), b), c), d), e), f), g), h), i) or j) and additionally a marker protein, e.g. GFP, whereby it is preferably linked with the polypeptide of the invention, e.g. encoded by a nucleic acid molecule selected from the group as depicted above under item a), b), c), d), e), f), g), h), i) or j).
In one embodiment the method of the invention is implemented with a membrane which comprises at least a functional equivalent or homologue of a polypeptide as shown in SEQ ID NO: 46 encoded by a nucleic acid molecule as depicted in SEQ ID NO: 173 or a homolog thereof.
In one embodiment the method of the invention is implemented with a membrane which comprises at least a functional equivalent or homologue of a polypeptide encoded by a nucleic acid molecule selected from the group as depicted above under item a), b), c), d), e), f), g), h), i) orj) and additionally a “promiscuous” G-protein, whose G subunits allow the G-proteins to couple with GPCRs that normally couple with G-proteins of other families, whereby the “promiscuous” G-protein is preferably linked with the polypeptide of the invention, e.g. encoded by a nucleic acid molecule selected from the group as depicted above under item a), b), c), d), e), f), g), h), i) or j).
In one embodiment the method of the invention is implemented with a membrane which comprises at least a functional equivalent or homologue of a polypeptide encoded by a nucleic acid molecule selected from the group as depicted above under item a), b), c), d), e), f), g), h), i) or j) and additionally a marker protein, e.g. GFP, and/or additionally a “promiscuous” G-protein, whereby marker protein and/or the “promiscuous” G-protein is preferably linked with the polypeptide of the invention, e.g. encoded by a nucleic acid molecule selected from the group as depicted above under item a), b), c), d), e), f), g), h), i) or j).
In one embodiment the method of the invention is implemented with a membrane which comprises at least a functional equivalent or homologue of a polypeptide as shown in SEQ ID NO: 46 encoded by a nucleic acid molecule as depicted in SEQ ID NO: 173 or a homolog thereof and additionally a “promiscuous” G-protein whereby the protein is preferably linked with the.
In one embodiment the “promiscuous” G-protein is a promiscuous G-alpha-16-protein or a homolog thereof.
In another embodiment the method of the invention is implemented with a membrane which comprises at least one polypeptide encoded by a nucleic acid molecule selected from the group consisting of:
a) a nucleic acid molecule encoding the polypeptide shown in SEQ ID NO: 228, 230, 232;
b) a nucleic acid molecule shown in SEQ ID NO: 227, 229, 231;
c) a nucleic acid molecule, which, as a result of the degeneracy of the genetic code, can be derived from a polypeptide sequence according to SEQ ID NO: 228, 230, 232;
d) a nucleic acid molecule having at least 50% identity with the nucleic acid molecule sequence of a polynucleotide comprising the nucleic acid molecule shown in SEQ ID NO: 227, 229, 231;
e) a nucleic acid molecule encoding a polypeptide having at least 50% identity with the amino acid sequence of the polypeptide encoded by the nucleic acid molecule of (a) to (c) and having the activity of a small-conductance Ca2+-activated potassium channel;
f) nucleic acid molecule which hybridizes with a nucleic acid molecule of (a) to (c) under stringent hybridization conditions;
g) a nucleic acid molecule encoding a polypeptide which can be isolated with the aid of monoclonal or polyclonal antibodies made against a polypeptide encoded by one of the nucleic acid molecules of (a) to (e) and having the activity of a small-conductance Ca2+-activated potassium channel;
h) a nucleic acid molecule encoding a polypeptide comprising the consensus sequence as shown in SEQ ID NO: 239 or one or more motifs selected from the group consisting of SEQ ID NO: 240, 241, 242, 243, 244, 245, 246, 247, 248, 249, 250, 251, 252 and 253;
i) nucleic acid molecule which comprises a polynucleotide, which is obtained by amplifying a cDNA library or a genomic library using the primers in SEQ ID NO: 233, 234, 235, 236; and 237, 238 respectively;
and
j) a nucleic acid molecule which is obtainable by screening a suitable nucleic acid library under stringent hybridization conditions with a probe comprising a complementary sequence of a nucleic acid molecule of (a) or (b) or with a fragment thereof, having at least 15 nt, preferably 20 nt, 30 nt, 50 nt, 100 nt, 200 nt or 500 nt of a nucleic acid molecule complementary to a nucleic acid molecule sequence characterized in (a) to (e) and encoding a polypeptide and having the activity of a small-conductance Ca2+-activated potassium channel.
In one embodiment the method of the invention is implemented with a membrane which comprises at least a functional equivalent or homologue of a polypeptide encoded by a nucleic acid molecule selected from the group as depicted above under item a), b), c), d), e), f), g), h), i) or j).
The term “functional equivalent” of a polypeptide as depicted above is a polypeptide which confers essentially the activity of a polypeptide as depicted in SEQ ID NO: 2, 6, 10, 14, 18, 22, 26 and/or 30, in the case of the shaker channel and/or a Hyperkinetic beta subunit SEQ ID NO:73, 75, 77, 79, 81, 83, 85 and/or 87, in the case of the G-protein coupled receptor SEQ ID NO: 130, 134, 138, 142, 146, 150, 154, 158, 162, 166, 170 and/or 174 and in the case of the SK-channel SEQ ID NO: 228, 230, 232.
The term “functional equivalent” of a nucleic acid molecule as depicted above is a polynucleotide which confers essentially the activity of a nucleic acid molecule as depicted in SEQ ID NO: 1, 5, 9, 13, 17, 21, 25 and/or 29, in the case of the shaker channel and/or a Hyperkinetic beta subunit SEQ ID NO: 72, 74, 76, 78, 80, 82, 84 and/or 86, in the case of the G-protein coupled receptor SEQ ID NO: 129, 133, 137, 141, 145, 149, 153, 157, 161, 165, 169 and/or 173 and in the case of the SK-channel SEQ ID NO: 227, 229, 231.
In accordance with the invention, a protein or polypeptide has the activity of a polypeptide as depicted in SEQ ID NO: 2, 6, 10, 14, 18, 22, 26 and/or 30, in the case of the shaker channel and/or a Hyperkinetic beta subunit SEQ ID NO: 73, 75, 77, 79, 81, 83, 85 and/or 87, in the case of the G-protein coupled receptor SEQ ID NO: 130, 134, 138, 142, 146, 150, 154, 158, 162, 166, 170 and/or 174 and in the case of the SK-channel SEQ ID NO:228, 230, 232 if the reduction, repression, decrease or inhibition of its activity mediates a decrease of potassium flux through the membrane.
In accordance with the invention, a nucleic acid molecule or polynucleotide has the activity of a nucleic acid molecule as depicted in SEQ ID NO: 1, 5, 9, 13, 17, 21, 25 and/or 29, in the case of the shaker channel and/or a Hyperkinetic beta subunit SEQ ID NO: 72, 74, 76, 78, 80, 82, 84 and/or 86, in the case of the G-protein coupled receptor SEQ ID NO: 129, 133, 137, 141, 145, 149, 153, 157, 161, 165, 169 and/or 173 and in the case of the SK-channel SEQ ID NO: 227, 229, 231 if the reduction, repression, decrease or inhibition of its expression mediates a decrease of potassium flux through the membrane.
Homologues (=homologs) of the polypeptide of the present invention, in particular homologues of a polypeptide which is encoded by or which is comprising a nucleic acid molecule as shown in SEQ ID NO: 1, 5, 9, 13, 17, 21, 25 and/or 29, or a polypeptide comprising the polypeptide, the consensus sequence as shown in SEQ ID NO: 33 and/or 34 respectively or one or more motifs selected from the group consisting of SEQ ID NO: 35, 36, 37, 38, 39, 40, 41, 42, 43, 44, 45, 46, 47, 48, 49, 50, 51, 52, 53, 54 and/or 55, and/or 56, 57, 58, 59, 60, 61, 62, 63, 64, 65, 66, 67, 68, 69, 70 and/or 71 respectively, can be derived from any organisms as long as the homologue confers the herein mentioned activity, i.e. it is a functional equivalent of said molecules.
In the case of the shaker channel and/or a Hyperkinetic beta subunit, homologues (=homologs) of the polypeptide of the present invention, in particular homologues of a polypeptide which is encoded by or which is comprising a nucleic acid molecule as shown in SEQ ID NO: 72, 74, 76, 78, 80, 82, 84 and/or 86, or a polypeptide comprising the polypeptide, the consensus sequence as shown in SEQ ID NO: 102 and/or 103 respectively or one or more motifs selected from the group consisting of SEQ ID NO: 104, 105, 106, 107, 108, 109, 110, 111, 112, 113, 114, 115, 116, 117, 118, 119, 120, 121, 122, 123, 124 and/or 125 and/or 126, 127 and/or 128 respectively, can be derived from any organisms as long as the homologue confers the herein mentioned activity, i.e. it is a functional equivalent of said molecules.
In the case of the G-protein coupled receptor, homologues (=homologs) of the polypeptide of the present invention, in particular homologues of a polypeptide which is encoded by or which is comprising a nucleic acid molecule as shown in SEQ ID NO: 129, 133, 137, 141, 145, 149, 153, 157, 161, 165, 169 and/or 173, or a polypeptide comprising the polypeptide, the consensus sequence as shown in SEQ ID NO: 177, 178 and/or 179 respectively or one or more motifs selected from the group consisting of SEQ ID NO: 180, 181, 182, 183, 184, 185, 186, 187, 188, 189 and/or 190, and/or 191, 192, 193, 194, 195, 196, 197, 198, 199, 200, 201, 202, 203, 204, 205, 206, 207 and/or 208, and/or 209, 210, 211, 212, 213, 214, 215, 216, 217, 218, 219, 220, 221, 222, 223, 224, 225 and/or 226 respectively, can be derived from any organisms as long as the homologue confers the herein mentioned activity, i.e. it is a functional equivalent of said molecules.
In the case of the SK-channel, homologues (=homologs) of the polypeptide of the present invention, in particular homologues of a polypeptide which is encoded by or which is comprising a nucleic acid molecule as shown in SEQ ID NO: 227, 229, 231, or a polypeptide comprising the polypeptide, the consensus sequence as shown in SEQ ID NO: 239 or one or more motifs selected from the group consisting of SEQ ID NO: 240, 241, 242, 243, 244, 245, 246, 247, 248, 249, 250, 251, 252 and 253, can be derived from any organisms as long as the homologue confers the herein mentioned activity, i.e. it is a functional equivalent of said molecules.
Further, according to the present invention, the term “homologue” relates to the sequence of an organism having preferably the highest or essentially the highest sequence homology to the herein mentioned or listed sequences of all expressed sequences of said organism.
The person skilled in the art knows how to find, identify and confirm, that a putative homologue has the same activity as described herein. If known, the biological function or activity in an organism essentially relates or corresponds to the activity or function as described for the genes mentioned in SEQ ID NO: 1, 5, 9, 13, 17, 21, 25 and/or 29, in the case of the shaker channel and/or a Hyperkinetic beta subunit SEQ ID NO: 72, 74, 76, 78, 80, 82, 84 and/or 86, in the case of the G-protein coupled receptor SEQ ID NO: 129, 133, 137, 141, 145, 149, 153, 157, 161, 165, 169 and/or 173, and in the case of the SK-channel SEQ ID NO: 227, 229, 231.
Accordingly, in one embodiment, the homologue or the functional equivalent comprises the sequence of a polypeptide encoded by a nucleic acid molecule comprising a sequence indicated in SEQ ID NO: 1, 5, 9, 13, 17, 21, 25 and/or 29 or a polypeptide sequence as depicted in SEQ ID NO: 2, 6, 10, 14, 18, 22, 26 and/or 30, a consensus sequence as shown in SEQ ID NO: 33 and/or 34 respectively or one or more motifs selected from the group consisting of SEQ ID NO: 35, 36, 37, 38, 39, 40, 41, 42, 43, 44, 45, 46, 47, 48, 49, 50, 51, 52, 53, 54 and/or 55, and/or 56, 57, 58, 59, 60, 61, 62, 63, 64, 65, 66, 67, 68, 69, 70 and/or 71 respectively or it is the expression product of a nucleic acid molecule comprising a polynucleotide indicated in SEQ ID NO: 2, 6, 10, 14, 18, 22, 26 and/or 30.
In the case of the shaker channel and/or a Hyperkinetic beta subunit, accordingly, in one embodiment, the homologue or the functional equivalent comprises the sequence of a polypeptide encoded by a nucleic acid molecule comprising a sequence indicated in SEQ ID NO: 72, 74, 76, 78, 80, 82, 84 and/or 86 or a polypeptide sequence as depicted in SEQ ID NO: 73, 75, 77, 79, 81, 83, 85 and/or 87, a consensus sequence as shown in SEQ ID NO: 102 and/or 103 respectively or one or more motifs selected from the group consisting of SEQ ID NO: 104, 105, 106, 107, 108, 109, 110, 111, 112, 113, 114, 115, 116, 117, 118, 119, 120, 121, 122, 123, 124 and/or 125 and/or 126, 127 and/or 128 respectively or it is the expression product of a nucleic acid molecule comprising a polynucleotide indicated in SEQ ID NO: 73, 75, 77, 79, 81, 83, 85 and/or 87.
In the case of the G-protein coupled receptor, accordingly, in one embodiment, the homologue or the functional equivalent comprises the sequence of a polypeptide encoded by a nucleic acid molecule comprising a sequence indicated in SEQ ID NO: 129, 133, 137, 141, 145, 149, 153, 157, 161, 165, 169 and/or 173 or a polypeptide sequence as depicted in SEQ ID NO: 130, 134, 138, 142, 146, 150, 154, 158, 162, 166, 170 and/or 174, a consensus sequence as shown in SEQ ID NO: 177, 178 and/or 179 respectively or one or more motifs selected from the group consisting of SEQ ID NO: 180, 181, 182, 183, 184, 185, 186, 187, 188, 189 and/or 190, and/or 191, 192, 193, 194, 195, 196, 197, 198, 199, 200, 201, 202, 203, 204, 205, 206, 207 and/or 208, and/or 209, 210, 211, 212, 213, 214, 215, 216, 217, 218, 219, 220, 221, 222, 223, 224, 225 and/or 226 respectively or it is the expression product of a nucleic acid molecule comprising a polynucleotide indicated in SEQ ID NO: 130, 134, 138, 142, 146, 150, 154, 158, 162, 166, 170 and/or 174.
In the case of the SK-channel, accordingly, in one embodiment, the homologue or the functional equivalent comprises the sequence of a polypeptide encoded by a nucleic acid molecule comprising a sequence indicated in SEQ ID NO: 227, 229, 231 or a polypeptide sequence as depicted in SEQ ID NO: 228, 230, 232, a consensus sequence as shown in SEQ ID NO: 239 or one or more motifs selected from the group consisting of SEQ ID NO: 240, 241, 242, 243, 244, 245, 246, 247, 248, 249, 250, 251, 252 and 253 or it is the expression product of a nucleic acid molecule comprising a polynucleotide indicated in SEQ ID NO: 228, 230, 232.
The herein disclosed information about sequence, activity, consensus sequence, polypeptide motifs and tests leads the person skilled in the art to the respective homologous or functional equivalent expression product in an organism.
In one embodiment, the homolog of any one of the polypeptides of the invention is derived from an insect and has a sequence identity of at least 50% and preferably has essentially the same or a similar activity of an insect voltage-gated potassium channel ShaI (Shaker cognate I or Shaker-like) and/or its accessory protein KChIP (potassium channel-interacting protein) respectively.
In another embodiment, the homolog of any one of the polypeptides of the invention is derived from an insect and has a sequence identity of at least 50% and preferably has essentially the same or a similar activity of a Shaker channel and/or a Hyperkinetic beta subunit, preferably H-kv beta subunit A or C subtype respectively.
In another embodiment, the homolog of any one of the polypeptides of the invention is derived from an insect and has a sequence identity of at least 50% and preferably has essentially the same or a similar activity of a octopamine receptor selected from the group consisting of oa2, preferably from Drosophila melanogaster, Oamb, Oct-beta-2R and Oct-beta-3R.
In a further embodiment, the homolog of any one of the polypeptides of the invention is derived from an insect and has a sequence identity of at least 50% and preferably has essentially the same or a similar activity of an insect small-conductance Ca2+-activated potassium channel.
In one embodiment, the homolog of any one of the polypeptides of the invention is derived from a insect, preferably from a insect selected from the group consisting of Pterygota, Neopetra, Hemiptera, Lepidoptera, Coleoptera, Diptera, Homoptera, Tenebrionoidea, Tenebrionidae, Tenebrio, Sternorrhyncha, Aphidina, Brachycera, Drosophilidae, Drosophilinae and Drosophila and has a sequence identity of at least 50% and preferably has essentially the same or a essentially similar activity of
i) an insect voltage-gated potassium channel ShaI (Shaker cognate I or Shaker-like) and/or its accessory protein KChIP (potassium channel-interacting protein) respectively, or
ii) a Shaker channel and/or a Hyperkinetic beta subunit, preferably H-kv beta subunit A or C subtype respectively, or
iii) a octopamine receptor selected from the group consisting of oa2, preferably from Drosophila melanogaster, Oamb, Oct-beta-2R and Oct-beta-3R, or
iv) an insect small-conductance Ca2+-activated potassium channel.
In one embodiment, the homolog of any one of the polypeptides of the invention is derived from
i) Drosophila melanogaster, southern armyworm, tribolium, brown plant hopper, or
ii) Drosophila melanogaster, southern armyworm, tribolium, green peach aphid, cotton aphid and/or black bean aphid, or
iii) Drosophila melanogaster, southern armyworm (Spodoptera eridania), Red Fluor Beetle (Tribolium castaneum), Green Peach Aphid (Myzus persicae), and Silverleaf Whitefly (Bemisia argentifolii), or
iv) a insect, preferably from a insect selected from the group consisting of Pterygota, Neopetra, Hemiptera, Lepidoptera, Coleoptera, Diptera, Homoptera, Tenebrionoidea, Tenebrionidae, Tenebrio, Sternorrhyncha, Aphidina, Brachycera, Drosophilidae, Drosophilinae and Drosophila and has a sequence identity of at least 50% and preferably has essentially the same or a essentially similar activity of an insect small-conductance Ca2+-activated potassium channel.
The functional equivalent or homologs of the polypeptide of the invention have the activity of
i) an insect voltage-gated potassium channel ShaI (Shaker cognate I or Shaker-like) and/or its accessory protein KChIP (potassium channel-interacting protein) respectively, or
ii) an insect Shaker channel and/or a Hyperkinetic beta subunit, preferably H-kv beta subunit A or C subtype respectively, or
iii) an insect octopamine receptor selected from the group consisting of oa2, preferably from Drosophila melanogaster, Oamb, Oct-beta-2R and Oct-beta-3R, or
iv) an insect small-conductance Ca2+-activated potassium channel
and an amino acid sequence that has at least an identity of 55%, 56%, 57%, 58%, 59%, 60%, 61%, 62% or 63% preferably at least 64%, 65%, 66%, 67%, 68% or 69% more preferably at least 70%, 71%, 72%, 73%, 74%, 75%, 76%, 77%, 78%, 79%, 80%, 81%, 82%, 83%, 84% or 85% most preferably 86%, 87%, 88%, 89% or 90% especially preferably at least 91%, 92%, 93%, 94%, 95%, 96%, 97%, 98% or 99% identity with a polypeptide as shown in the sequences selected from the group consisting of SEQ ID NO: 2, 6, 10, 14, 18, 22, 26 and/or 30, in the case of the shaker channel and/or a Hyperkinetic beta subunit SEQ ID NO: 73, 75, 77, 79, 81, 83, 85 and/or 87, in the case of the G-protein coupled receptor SEQ ID NO: 130, 134, 138, 142, 146, 150, 154, 158, 162, 166, 170 and/or 174, and in the case of the SK-channel SEQ ID NO: 228, 230, 232.
“Functional equivalents” describe, in the present context, nucleic acid sequences which hybridize under standard conditions with the nucleic acid sequence encoding a polypeptide with the biological activity of
i) an insect voltage-gated potassium channel ShaI (Shaker cognate I or Shaker-like) and/or its accessory protein KChIP (potassium channel-interacting protein) respectively, or
ii) an insect Shaker channel and/or a Hyperkinetic beta subunit, preferably H-kv beta subunit A or C subtype respectively, or
iii) an octopamine receptor selected from the group consisting of oa2, preferably from Drosophila melanogaster, Oamb, Oct-beta-2R and Oct-beta-3R, or
iv) an insect small-conductance Ca2+-activated potassium channel
or parts of the aforementioned nucleic acid sequence, which are capable of bringing about the expression, in a cell or an organism, of a polypeptide with the biological activity of a of
i) an insect voltage-gated potassium channel ShaI (Shaker cognate I or Shaker-like) and/or its accessory protein KChIP (potassium channel-interacting protein) respectively, or
ii) an insect Shaker channel and/or a Hyperkinetic beta subunit, preferably H-kv beta subunit A or C subtype respectively, or
iii) an insect octopamine receptor selected from the group consisting of oa2, preferably from Drosophila melanogaster, Oamb, Oct-beta-2R and Oct-beta-3R, or
iv) an insect small-conductance Ca2+-activated potassium channel.
To carry out the hybridization, it is advantageous to use short oligonucleotides with a length of approximately 10-50 bp, preferably 15-40 bp, for example of the conserved or other regions, which can be determined in the manner with which the skilled worker is familiar by comparisons with other related genes. However, longer fragments of the nucleic acids according to the invention with a length of 100-500 bp, or the complete sequences, may also be used for hybridization. Depending on the nucleic acid/oligonucleotide used, the length of the fragment or the complete sequence, or depending on which type of nucleic acid, i.e. DNA or RNA, is being used for the hybridization, these standard conditions vary. Thus, for example, the melting temperatures for DNA:DNA hybrids are approximately 10° C. lower than those of DNA:RNA hybrids of the same length.
Standard hybridization conditions are to be understood as meaning, depending on the nucleic acid, for example temperatures of between 42 and 58° C. in an aqueous buffer solution with a concentration of between 0.1 to 5×SSC (1×SSC=0.15 M NaCl, 15 mM sodium citrate, pH 7.2) or additionally in the presence of 50% formamide, such as, for example, 42° C. in 5×SSC, 50% formamide. The hybridization conditions for DNA:DNA hybrids are advantageously 0.1×SSC and temperatures of between approximately 20° C. and 65° C., preferably between approximately 30° C. and 45° C. In the case of DNA:RNA hybrids, the hybridization conditions are advantageously 0.1×SSC and temperatures of between approximately 30° C. and 65° C., preferably between approximately 45° C. and 55° C. These hybridization temperatures which have been stated are melting temperature values which have been calculated by way of example for a nucleic acid with a length of approx. 100 nucleotides and a G+C content of 50% in the absence of formamide. The experimental conditions for DNA hybridization are described in specialist textbooks of genetics such as, for example, in Sambrook et al., “Molecular Cloning”, Cold Spring Harbor Laboratory, 1989, and can be calculated using formulae with which the skilled worker is familiar, for example as a function of the length of the nucleic acids, the type of the hybrids or the G+C content. The skilled worker will find further information on hybridization in the following textbooks: Ausubel et al. (eds), 1985, Current Protocols in Molecular Biology, John Wiley & Sons, New York; Hames and Higgins (eds), 1985, Nucleic Acids Hybridization: A Practical Approach, IRL Press at Oxford University Press, Oxford; Brown (ed), 1991, Essential Molecular Biology: A Practical Approach, IRL Press at Oxford University Press, Oxford.
A functional equivalent is furthermore also understood as meaning in particular natural or artificial mutations of the respective nucleic acid sequences of the protein encoded by the nucleic acid sequences according to the invention and their homologs from other organisms.
Thus, the present invention also encompasses, for example, those nucleotide sequences which are obtained by modification of the nucleic acid sequence of SEQ ID NO: 1, 5, 9, 13, 17, 21, 25 and/or 29, in the case of the shaker channel and/or a Hyperkinetic beta subunit SEQ ID NO: 72, 74, 76, 78, 80, 82, 84 and/or 86, in the case of the G-protein coupled receptor SEQ ID NO: 129, 133, 137, 141, 145, 149, 153, 157, 161, 165, 169 and/or 173, and in the case of the SK-channel SEQ ID NO: 227, 229, 231. For example, such modifications can be generated by techniques with which the skilled worker is familiar, such as “Site Directed Mutagenesis”, “Error Prone PCR”, “DNA shuffling” (Nature 370, 1994, pp. 389-391) or “Staggered Extension Process” (Nature Biotechnol. 16, 1998, pp. 258-261). The purpose of such a modification can be, for example, the insertion of further cleavage sites for restriction enzymes, the removal of DNA in order to truncate the sequence, the substitution of nucleotides to optimize the codons, or the addition of further sequences. Proteins which are encoded via modified nucleic acid sequences must retain the desired functions despite a deviating nucleic acid sequence, which is the biological activity of
i) an insect voltage-gated potassium channel ShaI (Shaker cognate I or Shaker-like) and/or its accessory protein KChIP (potassium channel-interacting protein) respectively, or
ii) an insect Shaker channel and/or a Hyperkinetic beta subunit, preferably H-kv beta subunit A or C subtype respectively, or
iii) an insect octopamine receptor selected from the group consisting of oa2, preferably from Drosophila melanogaster, Oamb, Oct-beta-2R and Oct-beta-3R, or
iv) an insect small-conductance Ca2+-activated potassium channel.
Functional equivalents thus comprise naturally occurring variants of the herein-described sequences and artificial nucleic acid sequences, for example those which have been obtained by chemical synthesis and which are adapted to the codon usage, and also the amino acid sequences derived from them.
Nucleic acid molecules corresponding to natural variant homologues of the nucleic acid molecule comprising a polynucleotide shown in SEQ ID NO: 2, 6, 10, 14, 18, 22, 26 and/or 30, in the case of the shaker channel and/or a Hyperkinetic beta subunit SEQ ID NO: 73, 75, 77, 79, 81, 83, 85 and/or 87, in the case of the G-protein coupled receptor SEQ ID NO: 130, 134, 138, 142, 146, 150, 154, 158, 162, 166, 170 and/or 174, and in the case of the SK-channel SEQ ID NO: 228, 230, 232, such as the nucleic acid molecule of the invention, and which can also be a cDNA, can be isolated based on their homology to the nucleic acid molecules disclosed herein using the nucleic acid molecule shown in SEQ ID NO: 1, 5, 9, 13, 17, 21, 25 and/or 29, in the case of the shaker channel and/or a Hyperkinetic beta subunit SEQ ID NO: 72, 74, 76, 78, 80, 82, 84 and/or 86, in the case of the G-protein coupled receptor SEQ ID NO: 129, 133, 137, 141, 145, 149, 153, 157, 161, 165, 169 and/or 173, and in the case of the SK-channel SEQ ID NO: 227, 229, 231, e.g. the nucleic acid molecule of the invention, or a fragment thereof, as a hybridization probe according to standard hybridization techniques under stringent hybridization conditions.
Nucleic acid molecules which are advantageously for the process according to the invention can be isolated based on their homology to the nucleic acid molecules disclosed herein using the sequences or part thereof as hybridization probe and following standard hybridization techniques under stringent hybridization conditions.
The term “homology” means that the respective nucleic acid molecules or encoded proteins are functionally and/or structurally equivalent. The nucleic acid molecules that are homologous to the nucleic acid molecules described above and that are derivatives of said nucleic acid molecules are, for example, variations of said nucleic acid molecules which represent modifications having the same biological function, in particular encoding proteins with the same or substantially the same biological function. They may be naturally occurring variations, such as sequences from other plant varieties or species, or mutations. These mutations may occur naturally or may be obtained by mutagenesis techniques. The allelic variations may be naturally occurring allelic variants as well as synthetically produced or genetically engineered variants. Structural equivalents can for example be identified by testing the binding of said polypeptide to antibodies or computer based predictions. Structural equivalents have the similar immunological characteristic, e.g. comprise similar epitopes.
By “hybridizing” it is meant that such nucleic acid molecules hybridize under conventional hybridization conditions, preferably under stringent conditions such as described by, e.g., Sambrook (Molecular Cloning; A Laboratory Manual, 2nd Edition, Cold Spring Harbor Laboratory Press, Cold Spring Harbor, N.Y. (1989)) or in Current Protocols in Molecular Biology, John Wiley & Sons, N. Y. (1989), 6.3.1-6.3.6.
According to the invention, DNA as well as RNA molecules of the nucleic acid of the invention can be used as probes. Further, as template for the identification of functional homologues Northern blot assays as well as Southern blot assays can be performed. The Northern blot assay advantageously provides further information about the expressed gene product: e.g. expression pattern, occurrence of processing steps, like splicing and capping, etc. The Southern blot assay provides additional information about the chromosomal localization and organization of the gene encoding the nucleic acid molecule of the invention.
A preferred, nonlimiting example of stringent Southern blot hydridization conditions are hybridizations in 6× sodium chloride/sodium citrate (=SSC) at approximately 45° C., followed by one or more wash steps in 0.2×SSC, 0.1% SDS at 50 to 65° C., for example at 50° C., 55° C. or 60° C. The skilled worker knows that these hybridization conditions differ as a function of the type of the nucleic acid and, for example when organic solvents are present, with regard to the temperature and concentration of the buffer. The temperature under “standard hybridization conditions” differs for example as a function of the type of the nucleic acid between 42° C. and 58° C., preferably between 45° C. and 50° C. in an aqueous buffer with a concentration of 0.1×0.5×, 1×, 2×, 3×, 4× or 5×SSC (pH 7.2). If organic solvent(s) is/are present in the abovementioned buffer, for example 50% formamide, the temperature under standard conditions is approximately 40° C., 42° C. or 45° C. The hybridization conditions for DNA:DNA hybrids are preferably for example 0.1×SSC and 20° C., 25° C., 30° C., 35° C., 40° C. or 45° C., preferably between 30° C. and 45° C. The hybridization conditions for DNA:RNA hybrids are preferably for example 0.1×SSC and 30° C., 35° C., 40° C., 45° C., 50° C. or 55° C., preferably between 45° C. and 55° C. The abovementioned hybridization temperatures are determined for example for a nucleic acid approximately 100 bp (=base pairs) in length and a G+C content of 50% in the absence of formamide. The skilled worker knows to determine the hybridization conditions required with the aid of textbooks, for example the ones mentioned above, or from the following textbooks: Sambrook et al., “Molecular Cloning”, Cold Spring Harbor Laboratory, 1989; Hames and Higgins (Ed.) 1985, “Nucleic Acids Hybridization: A Practical Approach”, IRL Press at Oxford University Press, Oxford; Brown (Ed.) 1991, “Essential Molecular Biology: A Practical Approach”, IRL Press at Oxford University Press, Oxford.
A further example of one such stringent hybridization condition is hybridization at 4×SSC at 65° C., followed by a washing in 0.1×SSC at 65° C. for one hour. Alternatively, an exemplary stringent hybridization condition is in 50% formamide, 4×SSC at 42° C. Further, the conditions during the wash step can be selected from the range of conditions delimited by low-stringency conditions (approximately 2×SSC at 50° C.) and high-stringency conditions (approximately 0.2×SSC at 50° C., preferably at 65° C.) (20×SSC: 0.3M sodium citrate, 3M NaCl, pH 7.0). In addition, the temperature during the wash step can be raised from low-stringency conditions at room temperature, approximately 22° C., to higher-stringency conditions at approximately 65° C.
Both of the parameters salt concentration and temperature can be varied simultaneously, or else one of the two parameters can be kept constant while only the other is varied. Denaturants, for example formamide or SDS, may also be employed during the hybridization. In the presence of 50% formamide, hybridization is preferably effected at 42° C. Relevant factors like i) length of treatment, ii) salt conditions, iii) detergent conditions, iv) competitor DNAs, v) temperature and vi) probe selection can combined case by case so that not all possibilities can be mentioned herein.
Some examples of conditions for DNA hybridization (Southern blot assays) and wash step are shown hereinbelow:
(1) Hybridization conditions can be selected, for example, from the following conditions:

    
    
        a) 4×SSC at 65° C.,
        b) 6×SSC at 45° C.,
        c) 6×SSC, 100 mg/ml denatured fragmented fish sperm DNA at 68° C.,
        d) 6×SSC, 0.5% SDS, 100 mg/ml denatured salmon sperm DNA at 68° C.,
        e) 6×SSC, 0.5% SDS, 100 mg/ml denatured fragmented salmon sperm DNA, 50% formamide at 42° C.,
        f) 50% formamide, 4×SSC at 42° C.,
        g) 50% (vol/vol) formamide, 0.1% bovine serum albumin, 0.1% Ficoll, 0.1% polyvinylpyrrolidone, 50 mM sodium phosphate buffer pH 6.5, 750 mM NaCl, 75 mM sodium citrate at 42° C.,
        h) 2× or 4×SSC at 50° C. (low-stringency condition), or
        i) 30 to 40% formamide, 2× or 4×SSC at 42° C. (low-stringency condition).
    
    


(2) Wash steps can be selected, for example, from the following conditions:

    
    
        a) 0.015 M NaCl/0.0015 M sodium citrate/0.1% SDS at 50° C.
        b) 0.1×SSC at 65° C.
        c) 0.1×SSC, 0.5% SDS at 68° C.
        d) 0.1×SSC, 0.5% SDS, 50% formamide at 42° C.
        e) 0.2×SSC, 0.1% SDS at 42° C.
        f) 2×SSC at 65° C. (low-stringency condition).
        g) 0.2×SSC, 0.1% SDS at 60° C. (medium-high stringency conditions), or
        h) 0.1×SSC, 0.1% SDS at 60° C. (medium-high stringency conditions), or
        i) 0.2×SSC, 0.1% SDS at 65° C. (high stringency conditions), or
        h) 0.1×SSC, 0.1% SDS at 65° C. (high stringency conditions).
    
    


In one embodiment, the term “hybridizes under stringent conditions” is intended to describe conditions for hybridization and washing under which nucleotide sequences of at least 30%, 40%, 50% or 65% identical to each other typically remain hybridized to each other. Preferably, the conditions are such that sequences of at least about 70%, more preferably at least about 75% or 80%, and even more preferably of at least about 85%, 90% or 95% or more identical to each other typically remain hybridized to each other.
In one embodiment the nucleic acid molecule of the invention hybridizes under stringent conditions to a sequence shown in SEQ ID NO: 1, 5, 9, 13, 17, 21, 25 and/or 29, in the case of the shaker channel and/or a Hyperkinetic beta subunit SEQ ID NO: 72, 74, 76, 78, 80, 82, 84 and/or 86, in the case of the G-protein coupled receptor SEQ ID NO: 129, 133, 137, 141, 145, 149, 153, 157, 161, 165, 169 and/or 173, and in the case of the SK-channel SEQ ID NO: 227, 229, 231 and corresponds to a naturally-occurring nucleic acid molecule. As used herein, a “naturally-occurring” nucleic acid molecule refers to a RNA or DNA molecule having a nucleotide sequence that occurs in nature (e.g., encodes a natural protein).
In addition to naturally-occurring variants of the nucleic acid or protein sequence that may exist in the population, the skilled artisan will further appreciate that changes can be introduced by mutation into a nucleotide sequence of the nucleic acid molecule encoding the polypeptide, thereby leading to changes in the amino acid sequence of the encoded polypeptide and thereby altering the functional ability of the polypeptide, meaning preferably reducing, decreasing or deleting said activity. For example, nucleotide substitutions leading to amino acid substitutions at “essential” amino acid residues can be made in a sequence of the nucleic acid molecule to be reduced in the process of the invention, e.g. comprising the corresponding nucleic acid molecule as shown in SEQ ID NO: 1, 5, 9, 13, 17, 21, 25 and/or 29, in the case of the shaker channel and/or a Hyperkinetic beta subunit SEQ ID NO: 72, 74, 76, 78, 80, 82, 84 and/or 86, in the case of the G-protein coupled receptor SEQ ID NO: 129, 133, 137, 141, 145, 149, 153, 157, 161, 165, 169 and/or 173, and in the case of the SK-channel SEQ ID NO: 227, 229, 231. An “essential” amino acid residue is a residue that if altered from the wild-type sequence of one of the polypeptide lead to an altered activity of said polypeptide, whereas a “non-essential” amino acid residue is not required for the activity of the protein for example for the activity as an enzyme or channel. The alteration of “essential” residues often lead to a reduced, decreased or deleted activity of the polypeptides. Preferably amino acid of the polypeptide are changed in such a manner that the activity is reduced, decreased or deleted that means preferably essential amino acid residues and/or more non-essential residues are changed and thereby the activity is reduced after decreasing the expression or activity of the polypeptide. Other amino acid residues, however, (e.g., those that are not conserved or only semi-conserved in the domain having said activity) may not be essential for activity and thus are likely to be amenable to alteration without altering said activity are less preferred.
Preferably, the protein encoded by the nucleic acid molecule is at least about 60%, 70% or 80% identical to the sequence shown in SEQ ID NO: 2, 6, 10, 14, 18, 22, 26 and/or 30 or to a sequence comprising a consensus sequence as shown in SEQ ID NO: 33 and/or 34 respectively or one or more motifs selected from the group consisting of SEQ ID NO: 35, 36, 37, 38, 39, 40, 41, 42, 43, 44, 45, 46, 47, 48, 49, 50, 51, 52, 53, 54 and/or 55, and/or 56, 57, 58, 59, 60, 61, 62, 63, 64, 65, 66, 67, 68, 69, 70 and/or 71 respectively, more preferably at least about 85% identical to one of the sequences shown in SEQ ID NO: 2, 6, 10, 14, 18, 22, 26 and/or 30 or to a sequence comprising a consensus sequence as shown in SEQ ID NO: 33 and/or 34 respectively or one or more motifs selected from the group consisting of SEQ ID NO: 35, 36, 37, 38, 39, 40, 41, 42, 43, 44, 45, 46, 47, 48, 49, 50, 51, 52, 53, 54 and/or 55, and/or 56, 57, 58, 59, 60, 61, 62, 63, 64, 65, 66, 67, 68, 69, 70 and/or 71 respectively, even more preferably at least about 90%, 91%, 92%, 93%, 94%, 95% homologous to the sequence shown in SEQ ID NO: 2, 6, 10, 14, 18, 22, 26 and/or 30 or to a sequence comprising a consensus sequence as shown in SEQ ID NO: 33 and/or 34 respectively or one or more motifs selected from the group consisting of SEQ ID NO: 35, 36, 37, 38, 39, 40, 41, 42, 43, 44, 45, 46, 47, 48, 49, 50, 51, 52, 53, 54 and/or 55, and/or 56, 57, 58, 59, 60, 61, 62, 63, 64, 65, 66, 67, 68, 69, 70 and/or 71 respectively, and most preferably at least about 96%, 97%, 98%, or 99% identical to the sequence shown in SEQ ID NO: 2, 6, 10, 14, 18, 22, 26 and/or 30 or to a sequence comprising a consensus sequence as shown in SEQ ID NO: 33 and/or 34 respectively or one or more motifs selected from the group consisting of SEQ ID NO: 35, 36, 37, 38, 39, 40, 41, 42, 43, 44, 45, 46, 47, 48, 49, 50, 51, 52, 53, 54 and/or 55, and/or 56, 57, 58, 59, 60, 61, 62, 63, 64, 65, 66, 67, 68, 69, 70 and/or 71 respectively.
In the case of the shaker channel and/or a Hyperkinetic beta subunit, preferably, the protein encoded by the nucleic acid molecule is at least about 60%, 70% or 80% identical to the sequence shown in SEQ ID NO: 73, 75, 77, 79, 81, 83, 85 and/or 87 or to a sequence comprising a consensus sequence as shown in SEQ ID NO: 102 and/or 103 respectively or one or more motifs selected from the group consisting of SEQ ID NO: 104, 105, 106, 107, 108, 109, 110, 111, 112, 113, 114, 115, 116, 117, 118, 119, 120, 121, 122, 123, 124 and/or 125 and/or 126, 127 and/or 128 respectively, more preferably at least about 85% identical to one of the sequences shown in SEQ ID NO: 73, 75, 77, 79, 81, 83, 85 and/or 87 or to a sequence comprising a consensus sequence as shown in SEQ ID NO: 102 and/or 103 respectively or one or more motifs selected from the group consisting of SEQ ID NO: 104, 105, 106, 107, 108, 109, 110, 111, 112, 113, 114, 115, 116, 117, 118, 119, 120, 121, 122, 123, 124 and/or 125 and/or 126, 127 and/or 128 respectively, even more preferably at least about 90%, 91%, 92%, 93%, 94%, 95% homologous to the sequence shown in SEQ ID NO: 73, 75, 77, 79, 81, 83, 85 and/or 87 or to a sequence comprising a consensus sequence as shown in SEQ ID NO: 102 and/or 103 respectively or one or more motifs selected from the group consisting of SEQ ID NO: 104, 105, 106, 107, 108, 109, 110, 111, 112, 113, 114, 115, 116, 117, 118, 119, 120, 121, 122, 123, 124 and/or 125 and/or 126, 127 and/or 128 respectively, and most preferably at least about 96%, 97%, 98%, or 99% identical to the sequence shown in SEQ ID NO: 73, 75, 77, 79, 81, 83, 85 and/or 87 or to a sequence comprising a consensus sequence as shown in SEQ ID NO: 102 and/or 103 respectively or one or more motifs selected from the group consisting of SEQ ID NO: 104, 105, 106, 107, 108, 109, 110, 111, 112, 113, 114, 115, 116, 117, 118, 119, 120, 121, 122, 123, 124 and/or 125 and/or 126, 127 and/or 128 respectively.
In the case of the G-protein coupled receptor, preferably, the protein encoded by the nucleic acid molecule is at least about 60%, 70% or 80% identical to the sequence shown in SEQ ID NO: 130, 134, 138, 142, 146, 150, 154, 158, 162, 166, 170 and/or 174 or to a sequence comprising a consensus sequence as shown in SEQ ID NO: 177, 178 and/or 179 respectively or one or more motifs selected from the group consisting of SEQ ID NO: 180, 181, 182, 183, 184, 185, 186, 187, 188, 189 and/or 190, and/or 191, 192, 193, 194, 195, 196, 197, 198, 199, 200, 201, 202, 203, 204, 205, 206, 207 and/or 208, and/or 209, 210, 211, 212, 213, 214, 215, 216, 217, 218, 219, 220, 221, 222, 223, 224, 225 and/or 226 respectively, more preferably at least about 85% identical to one of the sequences shown in SEQ ID NO: 130, 134, 138, 142, 146, 150, 154, 158, 162, 166, 170 and/or 174 or to a sequence comprising a consensus sequence as shown in SEQ ID NO: 177, 178 and/or 179 respectively or one or more motifs selected from the group consisting of SEQ ID NO: 180, 181, 182, 183, 184, 185, 186, 187, 188, 189 and/or 190, and/or 191, 192, 193, 194, 195, 196, 197, 198, 199, 200, 201, 202, 203, 204, 205, 206, 207 and/or 208, and/or 209, 210, 211, 212, 213, 214, 215, 216, 217, 218, 219, 220, 221, 222, 223, 224, 225 and/or 226 respectively, even more preferably at least about 90%, 91%, 92%, 93%, 94%, 95% homologous to the sequence shown in SEQ ID NO: 130, 134, 138, 142, 146, 150, 154, 158, 162, 166, 170 and/or 174 or to a sequence comprising a consensus sequence as shown in SEQ ID NO: 177, 178 and/or 179 respectively or one or more motifs selected from the group consisting of SEQ ID NO: 180, 181, 182, 183, 184, 185, 186, 187, 188, 189 and/or 190, and/or 191, 192, 193, 194, 195, 196, 197, 198, 199, 200, 201, 202, 203, 204, 205, 206, 207 and/or 208, and/or 209, 210, 211, 212, 213, 214, 215, 216, 217, 218, 219, 220, 221, 222, 223, 224, 225 and/or 226 respectively, and most preferably at least about 96%, 97%, 98%, or 99% identical to the sequence shown in SEQ ID NO: 130, 134, 138, 142, 146, 150, 154, 158, 162, 166, 170 and/or 174 or to a sequence comprising a consensus sequence as shown in SEQ ID NO: 177, 178 and/or 179 respectively or one or more motifs selected from the group consisting of SEQ ID NO: 180, 181, 182, 183, 184, 185, 186, 187, 188, 189 and/or 190, and/or 191, 192, 193, 194, 195, 196, 197, 198, 199, 200, 201, 202, 203, 204, 205, 206, 207 and/or 208, and/or 209, 210, 211, 212, 213, 214, 215, 216, 217, 218, 219, 220, 221, 222, 223, 224, 225 and/or 226 respectively.
In the case of the SK-channel, preferably, the protein encoded by the nucleic acid molecule is at least about 60%, 70% or 80% identical to the sequence shown in SEQ ID NO: 228, 230, 232 or to a sequence comprising a consensus sequence as shown in SEQ ID NO: 239 or one or more motifs selected from the group consisting of SEQ ID NO: 240, 241, 242, 243, 244, 245, 246, 247, 248, 249, 250, 251, 252 and 253, more preferably at least about 85% identical to one of the sequences shown in SEQ ID NO: 228, 230, 232 or to a sequence comprising a consensus sequence as shown in SEQ ID NO: 239 or one or more motifs selected from the group consisting of SEQ ID NO: 240, 241, 242, 243, 244, 245, 246, 247, 248, 249, 250, 251, 252 and 253, even more preferably at least about 90%, 91%, 92%, 93%, 94%, 95% homologous to the sequence shown in SEQ ID NO: 228, 230, 232 or to a sequence comprising a consensus sequence as shown in SEQ ID NO: 239 or one or more motifs selected from the group consisting of SEQ ID NO: 240, 241, 242, 243, 244, 245, 246, 247, 248, 249, 250, 251, 252 and 253, and most preferably at least about 96%, 97%, 98%, or 99% identical to the sequence shown in SEQ ID NO: 228, 230, 232 or to a sequence comprising a consensus sequence as shown in SEQ ID NO: 239 or one or more motifs selected from the group consisting of SEQ ID NO: 240, 241, 242, 243, 244, 245, 246, 247, 248, 249, 250, 251, 252 and 253.
To determine the percentage homology (=identity) of two amino acid sequences or of two nucleic acid molecules, the sequences are written one underneath the other for an optimal comparison. Gaps may be inserted into the sequence of a protein or of a nucleic acid molecule in order to generate an optimal alignment with the other protein or the other nucleic acid. The amino acid residue or nucleotide at the corresponding amino acid position or nucleotide position is then compared between both polymers. If a position in one sequence is occupied by the same amino acid residue or the same nucleotide as in the corresponding position of the other sequence, the molecules are identical at this position. Amino acid or nucleotide “identity” as used in the present context corresponds to amino acid or nucleic acid “homology”. Generally the percentage homology between the two sequences is a function of the number of identical positions shared by the sequences (i.e. % homology=number of identical positions/total number of positions×100). The terms “homology” and “identity” are thus to be considered as synonyms for this description.
For the determination of the percentage homology (=identity) of two or more amino acid or of two or more nucleotide sequences several computer software programs have been developed. The homology of two or more sequences can be calculated with for example the software fasta, which presently has been used in the version fasta 3 (W. R. Pearson and D. J. Lipman (1988), Improved Tools for Biological Sequence Comparison. PNAS 85:2444-2448; W. R. Pearson (1990) Rapid and Sensitive Sequence Comparison with FASTP and FASTA, Methods in Enzymology 183:63-98; W. R. Pearson and D. J. Lipman (1988) Improved Tools for Biological Sequence Comparison. PNAS 85:2444-2448; W. R. Pearson (1990); Rapid and Sensitive Sequence Comparison with FASTP and FASTAMethods in Enzymology 183:63-98). Another useful program for the calculation of homologies of different sequences is the standard blast program, which is included in the Biomax pedant software (Biomax, Munich, Federal Republic of Germany). This leads unfortunately sometimes to suboptimal results since blast does not always include complete sequences of the subject and the query. Nevertheless as this program is very efficient it can be used for the comparison of a huge number of sequences. The following settings are typically used for such a comparisons of sequences: -p Program Name [String]; -d Database [String]; default=nr; -i Query File [File In]; default=stdin; -e Expectation value (E) [Real]; default=10.0; -m alignment view options: 0=pairwise; 1=query-anchored showing identities; 2=query-anchored no identities; 3=flat query-anchored, show identities; 4=flat query-anchored, no identities; 5=query-anchored no identities and blunt ends; 6=flat query-anchored, no identities and blunt ends; 7=XML Blast output; 8=tabular; 9 tabular with comment lines [Integer]; default=0; -o BLAST report Output File [File Out] Optional; default=stdout; -F Filter query sequence (DUST with blastn, SEG with others) [String]; default=T; -G Cost to open a gap (zero invokes default behavior) [Integer]; default=0; -E Cost to extend a gap (zero invokes default behavior) [Integer]; default=0; -X X dropoff value for gapped alignment (in bits) (zero invokes default behavior); blastn 30, megablast 20, tblastx 0, all others 15 [Integer]; default=0; -I Show GI's in deflines [T/F]; default=F; -q Penalty for a nucleotide mismatch (blastn only) [Integer]; default=−3; -r Reward for a nucleotide match (blastn only) [Integer]; default=1; -v Number of database sequences to show one-line descriptions for (V) [Integer]; default=500; -b Number of database sequence to show alignments for (B) [Integer]; default=250; -f Threshold for extending hits, default if zero; blastp 11, blastn 0, blastx 12, tblastn 13; tblastx 13, megablast 0 [Integer]; default=0; -g Perfom gapped alignment (not available with tblastx) [T/F]; default=T; -Q Query Genetic code to use [Integer]; default=1; -D DB Genetic code (for tblast[nx] only) [Integer]; default=1; -a Number of processors to use [Integer]; default=1; -O SeqAlign file [File Out] Optional; -J Believe the query defline [T/F]; default=F; -M Matrix [String]; default=BLOSUM62; -W Word size, default if zero (blastn 11, megablast 28, all others 3) [Integer]; default=0; -z Effective length of the database (use zero for the real size) [Real]; default=0; -K Number of best hits from a region to keep (off by default, if used a value of 100 is recommended) [Integer]; default=0; -P 0 for multiple hit, 1 for single hit [Integer]; default=0; -Y Effective length of the search space (use zero for the real size) [Real]; default=0; -S Query strands to search against database (for blast[nx], and tblastx); 3 is both, 1 is top, 2 is bottom [Integer]; default=3; -T Produce HTML output [T/F]; default=F; -I Restrict search of database to list of GI's [String] Optional; -U Use lower case filtering of FASTA sequence [T/F] Optional; default=F; -y X dropoff value for ungapped extensions in bits (0.0 invokes default behavior); blastn 20, megablast 10, all others 7 [Real]; default=0.0; -Z X dropoff value for final gapped alignment in bits (0.0 invokes default behavior); blastn/megablast 50, tblastx 0, all others 25 [Integer]; default=0; -R PSI-TBLASTN checkpoint file [File In] Optional; -n MegaBlast search [T/F]; default=F; -L Location on query sequence [String] Optional; -A Multiple Hits window size, default if zero (blastn/megablast 0, all others 40 [Integer]; default=0; -w Frame shift penalty (OOF algorithm for blastx) [Integer]; default=0; -t Length of the largest intron allowed in tblastn for linking HSPs (0 disables linking) [Integer]; default=0.
Results of high quality are reached by using the algorithm of Needleman and Wunsch or Smith and Waterman. Therefore programs based on said algorithms are preferred. Advantageously the comparisons of sequences can be done with the program PileUp (J. Mol. Evolution., 25, 351-360, 1987, Higgins et al., CABIOS, 5 1989: 151-153) or preferably with the programs Gap and BestFit, which are respectively based on the algorithms of Needleman and Wunsch [J. Mol. Biol. 48; 443-453 (1970)] and Smith and Waterman [Adv. Appl. Math. 2; 482-489 (1981)]. Both programs are part of the GCG software-package [Genetics Computer Group, 575 Science Drive, Madison, Wis., USA 53711 (1991); Altschul et al. (1997) Nucleic Acids Res. 25:3389 et seq.]. Therefore preferably the calculations to determine the perentages of sequence homology are done with the program Gap over the whole range of the sequences. The following standard adjustments for the comparison of nucleic acid sequences were used: gap weight: 50, length weight: 3, average match: 10.000, average mismatch: 0.000.
For example a sequence which has a 80% homology with sequence shown in SEQ ID NO.: 1 at the nucleic acid level is understood as meaning a sequence which, upon comparison with the sequence SEQ ID NO: 1 by the above Gap program algorithm with the above parameter set, has 80% homology.
Homology between two polypeptides is understood as meaning the identity of the amino acid sequence over in each case the entire sequence length which is calculated by comparison with the aid of the program algorithm Gap (Wisconsin Package Version 10.0, University of Wisconsin, Genetics Computer Group (GCG), Madison, USA), setting the following parameters: gap weight: 8; length weight: 2; average match: 2.912; average mismatch: −2.003.
For example a sequence which has a 80% homology with sequence SEQ ID NO: 2 at the protein level is understood as meaning a sequence which, upon comparison with the sequence SEQ ID NO: 2 by the above Gap program algorithm with the above parameter set, has 80% homology.
Functional equivalents derived from one of the polypeptides as shown in SEQ ID NO: 2, 6, 10, 14, 18, 22, 26 and/or 30 or comprising the consensus sequence as shown in SEQ ID NO: 33 and/or 34 respectively or one or more motifs selected from the group consisting of SEQ ID NO: 35, 36, 37, 38, 39, 40, 41, 42, 43, 44, 45, 46, 47, 48, 49, 50, 51, 52, 53, 54 and/or 55, and/or 56, 57, 58, 59, 60, 61, 62, 63, 64, 65, 66, 67, 68, 69, 70 and/or 71 respectively according to the invention by substitution, insertion or deletion have at least 30%, 35%, 40%, 45% or 50%, preferably at least 55%, 60%, 65% or 70% by preference at least 80%, especially preferably at least 85% or 90%, 91%, 92%, 93% or 94%, very especially preferably at least 95%, 97%, 98% or 99% homology with one of the polypeptides as shown in SEQ ID NO: 2, 6, 10, 14, 18, 22, 26 and/or 30 or with one of the polypeptides comprising a consensus sequence as shown in SEQ ID NO: 33 and/or 34 respectively or one or more motifs selected from the group consisting of SEQ ID NO: 35, 36, 37, 38, 39, 40, 41, 42, 43, 44, 45, 46, 47, 48, 49, 50, 51, 52, 53, 54 and/or 55, and/or 56, 57, 58, 59, 60, 61, 62, 63, 64, 65, 66, 67, 68, 69, 70 and/or 71 respectively and are distinguished by essentially the same properties as the polypeptide as shown in SEQ ID NO: 2, 6, 10, 14, 18, 22, 26 and/or 30.
Functional equivalents derived from the nucleic acid sequence as shown in SEQ ID NO: 1, 5, 9, 13, 17, 21, 25 and/or 29 according to the invention by substitution, insertion or deletion have at least 30%, 35%, 40%, 45% or 50%, preferably at least 55%, 60%, 65% or 70% by preference at least 80%, especially preferably at least 85% or 90%, 91%, 92%, 93% or 94%, very especially preferably at least 95%, 97%, 98% or 99% homology with one of the nucleic acids as shown in SEQ ID NO: 1, 5, 9, 13, 17, 21, 25 and/or 29 according to the invention and encode polypeptides having essentially the same properties as the polypeptide as shown in SEQ ID NO: 2, 6, 10, 14, 18, 22, 26 and/or 30.
In the case of theshaker channel and/or a Hyperkinetic beta subunit, functional equivalents derived from one of the polypeptides as shown in SEQ ID NO: 73, 75, 77, 79, 81, 83, 85 and/or 87 or comprising the consensus sequence as shown in SEQ ID NO: 102 and/or 103 respectively or one or more motifs selected from the group consisting of SEQ ID NO: 104, 105, 106, 107, 108, 109, 110, 111, 112, 113, 114, 115, 116, 117, 118, 119, 120, 121, 122, 123, 124 and/or 125 and/or 126, 127 and/or 128 respectively according to the invention by substitution, insertion or deletion have at least 30%, 35%, 40%, 45% or 50%, preferably at least 55%, 60%, 65% or 70% by preference at least 80%, especially preferably at least 85% or 90%, 91%, 92%, 93% or 94%, very especially preferably at least 95%, 97%, 98% or 99% homology with one of the polypeptides as shown in SEQ ID NO: 73, 75, 77, 79, 81, 83, 85 and/or 87 or with one of the polypeptides comprising a consensus sequence as shown in SEQ ID NO: 102 and/or 103 respectively or one or more motifs selected from the group consisting of SEQ ID NO: 104, 105, 106, 107, 108, 109, 110, 111, 112, 113, 114, 115, 116, 117, 118, 119, 120, 121, 122, 123, 124 and/or 125 and/or 126, 127 and/or 128 respectively and are distinguished by essentially the same properties as the polypeptide as shown in SEQ ID NO: 73, 75, 77, 79, 81, 83, 85 and/or 87.
In the case of theshaker channel and/or a Hyperkinetic beta subunit, functional equivalents derived from the nucleic acid sequence as shown in SEQ ID NO: 72, 74, 76, 78, 80, 82, 84 and/or 86 according to the invention by substitution, insertion or deletion have at least 30%, 35%, 40%, 45% or 50%, preferably at least 55%, 60%, 65% or 70% by preference at least 80%, especially preferably at least 85% or 90%, 91%, 92%, 93% or 94%, very especially preferably at least 95%, 97%, 98% or 99% homology with one of the nucleic acids as shown in SEQ ID NO: 72, 74, 76, 78, 80, 82, 84 and/or 86 according to the invention and encode polypeptides having essentially the same properties as the polypeptide as shown in SEQ ID NO: 73, 75, 77, 79, 81, 83, 85 and/or 87.
In the case of the G-protein coupled receptor, functional equivalents derived from one of the polypeptides as shown in SEQ ID NO: 130, 134, 138, 142, 146, 150, 154, 158, 162, 166, 170 and/or 174 or comprising the consensus sequence as shown in SEQ ID NO: 177, 178 and/or 179 respectively or one or more motifs selected from the group consisting of SEQ ID NO: 180, 181, 182, 183, 184, 185, 186, 187, 188, 189 and/or 190, and/or 191, 192, 193, 194, 195, 196, 197, 198, 199, 200, 201, 202, 203, 204, 205, 206, 207 and/or 208, and/or 209, 210, 211, 212, 213, 214, 215, 216, 217, 218, 219, 220, 221, 222, 223, 224, 225 and/or 226 respectively according to the invention by substitution, insertion or deletion have at least 30%, 35%, 40%, 45% or 50%, preferably at least 55%, 60%, 65% or 70% by preference at least 80%, especially preferably at least 85% or 90%, 91%, 92%, 93% or 94%, very especially preferably at least 95%, 97%, 98% or 99% homology with one of the polypeptides as shown in SEQ ID NO: 130, 134, 138, 142, 146, 150, 154, 158, 162, 166, 170 and/or 174 or with one of the polypeptides comprising a consensus sequence as shown in SEQ ID NO: 177, 178 and/or 179 respectively or one or more motifs selected from the group consisting of SEQ ID NO: 180, 181, 182, 183, 184, 185, 186, 187, 188, 189 and/or 190, and/or 191, 192, 193, 194, 195, 196, 197, 198, 199, 200, 201, 202, 203, 204, 205, 206, 207 and/or 208, and/or 209, 210, 211, 212, 213, 214, 215, 216, 217, 218, 219, 220, 221, 222, 223, 224, 225 and/or 226 respectively and are distinguished by essentially the same properties as the polypeptide as shown in SEQ ID NO: 130, 134, 138, 142, 146, 150, 154, 158, 162, 166, 170 and/or 174.
In the case of the G-protein coupled receptor, functional equivalents derived from the nucleic acid sequence as shown in SEQ ID NO: 129, 133, 137, 141, 145, 149, 153, 157, 161, 165, 169 and/or 173 according to the invention by substitution, insertion or deletion have at least 30%, 35%, 40%, 45% or 50%, preferably at least 55%, 60%, 65% or 70% by preference at least 80%, especially preferably at least 85% or 90%, 91%, 92%, 93% or 94%, very especially preferably at least 95%, 97%, 98% or 99% homology with one of the nucleic acids as shown in SEQ ID NO: 129, 133, 137, 141, 145, 149, 153, 157, 161, 165, 169 and/or 173 according to the invention and encode polypeptides having essentially the same properties as the polypeptide as shown in SEQ ID NO: 130, 134, 138, 142, 146, 150, 154, 158, 162, 166, 170 and/or 174.
In the case of the SK-channel, functional equivalents derived from one of the polypeptides as shown in SEQ ID NO: 228, 230, 232 or comprising the consensus sequence as shown in SEQ ID NO: 239 or one or more motifs selected from the group consisting of SEQ ID NO: 240, 241, 242, 243, 244, 245, 246, 247, 248, 249, 250, 251, 252 and 253 according to the invention by substitution, insertion or deletion have at least 30%, 35%, 40%, 45% or 50%, preferably at least 55%, 60%, 65% or 70% by preference at least 80%, especially preferably at least 85% or 90%, 91%, 92%, 93% or 94%, very especially preferably at least 95%, 97%, 98% or 99% homology with one of the polypeptides as shown in SEQ ID NO: 228, 230, 232 or with one of the polypeptides comprising a consensus sequence as shown in SEQ ID NO: 239 or one or more motifs selected from the group consisting of SEQ ID NO: 240, 241, 242, 243, 244, 245, 246, 247, 248, 249, 250, 251, 252 and 253 and are distinguished by essentially the same properties as the polypeptide as shown in SEQ ID NO: 228, 230, 232.
In the case of the SK-channel, functional equivalents derived from the nucleic acid sequence as shown in SEQ ID NO: 227, 229, 231 according to the invention by substitution, insertion or deletion have at least 30%, 35%, 40%, 45% or 50%, preferably at least 55%, 60%, 65% or 70% by preference at least 80%, especially preferably at least 85% or 90%, 91%, 92%, 93% or 94%, very especially preferably at least 95%, 97%, 98% or 99% homology with one of the nucleic acids as shown in SEQ ID NO: 227, 229, 231 according to the invention and encode polypeptides having essentially the same properties as the polypeptide as shown in SEQ ID NO: 228, 230, 232.
In one embodiment “homology” or “identity” between two nucleic acid sequences or polypeptide sequences is defined by the identity of the nucleic acid sequence/polypeptide sequence over in each case the entire sequence length, which is calculated by alignment with the aid of the program algorithm GAP (Wisconsin Package Version 10.0, University of Wisconsin, Genetics Computer Group (GCG), Madison, USA), setting the following parameters:
Gap Weight: 8 Length Weight: 2
Average Match: 2,912 Average Mismatch: −2,003
In the following text, the term identity is also used synonymously instead of the term “homologous” or “homology”.
“Mutations” comprise substitutions, additions, deletions, inversions or insertions of one or more nucleotide residues, which may also bring about changes in the corresponding amino acid sequence of the target protein by substitution, insertion or deletion of one or more amino acids.
In one embodiment the present invention is directed to an isolated nucleic acid molecule selected from the group consisting of:
a) a nucleic acid molecule encoding the polypeptide shown in SEQ ID NO: 2, 6, 10, 14, 18, 22, 26 and/or 30;
b) a nucleic acid molecule shown in SEQ ID NO: 1, 5, 9, 13, 17, 21, 25 and/or 29;
c) a nucleic acid molecule, which, as a result of the degeneracy of the genetic code, can be derived from a polypeptide sequence according to SEQ ID NO: 2, 6, 10, 14, 18, 22, 26 and/or 30;
d) a nucleic acid molecule having at least 50% identity with the nucleic acid molecule sequence of a polynucleotide comprising the nucleic acid molecule shown in SEQ ID NO: 1, 5, 9, 13, 17, 21, 25 and/or 29;
e) a nucleic acid molecule encoding a polypeptide having at least 50% identity with the amino acid sequence of the polypeptide encoded by the nucleic acid molecule of (a) to (c) and having the activity of a voltage-gated potassium channel ShaI (Shaker cognate I or Shaker-like) and/or its accessory protein KChIP (potassium channel-interacting protein) respectively;
f) nucleic acid molecule which hybridizes with a nucleic acid molecule of (a) to (c) under stringent hybridization conditions;
g) a nucleic acid molecule encoding a polypeptide which can be isolated with the aid of monoclonal or polyclonal antibodies made against a polypeptide encoded by one of the nucleic acid molecules of (a) to (e) and having the activity of a voltage-gated potassium channel ShaI (Shaker cognate I or Shaker-like) and/or its accessory protein KChIP (potassium channel-interacting protein) respectively;
h) a nucleic acid molecule encoding a polypeptide comprising the consensus sequence as shown in SEQ ID NO: 33 and/or 34 respectively or one or more motifs selected from the group consisting of SEQ ID NO: 35, 36, 37, 38, 39, 40, 41, 42, 43, 44, 45, 46, 47, 48, 49, 50, 51, 52, 53, 54 and/or 55, and/or 56, 57, 58, 59, 60, 61, 62, 63, 64, 65, 66, 67, 68, 69, 70 and/or 71 respectively;
i) nucleic acid molecule which comprises a polynucleotide, which is obtained by amplifying a cDNA library or a genomic library using the primers in SEQ ID NO: 7, 8; 9, 10; 11, 12; respectively
and
j) a nucleic acid molecule which is obtainable by screening a suitable nucleic acid library under stringent hybridization conditions with a probe comprising a complementary sequence of a nucleic acid molecule of (a) or (b) or with a fragment thereof, having at least 15 nt, preferably 20 nt, 30 nt, 50 nt, 100 nt, 200 nt or 500 nt of a nucleic acid molecule complementary to a nucleic acid molecule sequence characterized in (a) to (e) and encoding a polypeptide and having the activity of a voltage-gated potassium channel ShaI (Shaker cognate I or Shaker-like) and/or its accessory protein KChIP (potassium channel-interacting protein) respectively.
In the case of theshaker channel and/or a Hyperkinetic beta subunit, in one embodiment the present invention is directed to an isolated nucleic acid molecule selected from the group consisting of:
a) a nucleic acid molecule encoding a polypeptide comprising the polypeptide shown in SEQ ID NO: 73, 75, 77, 79, 81, 83, 85 and/or 87;
b) a nucleic acid molecule comprising the nucleic acid molecule shown in SEQ ID NO: 72, 74, 76, 78, 80, 82, 84 and/or 86;
c) a nucleic acid molecule, which, as a result of the degeneracy of the genetic code, can be derived from a polypeptide comprising a polypeptide sequence according to SEQ ID NO: 73, 75, 77, 79, 81, 83, 85 and/or 87;
d) a nucleic acid molecule having at least 50% identity with the nucleic acid molecule sequence of a polynucleotide comprising the nucleic acid molecule shown in SEQ ID NO: 72, 74, 76, 78, 80, 82, 84 and/or 86;
e) a nucleic acid molecule encoding a polypeptide having at least 50% identity with the amino acid sequence of the polypeptide encoded by the nucleic acid molecule of (a) to (c) and having the activity of a Shaker channel and/or a Hyperkinetic beta subunit, preferably H-kv beta subunit A or C subtype respectively;
f) nucleic acid molecule which hybridizes with a nucleic acid molecule of (a) to (c) under stringent hybridization conditions;
g) a nucleic acid molecule encoding a polypeptide which can be isolated with the aid of monoclonal or polyclonal antibodies made against a polypeptide encoded by one of the nucleic acid molecules of (a) to (e) and having the activity of a Shaker channel and/or a Hyperkinetic beta subunit, preferably H-kv beta subunit A or C subtype respectively;
h) a nucleic acid molecule encoding a polypeptide comprising the consensus sequence as shown in SEQ ID NO: 102 and/or 103 respectively or one or more motifs selected from the group consisting of SEQ ID NO: 104, 105, 106, 107, 108, 109, 110, 111, 112, 113, 114, 115, 116, 117, 118, 119, 120, 121, 122, 123, 124 and/or 125 and/or 126, 127 and/or 128 respectively;
i) nucleic acid molecule which comprises a polynucleotide, which is obtained by amplifying a cDNA library or a genomic library using the primers in SEQ ID NO: 88, 89; 90, 91; 92, 93; 94, 95; 96, 97; 98, 99 and/or 100, 101 respectively
and
j) a nucleic acid molecule which is obtainable by screening a suitable nucleic acid library under stringent hybridization conditions with a probe comprising a complementary sequence of a nucleic acid molecule of (a) or (b) or with a fragment thereof, having at least 15 nt, preferably 20 nt, 30 nt, 50 nt, 100 nt, 200 nt or 500 nt of a nucleic acid molecule complementary to a nucleic acid molecule sequence characterized in (a) to (e) and encoding a polypeptide and having the activity of a Shaker channel and/or a Hyperkinetic beta subunit, preferably H-kv beta subunit A or C subtype respectively.
In the case of the G-protein coupled receptor, in one embodiment the present invention is directed to an isolated nucleic acid molecule selected from the group consisting of:
a) a nucleic acid molecule encoding the polypeptide shown in SEQ ID NO: 130, 134, 138, 142, 146, 150, 154, 158, 162, 166, 170 and/or 174;
b) a nucleic acid molecule shown in SEQ ID NO: 129, 133, 137, 141, 145, 149, 153, 157, 161, 165, 169 and/or 173;
c) a nucleic acid molecule, which, as a result of the degeneracy of the genetic code, can be derived from a polypeptide comprising a polypeptide sequence according to SEQ ID NO: 130, 134, 138, 142, 146, 150, 154, 158, 162, 166, 170 and/or 174;
d) a nucleic acid molecule having at least 50% identity with the nucleic acid molecule sequence of a polynucleotide comprising the nucleic acid molecule shown in SEQ ID NO: 129, 133, 137, 141, 145, 149, 153, 157, 161, 165, 169 and/or 173;
e) a nucleic acid molecule encoding a polypeptide having at least 50% identity with the amino acid sequence of the polypeptide encoded by the nucleic acid molecule of (a) to (c) and having the activity of a octopamine receptor selected from the group consisting of oa2, preferably from Drosophila melanogaster, Oamb, Oct-beta-2R and Oct-beta-3R;
f) nucleic acid molecule which hybridizes with a nucleic acid molecule of (a) to (c) under stringent hybridization conditions;
g) a nucleic acid molecule encoding a polypeptide which can be isolated with the aid of monoclonal or polyclonal antibodies made against a polypeptide encoded by one of the nucleic acid molecules of (a) to (e) and having the activity of a octopamine receptor selected from the group consisting of oa2, preferably from Drosophila melanogaster, Oamb, Oct-beta-2R and Oct-beta-3R;
h) a nucleic acid molecule encoding a polypeptide comprising the consensus sequence as shown in SEQ ID NO: 177, 178 and/or 179 respectively or one or more motifs selected from the group consisting of SEQ ID NO: 180, 181, 182, 183, 184, 185, 186, 187, 188, 189 and/or 190, and/or 191, 192, 193, 194, 195, 196, 197, 198, 199, 200, 201, 202, 203, 204, 205, 206, 207 and/or 208, and/or 209, 210, 211, 212, 213, 214, 215, 216, 217, 218, 219, 220, 221, 222, 223, 224, 225 and/or 226 respectively,
i) nucleic acid molecule which comprises a polynucleotide, which is obtained by amplifying a cDNA library or a genomic library using the primers in SEQ ID NO: 131, 132; 135, 136; 139, 140; 143, 144; 147, 148; 151, 152; 155, 156, 159, 160; 163, 164; 167, 168; 171, 172 and/or 175, 176
and
j) a nucleic acid molecule which is obtainable by screening a suitable nucleic acid library under stringent hybridization conditions with a probe comprising a complementary sequence of a nucleic acid molecule of (a) or (b) or with a fragment thereof, having at least 15 nt, preferably 20 nt, 30 nt, 50 nt, 100 nt, 200 nt or 500 nt of a nucleic acid molecule complementary to a nucleic acid molecule sequence characterized in (a) to (e) and encoding a polypeptide and having the activity of a octopamine receptor selected from the group consisting of oa2, preferably from Drosophila melanogaster, Oamb, Oct-beta-2R and Oct-beta-3R.
In the case of the SK-channel, in one embodiment the present invention is directed to an isolated nucleic acid molecule selected from the group consisting of:
a) a nucleic acid molecule encoding the polypeptide shown in SEQ ID NO: 230, 232;
b) a nucleic acid molecule shown in SEQ ID NO: 229, 231;
c) a nucleic acid molecule, which, as a result of the degeneracy of the genetic code, can be derived from a polypeptide sequence according to SEQ ID NO: 230, 232;
d) a nucleic acid molecule having at least 50% identity with the nucleic acid molecule sequence of a polynucleotide comprising the nucleic acid molecule shown in SEQ ID NO: 229, 231;
e) a nucleic acid molecule encoding a polypeptide having at least 50% identity with the amino acid sequence of the polypeptide encoded by the nucleic acid molecule of (a) to (c) and having the activity of a small-conductance Ca2+-activated potassium channel;
f) nucleic acid molecule which hybridizes with a nucleic acid molecule of (a) to (c) under stringent hybridization conditions;
g) a nucleic acid molecule encoding a polypeptide which can be isolated with the aid of monoclonal or polyclonal antibodies made against a polypeptide encoded by one of the nucleic acid molecules of (a) to (e) and having the activity of a small-conductance Ca2+-activated potassium channel;
h) a nucleic acid molecule encoding a polypeptide comprising the consensus sequence as shown in SEQ ID NO: 239 or one or more motifs selected from the group consisting of SEQ ID NO: 240, 241, 242, 243, 244, 245, 246, 247, 248, 249, 250, 251, 252 and 253;
i) nucleic acid molecule which comprises a polynucleotide, which is obtained by amplifying a cDNA library or a genomic library using the primers in SEQ ID NO: 233, 234, 235, 236; and 237, 238; respectively
and
j) a nucleic acid molecule which is obtainable by screening a suitable nucleic acid library under stringent hybridization conditions with a probe comprising a complementary sequence of a nucleic acid molecule of (a) or (b) or with a fragment thereof, having at least 15 nt, preferably 20 nt, 30 nt, 50 nt, 100 nt, 200 nt or 500 nt of a nucleic acid molecule complementary to a nucleic acid molecule sequence characterized in (a) to (e) and encoding a polypeptide and having the activity of a small-conductance Ca2+-activated potassium channel.
In one embodiment the present invention is directed to nucleic acid construct comprising the isolated nucleic acid molecule of the invention.
In one embodiment the present invention is directed to a vector comprising the nucleic acid construct or the isolated nucleic acid molecule of the invention.
In one embodiment the present invention is directed to a transgenic host cell, comprising the vector, the nucleic acid construct or the isolated nucleic acid molecule of the invention by way of transfection/transformation.
An “isolated” polynucleotide or nucleic acid molecule is separated from other polynucleotides or nucleic acid molecules, which are present in the natural source of the nucleic acid molecule. An isolated nucleic acid molecule may be a chromosomal fragment of several kb, or preferably, a molecule only comprising the coding region of the gene. Accordingly, an isolated nucleic acid molecule may comprise chromosomal regions, which are adjacent 5′ and 3′ or further adjacent chromosomal regions, but preferably comprises no such sequences which naturally flank the nucleic acid molecule sequence in the genomic or chromosomal context in the organism from which the nucleic acid molecule originates (for example sequences which are adjacent to the regions encoding the 5′- and 3′-UTRs of the nucleic acid molecule). In various embodiments, the isolated nucleic acid molecule used in the process according to the invention may, for example comprise less than approximately 5 kb, 4 kb, 3 kb, 2 kb, 1 kb, 0.5 kb or 0.1 kb nucleotide sequences which naturally flank the nucleic acid molecule in the genomic DNA of the cell from which the nucleic acid molecule originates.
The nucleic acid molecules used in the process or a part thereof can be isolated using molecular-biological standard techniques and the sequence information provided herein. Also, for example a homologous sequence or homologous, conserved sequence regions at the DNA or amino acid level can be identified with the aid of comparison algorithms. The former can be used as hybridization probes under standard hybridization techniques (for example those described in Sambrook et al., Molecular Cloning: A Laboratory Manual. 2nd Ed., Cold Spring Harbor Laboratory, Cold Spring Harbor Laboratory Press, Cold Spring Harbor, N.Y., 1989) for isolating further nucleic acid sequences useful in this process.
A nucleic acid molecule encompassing a complete sequence of a molecule which activity is to be reduced in the process of the present invention or a part thereof may additionally be isolated by polymerase chain reaction, oligonucleotide primers based on this sequence or on parts thereof being used. For example, a nucleic acid molecule comprising the complete sequence or part thereof can be isolated by polymerase chain reaction using oligonucleotide primers which have been generated on the basis of this every sequence. For example, mRNA can be isolated from cells, for example by means of the guanidinium thiocyanate extraction method of Chirgwin et al. (1979) Biochemistry 18:5294-5299, and cDNA can be generated by means of reverse transcriptase (for example Moloney MLV reverse transcriptase, available from Gibco/BRL, Bethesda, Md., or AMV reverse transcriptase, obtainable from Seikagaku America, Inc., St. Petersburg, Fla.).
Synthetic oligonucleotide primers for the amplification by means of polymerase chain reaction can be generated on the basis of a sequence shown herein. Such primers can be used to amplify nucleic acids sequences for example from cDNA libaries or from genomic libraries and identify nucleic acid molecules, which are useful in the inventive process.
Moreover, it is possible to identify conserved regions from various organisms by carrying out protein sequence alignments with the polypeptide encoded by the nucleic acid molecule to be reduced according to the process of the invention, in particular with the sequences encoded by the nucleic acid molecule shown in SEQ ID NO: 1, 5, 9, 13, 17, 21, 25 and/or 29, in the case of the shaker channel and/or a Hyperkinetic beta subunit SEQ ID NO: 72, 74, 76, 78, 80, 82, 84 and/or 86, in the case of the G-protein coupled receptor SEQ ID NO: 129, 133, 137, 141, 145, 149, 153, 157, 161, 165, 169 and/or 173, and in the case of the SK-channel SEQ ID NO: 227, 229, 231, from which conserved regions, and in turn, degenerate primers can be derived.
Conserved regions are those, which show a very little variation in the amino acid in one particular position of several homologs from different origin. The consenus sequence and polypeptide motifs shown herein are derived from said aligments. Moreover, it is possible to identify conserved regions from various organisms by carrying out protein sequence alignments with the polypeptide encoded by the nucleic acid molecule to be reduced according to the process of the invention, in particular with the sequences encoded by the polypeptide molecule shown in SEQ ID NO: 2, 6, 10, 14, 18, 22, 26 and/or 30, in the case of the shaker channel and/or a Hyperkinetic beta subunit SEQ ID NO: 73, 75, 77, 79, 81, 83, 85 and/or 87, in the case of the G-protein coupled receptor SEQ ID NO: 130, 134, 138, 142, 146, 150, 154, 158, 162, 166, 170 and/or 174, and in the case of the SK-channel SEQ ID NO: 228, 230, 232 from which conserved regions, and in turn, degenerate primers can be derived.
Conserved regions are those, which show a very little variation in the amino acid in one particular position of several homologs from different origin. The consenus sequences and polypeptide motifs shown herein are derived from said aligments. In one advantageous embodiment, in the method of the present invention the activity of a polypeptide is decreased comprising or consisting of a consensus sequence as shown in SEQ ID NO: 33 and/or 34, in the case of the shaker channel and/or a Hyperkinetic beta subunit SEQ ID NO: 102 and/or 103, in the case of the G-protein coupled receptor SEQ ID NO: 177, 178 and/or 179, and in the case of the SK-channel SEQ ID NO: 239 respectively or one or more motifs selected from the group consisting of SEQ ID NO: 35, 36, 37, 38, 39, 40, 41, 42, 43, 44, 45, 46, 47, 48, 49, 50, 51, 52, 53, 54 and/or 55, and/or 56, 57, 58, 59, 60, 61, 62, 63, 64, 65, 66, 67, 68, 69, 70 and/or 71, in the case of the shaker channel and/or a Hyperkinetic beta subunit SEQ ID NO: 104, 105, 106, 107, 108, 109, 110, 111, 112, 113, 114, 115, 116, 117, 118, 119, 120, 121, 122, 123, 124 and/or 125 and/or 126, 127 and/or 128, in the case of the G-protein coupled receptor SEQ ID NO: 180, 181, 182, 183, 184, 185, 186, 187, 188, 189 and/or 190, and/or 191, 192, 193, 194, 195, 196, 197, 198, 199, 200, 201, 202, 203, 204, 205, 206, 207 and/or 208, and/or 209, 210, 211, 212, 213, 214, 215, 216, 217, 218, 219, 220, 221, 222, 223, 224, 225 and/or 226, and in the case of the SK-channel SEQ ID NO: 240, 241, 242, 243, 244, 245, 246, 247, 248, 249, 250, 251, 252 and 253 respectively and in one another embodiment, the present invention relates to a polypeptide comprising or consisting of the consensus sequence as shown in SEQ ID NO: 33 and/or 34, in the case of the shaker channel and/or a Hyperkinetic beta subunit SEQ ID NO: 102 and/or 103, in the case of the G-protein coupled receptor SEQ ID NO: 177, 178 and/or 179, and in the case of the SK-channel SEQ ID NO: 239 respectively or one or more motifs selected from the group consisting of SEQ ID NO: 35, 36, 37, 38, 39, 40, 41, 42, 43, 44, 45, 46, 47, 48, 49, 50, 51, 52, 53, 54 and/or 55, and/or 56, 57, 58, 59, 60, 61, 62, 63, 64, 65, 66, 67, 68, 69, 70 and/or 71, in the case of the shaker channel and/or a Hyperkinetic beta subunit SEQ ID NO: 104, 105, 106, 107, 108, 109, 110, 111, 112, 113, 114, 115, 116, 117, 118, 119, 120, 121, 122, 123, 124 and/or 125 and/or 126, 127 and/or 128, in the case of the G-protein coupled receptor SEQ ID NO: 180, 181, 182, 183, 184, 185, 186, 187, 188, 189 and/or 190, and/or 191, 192, 193, 194, 195, 196, 197, 198, 199, 200, 201, 202, 203, 204, 205, 206, 207 and/or 208, and/or 209, 210, 211, 212, 213, 214, 215, 216, 217, 218, 219, 220, 221, 222, 223, 224, 225 and/or 226, and in the case of the SK-channel SEQ ID NO: 240, 241, 242, 243, 244, 245, 246, 247, 248, 249, 250, 251, 252 and 253 respectively whereby 20 or less, preferably 15 or 10, preferably 9, 8, 7, or 6, more preferred 5 or 4, even more preferred 3, even more preferred 2, even more preferred 1, most preferred 0 of the amino acids positions indicated can be replaced by any amino acid. In one embodiment not more than 15%, preferably 10%, even more preferred 5%, 4%, 3%, or 2%, most preferred 1% or 0% of the amino acid position indicated by a letter are/is replaced another amino acid. In one embodiment 20 or less, preferably 15 or 10, preferably 9, 8, 7, or 6, more preferred 5 or 4, even more preferred 3, even more preferred 2, even more preferred 1, most preferred 0 amino acids are inserted into a consensus sequence or protein motif.
The consensus sequence was derived from a multiple alignment of the sequences as shown in SEQ ID NO 2, 6, 10, 14, 18, 22, 26 and/or 30, in the case of the shaker channel and/or a Hyperkinetic beta subunit SEQ ID NO 73, 75, 77, 79, 81, 83, 85 and/or 87, in the case of the G-protein coupled receptor SEQ ID NO 130, 134, 138, 142, 146, 150, 154, 158, 162, 166, 170 and/or 174, and in the case of the SK-channel SEQ ID NO 228, 230, 232. The letters represent the one letter amino acid code and indicate that the amino acids are conserved in all aligned proteins. The letter X stands for amino acids, which are not conserved in all sequences.
Conserved domains were identified from all sequences and are described using a subset of the standard Prosite notation, e.g the pattern Y-x(21,23)-[FW] means that a conserved tyrosine is separated by minimum 21 and maximum 23 amino acid residues from either a phenylalanine or tryptophane.
Conserved patterns were identified with the software tool MEME version 3.5.1 or manually. MEME was developed by Timothy L. Bailey and Charles Elkan, Dept. of Computer Science and Engeneering, University of California, San Diego, USA and is described by Timothy L. Bailey and Charles Elkan [Fitting a mixture model by expectation maximization to discover motifs in biopolymers, Proceedings of the Second International Conference on Intelligent Systems for Molecular Biology, pp. 28-36, AAAI Press, Menlo Park, Cali., 1994]. The source code for the stand-alone program is public available from the San Diego Supercomputer center (http://meme.sdsc.edu).
For identifying common motifs in all sequences with the software tool MEME, the following settings were used: -maxsize 500000, -nmotifs 15, -evt 0.001, -maxw 60, -distance 1e-3, -minsites number of sequences used for the analysis. Input sequences for MEME were non-aligned sequences in Fasta format. Other parameters were used in the default settings in this software version.
Prosite patterns for conserved domains were generated with the software tool Pratt version 2.1 or manually. Pratt was developed by Inge Jonassen, Dept. of Informatics, University of Bergen, Norway and is described by Jonassen et al. [I. Jonassen, J. F. Collins and D. G. Higgins, Finding flexible patterns in unaligned protein sequences, Protein Science 4 (1995), pp. 1587-1595; I. Jonassen, Efficient discovery of conserved patterns using a pattern graph, Submitted to CABIOS February 1997]. The source code (ANSI C) for the stand-alone program is public available, e.g. at establisched Bioinformatic centers like EBI (European Bioinformatics Institute).
For generating patterns with the software tool Pratt, following settings were used: PL (max Pattern Length): 100, PN (max Nr of Pattern Symbols): 100, PX (max Nr of consecutive x′s): 30, FN (max Nr of flexible spacers): 5, FL (max Flexibility): 30, FP (max Flex.Product): 10, ON (max number patterns): 50. Input sequences for Pratt were distinct regions of the protein sequences exhibiting high similarity as identified from software tool MEME. The minimum number of sequences, which have to match the generated patterns (CM, min Nr of Seqs to Match) was set to at least 80% of the provided sequences. Parameters not mentioned here were used in their default settings.
The Prosite patterns of the conserved domains can be used to search for protein sequences matching this pattern. Various establisched Bioinformatic centers provide public internet portals for using those patterns in database searches (e.g. PIR [Protein Information Resource, located at Georgetown University Medical Center] or ExPASy [Expert Protein Analysis System]). Alternatively, stand-alone software is available, like the program Fuzzpro, which is part of the EMBOSS software package. For example, the program Fuzzpro not only allows to search for an exact pattern-protein match but also allows to set various ambiguities in the performed search.
The alignment was performed with the software ClustalW (version 1.83) and is described by Thompson et al. [Thompson, J. D., Higgins, D. G. and Gibson, T. J. (1994) CLUSTAL W: improving the sensitivity of progressive multiple sequence alignment through sequence weighting, positions-specific gap penalties and weight matrix choice. Nucleic Acids Research, 22:4673-4680]. The source code for the stand-alone program is public available from the European Molecular Biology Laboratory; Heidelberg, Germany. The analysis was performed using the default parameters of ClustalW v1.83 (gap open penalty: 10.0; gap extension penalty: 0.2; protein matrix: Gonnet; pprotein/DNA endgap: −1; protein/DNA gapdist: 4).
Degenerate primers, designed as described above, can then be utilized by PCR for the amplification of fragments of novel coding regions coding for proteins having above-mentioned activity.
These fragments can then be utilized as hybridization probe for isolating the complete gene sequence. As an alternative, the missing 5′ and 3′ sequences can be isolated by means of RACE-PCR. A nucleic acid molecule according to the invention can be amplified using cDNA or, as an alternative, genomic DNA as template and suitable oligonucleotide primers, following standard PCR amplification techniques. The nucleic acid molecule amplified thus can be cloned into a suitable vector and characterized by means of DNA sequence analysis. Oligonucleotides, which correspond to one of the nucleic acid molecules used in the process, can be generated by standard synthesis methods, for example using an automatic DNA synthesizer.
Nucleic acid molecules which are advantageously for the process according to the invention can be isolated based on their homology to the nucleic acid molecules disclosed herein using the sequences or part thereof as hybridization probe and following standard hybridization techniques under stringent hybridization conditions.
In one embodiment the present invention is directed to a polypeptide encoded by a nucleic acid molecule selected from the group consisting of:

    
    
        a) a nucleic acid molecule encoding the polypeptide shown in SEQ ID NO: 2, 6, 10, 14, 18, 22, 26 and/or 30;
        b) a nucleic acid molecule shown in SEQ ID NO: 1, 5, 9, 13, 17, 21, 25 and/or 29;
        c) a nucleic acid molecule, which, as a result of the degeneracy of the genetic code, can be derived from a polypeptide sequence according to SEQ ID NO: 2, 6, 10, 14, 18, 22, 26 and/or 30;
        d) a nucleic acid molecule having at least 50% identity with the nucleic acid molecule sequence of a polynucleotide comprising the nucleic acid molecule shown in SEQ ID NO: 1, 5, 9, 13, 17, 21, 25 and/or 29;
        e) a nucleic acid molecule encoding a polypeptide having at least 50% identity with the amino acid sequence of the polypeptide encoded by the nucleic acid molecule of (a) to (c) and having the activity of a voltage-gated potassium channel ShaI (Shaker cognate I or Shaker-like) and/or its accessory protein KChIP (potassium channel-interacting protein) respectively;
        f) nucleic acid molecule which hybridizes with a nucleic acid molecule of (a) to (c) under stringent hybridization conditions;
        g) a nucleic acid molecule encoding a polypeptide which can be isolated with the aid of monoclonal or polyclonal antibodies made against a polypeptide encoded by one of the nucleic acid molecules of (a) to (e) and having the activity of a voltage-gated potassium channel ShaI (Shaker cognate I or Shaker-like) and/or its accessory protein KChIP (potassium channel-interacting protein) respectively;
        h) a nucleic acid molecule encoding a polypeptide comprising the consensus sequence as shown in SEQ ID NO: 33 and/or 34 respectively or one or more motifs selected from the group consisting of SEQ ID NO: 35, 36, 37, 38, 39, 40, 41, 42, 43, 44, 45, 46, 47, 48, 49, 50, 51, 52, 53, 54 and/or 55, and/or 56, 57, 58, 59, 60, 61, 62, 63, 64, 65, 66, 67, 68, 69, 70 and/or 71 respectively;
        i) nucleic acid molecule which comprises a polynucleotide, which is obtained by amplifying a cDNA library or a genomic library using the primers in SEQ ID NO: 7, 8; 9, 10; 11, 12; respectively and
        j) a nucleic acid molecule which is obtainable by screening a suitable nucleic acid library under stringent hybridization conditions with a probe comprising a complementary sequence of a nucleic acid molecule of (a) or (b) or with a fragment thereof, having at least 15 nt, preferably 20 nt, 30 nt, 50 nt, 100 nt, 200 nt or 500 nt of a nucleic acid molecule complementary to a nucleic acid molecule sequence characterized in (a) to (e) and encoding a polypeptide and having the activity of a voltage-gated potassium channel ShaI (Shaker cognate I or Shaker-like) and/or its accessory protein KChIP (potassium channel-interacting protein) respectively.
    
    


In the case of theshaker channel and/or a Hyperkinetic beta subunit, in one embodiment the present invention is directed to a polypeptide encoded by a nucleic acid molecule selected from the group consisting of:

    
    
        a) a nucleic acid molecule encoding the polypeptide shown in SEQ ID NO: 73, 75, 77, 79, 81, 83, 85 and/or 87;
        b) a nucleic acid molecule shown in SEQ ID NO: 72, 74, 76, 78, 80, 82, 84 and/or 86;
        c) a nucleic acid molecule, which, as a result of the degeneracy of the genetic code, can be derived from a polypeptide sequence according to SEQ ID NO: 73, 75, 77, 79, 81, 83, 85 and/or 87;
        d) a nucleic acid molecule having at least 50% identity with the nucleic acid molecule sequence of a polynucleotide comprising the nucleic acid molecule shown in SEQ ID NO: 72, 74, 76, 78, 80, 82, 84 and/or 86;
        e) a nucleic acid molecule encoding a polypeptide having at least 50% identity with the amino acid sequence of the polypeptide encoded by the nucleic acid molecule of (a) to (c) and having the activity of a Shaker channel and/or a Hyperkinetic beta subunit, preferably H-kv beta subunit A or C subtype respectively;
        f) nucleic acid molecule which hybridizes with a nucleic acid molecule of (a) to (c) under stringent hybridization conditions;
        g) a nucleic acid molecule encoding a polypeptide which can be isolated with the aid of monoclonal or polyclonal antibodies made against a polypeptide encoded by one of the nucleic acid molecules of (a) to (e) and having the activity of a Shaker channel and/or a Hyperkinetic beta subunit, preferably H-kv beta subunit A or C subtype respectively;
        h) a nucleic acid molecule encoding a polypeptide comprising the consensus sequence as shown in SEQ ID NO: 102 and/or 103 respectively or one or more motifs selected from the group consisting of SEQ ID NO: 104, 105, 106, 107, 108, 109, 110, 111, 112, 113, 114, 115, 116, 117, 118, 119, 120, 121, 122, 123, 124 and/or 125 and/or 126, 127 and/or 128 respectively;
        i) nucleic acid molecule which comprises a polynucleotide, which is obtained by amplifying a cDNA library or a genomic library using the primers in SEQ ID NO: 88, 89; 90, 91; 92, 93; 94, 95; 96, 97; 98, 99 and/or 100, 101 respectively and
        j) a nucleic acid molecule which is obtainable by screening a suitable nucleic acid library under stringent hybridization conditions with a probe comprising a complementary sequence of a nucleic acid molecule of (a) or (b) or with a fragment thereof, having at least 15 nt, preferably 20 nt, 30 nt, 50 nt, 100 nt, 200 nt or 500 nt of a nucleic acid molecule complementary to a nucleic acid molecule sequence characterized in (a) to (e) and encoding a polypeptide and having the activity of a Shaker channel and/or a Hyperkinetic beta subunit, preferably H-kv beta subunit A or C subtype respectively.
    
    


In the case of the G-protein coupled receptor, in one embodiment the present invention is directed to a polypeptide encoded by a nucleic acid molecule selected from the group consisting of:

    
    
        a) a nucleic acid molecule encoding the polypeptide shown in SEQ ID NO: 130, 134, 138, 142, 146, 150, 154, 158, 162, 166, 170 and/or 174;
        a nucleic acid molecule shown in SEQ ID NO: 129, 133, 137, 141, 145, 149, 153, 157, 161, 165, 169 and/or 173
        a nucleic acid molecule, which, as a result of the degeneracy of the genetic code, can be derived from a polypeptide sequence according to SEQ ID NO: 130, 134, 138, 142, 146, 150, 154, 158, 162, 166, 170 and/or 174;
        d) a nucleic acid molecule having at least 50% identity with the nucleic acid molecule sequence of a polynucleotide comprising the nucleic acid molecule shown in SEQ ID NO: 129, 133, 137, 141, 145, 149, 153, 157, 161, 165, 169 and/or 173;
        e) a nucleic acid molecule encoding a polypeptide having at least 50% identity with the amino acid sequence of the polypeptide encoded by the nucleic acid molecule of (a) to (c) and having the activity of a octopamine receptor selected from the group consisting of oa2, preferably from Drosophila melanogaster, Oamb, Oct-beta-2R and Oct-beta-3R;
        f) nucleic acid molecule which hybridizes with a nucleic acid molecule of (a) to (c) under stringent hybridization conditions;
        g) a nucleic acid molecule encoding a polypeptide which can be isolated with the aid of monoclonal or polyclonal antibodies made against a polypeptide encoded by one of the nucleic acid molecules of (a) to (e) and having the activity of a octopamine receptor selected from the group consisting of oa2, preferably from Drosophila melanogaster, Oamb, Oct-beta-2R and Oct-beta-3R;
        h) a nucleic acid molecule encoding a polypeptide comprising the consensus sequence as shown in SEQ ID NO: 177, 178 and/or 179 respectively or one or more motifs selected from the group consisting of SEQ ID NO: 180, 181, 182, 183, 184, 185, 186, 187, 188, 189 and/or 190, and/or 191, 192, 193, 194, 195, 196, 197, 198, 199, 200, 201, 202, 203, 204, 205, 206, 207 and/or 208, and/or 209, 210, 211, 212, 213, 214, 215, 216, 217, 218, 219, 220, 221, 222, 223, 224, 225 and/or 226 respectively;
        i) nucleic acid molecule which comprises a polynucleotide, which is obtained by amplifying a cDNA library or a genomic library using the primers in SEQ ID NO: 131, 132; 135, 136; 139, 140; 143, 144; 147, 148; 151, 152; 155, 156, 159, 160; 163, 164; 167, 168; 171, 172 and/or 175, 176 and
        j) a nucleic acid molecule which is obtainable by screening a suitable nucleic acid library under stringent hybridization conditions with a probe comprising a complementary sequence of a nucleic acid molecule of (a) or (b) or with a fragment thereof, having at least 15 nt, preferably 20 nt, 30 nt, 50 nt, 100 nt, 200 nt or 500 nt of a nucleic acid molecule complementary to a nucleic acid molecule sequence characterized in (a) to (e) and encoding a polypeptide and having the activity of a octopamine receptor selected from the group consisting of oa2, preferably from Drosophila melanogaster, Oamb, Oct-beta-2R and Oct-beta-3R.
    
    


In the case of the SK-channel, in one embodiment the present invention is directed to a polypeptide encoded by a nucleic acid molecule selected from the group consisting of:

    
    
        a) a nucleic acid molecule encoding the polypeptide shown in SEQ ID NO: 230, 232;
        b) a nucleic acid molecule shown in SEQ ID NO: 229, 231;
        c) a nucleic acid molecule, which, as a result of the degeneracy of the genetic code, can be derived from a polypeptide sequence according to SEQ ID NO: 230, 232;
        d) a nucleic acid molecule having at least 50% identity with the nucleic acid molecule sequence of a polynucleotide comprising the nucleic acid molecule shown in SEQ ID NO: 229, 231;
        e) a nucleic acid molecule encoding a polypeptide having at least 50% identity with the amino acid sequence of the polypeptide encoded by the nucleic acid molecule of (a) to (c) and having the activity of a small-conductance Ca2+-activated potassium channel;
        f) nucleic acid molecule which hybridizes with a nucleic acid molecule of (a) to (c) under stringent hybridization conditions;
        g) a nucleic acid molecule encoding a polypeptide which can be isolated with the aid of monoclonal or polyclonal antibodies made against a polypeptide encoded by one of the nucleic acid molecules of (a) to (e) and having the activity of a small-conductance Ca2+-activated potassium channel;
        h) a nucleic acid molecule encoding a polypeptide comprising the consensus sequence as shown in SEQ ID NO: 239 or one or more motifs selected from the group consisting of SEQ ID NO: 240, 241, 242, 243, 244, 245, 246, 247, 248, 249, 250, 251, 252 and 253;
        i) nucleic acid molecule which comprises a polynucleotide, which is obtained by amplifying a cDNA library or a genomic library using the primers in SEQ ID NO: 233, 234, 235, 236; and 237, 238; respectively and
        j) a nucleic acid molecule which is obtainable by screening a suitable nucleic acid library under stringent hybridization conditions with a probe comprising a complementary sequence of a nucleic acid molecule of (a) or (b) or with a fragment thereof, having at least 15 nt, preferably 20 nt, 30 nt, 50 nt, 100 nt, 200 nt or 500 nt of a nucleic acid molecule complementary to a nucleic acid molecule sequence characterized in (a) to (e) and encoding a polypeptide and having the activity of a small-conductance Ca2+-activated potassium channel.
    
    


In one embodiment the present invention is directed to a membrane comprising the polypeptide of the invention, whereby the membrane has not shown endogenous activity of the polypeptide of the invention.
In one embodiment the present invention is directed to a host cell comprising the polypeptide of the invention, whereby the membrane of the host has not shown endogenous activity of the polypeptide of the invention.
The wording “the membrane has not shown originally the activity of the polypeptide of the invention” means, that a polypeptide of the invention is not part of the natural occuring membrane, but it was assembled into the membrane according to a method of the invention.
In one embodiment the method of the present invention comprise the expression of a gene coding for a polypeptide with the activity of
i) an insect voltage-gated potassium channel ShaI (Shaker cognate I or Shaker-like) and/or its accessory protein KChIP (potassium channel-interacting protein) respectively in the membrane of a host cell, or
ii) an insect Shaker channel and/or a Hyperkinetic beta subunit, preferably H-kv beta subunit A or C subtype respectively in the membrane of a host cell, or
iii) an insect octopamine receptor selected from the group consisting of oa2, preferably from Drosophila melanogaster, Oamb, Oct-beta-2R and Oct-beta-3R in the membrane of a host cell, or
iv) an insect small-conductance Ca2+-activated potassium channel in the membrane of a host cell.
In one embodiment the host cell is a mammalian cell.
In one embodiment the host cell is a cell that in its native state has low or uninteresting electric activity. In contrast, a host cell expressing the polypeptide of the invention shows conductivity selected from the group of intervals 2-20 pS, 3-20 pS, 4-15 pS, 5-12 pS and 5-10 pS.
In one embodiment the host cell is selected from the group consisting of CHO-cells, HEK293, COS, HeLa, NIH3T3, BAK21, Jurkat, CV-1, HepC-2-, Xenopus oocyte; Sf9, S2, Sf21, Hi5, Pc12, U2OS.
For the production of the host cell of the invention comprising the polypeptide of the invention with the activity of
i) a ion channel and/or its accessory protein, a nucleotide sequence encoding the polypeptide, or
ii) a ion channel and/or a Hyperkinetic beta subunit, preferably H-kv beta subunit A or C subtype, a nucleotide sequence encoding the polypeptide, or
iii) an octopamine receptor and preferably additionally a marker protein, e.g. GFP, and/or additionally a “promiscuous” G-protein at least one nucleotide sequence encoding the polypeptides, or
iv) a ion channel a nucleotide sequence encoding the polypeptide is introduced into the host cell where it is recombinantly produced.
is introduced into the host cell where it is recombinantly produced.
In one embodiment the host cell is a microorganism in which the nucleotide sequence encoding the polypeptide with the activity of
i) a potassium ion channel of the invention and/or its accessory protein, or
ii) a potassium ion channel of the invention and/or a Hyperkinetic beta subunit, preferably H-kv beta subunit A or C subtype, or
iii) an octopamine receptor of the invention and preferably additionally a marker protein, e.g. GFP, and/or additionally a “promiscuous” G-protein, or
iv) a channel of the invention
is introduce in order to manifold said nucleotide sequence according to the general cloning techniques as are described, for example, in T. Maniatis, E. F. Fritsch and J. Sambrook, “Molecular Cloning: A Laboratory Manual”, Cold Spring Harbor Laboratory, Cold Spring Harbor, N.Y. (1989).
The above mentioned nucleic acid molecules can be cloned into a nucleic acid constructs or vectors according to the invention in combination together with further genes, or else different genes are introduced by transforming several nucleic acid constructs or vectors (including plasmids) into a host cell.
Accordingly, the invention also relates to a nucleic acid construct, preferably to an expression construct, comprising the nucleic acid molecule or molecules used in the process of the present invention or a fragment thereof functionally linked to one or more regulatory elements or signals. Furthermore the invention also relates to a nucleic acid constructs for the production of homologous recombination events, comprising the nucleic acids molecule used in the process of the present invention or parts thereof.
The nucleic acid construct can also comprise further genes, which are to be introduced into the host cells.
As described herein, regulator sequences or factors can have a positive effect on preferably the expression of the constructs introduced, thus increasing it. Thus, an enhancement of the regulator elements may advantageously take place at the transcriptional level by using strong transcription signals such as promoters and/or enhancers. In addition, however, an enhancement of translation is also possible, for example by increasing RNA stability.
Thus, the nucleic acid construct of the invention can be used as expression cassette and thus can be used directly for introduction into the host cell, or else they may be introduced into a vector. Accordingly in one embodiment the nucleic acid construct is an expression cassette comprising a microorganism promoter or a microorganism terminator or both. In another embodiment the expression cassette encompasses a eukaryotic promoter or a eukaryotic terminator or both.
If it is intended to transform the host cell with several constructs or vectors, the marker of a preceding transformation must be removed or a further marker employed in a following transformation. The markers can be removed from the host cell as described in the state of art via methods with which the skilled worker is familiar.
In one embodiment, the nucleic acid sequences used in the method according to the invention can be advantageously linked operably to one or more regulatory signals in order to increase gene expression.
These regulatory sequences are intended to enable the specific expression of nucleic acid molecules, e.g. the genes or gene fragments or of the gene products or the nucleic acid used in the process of the invention. Depending on the host organism for example eukaryotic cell or microorganism, this may mean, for example, that the gene or gene constructs is expressed and/or overexpressed after induction only, or that it is expressed and/or overexpressed constitutive. These regulatory sequences are, for example, sequences to which the inductors or repressors bind and which thus regulate the expression of the nucleic acid. Moreover, the gene construct can advantageously also comprise one or more of what are known as enhancer sequences in operable linkage with the promoter, and these enable an increased expression of the nucleic acid sequence. Also, it is possible to insert additional advantageous sequences at the 3′ end of the DNA sequences, such as, for example, further regulatory elements or terminators.
The term “increased expression” or “overexpression” as used herein means any form of expression that is additional to the original wild-type expression level.
Methods for increasing expression of genes or gene products are well documented in the art and include, for example, overexpression driven by appropriate promoters, the use of transcription enhancers or translation enhancers. Isolated nucleic acids which serve as promoter or enhancer elements may be introduced in an appropriate position (typically upstream) of a non-heterologous form of a polynucleotide so as to upregulate expression of a nucleic acid encoding the polypeptide of interest.
In the case of theshaker channel and/or a Hyperkinetic beta subunit, in one embodiment of the invention a Kozak sequence is used.
The nucleic acid molecules, which encode proteins according to the invention and nucleic acid molecules, which encode other polypeptides may be present in one nucleic acid construct or vector or in several ones. In one embodiment, only one copy of the nucleic acid molecule for use in the process of the invention or its encoding genes is present in the nucleic acid construct or vector. Several vectors or nucleic acid construct or vector can be expressed together in the host organism. The nucleic acid molecule or the nucleic acid construct according to the invention can be inserted in a vector and be present in the cell in a free form. If a stable transformation is preferred, a vector is used, which is stably duplicated over several generations or which or a part of which is else be inserted into the genome. In the case of mammalian cells, integration into the nuclear genome may have taken place. For the insertion of more than one constructs in the host genome the constructs to be expressed might be present together in one vector, for example in above-described vectors bearing a plurality of constructs.
As a rule, regulatory sequences for the expression rate of a constructs are located upstream (5′), within, and/or downstream (3′) relative to the sequence of the nucleic acid molecule to be regulated. They control in particular transcription and/or translation and/or the transcript stability. The expression level is dependent on the conjunction of further cellular regulatory systems, such as the protein biosynthesis and degradation systems of the cell.
Regulatory sequences include transcription and translation regulating sequences or signals, e.g. sequences located upstream (5′), which concern in particular the regulation of transcription or translation initiation, such as promoters or start codons, and sequences located downstream (3′), which concern in particular the regulation of transcription or translation termination and transcript stability, such as polyadenylation signals or stop codons.
Promoters, which are particularly advantageous, are constitutive, tissue or compartment specific and inducible promoters. In general, “promoter” is understood as meaning, in the present context, a regulatory sequence in a nucleic acid molecule, which mediates the expression of a coding sequence segment of a nucleic acid molecule. In principle, it is possible to use natural promoters together with their regulatory sequences. Some promoters for mammalian cells are for example CMV, SV40, TK, Beta-actin.
The nucleic acid construct is advantageously constructed in such a way that a promoter is followed by a suitable cleavage site for insertion of the nucleic acid to be expressed, advantageously in a polylinker, followed, if appropriate, by a terminator located behind the polylinker. If appropriate, this order is repeated several times so that several genes are combined in one construct and thus can be introduced into the transgenic plant in order to be expressed. The sequence is a for example repeated up to three times. For the expression, the nucleic acid sequences are inserted via the suitable cleavage site, for example in the polylinker behind the promoter. It is advantageous for each nucleic acid sequence to have its own promoter and, if appropriate, its own terminator, as mentioned above. However, it is also possible to insert several nucleic acid sequences behind a promoter and, if appropriate, before a terminator, in particular, if a polycistronic transcription is possible in the host or target cells. In this context, the insertion site, or the sequence of the nucleic acid molecules inserted, in the nucleic acid construct is not decisive, that is to say a nucleic acid molecule can be inserted in the first or last position in the cassette without this having a substantial effect on the expression. However, it is also possible to use only one promoter type in the construct.
One embodiment of the present invention also relates to a method for generating a vector, which comprises the insertion, into a vector, of the nucleic acid molecule characterized herein, the nucleic acid molecule according to the invention or the expression cassette according to the invention. The vector can, for example, be introduced into a cell, e.g. a microorganism or a mammalian cell, as described herein for the nucleic acid construct, or below under transformation or transfection or shown in the examples. A transient or stable transformation of the host or target cell is possible, however, a stable transformation is preferred.
The vector according to the invention is preferably a vector, which is suitable for expressing the polypeptide according to the invention in a cell, preferable a mammalian cell. The method can thus also encompass one or more steps for integrating regulatory signals into the vector, in particular signals, which mediate the expression in an organism such as a microorganism or mammalian cell.
Accordingly, the present invention also relates to a vector comprising the nucleic acid molecule characterized herein as part of a nucleic acid construct suitable for plant expression or the nucleic acid molecule according to the invention.
A advantageous vector used in the process of the invention, e.g. the vector of the invention, comprises a nucleic acid molecule which encodes a nucleic acid molecule which is used in the method of the invention, or a nucleic acid construct suitable for the expression in a cell comprising the nucleic acid molecules usable in the method of the invention as described above, either alone or in combination with further genes such as marker or selection genes.
Accordingly, the recombinant expression vectors which are advantageously used in the method of the invention comprise the nucleic acid molecules used in the method according to the invention or the nucleic acid construct according to the invention in a form which is suitable for expressing a nucleic acid molecule comprising a polynucleotide as shown in SEQ ID NO 2, 6, 10, 14, 18, 22, 26 and/or 30, in the case of the shaker channel and/or a Hyperkinetic beta subunit SEQ ID NO 73, 75, 77, 79, 81, 83, 85 and/or 87, in the case of the G-protein coupled receptor SEQ ID NO 130, 134, 138, 142, 146, 150, 154, 158, 162, 166, 170 and/or 174, and in the case of the SK-channel SEQ ID NO 228, 230, 232, or a homologue thereof and/or in the same time expressing, in a host cell, additional genes, which are accompanied by the nucleic acid molecules according to the invention or described herein. Accordingly, the recombinant expression vectors comprise one or more regulatory signals selected on the basis of the host cells to be used for the expression, in operable linkage with the nucleic acid sequence to be expressed.
In accordance with the invention, the term “vector” refers to a nucleic acid molecule, which is capable of transporting another nucleic acid to which it is linked. One type of vector is a “plasmid”, which means a circular double-stranded DNA loop into which additional DNA segments can be ligated. A further type of vector is a viral vector, it being possible to ligate additional DNA segments into the viral genome. Certain vectors are capable of autonomous replication in a host cell into which they have been introduced (for example bacterial vectors with bacterial replication origin). Other preferred vectors are advantageously completely or partly integrated into the genome of a host cell when they are introduced into the host cell and thus replicate together with the host genome. Moreover, certain vectors are capable of controlling the expression of genes with which they are in operable linkage. In the present context, these vectors are referred to as “expression vectors”. As mentioned above, they are capable of autonomous replication or may be integrated partly or completely into the host genome. Expression vectors, which are suitable for DNA recombination techniques usually, take the form of plasmids. In the present description, “plasmid” and “vector” can be used interchangeably since the plasmid is the most frequently used form of a vector. However, the invention is also intended to encompass these other forms of expression vectors, such as viral vectors, which exert similar functions. The term vector is furthermore also to encompass other vectors which are known to the skilled worker, such as phages, viruses such as SV40, CMV, TMV, transposons, IS elements, phasmids, phagemids, cosmids, and linear or circular DNA.
In a recombinant expression vector, “operable linkage” means that the nucleic acid molecule of interest is linked to the regulatory signals in such a way that expression of the genes is possible: they are linked to one another in such a way that the two sequences fulfill the predicted function assigned to the sequence (for example in an in-vitro transcription/translation system, or in a host cell if the vector is introduced into the host cell).
The term “regulatory sequence” is intended to comprise promoters, enhancers and other expression control elements (for example polyadenylation signals). These regulatory sequences are described, for example, in Goeddel: Gene Expression Technology: Methods in Enzymology 185, Academic Press, San Diego, Calif. (1990), or see: Gruber and Crosby, in: Methods in Plant Molecular Biology and Biotechnolgy, CRC Press, Boca Raton, Fla., Ed.: Glick and Thompson, chapter 7, 89-108, including the references cited therein. Regulatory sequences encompass those, which control the constitutive expression of a nucleotide sequence in many types of host cells and those which control the direct expression of the nucleotide sequence in specific host cells only, and under specific conditions. The skilled worker knows that the design of the expression vector may depend on factors such as the selection of the host cell to be transformed, the extent to which the protein amount is reduced, and the like. A preferred selection of regulatory sequences is described above, for example promoters, terminators, enhancers and the like. The term regulatory sequence is to be considered as being encompassed by the term regulatory signal. Several advantageous regulatory sequences, in particular promoters and terminators are described above. In general, the regulatory sequences described as advantageous for nucleic acid construct suitable for expression are also applicable for vectors.
As an alternative, the nucleic acid sequences can be expressed in insect cells using baculovirus expression vectors. Baculovirus vectors which are available for expressing proteins in cultured insect cells (for example Sf9 cells) encompass the pAc series (Smith et al. (1983) Mol. Cell Biol. 3:2156-2165) and the pVL series (Lucklow and Summers (1989) Virology 170:31-39). The abovementioned vectors are only a small overview of potentially suitable vectors. Further plasmids are known to the skilled worker and are described, for example, in: Cloning Vectors (Ed. Pouwels, P. H., et al., Elsevier, Amsterdam-New York-Oxford, 1985, ISBN 0 444 904018). Further suitable expression systems for prokaryotic and eukaryotic cells, see the chapters 16 and 17 by Sambrook, J., Fritsch, E. F., and Maniatis, T., Molecular Cloning: A Laboratory Manual, 2nd Edition, Cold Spring Harbor Laboratory, Cold Spring Harbor Laboratory Press, Cold Spring Harbor, N.Y., 1989.
Accordingly, one embodiment of the invention relates to a vector comprising a nucleic acid molecule for use in the process according to the invention or a nucleic acid construct for use in the method of the invention, e.g. the nucleic acid molecule or the nucleic acid construct of the invention. Said vector is useful for the transfection or transformation of host cells in order to provide the expression of the polypeptide according to the invention. Advantageously said nucleic acid molecule is in an operable linkage with regulatory sequences for the expression in a prokaryotic or eukaryotic, or in a prokaryotic and an eukaryotic host. Furthermore vectors which are suitable for homologous recombination are also within the scope of the invention.
Accordingly, one embodiment of the invention relates to a host cell, which has been transformed stably or transiently with the vector usable in the process of the invention, in particular with the vector according to the invention or the nucleic acid molecule according to the invention or the nucleic acid construct according to the invention, whereby the membrane of the host has not shown endogenously the activity of the polypeptide of the invention.
A further embodiment of the invention also relates to a method for the generation of a transgenic host cell, e.g. a eukaryotic or prokaryotic host or host cell, preferably a transgenic mammalian cell which comprises introducing, into the host cell, the nucleic acid construct according to the invention, the vector according to the invention, or the nucleic acid molecule according to the invention, whereby the membrane of the host has not shown endogenously the activity of the polypeptide of the invention.
Vector DNA can be introduced into prokaryotic or eukaryotic cells via conventional transformation or transfection techniques. The terms “transformation” and “transfection” include conjugation and transduction and, as used in the present context, are intended to encompass a multiplicity of prior-art methods for introducing foreign nucleic acid molecules (for example DNA) into a host cell, including calcium phosphate coprecipitation or calcium chloride coprecipitation, DEAE-dextran-mediated transfection, PEG-mediated transfection, lipofection, natural competence, chemically mediated transfer, electroporation or particle bombardment. Suitable methods for the transformation or transfection of host cells, including plant cells, can be found in Sambrook et al. (Molecular Cloning: A Laboratory Manual., 2nd Ed., Cold Spring Harbor Laboratory, Cold Spring Harbor Laboratory Press, Cold Spring Harbor, N.Y., 1989) and in other laboratory handbooks such as Methods in Molecular Biology, 1995, Vol. 44, Agrobacterium protocols, Ed.: Gartland and Davey, Humana Press, Totowa, N.J.
To select for the successful transfer of a nucleic acid molecule, vector or nucleic acid construct into a host organism, it is advantageous to use marker genes as have already been described above in detail. It is known of the stable or transient integration of nucleic acids into plant cells that only a minority of the cells takes up the foreign DNA and, if desired, integrates it into its genome, depending on the expression vector used and the transfection technique used. To identify and select these integrants, a gene encoding for a selectable marker (as described above, for example resistance to antibiotics) is usually introduced into the host cells together with the gene of interest. Preferred selectable markers are, for example, markers, which encode genes involved in a resistance, preferably gene for resistance to antibiotics, or in biosynthetic pathways of, for example, sugars or amino acids, such as 1-galactosidase, ura3 or ilv2. Markers, which encode genes such as luciferase, gfp or other fluorescence genes, are likewise suitable. These markers and the aforementioned markers can be used in mutants in whom these genes are not functional since, for example, they have been deleted by conventional methods. Furthermore, nucleic acid molecules, which encode a selectable marker, can be introduced into a host cell on the same vector as those, which encode the nucleotide acid molecule used in the process or else in a separate vector. Cells which have been transfected stably with the nucleic acid molecule introduced can be identified for example by selection (for example, cells which have integrated the selectable marker survive whereas the other cells die).
“Reporter genes” encode readily quantifiable proteins, as the above mentioned marker genes. The transformation efficacy or the expression site or timing can be assessed by means of these genes via growth assay, fluorescence assay, chemoluminescence assay, bioluminescence assay or resistance assay or via a photometric measurement (intrinsic color) or enzyme activity. Very especially preferred in this context are reporter proteins (Schenborn E, Groskreutz D. Mol Biotechnol. 1999; 13(1):29-44) such as the “green fluorescent protein” (GFP) (Gerdes H H and Kaether C, FEBS Lett. 1996; 389(1):44-47; Chui W L et al., Curr Biol 1996, 6:325-330; Leffel S M et al., Biotechniques. 23(5):912-8, 1997), chloramphenicol acetyltransferase, a luciferase (Giacomin, Plant Sci 1996, 116:59-72; Scikantha, J Bact 1996, 178:121; Millar et al., Plant Mol Biol Rep 1992 10:324-414), and luciferase genes in general, beta-galactosidase or beta-glucuronidase (Jefferson et al., EMBO J. 1987, 6, 3901-3907) or the Ura3 gene.
“Selection markers” confer resistance to antibiotics or other toxic compounds: examples which may be mentioned in this context are the neomycin phosphotransferase gene, which confers resistance to the aminoglycoside antibiotics neomycin (G 418), kanamycin, paromycin (Deshayes A et al., EMBO J. 4 (1985) 2731-2737), the sul gene encoding a mutated dihydropteroate synthase (Guerineau F et al., Plant Mol Biol. 1990; 15(1):127-136), the hygromycin B phosphotransferase gene (Gen Bank Accession NO: K 01193) and the she ble resistance gene, which confers resistance to the bleomycin antibiotics, e.g. zeocin. Further examples of selection marker genes are genes which confer resistance to 2-deoxyglucose-6-phosphate (WO 98/45456) or phosphinothricin and the like, or those which confer a resistance to antimetabolites, for example the dhfr gene (Reiss, Plant Physiol. (Life Sci. Adv.) 13 (1994) 142-149). Examples of other genes which are suitable are trpB or hisD (Hartman S C and Mulligan R C, Proc Natl Acad Sci USA. 85 (1988) 8047-8051). Another suitable gene is the mannose phosphate isomerase gene (WO 94/20627), the ODC (ornithine decarboxylase) gene (McConlogue, 1987 in: Current Communications in Molecular Biology, Cold Spring Harbor Laboratory, Ed.) or the Aspergillus terreus deaminase (Tamura K et al., Biosci Biotechnol Biochem. 59 (1995) 2336-2338).
In one embodiment the vector or nucleic acid construct of the invention comprises a nucleic acid sequence coding fo an affinity tag. “Affinity tag”: this refers to a peptide or polypeptide whose coding nucleic acid sequence can be fused to the nucleic acid sequence encoding the polypeptide of the invention either directly or by means of a linker, using customary cloning techniques. The affinity tag serves for the isolation, concentration and/or specific purification of the recombinant target protein by means of affinity chromatography from total cell extracts. The abovementioned linker can advantageously contain a protease cleavage site (for example for thrombin or factor Xa), whereby the affinity tag can be cleaved from the target protein when required. Examples of usual affinity tags are the “His tag” for example from Quiagen, Hilden, “Strep tag”, the “Myc tag” (Invitrogen, Carlsberg), the tag from New England Biolabs which consists of a chitin-binding domain and an intein, the maltose-binding protein (pMal) from New England Biolabs, and what is known as the CBD tag from Novagen. In this context, the affinity tag can be attached to the 5′ or the 3′ end of the coding nucleic acid sequence with the sequence encoding the target protein.
The nucleic acid molecules of the invention can be used for generating hybridization probes via which functional equivalents of the nucleic acid sequences according to the invention can be isolated. The generation of these probes and the experimental procedure are known. For example, this involves the specific generation of radioactive or nonradioactive probes by means of PCR and the use of suitably labeled oligonucleotides, followed by hybridization experiments. The techniques required for this purpose are mentioned, for example, in T. Maniatis, E. F. Fritsch and J. Sambrook, Molecular Cloning: A Laboratory Manual, Cold Spring Harbor Laboratory, Cold Spring Harbor, N.Y. (1989). The probes in question can furthermore be modified by standard techniques (Lit. SDM or random mutagenesis) in such a way that they can be employed for further purposes, for example as probe which hybridizes specifically with mRNA and the corresponding coding sequences, in order to analyze the corresponding sequences in other organisms. The probe can be used for example for screening a genomic library or a cDNA library of the insect in question or in a computer search for analogous sequences in electronic databases.
“Genetic control sequence”: the term “genetic control sequence” is considered as equivalent to the term “regulatory sequence” and describes sequences which have an effect on the transcription and, if appropriate, translation of the nucleic acids according to the invention in prokaryotic or eukaryotic organisms. Examples are promoters, terminators or what are known as “enhancer” sequences. In addition to these control sequences, or instead of these sequences, the natural regulation of these sequences may still be present before the actual structural genes and may, if appropriate, have been genetically modified in such a way that the natural regulation is switched off and the expression of the target gene has been modified, that is to say increased or reduced. The choice of the control sequence depends on the host organism or starting organism. Genetic control sequences furthermore also comprise the 5′-untranslated region, introns or the noncoding 3′ region of genes. Control sequences are furthermore understood as meaning those which make possible homologous recombination or insertion into the genome of a host organism or which permit removal from the genome.
“Knock-out transformants” refers to individual transgenic organism in which a specific gene has been inactivated, respectively the activity of a specific gene has been decreased, doew regulated, reduced or deleted in a targeted fashion by means of transformation.
“Natural genetic environment” refers to the natural chromosomal locus in the organism of origin. In the case of a genomic library, the natural genetic environment of the nucleic acid sequence is preferably retained at least in part. The environment flanks the nucleic acid sequence at least 5′ or 3′ and has a sequence length of at least 50 bp, preferably at least 100 bp, especially preferably at least 500 bp, very especially preferably at least 1 000 bp, and most preferably at least 5 000 bp.
“Reaction time” refers to the time required for carrying out an activity assay until a significant finding regarding an activity is obtained; it depends both on the specific activity of the protein employed in the assay and on the method used and the sensitivity of the apparatus used. The skilled worker is familiar with the determination of the reaction times. In the case of methods for identifying fungicidally active compounds which are based on photometry, the reaction times are generally between >0 and 360 minutes.
“Recombinant DNA” describes a combination of DNA sequences which can be generated by recombinant DNA technology.
“Recombinant DNA technology”: generally known techniques for fusing DNA sequences (for example described in Sambrook et al., 1989, Cold Spring Harbor, N.Y., Cold Spring Harbor Laboratory Press).
“Origin of Replication” ensure the multiplication of the expression cassettes or vectors according to the invention in microorganisms and yeasts, for example the pBR322 ori, ColE1 or the P15A on in E. coli (Sambrook et al.: Molecular Cloning. A Laboratory Manual, 2nd ed. Cold Spring Harbor Laboratory Press, Cold Spring Harbor, N.Y., 1989) and the ARS1 on in yeast (Nucleic Acids Research, 2000, 28(10): 2060-2068).
“Target/target protein”: the polypeptide of the invention, a protein, with the activity of
i) a potassium ion channel and/or its accessory protein respectively, or
ii) a potassium ion channel and/or a Hyperkinetic beta subunit, preferably H-kv beta subunit A or C subtype, or
iii) a protein, with the activity of an octopamine receptor, or

    
    
        iv) a potassium ion channel.
    
    


All of the targets or sites of action share the characteristic that the functional presence of the target protein is essential for the survival of the insect.
“Transformation” describes a process for introducing heterologous DNA into a prokaryotic or eukaryotic cell. A transformed cell describes not only the product of the transformation process per se, but also all of the transgenic progeny of the transgenic organism generated by the transformation process.
“Transgenic”: referring to a nucleic acid sequence, an expression cassette or a vector comprising a nucleic acid sequence according to the invention or an organism transformed with a nucleic acid sequence according to the invention, expression cassette or vector, the term transgenic describes all those constructs which have been generated by genetic engineering methods in which either the nucleic acid sequence of the target protein or a genetic control sequence linked operably to the nucleic acid sequence of the target protein or a combination of the abovementioned possibilities are not in their natural genetic environment or have been modified by recombinant methods. In this context, the modification can be achieved, for example, by mutating one or more nucleotide residues of the nucleic acid sequence in quest.
In one embodiment the present invention is directed to the use of the polypeptide, the membrane or the host cell of the invention as insecticidal target.
The deletion of the gene coding for insect voltage-gated potassium channel ShaI (Shaker cognate I or Shaker-like) and/or its accessory protein KChIP (potassium channel-interacting protein) respectively in Drosophila melanogaster, or the inhibition of the activity of the voltage-gated potassium channel ShaI (Shaker cognate I or Shaker-like) and/or its accessory protein KChIP (potassium channel-interacting protein) respectively is lethal for Drosophila melanogaster. 
Accordingly, in one embodiment the present invention is directed to the use of a polypeptide encoded by a nucleic acid molecule selected from the group consisting of:

    
    
        a) a nucleic acid molecule encoding the polypeptide shown in SEQ ID NO: 2, 6, 10, 14, 18, 22, 26 and/or 30;
        b) a nucleic acid molecule shown in SEQ ID NO: 1, 5, 9, 13, 17, 21, 25 and/or 29;
        c) a nucleic acid molecule, which, as a result of the degeneracy of the genetic code, can be derived from a polypeptide sequence according to SEQ ID NO: 2, 6, 10, 14, 18, 22, 26 and/or 30;
        d) a nucleic acid molecule having at least 50% identity with the nucleic acid molecule sequence of a polynucleotide comprising the nucleic acid molecule shown in SEQ ID NO: 2, 6, 10, 14, 18, 22, 26 and/or 30;
        e) a nucleic acid molecule encoding a polypeptide having at least 50% identity with the amino acid sequence of the polypeptide encoded by the nucleic acid molecule of (a) to (c) and having the activity of a voltage-gated potassium channel ShaI (Shaker cognate I or Shaker-like) and/or its accessory protein KChIP (potassium channel-interacting protein) respectively;
        f) nucleic acid molecule which hybridizes with a nucleic acid molecule of (a) to (c) under stringent hybridization conditions;
        g) a nucleic acid molecule encoding a polypeptide which can be isolated with the aid of monoclonal or polyclonal antibodies made against a polypeptide encoded by one of the nucleic acid molecules of (a) to (e) and having the activity of a voltage-gated potassium channel ShaI (Shaker cognate I or Shaker-like) and/or its accessory protein KChIP (potassium channel-interacting protein) respectively;
        h) a nucleic acid molecule encoding a polypeptide comprising the consensus sequence as shown in SEQ ID NO: 33 and/or 34 respectively or one or more motifs selected from the group consisting of SEQ ID NO: 35, 36, 37, 38, 39, 40, 41, 42, 43, 44, 45, 46, 47, 48, 49, 50, 51, 52, 53, 54 and/or 55, and/or 56, 57, 58, 59, 60, 61, 62, 63, 64, 65, 66, 67, 68, 69, 70 and/or 71 respectively;
        i) nucleic acid molecule which comprises a polynucleotide, which is obtained by amplifying a cDNA library or a genomic library using the primers in SEQ ID NO: 7, 8; 9, 10; 11, 12; respectively; and
        j) a nucleic acid molecule which is obtainable by screening a suitable nucleic acid library under stringent hybridization conditions with a probe comprising a complementary sequence of a nucleic acid molecule of (a) or (b) or with a fragment thereof, having at least 15 nt, preferably 20 nt, 30 nt, 50 nt, 100 nt, 200 nt or 500 nt of a nucleic acid molecule complementary to a nucleic acid molecule sequence characterized in (a) to (e) and encoding a polypeptide and having the activity of a voltage-gated potassium channel ShaI (Shaker cognate I or Shaker-like) and/or its accessory protein KChIP (potassium channel-interacting protein) respectively, or a homologue thereof as insecticidal target.
    
    


In the case of theshaker channel and/or a Hyperkinetic beta subunit, the deletion of the gene coding for insect Shaker channel and/or a Hyperkinetic beta subunit, preferably H-kv beta subunit A or C subtype respectively in Drosophila melanogaster, or the inhibition of the activity of the Shaker channel and/or a Hyperkinetic beta subunit, preferably H-kv beta subunit A or C subtype respectively is lethal for Drosophila melanogaster. 
Accordingly, in one embodiment the present invention is directed to the use of a polypeptide encoded by a nucleic acid molecule selected from the group consisting of:

    
    
        a) a nucleic acid molecule encoding a polypeptide comprising the polypeptide shown in SEQ ID NO: 73, 75, 77, 79, 81, 83, 85 and/or 87;
        b) a nucleic acid molecule comprising the nucleic acid molecule shown in SEQ ID NO: 72, 74, 76, 78, 80, 82, 84 and/or 86;
        c) a nucleic acid molecule, which, as a result of the degeneracy of the genetic code, can be derived from a polypeptide comprising a polypeptide sequence according to SEQ ID NO: 73, 75, 77, 79, 81, 83, 85 and/or 87;
        d) a nucleic acid molecule having at least 50% identity with the nucleic acid molecule sequence of a polynucleotide comprising the nucleic acid molecule shown in SEQ ID NO: 73, 75, 77, 79, 81, 83, 85 and/or 87;
        e) a nucleic acid molecule encoding a polypeptide having at least 50% identity with the amino acid sequence of the polypeptide encoded by the nucleic acid molecule of (a) to (c) and having the activity of a Shaker channel and/or a Hyperkinetic beta subunit, preferably H-kv beta subunit A or C subtype respectively;
        f) nucleic acid molecule which hybridizes with a nucleic acid molecule of (a) to (c) under stringent hybridization conditions;
        g) a nucleic acid molecule encoding a polypeptide which can be isolated with the aid of monoclonal or polyclonal antibodies made against a polypeptide encoded by one of the nucleic acid molecules of (a) to (e) and having the activity of a Shaker channel and/or a Hyperkinetic beta subunit, preferably H-kv beta subunit A or C subtype respectively;
        h) a nucleic acid molecule encoding a polypeptide comprising the consensus sequence as shown in SEQ ID NO: 102 and/or 103 respectively or one or more motifs selected from the group consisting of SEQ ID NO: 104, 105, 106, 107, 108, 109, 110, 111, 112, 113, 114, 115, 116, 117, 118, 119, 120, 121, 122, 123, 124 and/or 125 and/or 126, 127 and/or 128 respectively;
        i) nucleic acid molecule which comprises a polynucleotide, which is obtained by amplifying a cDNA library or a genomic library using the primers in SEQ ID NO: 88, 89; 90, 91; 92, 93; 94, 95; 96, 97; 98, 99 and/or 100, 101 respectively;
        and
        j) a nucleic acid molecule which is obtainable by screening a suitable nucleic acid library under stringent hybridization conditions with a probe comprising a complementary sequence of a nucleic acid molecule of (a) or (b) or with a fragment thereof, having at least 15 nt, preferably 20 nt, 30 nt, 50 nt, 100 nt, 200 nt or 500 nt of a nucleic acid molecule complementary to a nucleic acid molecule sequence characterized in (a) to (e) and encoding a polypeptide and having the activity of a Shaker channel and/or a Hyperkinetic beta subunit, preferably H-kv beta subunit A or C subtype respectively,
        or a homologue thereof as insecticidal target.
    
    


In the case of the G-protein coupled receptor, the deletion of the gene coding for insect octopamine receptor selected from the group consisting of oa2, preferably from Drosophila melanogaster, Oamb, Oct-beta-2R and Oct-beta-3R in Drosophila melanogasteror othe insects, or the inhibition of the activity of the octopamine receptor selected from the group consisting of oa2, preferably from Drosophila melanogaster, Oamb, Oct-beta-2R and Oct-beta-3R is lethal for Drosophila melanogaster or other insect respectively.
Accordingly, in one embodiment the present invention is directed to the use of a polypeptide encoded by a nucleic acid molecule selected from the group consisting of:

    
    
        a) a nucleic acid molecule encoding the polypeptide shown in SEQ ID NO: 130, 134, 138, 142, 146, 150, 154, 158, 162, 166, 170 and/or 174;
        b) a nucleic acid molecule shown in SEQ ID NO: 129, 133, 137, 141, 145, 149, 153, 157, 161, 165, 169 and/or 173;
        c) a nucleic acid molecule, which, as a result of the degeneracy of the genetic code, can be derived from a polypeptide comprising a polypeptide sequence according to SEQ ID NO: 130, 134, 138, 142, 146, 150, 154, 158, 162, 166, 170 and/or 174;
        d) a nucleic acid molecule having at least 50% identity with the nucleic acid molecule sequence of a polynucleotide comprising the nucleic acid molecule shown in SEQ ID NO: 130, 134, 138, 142, 146, 150, 154, 158, 162, 166, 170 and/or 174;
        e) a nucleic acid molecule encoding a polypeptide having at least 50% identity with the amino acid sequence of the polypeptide encoded by the nucleic acid molecule of (a) to (c) and having the activity of a octopamine receptor selected from the group consisting of oa2, preferably from Drosophila melanogaster, Oamb, Oct-beta-2R and Oct-beta-3R;
        f) nucleic acid molecule which hybridizes with a nucleic acid molecule of (a) to (c) under stringent hybridization conditions;
        g) a nucleic acid molecule encoding a polypeptide which can be isolated with the aid of monoclonal or polyclonal antibodies made against a polypeptide encoded by one of the nucleic acid molecules of (a) to (e) and having the activity of a octopamine receptor selected from the group consisting of oa2, preferably from Drosophila melanogaster, Oamb, Oct-beta-2R and Oct-beta-3R;
        h) a nucleic acid molecule encoding a polypeptide comprising the consensus sequence as shown in SEQ ID NO: 177, 178 and/or 179 respectively or one or more motifs selected from the group consisting of SEQ ID NO: 180, 181, 182, 183, 184, 185, 186, 187, 188, 189 and/or 190, and/or 191, 192, 193, 194, 195, 196, 197, 198, 199, 200, 201, 202, 203, 204, 205, 206, 207 and/or 208, and/or 209, 210, 211, 212, 213, 214, 215, 216, 217, 218, 219, 220, 221, 222, 223, 224, 225 and/or 226 respectively;
        i) nucleic acid molecule which comprises a polynucleotide, which is obtained by amplifying a cDNA library or a genomic library using the primers in SEQ ID NO: 131, 132; 135, 136; 139, 140; 143, 144; 147, 148; 151, 152; 155, 156, 159, 160; 163, 164; 167, 168; 171, 172 and/or 175, 176;
        and
        j) a nucleic acid molecule which is obtainable by screening a suitable nucleic acid library under stringent hybridization conditions with a probe comprising a complementary sequence of a nucleic acid molecule of (a) or (b) or with a fragment thereof, having at least 15 nt, preferably 20 nt, 30 nt, 50 nt, 100 nt, 200 nt or 500 nt of a nucleic acid molecule complementary to a nucleic acid molecule sequence characterized in (a) to (e) and encoding a polypeptide and having the activity of a octopamine receptor selected from the group consisting of oa2, preferably from Drosophila melanogaster, Oamb, Oct-beta-2R and Oct-beta-3R,
        or a homologue thereof as insecticidal target.
    
    


In the case of the SK-channel, the deletion of the gene coding for insect small-conductance Ca2+-activated potassium channel in Drosophila melanogaster, or the inhibition of the activity of the insect small-conductance Ca2+-activated potassium channel is lethal for Drosophila melanogaster. 
Accordingly, in one embodiment the present invention is directed to the use of a polypeptide encoded by a nucleic acid molecule selected from the group consisting of:

    
    
        a) a nucleic acid molecule encoding the polypeptide shown in SEQ ID NO: 228, 230, 232;
        b) a nucleic acid molecule shown in SEQ ID NO: 227, 229, 231;
        c) a nucleic acid molecule, which, as a result of the degeneracy of the genetic code, can be derived from a polypeptide sequence according to SEQ ID NO: 228, 230, 232;
        d) a nucleic acid molecule having at least 50% identity with the nucleic acid molecule sequence of a polynucleotide comprising the nucleic acid molecule shown in SEQ ID NO: 228, 230, 232;
        e) a nucleic acid molecule encoding a polypeptide having at least 50% identity with the amino acid sequence of the polypeptide encoded by the nucleic acid molecule of (a) to (c) and having the activity of a small-conductance Ca2+-activated potassium channel;
        f) nucleic acid molecule which hybridizes with a nucleic acid molecule of (a) to (c) under stringent hybridization conditions;
        g) a nucleic acid molecule encoding a polypeptide which can be isolated with the aid of monoclonal or polyclonal antibodies made against a polypeptide encoded by one of the nucleic acid molecules of (a) to (e) and having the activity of a small-conductance Ca2+-activated potassium channel;
        h) a nucleic acid molecule encoding a polypeptide comprising the consensus sequence as shown in SEQ ID NO: 239 or one or more motifs selected from the group consisting of SEQ ID NO: 240, 241, 242, 243, 244, 245, 246, 247, 248, 249, 250, 251, 252 and 253;
        i) nucleic acid molecule which comprises a polynucleotide, which is obtained by amplifying a cDNA library or a genomic library using the primers in SEQ ID NO: 233, 234, 235, 236; and 237, 238; respectively;
        and
        j) a nucleic acid molecule which is obtainable by screening a suitable nucleic acid library under stringent hybridization conditions with a probe comprising a complementary sequence of a nucleic acid molecule of (a) or (b) or with a fragment thereof, having at least 15 nt, preferably 20 nt, 30 nt, 50 nt, 100 nt, 200 nt or 500 nt of a nucleic acid molecule complementary to a nucleic acid molecule sequence characterized in (a) to (e) and encoding a polypeptide and having the activity of a small-conductance Ca2+-activated potassium channel,
        or a homologue thereof as insecticidal target.
    
    


The invention furthermore relates to nucleic acid construct or expression cassettes comprising
a) genetic control sequences in operable linkage with a nucleic acid sequence encompassing
i) a nucleic acid sequence with the nucleic acid sequence shown in SEQ ID NO 1, 5, 9, 13, 17, 21, 25 and/or 29, in the case of the shaker channel and/or a Hyperkinetic beta subunit SEQ ID NO 72, 74, 76, 78, 80, 82, 84 and/or 86, in the case of the G-protein coupled receptor SEQ ID NO 129, 133, 137, 141, 145, 149, 153, 157, 161, 165, 169 and/or 173, and in the case of the SK-channel SEQ ID NO 227, 229, 231; or
ii) a nucleic acid sequence which, on the basis of the degeneracy of the genetic code, can be derived from the amino acid sequence shown in SEQ ID NO 2, 6, 10, 14, 18, 22, 26 and/or 30, in the case of the shaker channel and/or a Hyperkinetic beta subunit SEQ ID NO 73, 75, 77, 79, 81, 83, 85 and/or 87, in the case of the G-protein coupled receptor SEQ ID NO 130, 134, 138, 142, 146, 150, 154, 158, 162, 166, 170 and/or 174, and in the case of the SK-channel SEQ ID NO 228, 230, 232 by back translation; or
iii) a functional equivalent of the nucleic acid sequence SEQ ID NO 1, 5, 9, 13, 17, 21, 25 and/or 29, in the case of the shaker channel and/or a Hyperkinetic beta subunit SEQ ID NO 72, 74, 76, 78, 80, 82, 84 and/or 86, in the case of the G-protein coupled receptor SEQ ID NO 129, 133, 137, 141, 145, 149, 153, 157, 161, 165, 169 and/or 173, and in the case of the SK-channel SEQ ID NO 227, 229, 231; and
b) additional functional elements; or
c) a combination of a) and b)
and to the use of the nucleic acid construct or the expression cassettes comprising
a) genetic control sequences in operable linkage with a nucleic acid sequence according to the invention;
b) additional functional elements; or
c) a combination of a) and b) in “in vitro” or “in vivo” assay systems.
The invention furthermore relates to the use of the abovementioned embodiments of the nucleic acid construct or the expression cassettes for expressing the polypeptide of the invention for in-vitro or in-vivo assay systems.
In one embodiment the present invention is directed to the use of the polypeptide, the membrane or the host cell of the invention for identifying compounds with insecticidal activity.
The present invention furthermore relates to the use of a polypeptide of the invention in a method for identifying insecticidal compounds.
A preferred embodiment of the method according to the invention comprises the following steps:
i. bringing a polypeptide of the invention into contact with one or more test compounds under conditions which permit the test compound(s) to bind to the polypeptide of the invention,
ii. detecting whether the test compound binds the polypeptide of the invention set forth in i); or
iii. detecting whether the test compound reduces or inhibits or blocks the activity of the polypeptide of the invention set forth in i); or
iv. detecting whether the test compound reduces or inhibits or blocks the transcription, translation or expression of the polypeptide with the activity of
1. the voltage-gated potassium channel ShaI (Shaker cognate I or Shaker-like) and/or its accessory protein KChIP (potassium channel-interacting protein) respectively set forth in I), or
2. the Shaker channel and/or a Hyperkinetic beta subunit, preferably H-kv beta subunit A or C subtype respectively set forth in i), or
3. the octopamine receptor selected from the group consisting of oa2, preferably from Drosophila melanogaster, Oamb, Oct-beta-2R and Oct-beta-3R set forth in i), or
4. the insect small-conductance Ca2+-activated potassium channel set forth in i).
The detection in accordance with step ii or iii of the above method regarding the activity of the polypeptide of the invention can be assessed using a variety of in vitro and in vivo assays, e. g., measuring current, measuring membrane potential, measuring ion flow, e. g., potassium or rubidium, measuring potassium concentration, measuring second messengers and transcription levels, using potassium-dependent yeast growth assays, and using e. g., voltage-sensitive dyes, radioactive tracers, and patch-clamp electrophysiology.
In the case of the G-protein coupled receptor, the detection in accordance with step ii or iii of the above method regarding the activity of the polypeptide of the invention can be assessed using a variety of in vitro and in vivo assays, e. g., measuring current, measuring membrane potential, measuring ion flow, e. g., calcium, preferably measuring calcium concentration, measuring second messengers and transcription levels, using potassium-dependent yeast growth assays, and using e. g., calcium concentration sensitive dyes or radioactive tracers.
The detection in accordance with step ii or iii of the above method can be effected using techniques which identify the interaction between protein and ligand. In this context, either the test compound or the polypeptide can contain a detectable label such as, for example, a fluorescent label, a radioisotope, a chemiluminescent label or an membrane or potentiometric label. Examples of labels are selected from the group consisting blue membrane potential dye from Molecular Devices, ANEP (AminoNaphthylEthenylPyridinium) dyes like di-4-ANEPPS, di-8-ANEPPS, di-2-ANEPEQ+, di-8-ANEPPQ, di-12-ANEPPQ; RH dyes (originally synthesized by Rina Hildesheim), including a serie of dialkylaminophenylpolyenylpyridinium dyes from Molecular Probes like RH 414 (T-1111), RH 795 (R-649) and RH 237 (S-1109). RH 421 (S-1108), or other dyes from Molecular Probes based on Carbocyanine and Oxonol as described in the Seventh Edition of Molecular Probes' Handbook of Fluorescent Probes and Research Chemicals published in 1999.
In the case of theshaker channel and/or a Hyperkinetic beta subunit, in one embodiment of the invention the method of detection whether the test compound reduces or inhibits or blocks the activity of the polypeptide of the invention comprises subjecting CHO-cells stably transfected with a insect Shaker channel and/or a Hyperkinetic beta subunit, preferably H-kv beta subunit A or C subtype respectively, preferably selected from the group consisting of SEQ ID NOs: 72, 74, 76, 78, 80, 82, 84 and/or 86, to loading with at least one of the above mentioned dyes, preferably blue membrane potential dye from Molecular Devices, for 0.1-3 hours, preferably 0.5-1 hours, preferably 0.45 hours,
activating the Shaker channel and/or a Hyperkinetic beta subunit, preferably H-kv beta subunit A or C subtype respectively by increasing the extrcellular level of KCl, in a concentration of the EC50 value of 1-120 mM, preferably 10-60 mM, more preferably 50 mM KCl,
adding the compound suspected to have the ability to inhibit the activity of the channel, preferably in a concentration of 1 μM-100 mM, 10-10000 μM, preferably 100-1000 μM
measuring the luminescence, fluorescence,
comparing the data of the luminescence/fluorescence of the dye with a control and determining whether the tested compound has the ability to inhibit the activity of the channel.
In the case of the G-protein coupled receptor, the detection in accordance with step ii or iii of the above method can be effected using techniques which identify the interaction between protein and ligand. In this context, either the test compound or the polypeptide can contain a detectable label such as, for example, a fluorescent label, a radioisotope, a chemiluminescent label or an membrane or potentiometric label. Examples of labels are selected from the group of calcium concentration sensitive dyes, e.g. Fluo-4 Calcium CrimsonTM, Calcium GreenTM, Calcium OrangeTM, Calcium YellowTM, Fura RedTM, Oregon Green®, Rhod-3, X-rhod-5F, Fura-2, bis-fura-2, fluo-5F, fluo-5N, fura dextran, fura-4F, fura-5F, fura-6F, fura-FF, fura-FF, quin-2, rhod dextran, rhod-2, rhod-5N or rhod-FF or other dyes from Molecular Probes based on Carbocyanine and Oxonol as described in the Seventh Edition of Molecular Probes' Handbook of Fluorescent Probes and Research Chemicals published in 1999.
In the case of the SK-channel, in one embodiment of the invention the method of detection whether the test compound reduces or inhibits or blocks the activity of the polypeptide of the invention comprises subjecting CHO-cells stably transfected with a insect small-conductance Ca2+-activated potassium channel, preferably selected from the group consisting of SEQ ID NOs: 227, 229, 231, to loading with at least one of the above mentioned dyes, preferably blue membrane potential dye from Molecular Devices, for 2-6 hours, preferably −5 hours, preferably 4 hours,
activating the small-conductance Ca2+-activated potassium channel with a ionophore, preferably ionomycin, in a concentration of the EC50 value of 100-500 nM, more preferably 200 nM, meaning incubation the cells in a concentration of 1-5 μM, adding the compound suspected to have the ability to inhibit the activity of the channel in a concentration of 5-50 ρM, 5-20 μM, preferably 10 μM measuring the luminescence, fluorescence
comparing the data the luminescence/fluorescence of the dye with a control and determining whether the tested compound has the ability to inhibit the activity of the channel.
In one embodiment of the invention the method of detection whether the test compound reduces or inhibits or blocks the activity of the polypeptide of the invention comprises subjecting CHO-cells stably transfected with a insect voltage-gated potassium channel ShaI (Shaker cognate I or Shaker-like) and/or its accessory protein KChIP (potassium channel-interacting protein) respectively, preferably selected from the group consisting of SEQ ID NOs: 1, 5, 9, 13, 17, 21, 25 and/or 29, to loading with at least one of the above mentioned dyes, preferably blue membrane potential dye from Molecular Devices, for 0.5-3 hours, preferably 1.5 hours, preferably 2-2.5 hours,
activating the voltage-gated potassium channel ShaI (Shaker cognate I or Shaker-like) and/or its accessory protein KChIP (potassium channel-interacting protein) respectively by increasing the extrcellular level of KCl, in a concentration of the EC50 value of 10-120 mM, preferably 10-60 mM, more preferably 30 mM KCl, adding the compound suspected to have the ability to inhibit the activity of the channel in a concentration of 1-200 μM, 5-100 μM, preferably 25-30 μM
measuring the luminescence, fluorescence
comparing the data the luminescence/fluorescence of the dye with a control and determining whether the tested compound has the ability to inhibit the activity of the channel.
The compound suspected of having the ability to inhibit the activity of the polypeptide of the invention is added directly to the bath solution.
Alternatively the detection in accordance with step ii or iii of the above method can be effected using the patch clamp technique.
Several variations of the basic technique can be applied selected from the group consisting of inside-out, outside-out, cell-attached, both excised patch, whole-cell patch and perforated patch techniques.
The subsequent detection depends on the label and is known to the skilled worker.
In one embodiment of the invention the method of detection whether the test compound reduces or inhibits or blocks the activity of the polypeptide of the invention comprises subjecting CHO-cells stably transfected with a insect voltage-gated potassium channel ShaI (Shaker cognate I or Shaker-like) and/or its accessory protein KChIP (potassium channel-interacting protein) respectively, preferably selected from the group consisting of SEQ ID NOs: 2, 6, 10, 14, 18, 22, 26 and/or 30, to the whole-cell configuration of the patch-clamp technique at room temperature (22-25° C.), using borosilicate glass capillaries with a resistances of 2-3 MOhm when filled with pipette solution and measured in bath solution, preferably compensating liquid junction potential between bath and pipette solution, measuring membrane current under whole-cell clamp, sampled at 2 kHz and filtered at 1 kHz, holding the cells at −70 mV and applying a family of 400 ms test voltage pulses starting from −100 to +130 mV in 10 mV increments every 2 sec, measuring the amplitude, as measured for the current-voltage relationship, and defining as the maximal outward current at a given depolarizing potential.
In the case of theshaker channel and/or a Hyperkinetic beta subunit, in one embodiment of the invention the method of detection whether the test compound reduces or inhibits or blocks the activity of the polypeptide of the invention comprises subjecting CHO-cells stably transfected with a insect Shaker channel and/or a Hyperkinetic beta subunit, preferably H-kv beta subunit A or C subtype respectively, preferably selected from the group consisting of SEQ ID NOs: 73, 75, 77, 79, 81, 83, 85 and/or 87, to the whole-cell configuration of the patch-clamp technique at room temperature (22-25° C.). The whole-cell voltage-clamp method of the invention comprises for data acquisition and further analysis, using the EPC10 digitally controlled amplifier in combination with PATCHMASTER software (HEKA Electronics, Lambrect, Germany). The EPC10 provides automatic subtraction of capacitance and leakage currents by mean of prepulse. The data are filtered at 66.7 KHz (−3 dB, 8-pole Bessel lowpass) and digitized at 5 μs per point. The input resistance of the patch pipettes is 2.0-4.0 MS/and the capacitances of the cells were 15.3±2.1 pF (n=45); the residual series resistances (after up to 80% compensation) are 4.2±0.4 MΩ. Correction for liquid junction potential is routinely applied. Membrane potential is clamped at −100 mV and currents are elicited by 50 ms depolarization pulses (0.1 Hz) from −60 mV to +100 mV (or +60 mV).
The compound suspected of having the ability to inhibit the activity of the polypeptide of the invention is added directly to the bath solution.
In one embodiment the subtraction of residual capacitance and leak current is performed with an on-line P/4 protocol by pClamp.
In the case of the G-protein coupled receptor, in one embodiment of the invention the method of detection whether the test compound reduces or inhibits or blocks the activity of the polypeptide of the invention comprises subjecting CHO-cells stably expressing the G-alpha-16 promiscuous G protein and stably or transiently expressing a octopamine receptor selected from the group consisting of oa2, preferably from Drosophila melanogaster, Oamb, Oct-beta-2R and Oct-beta-3R, preferably selected from the group consisting of SEQ ID NOs: 129, 133, 137, 141, 145, 149, 153, 157, 161, 165, 169 and/or 173, to
loading with at least one of the above mentioned dyes, preferably Fluo-4, for 0.1-3 hours, preferably 0.5-2 hours, preferably 1 hour, placing into the FLIPR and run using a two-addition protocol, whereby the test compounds, the compound suspected to have the ability to block or to activate the octopamine receptor, is to be added in the first addition and allowed to incubate for three minutes,
the activator octopamine is then to be introduced in the second addition at an EC80 concentration and the fluorescence read for two minutes. Controls will to be run for both additions. An increase in fluorescence above baseline in the first addition will indicate a possible activator and a reduced response or no increase in the second addition may indicate a possible inhibitor.
The subsequent detection depends on the label and is known to the skilled worker.
In the case of the SK-channel, in one embodiment of the invention the method of detection whether the test compound reduces or inhibits or blocks the activity of the polypeptide of the invention comprises subjecting CHO-cells stably transfected with a insect small-conductance Ca2+-activated potassium channel, preferably selected from the group consisting of SEQ ID NOs: 228, 230, 232, to the whole-cell configuration of the patch-clamp technique at room temperature (22-25° C.), using borosilicate glass capillaries with a resistances of 2-3 MOhm when filled with pipette solution and measured in bath solution, preferably compensating liquid junction potential between bath and pipette solution, measuring membrane current under whole-cell clamp, sampled at 2 kHz and filtered at 1 kHz, holding the cells at −70 mV and applying a family of 400 ms test voltage pulses starting from −100 to +130 mV in 10 mV increments every 2 sec, measuring the amplitude, as measured for the current-voltage relationship, and defining as the maximal outward current at a given depolarizing potential.
It is also possible, in the method according to the invention, to employ a plurality of test compounds in a method according to the invention. If a group of test compounds affects the target, then it is either possible directly to isolate the individual test compounds or to divide the group of test compounds into a variety of subgroups, for example when it consists of a multiplicity of different components, in order to reduce the number of the different test compounds in the method according to the invention. The method according to the invention is then repeated with the individual test compound or the relevant subgroup of test compounds. Depending on the complexity of the sample, the above-described steps can be carried out repeatedly, preferably until the subgroup identified in accordance with the method according to the invention only comprises a small number of test compounds, or indeed just one test compound.
The method according to the invention can advantageously be carried out as an HTS procedure.
HTS makes possible the simultaneous testing of a multiplicity of different compounds.
The quality of a high throughput screen is determined by two factors, the relative size of the assay window and the stability of this assay window from control experiment to control experiment (i.e. stability of assay signal across  ̃20 assay screening plates of controls). This is expressed as the z′ factor for the screen and the equation:

Z′=1−((3σmax+3σmin)/(Iμmax−μminI)).

In the case of the SK-channel, the quality of a high throughput screen is determined by two factors, the relative size of the assay window, in this case signal from a fully activated channel minus a the signal from an unactivated channel (usually expressed in arbitrary units). The second factor is the stability of this assay window from control experiment to control experiment (i.e. stability of assay signal across  ̃20 assay screening plates of controls). This is expressed as the z′ factor for the screen and the equation:

Z′=1−[3*(σmax+σmin)/Abs(Ave(MAX)−Ave(MIN))].

A calculated Z′>0.5 is considered an excellent assay.
In the case of theshaker channel and/or a Hyperkinetic beta subunit, alternatively for the calculation of the Z′ factor the following formula is used:




  
    Z
    ′
  
  =
  
    1
    -
    
      (
      
        
          3
          *
          
            (
            
              
                
                  ST
                  .
                  DEV
                
                ⁢
                
                  
                
                ⁢
                agonist
              
              +
              
                
                  ST
                  .
                  DEV
                
                ⁢
                
                  
                
                ⁢
                Tyrode
              
            
          
        
        
          
            MEAN
            ⁢
            
              
            
            ⁢
            agonist
          
          -
          
            MEAN
            ⁢
            
              
            
            ⁢
            Tyrode
          
        
      
      )
    
  




In this context preferred embodiments which are also suitable for high-throughput screening methods (HTS) in connection with the present invention, must be mentioned in particular:
1. In accordance with a preferred embodiment, the detection of step ii (and in the case of the SK-channel, variant 3) of the method according to the invention encompasses the following steps: Fluorescent resonance energy transfer (FRET) is based on the irradiation-free energy transfer between two spatially adjacent fluorescent molecules under suitable conditions. A prerequisite is that the emission spectrum of the donor molecule overlaps with the excitation spectrum of the acceptor molecule. By fluorescently labeling UGP and the test compounds, the binding can be measured by means of FRET (Cytometry 34, 1998, pp. 159-179). As an alternative, the method according to the invention may also take the form of the “displacement assay” described under 1. An especially suitable embodiment of FRET technology is “Homogeneous Time Resolved Fluorescence” (HTRF) as can be obtained from Packard BioScience. The compounds which are identified in this manner may be suitable as inhibitors.
2. In accordance with a preferred embodiment, the detection of step ii (and in the case of the SK-channel, variant 3) of the method according to the invention comprises the following steps: The measurement of surface plasmon resonance is based on the change in the refractive index at a surface when a chemical compound binds to a protein which is immobilized to said surface. Since the change in the refractive index is identical for virtually all proteins and polypeptides for a defined change in the mass concentration at the surface, this method can be applied to any protein in principle (Lindberg et al. Sensor Actuators 4 (1983) 299-304; Malmquist Nature 361 (1993) 186-187). The measurement can be carried out for example with the automatic analyzer based on surface plasmon resonance which is available from Biacore (Freiburg) at a throughput of, currently, up to 384 samples per day. A method according to the invention can be designed directly for measuring the binding of the test compound to the UGP. As an alternative, the method according to the invention may also take the form of the “displacement assay” described under 1. The compounds identified in this manner may be suitable as inhibitors.
3. In accordance with a preferred embodiment, the detection of step ii of the method according to the invention comprises the use of FLIPR Membrane Potential Assay Kits from Molecular Devices as disclosed in the examples. The method is based on the application of voltage-sensitive dyes on the FLIPR Fluorometric Imaging Plate Reader system, while showing good correlation with manual patch clamping data.
4. In accordance with a preferred embodiment, the detection of step ii of the method according to the invention comprises the use of BIOMOL Compound Screening or the BioFocus compound screening, using two fluid addition method to permit the detection of activators and antagonists in a single experiment, or subsequent BIOMOL and BioFocus validation screening as disclosed in the examples.
In the case of theshaker channel and/or a Hyperkinetic beta subunit, the method of the invention puts a functional cellular-based assay for the Drosophila melanogaster Shaker channel at disposal, preferably developed in CHO-K1 cells by stable pure clone selection and functional characterization with Membrane Potential sensitive dye at FLIPR, preferably for FLIPR384 and/or FLIPRTETRA or both experiments, and electrophysiological techniques.
The generated assay completely fulfil the HTS requirements showing very high signal quality and reproducibility.
For the purposes of high-throughput screening, the following parameters are to be mentioned:
Activator KCl concentration: 50 mM ( ̃EC80) in Activation buffer
Reference Z′: 0.60
Minimal acceptable Z′ for single assay plate: 0.45
% Inhibition Threshold for Hit: 40%
All of the substances identified via the above mentioned methods can subsequently be checked for their insecticidal action in another embodiment of the method according to the invention.
Furthermore, there exists the possibility of detecting further candidates for insecticidal active ingredients by molecular modeling via elucidation of the three-dimensional structure of the polypeptide of the invention by x-ray structure analysis. The preparation of protein crystals required for x-ray structure analysis, and the relevant measurements and subsequent evaluations of these measurements, the detection of a binding site in the protein, and the prediction of potential inhibitor structures are known to the skilled worker. In principle, an optimization of the active compounds identified by the abovementioned methods is also possible via molecular modeling.
In one embodiment the activity of the polypeptide of the invention incubated with the test compound is compared with the activity of a control a wild type cell or a polypeptide of the invention which has not been incubated with a test compound in step iii.
In this context, compounds are selected in step (iii) which result in a significant decrease in the activity of the polypeptide of the invention, a reduction of at least 10%, advantageously at least 20%, 25%, 29% preferably at least 30%, especially preferably at least 50% and very especially preferably at least 70%, 80%, 90%, 95% 96%, 97%, 98%, 995, or 100% reduction (inhibition), being achieved.
The invention furthermore relates to compounds identified by the methods according to the invention. These compounds are hereinbelow referred to as “selected compounds”. They have a molecular weight of less than 1 000 g/mol, advantageously less than 500 g/mol, preferably less than 400 g/mol, especially preferably less than 300 g/mol. Insecticidal active compounds have a Ki value of less than 1 μM, preferably less than 1 μM, especially preferably less than 0.1 μM, very especially preferably less than 0.01 μM.
Substances identified via the above mentioned methods and/or as shown in the examples are depicted in table I:













Structure
% inhibition
Mol Formula
Mol Weight











80-100%
C6H8ClN7O
 229.627 g/mol


Amiloride, 3,5-diamino-6-chloro-N-(diaminomethylene)pyrazine-





2-carboxamide





CAS number 2016-88-8





 







C16H15F3N2O4
  356.3 g/mol


Bay K8644





 







C26H29N3O6
 479.525 g/mol


 


Nicardipine





 







C20H24N2O2
 324.417 g/mol


Quinidine, Synonyms (2-ethenyl-4-azabicyclo[2.2.2]oct-5-yl)-





(6-methoxyquinolin-4-yl)-methanol 6′-methoxycinchonan-9-ol





6′-methoxy-a-(5-vinyl-2-quinuclidinyl)-4-quinolinemethanol





 







C13H9F3N2O2
282.21797 g/mol


Niflumic acid





 






42.6
C15H13NO
 223.28


 






35.2
C25H21NO5
 415.45


 






32.5
C17H13ClF3NO
 339.75


 






39.7
C17H14N2O
 262.31


 






56.8
C14H13NOS
 243.33


 






52.0
C15H15NOS
 257.36


 






38.3
C15H14ClNO2S
 307.8


 






56.8
C20H14ClFN2O
 352.8


 






55.5
C23H17FN2O
 356.4


 






60.3
C22H17F4NO4
 435.38


 






32.0
C17H15ClFNO2
 319.77


 






52.6
C16H12ClF2NO
 307.73


 






46.0
C20H17ClN2OS
 368.89


 






41.5
C21H15ClFNO4
 399.81


 






32.1
C23H15F3N2O2
 408.38


 






31.4
C20H15F3N2O2S
 404.41


 






39.1
C21H15F3N2O2
 384.36


 






50.4
C21H17F3N2O3
 402.38


 






29.1
C22H17F3N2O2
 398.39


 






40.1
C21H17F3N2O3
 402.38


 






34.0
C20H14F4N2O2S
 422.4


 






33.5
C17H13F4NO2
 339.29


 






37.8
C19H15F3N2O2S2
 424.47


 






42.5
C19H15FN2OS
 338.41


 






29.3
C20H17FN2OS
 352.43


 






48.5
C21H17FN2O
 332.38


 






57.5
C23H19FN2O2
 374.42


 






37.5
C19H14F2N2OS
 356.4


 






42.7
C21H13F5N2O
 404.34


 






54.9
C19H14F2N2O2
 340.33


 






54.1
C22H16F2N2O2
 378.38


 






58.0
C21H15FN2OS
 362.43


 






33.0
C20H17ClN2O2
 352.82


 






33.3
C22H17ClN2O2
 376.85


 






36.4
C20H14Cl2N2O
 369.25


 






31.2
C18H13F3N2O2
 346.31


 






63.3
C23H17ClN2O
 372.86


 






38.7
C18H16ClFN2O4
 378.79


 






29.6
C21H21N3O2
 347.42


 






52.7
C19H19FN2O3
 342.37


 






38.7
C18H14ClN3O2
 339.78


 






31.5
C18H15ClN2O4
 358.78


 






30.2
C17H14F2N2O2
 316.31


 






36.2
C24H22FN3O3
 419.46


 






33.3
C24H23N3O2
 385.47


 






41.6
C19H17FN2O3
 340.36


 






41.3
C19H20FN3O2
 341.39


 






38.0
C19H15Cl2FN2O2S
 425.31


 






30.2
C20H16F3N3O5S
 467.43


 






35.0
C22H18F3N3O2S
 445.47


 






33.3
C23H20F3N3O2S
 459.49


 






30.2
C20H16F4N2O3S
 440.42


 






35.2
C21H19FN2O2
 350.4









The selected compounds are suitable for controlling insect pests as those mentioned above. Examples of those insects are the selected from the group consisting of Pterygota, Neopetra, Hemiptera, Coleoptera, Diptera, Homoptera, Tenebrionoidea, Tenebrionidae, Tenebrio, Sternorrhyncha, Aphidina, Brachycera, Drosophilidae, Drosophilinae and Drosophila, preferably in the case of the SK-channel Green Peach Aphid (Myzus persica) and/or Red Flower Beetle (Tribolium castaneum).
The selected compounds are suitable for controlling insect pests in agriculture, for protection of crops, forests, urban trees, rangelands, postharvest systems (e.g. stored grains) and natural areas against insect pests as well as in storage of grains and/or food.
The selected compounds are suitable for preventing Infestation, meaning to impede the growth of a pest population that it becomes so large it becomes harmful or unpleasant.
According to the invention, a insect pest is any insect that is undesirable or causes harm to people, property, or the environment. An organism may be a pest in one place but not in another; for example, termites in a house vs. those that recycle dead trees in a forest.
The selected compounds can also be present in the form of their useful salts. Useful salts which are suitable are mainly the salts of those cations, or the acid addition salts of those acids, whose cations, or anions, do not adversely affect the insecticidal action of the insecticidal active compounds identified via the methods according to the invention.
All of the compounds identified via the above methods can, if they contain chiral centers, be subject matter of the present invention in the form of pure enantiomers or diastereomers or in the form of their mixtures and as racemates.
The selected compounds can be chemically synthesized substances or substances produced by microorganisms and can be found, for example, in cell extracts of, for example, plants, animals or microorganisms. The reaction mixture can be a cell-free extract or comprise a cell or cell culture. Suitable methods are known to the SKilled worker and are described generally for example in Alberts, Molecular Biology the cell, 3rd Edition (1994), for example chapter 17.
Possible test compounds can be expression libraries such as, for example, cDNA expression libraries, peptides, proteins, nucleic acids, antibodies, small organic substances, hormones, PNAs or the like (Milner, Nature Medicin 1 (1995), 879-880; Hupp, Cell. 83 (1995), 237-245; Gibbs, Cell. 79 (1994), 193-198 and references cited therein).
Compounds with an insecticidal activity according to the invention are selected from the group consisting of TMB-8, Nifedipine, Nitrndipine, Tetrandrine, Verapamil, Methoxy Verapamil, YS035, Propafenone, Quinidine, Sulfonamides, Thiazolidinones, Indolones, Isoxazolylamides.
For use in a method according to the present invention, the compounds can be converted into the customary formulations, e.g. solutions, emulsions, suspensions, dusts, powders, pastes, granules and directly sprayable solutions. The use form depends on the particular purpose and application method. Formulations and application methods are chosen to ensure in each case a fine and uniform distribution of the compound of the formula I according to the present invention.
The formulations are prepared in a known manner (see e.g. for review U.S. Pat. No. 3,060,084, EP-A 707 445 (for liquid concentrates), Browning, “Agglomeration”, Chemical Engineering, Dec. 4, 1967, 147-48, Perry's Chemical Engineer's Handbook, 4th Ed., McGraw-Hill, New York, 1963, pages 8-57 and et seq. WO 91/13546, U.S. Pat. No. 4,172,714, U.S. Pat. No. 4,144,050, U.S. Pat. No. 3,920,442, U.S. Pat. No. 5,180,587, U.S. Pat. No. 5,232,701, U.S. Pat. No. 5,208,030, GB 2,095,558, U.S. Pat. No. 3,299,566, Klingman, Weed Control as a Science, John Wiley and Sons, Inc., New York, 1961, Hance et al., Weed Control Handbook, 8th Ed., Blackwell Scientific Publications, Oxford, 1989 and Mollet, H., Grubemann, A., Formulation technology, Wiley VCH Verlag GmbH, Weinheim (Germany), 2001, 2. D. A. Knowles, Chemistry and Technology of Agrochemical Formulations, Kluwer Academic Publishers, Dordrecht, 1998 (ISBN 0-7514-0443-8), for example by extending the active compound with auxiliaries suitable for the formulation of agrochemicals, such as solvents and/or carriers, if desired emulsifiers, surfactants and dispersants, preservatives, antifoaming agents, anti-freezing agents, for seed treatment formulation also optionally colorants and/or binders and/or gelling agents.
Solvents/carriers, which are suitable, are e.g.:

    
    
        solvents such as water, aromatic solvents (for example Solvesso products, xylene and the like), paraffins (for example mineral fractions), alcohols (for example methanol, butanol, pentanol, benzyl alcohol), ketones (for example cyclohexanone, gamma-butyrolactone), pyrrolidones (N-metyhl-pyrrolidone (NMP), N-octylpyrrolidone NOP), acetates (glycol diacetate), alkyl lactates, lactones such as g-butyrolactone, glycols, fatty acid dimethylamides, fatty acids and fatty acid esters, triglycerides, oils of vegetable or animal origin and modified oils such as alkylated plant oils. In principle, solvent mixtures may also be used.
        carriers such as ground natural minerals and ground synthetic minerals, such as silica gels, finely divided silicic acid, silicates, talc, kaolin, attaclay, limestone, lime, chalk, bole, loess, clay, dolomite, diatomaceous earth, calcium sulfate and magnesium sulfate, magnesium oxide, ground synthetic materials, fertilizers, such as, for example, ammonium sulfate, ammonium phosphate, ammonium nitrate, ureas and products of vegetable origin, such as cereal meal, tree bark meal, wood meal and nutshell meal, cellulose powders and other solid carriers.
    
    


Suitable emulsifiers are nonionic and anionic emulsifiers (for example polyoxyethylene fatty alcohol ethers, alkylsulfonates and arylsulfonates).
Examples of dispersants are lignin-sulfite waste liquors and methylcellulose.
Suitable surfactants are alkali metal, alkaline earth metal and ammonium salts of lignosulfonic acid, naphthalenesulfonic acid, phenolsulfonic acid, dibutylnaphthalenesulfonic acid, alkylarylsulfonates, alkyl sulfates, alkylsulfonates, fatty alcohol sulfates, fatty acids and sulfated fatty alcohol glycol ethers, furthermore condensates of sulfonated naphthalene and naphthalene derivatives with formaldehyde, condensates of naphthalene or of naphthalenesulfonic acid with phenol and formaldehyde, polyoxyethylene octylphenyl ether, ethoxylated isooctylphenol, octylphenol, nonylphenol, alkylphenyl polyglycol ethers, tributylphenyl polyglycol ether, tristearylphenyl polyglycol ether, alkylaryl polyether alcohols, alcohol and fatty alcohol/ethylene oxide condensates, ethoxylated castor oil, polyoxyethylene alkyl ethers, ethoxylated polyoxypropylene, lauryl alcohol polyglycol ether acetal, sorbitol esters,
Also anti-freezing agents such as glycerin, ethylene glycol, propylene glycol and bactericides such as can be added to the formulation.
Suitable antifoaming agents are for example antifoaming agents based on silicon or magnesium stearate.
Suitable preservatives are for example dichlorophen and benzyl alcohol hemiformal
Suitable thickeners are compounds which confer a pseudoplastic flow behavior to the formulation, i.e. high viscosity at rest and low viscosity in the agitated stage. Mention may be made, in this context, for example, of commercial thickeners based on polysaccharides, such as Xanthan Gum® (Kelzan® from Kelco), Rhodopol®23 (Rhone Poulenc) or Veegum® (from R.T. Vanderbilt), or organic phyllosilicates, such as Attaclay® (from Engelhardt). Antifoam agents suitable for the dispersions according to the invention are, for example, silicone emulsions (such as, for example, Silikon® SRE, Wacker or Rhodorsil® from Rhodia), long-chain alcohols, fatty acids, organofluorine compounds and mixtures thereof. Biocides can be added to stabilize the compositions according to the invention against attack by microorganisms. Suitable biocides are, for example, based on isothiazolones such as the compounds marketed under the trademarks Proxel® from Avecia (or Arch) or Acticide® RS from Thor Chemie and Kathon® MK from Rohm & Haas. Suitable antifreeze agents are organic polyols, for example ethylene glycol, propylene glycol or glycerol. These are usually employed in amounts of not more than 10% by weight, based on the total weight of the active compound composition. If appropriate, the active compound compositions according to the invention may comprise 1 to 5% by weight of buffer, based on the total amount of the formulation prepared, to regulate the pH, the amount and type of the buffer used depending on the chemical properties of the active compound or the active compounds. Examples of buffers are alkali metal salts of weak inorganic or organic acids, such as, for example, phosphoric acid, boronic acid, acetic acid, propionic acid, citric acid, fumaric acid, tartaric acid, oxalic acid and succinic acid.
Substances which are suitable for the preparation of directly sprayable solutions, emulsions, pastes or oil dispersions are mineral oil fractions of medium to high boiling point, such as kerosene or diesel oil, furthermore coal tar oils and oils of vegetable or animal origin, aliphatic, cyclic and aromatic hydrocarbons, for example toluene, xylene, paraffin, tetrahydronaphthalene, alkylated naphthalenes or their derivatives, methanol, ethanol, propanol, butanol, cyclohexanol, cyclohexanone, isophorone, strongly polar solvents, for example dimethyl sulfoxide, N-methylpyrrolidone and water.
Powders, materials for spreading and dusts can be prepared by mixing or concomitantly grinding the active substances with a solid carrier.
Granules, for example coated granules, impregnated granules and homogeneous granules, can be prepared by binding the active ingredients to solid carriers. Examples of solid carriers are mineral earths such as silica gels, silicates, talc, kaolin, attaclay, limestone, lime, chalk, bole, loess, clay, dolomite, diatomaceous earth, calcium sulfate, magnesium sulfate, magnesium oxide, ground synthetic materials, fertilizers, such as, for example, ammonium sulfate, ammonium phosphate, ammonium nitrate, ureas, and products of vegetable origin, such as cereal meal, tree bark meal, wood meal and nutshell meal, cellulose powders and other solid carriers.
In general, the formulations comprise from 0.01 to 95% by weight, preferably from 0.1 to 90% by weight, of the active ingredient. The active ingredients are employed in a purity of from 90% to 100%, preferably 95% to 100% (according to NMR spectrum).
For seed treatment purposes, respective formulations can be diluted 2-10 fold leading to concentrations in the ready to use preparations of 0.01 to 60% by weight active compound by weight, preferably 0.1 to 40% by weight.
The compound identified according to the method of the invention can be used as such, in the form of their formulations or the use forms prepared therefrom, for example in the form of directly sprayable solutions, powders, suspensions or dispersions, emulsions, oil dispersions, pastes, dustable products, materials for spreading, or granules, by means of spraying, atomizing, dusting, spreading or pouring. The use forms depend entirely on the intended purposes; they are intended to ensure in each case the finest possible distribution of the active compounds according to the invention.
The following are examples of formulations:
1. Products for dilution with water. For seed treatment purposes, such products may be applied to the seed diluted or undiluted.
A) Water-soluble concentrates (SL, LS)
10 parts by weight of the active compound is dissolved in 90 parts by weight of water or a water-soluble solvent. As an alternative, wetters or other auxiliaries are added.
The active compound dissolves upon dilution with water, whereby a formulation with 10% (w/w) of active compound is obtained.
B) Dispersible concentrates (DC)
20 parts by weight of the active compound is dissolved in 70 parts by weight of cyclohexanone with addition of 10 parts by weight of a dispersant, for example polyvinylpyrrolidone. Dilution with water gives a dispersion, whereby a formulation with 20% (w/w) of active compounds is obtained.
C) Emulsifiable concentrates (EC)
15 parts by weight of the active compounds is dissolved in 7 parts by weight of xylene with addition of calcium dodecylbenzenesulfonate and castor oil ethoxylate (in each case 5 parts by weight). Dilution with water gives an emulsion, whereby a formulation with 15% (w/w) of active compounds is obtained.
D) Emulsions (EW, EO, ES)
25 parts by weight of the active compound is dissolved in 35 parts by weight of xylene with addition of calcium dodecylbenzenesulfonate and castor oil ethoxylate (in each case 5 parts by weight). This mixture is introduced into 30 parts by weight of water by means of an emulsifier machine (e.g. Ultraturrax) and made into a homogeneous emulsion. Dilution with water gives an emulsion, whereby a formulation with 25% (w/w) of active compound is obtained.
E) Suspensions (SC, OD, FS)
In an agitated ball mill, 20 parts by weight of the active compound is comminuted with addition of 10 parts by weight of dispersants, wetters and 70 parts by weight of water or of an organic solvent to give a fine active compound suspension. Dilution with water gives a stable suspension of the active compound, whereby a formulation with 20% (w/w) of active compound is obtained.
F) Water-dispersible granules and water-soluble granules (WG, SG)
50 parts by weight of the active compound is ground finely with addition of 50 parts by weight of dispersants and wetters and made as water-dispersible or water-soluble granules by means of technical appliances (for example extrusion, spray tower, fluidized bed). Dilution with water gives a stable dispersion or solution of the active compound, whereby a formulation with 50% (w/w) of active compound is obtained.
G) Water-dispersible powders and water-soluble powders (WP, SP, SS, WS)
75 parts by weight of the active compound are ground in a rotor-stator mill with addition of 25 parts by weight of dispersants, wetters and silica gel. Dilution with water gives a stable dispersion or solution of the active compound, whereby a formulation with 75% (w/w) of active compound is obtained.
H) Gel-Formulation (GF)
In an agitated ball mill, 20 parts by weight of the active compound is comminuted with addition of 10 parts by weight of dispersants, 1 part by weight of a gelling agent wetters and 70 parts by weight of water or of an organic solvent to give a fine active compound suspension. Dilution with water gives a stable suspension of the active compound, whereby a formulation with 20% (w/w) of active compound is obtained.
2. Products to be applied undiluted for foliar applications. For seed treatment purposes, such products may be applied to the seed diluted or undiluted.
I) Dustable powders (DP, DS)
5 parts by weight of the active compound are ground finely and mixed intimately with 95 parts by weight of finely divided kaolin. This gives a dustable product having 5% (w/w) of active compound.
J) Granules (GR, FG, GG, MG)
0.5 part by weight of the active compound is ground finely and associated with 95.5 parts by weight of carriers, whereby a formulation with 0.5% (w/w) of active compound is obtained. Current methods are extrusion, spray-drying or the fluidized bed. This gives granules to be applied undiluted for foliar use.
K) ULV solutions (UL)
10 parts by weight of the active compound is dissolved in 90 parts by weight of an organic solvent, for example xylene. This gives a product having 10% (w/w) of active compound, which is applied undiluted for foliar use.
Aqueous use forms can be prepared from emulsion concentrates, pastes or wettable powders (sprayable powders, oil dispersions) by adding water. To prepare emulsions, pastes or oil dispersions, the substances, as such or dissolved in an oil or solvent, can be homogenized in water by means of a wetter, tackifier, dispersant or emulsifier. Alternatively, it is possible to prepare concentrates composed of active substance, wetter, tackifier, dispersant or emulsifier and, if appropriate, solvent or oil, and such concentrates are suitable for dilution with water.
The active ingredient concentrations in the ready-to-use products can be varied within relatively wide ranges. In general, they are from 0.0001 to 10%, preferably from 0.01 to 1%.
The active ingredients may also be used successfully in the ultra-low-volume process (ULV), it being possible to apply formulations comprising over 95% by weight of active ingredient, or even to apply the active ingredient without additives.
In the method of this invention compounds identified according to the method of the invention may be applied with other active ingredients, for example with other pesticides, insecticides, herbicides, fertilizers such as ammonium nitrate, urea, potash, and superphosphate, phytotoxicants and plant growth regulators, safeners and nematicides. These additional ingredients may be used sequentially or in combination with the above-described compositions, if appropriate also added only immediately prior to use (tank mix). For example, the plant(s) may be sprayed with a composition of this invention either before or after being treated with other active ingredients.
The following list M of pesticides together with which the compounds according to the invention can be used and with which potential synergistic effects might be produced, is intended to illustrate the possible combinations, but not to impose any limitation:
M.1. Organo(thio)phosphates: acephate, azamethiphos, azinphos-ethyl, azinphosmethyl, chlorethoxyfos, chlorfenvinphos, chlormephos, chlorpyrifos, chlorpyrifosmethyl, coumaphos, cyanophos, demeton-S-methyl, diazinon, dichlorvos/DDVP, dicrotophos, dimethoate, dimethylvinphos, disulfoton, EPN, ethion, ethoprophos, famphur, fenamiphos, fenitrothion, fenthion, flupyrazophos, fosthiazate, heptenophos, isoxathion, malathion, mecarbam, methamidophos, methidathion, mevinphos, monocrotophos, naled, omethoate, oxydemeton-methyl, parathion, parathion-methyl, phenthoate, phorate, phosalone, phosmet, phosphamidon, phoxim, pirimiphos-methyl, profenofos, propetamphos, prothiofos, pyraclofos, pyridaphenthion, quinalphos, sulfotep, tebupirimfos, temephos, terbufos, tetrachlorvinphos, thiometon, triazophos, trichlorfon, vamidothion;
M.2. Carbamates: aldicarb, alanycarb, bendiocarb, benfuracarb, butocarboxim, butoxycarboxim, carbaryl, carbofuran, carbosulfan, ethiofencarb, fenobucarb, formetanate, furathiocarb, isoprocarb, methiocarb, methomyl, metolcarb, oxamyl, pirimicarb, propoxur, thiodicarb, thiofanox, trimethacarb, XMC, xylylcarb, triazamate;
M.3. Pyrethroids: acrinathrin, allethrin, d-cis-trans allethrin, d-trans allethrin, bifenthrin, bioallethrin, bioallethrin S-cylclopentenyl, bioresmethrin, cycloprothrin, cyfluthrin, beta-, yfluthrin, cyhalothrin, lambda-cyhalothrin, gamma-cyhalothrin, cypermethrin, alphacypermethrin, beta-cypermethrin, theta-cypermethrin, zeta-cypermethrin, cyphenothrin, deltamethrin, empenthrin, esfenvalerate, etofenprox, fenpropathrin, fenvalerate, flucythrinate, flumethrin, tau-fluvalinate, halfenprox, imiprothrin, permethrin, phenothrin, prallethrin, resmethrin, RU 15525, silafluofen, tefluthrin, tetramethrin, tralomethrin, transfluthrin, ZXI 8901;
M.4. Juvenile hormone mimics: hydroprene, kinoprene, methoprene, fenoxycarb, pyriproxyfen;
M.5. Nicotinic receptor agonists/antagonists compounds: acetamiprid, bensultap, cartap hydrochloride, clothianidin, dinotefuran, imidacloprid, thiamethoxam, nitenpyram, nicotine, spinosad (allosteric agonist), thiacloprid, thiocyclam, thiosultap-sodium and AKD1022.
M.6. GABA gated chloride channel antagonist compounds: chlordane, endosulfan, gamma-HCH (lindane); acetoprole, ethiprole, fipronil, pyrafluprole, pyriprole, vaniliprole, the phenylpyrazole compound of formula M6.1




M.7. Chloride channel activators: abamectin, emamectin benzoate, milbemectin, lepimectin;
M.8. METI I compounds: fenazaquin, fenpyroximate, pyrimidifen, pyridaben, tebufenpyrad, tolfenpyrad, flufenerim, rotenone;
M.9. METI II and III compounds: acequinocyl, fluacyprim, hydramethylnon;
M.10. Uncouplers of oxidative phosphorylation: chlorfenapyr, DNOC;
M.11. Inhibitors of oxidative phosphorylation: azocyclotin, cyhexatin, diafenthiuron, fenbutatin oxide, propargite, tetradifon;
M.12. Moulting disruptors: cyromazine, chromafenozide, halofenozide, methoxyfenozide, tebufenozide;
M.13. Synergists: piperonyl butoxide, tribufos;
M.14. Sodium channel blocker compounds: indoxacarb, metaflumizone;
M.15. Fumigants: methyl bromide, chloropicrin sulfuryl fluoride;
M.16. Selective feeding blockers: crylotie, pymetrozine, flonicamid;
M.17. Mite growth inhibitors: clofentezine, hexythiazox, etoxazole;
M.18. Chitin synthesis inhibitors: buprofezin, bistrifluron, chlorfluazuron, diflubenzuron, flucycloxuron, flufenoxuron, hexaflumuron, lufenuron, novaluron, noviflumuron, teflubenzuron, triflumuron;
M.19. Lipid biosynthesis inhibitors: spirodiclofen, spiromesifen, spirotetramat;
M.20. octapaminergic agonsits: amitraz;
M.21. ryanodine receptor modulators: flubendiamide;
M.22. Various: aluminium phosphide, amidoflumet, benclothiaz, benzoximate, bifenazate, borax, bromopropylate, cyanide, cyenopyrafen, cyflumetofen, chinomethionate, dicofol, fluoroacetate, phosphine, pyridalyl, pyrifluquinazon, sulfur, tartar emetic; pyrimidinyl alkynylether compounds M22.1 or thiadiazolyl alkynylether compounds M22.2:




wherein RM-22 is methyl or ethyl and Het* is 3,3-dimethylpyrrolidin-1-yl, 3-methylpiperidin-1-yl, 3,5-dimethylpiperidin-1-yl, 3-trifluormethylpiperidin-1-yl, hexahydroazepin-1-yl, 2,6-dimethylhexahydroazepin-1-yl or 2,6-dimethylmorpholin-4-yl.

M.23. N—R′-2,2-dihalo-1-R′′cyclo-propanecarboxamide-2-(2,6-dichloro-alpha, alpha, alpha-tri-fluoro-p-tolyl)hydrazone or N—R′-2,2-di(R′′′)propionamide-2-(2,6-dichloro-alpha, alpha, alpha-trifluoro-p-tolyl)-hydrazone, wherein R′ is methyl or ethyl, halo is chloro or bromo, R′′ is hydrogen or methyl and R′′′ is methyl or ethyl;
M.24. Anthranilamides: chloranthraniliprole, the compound of formula M24 1




M.25. Malononitrile compounds: CF3(CH2)2C(CN)2CH2(CF2)3CF2H, CF3(CH2)2C(CN)2CH2(CF2)5CF2H, CF3(CH2)2C(CN)2(CH2)2C(CF3)2F, CF3(CH2)2C(CN)2(CH2)2(CF2)3CF3, CF2H(CF2)3CH2C(CN)2CH2(CF2)3CF2H, CF3(CH2)2C(CN)2CH2(CF2)3CF3, CF3(CF2)2CH2C(CN)2CH2(CF2)3CF2H, CF3CF2CH2C(CN)2CH2(CF2)3CF2H, 2-(2,2,3,3,4,4,5,5-octafluoropentyl)-2-(3,3,4,4,4-pentafluorobutyl)-malonodinitrile, and CF2HCF2CF2CF2CH2C(CN)2CH2CH2CF2CF3;
M.26. Microbial disruptors: Bacillus thuringiensis subsp. Israelensi, Bacillus sphaericus, Bacillus thuringiensis subsp. Aizawai, Bacillus thuringiensis subsp. Kurstaki, Bacillus thuringiensis subsp. Tenebrionis; 
The commercially available compounds of the group A may be found in The Pesticide Manual, 13th Edition, British Crop Protection Council (2003) among other publications.
Thioamides of formula M6.1 and their preparation have been described in WO 98/28279. Lepimectin is known from Agro Project, PJB Publications Ltd, November 2004. Benclothiaz and its preparation have been described in EP-A1 454621. Methidathion and Paraoxon and their preparation have been described in Farm Chemicals Handbook, Volume 88, Meister Publishing Company, 2001. Acetoprole and its preparation have been described in WO 98/28277. Metaflumizone and its preparation have been described in EP-A 1 462 456. Flupyrazofos has been described in Pesticide Science 54, 1988, p. 237-243 and in U.S. Pat. No. 4,822,779. Pyrafluprole and its preparation have been described in JP 2002193709 and in WO 01/00614. Pyriprole and its preparation have been described in WO 98/45274 and in U.S. Pat. No. 6,335,357. Amidoflumet and its preparation have been described in U.S. Pat. No. 6,221,890 and in JP 21010907. Flufenerim and its preparation have been described in WO 03/007717 and in WO 03/007718. AKD 1022 and its preparation have been described in U.S. Pat. No. 6,300,348. Chloranthraniliprole has been described in WO 01/70671, WO 03/015519 and WO 05/118552. Anthranilamide derivatives of formula M24.1 have been described in WO 01/70671, WO 04/067528 and WO 05/118552. Cyflumetofen and its preparation have been described in WO 04/080180. The aminoquinazolinone compound pyrifluquinazon has been described in EPA 109 7932. The alkynylether compounds M22.1 and M22.2 are described e.g. in JP 2006131529. The malononitrile compounds CF3(CH2)2C(CN)2CH2(CF2)3CF2H, CF3(CH2)2C(CN)2CH2(CF2)5CF2H, CF3(CH2)2C(CN)2(CH2)2C(CF3)2F, CF3(CH2)2C(CN)2(CH2)2(CF2)3CF3, CF2H(CF2)3CH2C(CN)2CH2(CF2)3CF2H, CF3(CH2)2C(CN)2CH2(CH2)3CF3, CF3(CF2)2CH2C(CN)2CH2(CF2)3CF2H, CF3CF2CH2C(CN)2CH2(CF2)3CF2H, 2-(2,2,3,3,4,4,5,5-octafluoropentyl)-2-(3,3,4,4,4-pentafluorobutyl)-malonodinitrile, and CF2HCF2CF2CF2CH2C(CN)2CH2CH2CF2CF3 have been described in WO 05/63694.
Fungicidal mixing partners are those selected from the group F consisting of
F.1 acylalanines such as benalaxyl, metalaxyl, ofurace, oxadixyl;
F.2 amine derivatives such as aldimorph, dodine, dodemorph, fenpropimorph, fenpropidin, guazatine, iminoctadine, spiroxamin, tridemorph;
F.3 anilinopyrimidines such as pyrimethanil, mepanipyrim or cyrodinyl;
F.4 antibiotics such as cycloheximid, griseofulvin, kasugamycin, natamycin, polyoxin or streptomycin;
F.5 azoles such as bitertanol, bromoconazole, cyproconazole, difenoconazole, dinitroconazole, epoxiconazole, fenbuconazole, fluquiconazole, flusilazole, hexaconazole, imazalil, metconazole, myclobutanil, penconazole, propiconazole, prochloraz, prothioconazole, tebuconazole, triadimefon, triadimenol, triflumizol, triticonazole, flutriafol;
F.6 dicarboximides such as iprodion, myclozolin, procymidon, vinclozolin;
F.7 dithiocarbamates such as ferbam, nabam, maneb, mancozeb, metam, metiram, propineb, polycarbamate, thiram, ziram, zineb;
F.8 heterocyclic compounds such as anilazine, benomyl, boscalid, carbendazim, carboxin, oxycarboxin, cyazofamid, dazomet, dithianon, famoxadon, fenamidon, fenarimol, fuberidazole, flutolanil, furametpyr, isoprothiolane, mepronil, nuarimol, probenazole, proquinazid, pyrifenox, pyroquilon, quinoxyfen, silthiofam, thiabendazole, thifluzamid, thiophanate-methyl, tiadinil, tricyclazole, triforine;
F.9 copper fungicides such as Bordeaux mixture, copper acetate, copper oxychloride, basic copper sulfate;
F.10 nitrophenyl derivatives such as binapacryl, dinocap, dinobuton, nitrophthalisopropyl;
F.11 phenylpyrroles such as fenpiclonil or fludioxonil;
F.12 strobilurins such as azoxystrobin, dimoxystrobin, fluoxastrobin, kresoxim-methyl, metominostrobin, orysastrobin, picoxystrobin or trifloxystrobin;
F.13 sulfenic acid derivatives such as captafol, captan, dichlofluanid, folpet, tolylfluanid;
F.14 cinnemamides and analogs such as dimethomorph, flumetover or flumorph;
F.15 sulfur, and other fungicides such as acibenzolar-S-methyl, benthiavalicarb, carpropamid, chlorothalonil, cyflufenamid, cymoxanil, dazomet, diclomezin, diclocymet, diethofencarb, edifenphos, ethaboxam, fenhexamid, fentin-acetate, fenoxanil, ferimzone, fluazinam, fosetyl, fosetyl-aluminum, iprovalicarb, hexachlorobenzene, metrafenon, pencycuron, propamocarb, phthalide, toloclofos-methyl, quintozene, zoxamid.
Applications
The animal pest, i.e. the insects, the plant, soil or water in which the plant is growing can be contacted with the present compound(s) identified according to the method of the invention or composition(s) containing them by any application method known in the art. As such, “contacting” includes both direct contact (applying the compounds/compositions directly on the animal pest or plant—typically to the foliage, stem or roots of the plant) and indirect contact (applying the compounds/compositions to the locus of the animal pest or plant).
The compounds of formula identified according to the method of the invention or the insecticidal compositions comprising them may be used to protect growing plants and crops from attack or infestation by animal pests, especially insects, acaridae or arachnids by contacting the plant/crop with a insecticidally effective amount of compounds identified according to the method of the invention. The term “crop” refers both to growing and harvested crops.
Moreover, animal pests may be controlled by contacting the target pest, its food supply, habitat, breeding ground or its locus with a insecticidally effective amount of compounds identified according to the method of the invention. As such, the application may be carried out before or after the infection of the locus, growing crops, or harvested crops by the pest.
The compounds of the invention can also be applied preventively to places at which occurrence of the pests is expected.
The compounds identified according to the method of the invention may be also used to protect growing plants from attack or infestation by pests by contacting the plant with a insecticidally effective amount of compounds identified according to the method of the invention. As such, “contacting” includes both direct contact (applying the compounds/compositions directly on the pest and/or plant—typically to the foliage, stem or roots of the plant) and indirect contact (applying the compounds/compositions to the locus of the pest and/or plant).
“Locus” means a habitat, breeding ground, plant, seed, soil, area, material or environment in which a pest or parasite is growing or may grow.
In general, “insecticidally effective amount” means the amount of active ingredient needed to achieve an observable effect on growth, death, retardation, prevention, and removal, destruction, or otherwise diminishing the occurrence and activity of the target organism. The insecticidally effective amount can vary for the various compounds/compositions used in the invention. A insecticidally effective amount of the compositions will also vary according to the prevailing conditions such as desired insecticidal effect and duration, weather, target species, locus, mode of application, and the like.
In the case of soil treatment or of application to the pests dwelling place or nest, the quantity of active ingredient ranges from 0.0001 to 500 g per 100 m2, preferably from 0.001 to 20 g per 100 m2.
Customary application rates in the protection of materials are, for example, from 0.01 g to 1000 g of active compound per m2 treated material, desirably from 0.1 g to 50 g per m2.
Insecticidal compositions for use in the impregnation of materials typically contain from 0.001 to 95 weight %, preferably from 0.1 to 45 weight %, and more preferably from 1 to 25 weight % of at least one repellent and/or insecticide.
For use in treating crop plants, the rate of application of the active ingredients of this invention may be in the range of 0.1 g to 4000 g per hectare, desirably from 25 g to 600 g per hectare, more desirably from 50 g to 500 g per hectare.
The compounds of formula I are effective through both contact (via soil, glass, wall, bed net, carpet, plant parts or animal parts), and ingestion (bait, or plant part).
The compounds of the invention may also be applied against non-crop insect pests, such as ants, termites, wasps, flies, mosquitos, crickets, or cockroaches. For use against said non-crop pests, compounds of formula I are preferably used in a bait composition.
The bait can be a liquid, a solid or a semisolid preparation (e.g. a gel). Solid baits can be formed into various shapes and forms suitable to the respective application e.g. granules, blocks, sticks, disks. Liquid baits can be filled into various devices to ensure proper application, e.g. open containers, spray devices, droplet sources, or evaporation sources. Gels can be based on aqueous or oily matrices and can be formulated to particular necessities in terms of stickyness, moisture retention or aging characteristics.
The bait employed in the composition is a product, which is sufficiently attractive to incite insects such as ants, termites, wasps, flies, mosquitos, crickets etc. or cockroaches to eat it. The attractiveness can be manipulated by using feeding stimulants or sex pheromones. Food stimulants are chosen, for example, but not exclusively, from animal and/or plant proteins (meat-, fish- or blood meal, insect parts, egg yolk), from fats and oils of animal and/or plant origin, or mono-, oligo- or polyorganosaccharides, especially from sucrose, lactose, fructose, dextrose, glucose, starch, pectin or even molasses or honey. Fresh or decaying parts of fruits, crops, plants, animals, insects or specific parts thereof can also serve as a feeding stimulant. Sex pheromones are known to be more insect specific. Specific pheromones are described in the literature and are known to those skilled in the art.
For use in bait compositions, the typical content of active ingredient is from 0.001 weight % to 15 weight %, desirably from 0.001 weight % to 5% weight % of active compound.
Formulations of compounds identified according to the method of the invention as aerosols (e.g in spray cans), oil sprays or pump sprays are highly suitable for the non-professional user for controlling pests such as flies, fleas, ticks, mosquitos or cockroaches. Aerosol recipes are preferably composed of the active compound, solvents such as lower alcohols (e.g. methanol, ethanol, propanol, butanol), ketones (e.g. acetone, methyl ethyl ketone), paraffin hydrocarbons (e.g. kerosenes) having boiling ranges of approximately 50 to 250° C., dimethylformamide, N-methylpyrrolidone, di-methyl sulfoxide, aromatic hydrocarbons such as toluene, xylene, water, furthermore auxiliaries such as emulsifiers such as sorbitol monooleate, oleyl ethoxylate having 3-7 mol of ethylene oxide, fatty alcohol ethoxylate, perfume oils such as ethereal oils, esters of medium fatty acids with lower alcohols, aromatic carbonyl compounds, if appropriate stabilizers such as sodium benzoate, amphoteric surfactants, lower epoxides, triethyl orthoformate and, if required, propellants such as propane, butane, nitrogen, compressed air, dimethyl ether, carbon dioxide, nitrous oxide, or mixtures of these gases.
The oil spray formulations differ from the aerosol recipes in that no propellants are used.
For use in spray compositions, the content of active ingredient is from 0.001 to 80 weights %, preferably from 0.01 to 50 weight % and most preferably from 0.01 to 15 weight %.
The compounds identified according to the method of the invention and its respective compositions can also be used in mosquito and fumigating coils, smoke cartridges, vaporizer plates or long-term vaporizers and also in moth papers, moth pads or other heat-independent vaporizer systems.
Methods to control infectious diseases transmitted by insects (e.g. malaria, dengue and yellow fever, lymphatic filariasis, and leishmaniasis) with compounds of formula I and its respective compositions also comprise treating surfaces of huts and houses, air spraying and impregnation of curtains, tents, clothing items, bed nets, tsetse-fly trap or the like. Insecticidal compositions for application to fibers, fabric, knitgoods, nonwovens, netting material or foils and tarpaulins preferably comprise a mixture including the insecticide, optionally a repellent and at least one binder. Suitable repellents for example are N,N-Diethyl-meta-toluamide (DEET), N,N-diethylphenylacetamide (DEPA), 1-(3-cyclohexan-1-yl-carbonyl)-2-methylpiperine, (2-hydroxymethylcyclohexyl)acetic acid lactone, 2-ethyl-1,3-hexandiol, indalone, Methylneodecanamide (MNDA), a pyrethroid not used for insect control such as {(+/−)-3-allyl-2-methyl-4-oxocyclopent-2-(+)-enyl-(+)-trans-chrysantemate (Esbiothrin), a repellent derived from or identical with plant extracts like limonene, eugenol, (+)-Eucamalol (1), (−)-1-epi-eucamalol or crude plant extracts from plants like Eucalyptus maculate, Vitex rotundifolia, Cymbopogan martinii, Cymbopogan citratus (lemon grass), Cymopogan nartdus (citronella). Suitable binders are selected for example from polymers and copolymers of vinyl esters of aliphatic acids (such as such as vinyl acetate and vinyl versatate), acrylic and methacrylic esters of alcohols, such as butyl acrylate, 2-ethylhexylacrylate, and methyl acrylate, mono- and di-ethylenically unsaturated hydrocarbons, such as styrene, and aliphatic diens, such as butadiene.
The impregnation of curtains and bednets is done in general by dipping the textile material into emulsions or dispersions of the insecticide or spraying them onto the nets.
The compounds identified according to the method of the invention and its compositions can be used for protecting wooden materials such as trees, board fences, sleepers, etc. and buildings such as houses, outhouses, factories, but also construction materials, furniture, leathers, fibers, vinyl articles, electric wires and cables etc. from ants and/or termites, and for controlling ants and termites from doing harm to crops or human being (e.g. when the pests invade into houses and public facilities). The compounds identified according to the method of the invention are applied not only to the surrounding soil surface or into the under-floor soil in order to protect wooden materials but it can also be applied to lumbered articles such as surfaces of the under-floor concrete, alcove posts, beams, plywoods, furniture, etc., wooden articles such as particle boards, half boards, etc. and vinyl articles such as coated electric wires, vinyl sheets, heat insulating material such as styrene foams, etc. In case of application against ants doing harm to crops or human beings, the ant controller of the present invention is applied to the crops or the surrounding soil, or is directly applied to the nest of ants or the like.
Seed Treatment
The compounds identified according to the method of the invention are also suitable for the treatment of seeds in order to protect the seed from insect pest, in particular from soil-living insect pests and the resulting plant's roots and shoots against soil pests and foliar insects.
The compounds identified according to the method of the invention are particularly useful for the protection of the seed from soil pests and the resulting plant's roots and shoots against soil pests and foliar insects. The protection of the resulting plant's roots and shoots is preferred. More preferred is the protection of resulting plant's shoots from piercing and sucking insects, wherein the protection from aphids is most preferred, specially Green Peach Aphid (Myzus persica) and/or Red Flower Beetle (Tribolium castaneum)
The present invention therefore comprises a method for the protection of seeds from insects, in particular from soil insects and of the seedlings' roots and shoots from insects, in particular from soil and foliar insects, said method comprising contacting the seeds before sowing and/or after pregermination with a compound identified according to the method of the invention. Particularly preferred is a method, wherein the plant's roots and shoots are protected, more preferably a method, wherein the plants shoots are protected form piercing and sucking insects, most preferably a method, wherein the plants shoots are protected from aphids.
The term seed embraces seeds and plant propagules of all kinds including but not limited to true seeds, seed pieces, suckers, corms, bulbs, fruit, tubers, grains, cuttings, cut shoots and the like and means in a preferred embodiment true seeds.
The term seed treatment comprises all suitable seed treatment techniques known in the art, such as seed dressing, seed coating, seed dusting, seed soaking and seed pelleting.
The present invention also comprises seeds coated with or containing the active compound.
The term “coated with and/or containing” generally signifies that the active ingredient is for the most part on the surface of the propagation product at the time of application, although a greater or lesser part of the ingredient may penetrate into the propagation product, depending on the method of application. When the said propagation product is (re)planted, it may absorb the active ingredient.
Suitable seed is seed of cereals, root crops, oil crops, vegetables, spices, ornamentals, for example seed of durum and other wheat, barley, oats, rye, maize (fodder maize and sugar maize/sweet and field corn), soybeans, oil crops, crucifers, cotton, sunflowers, bananas, rice, oilseed rape, turnip rape, sugarbeet, fodder beet, egg-plants, potatoes, grass, lawn, turf, fodder grass, tomatoes, leeks, pumpkin/squash, cabbage, iceberg lettuce, pepper, cucumbers, melons, Brassica species, melons, beans, peas, garlic, onions, carrots, tuberous plants such as potatoes, sugar cane, tobacco, grapes, petunias, geranium/pelargoniums, pansies and impatiens.
In addition, the active compound may also be used for the treatment seeds from plants, which tolerate the action of herbicides or fungicides or insecticides owing to breeding, including genetic engineering methods.
For example, the active compound can be employed in treatment of seeds from plants, which are resistant to herbicides from the group consisting of the sulfonylureas, imidazolinones, glufosinate-ammonium or glyphosate-isopropylammonium and analogous active substances (see for example, EP-A-0242236, EP-A-242246) (WO 92/00377) (EP-A-0257993, U.S. Pat. No. 5,013,659) or in transgenic crop plants, for example cotton, with the capability of producing Bacillus thuringiensis toxins (Bt toxins) which make the plants resistant to certain pests (EP-A-0142924, EP-A-0193259), Furthermore, the active compound can be used also for the treatment of seeds from plants, which have modified characteristics in comparison with existing plants consist, which can be generated for example by traditional breeding methods and/or the generation of mutants, or by recombinant procedures). For example, a number of cases have been described of recombinant modifications of crop plants for the purpose of modifying the starch synthesized in the plants (e.g. WO 92/11376, WO 92/14827, WO 91/19806) or of transgenic crop plants having a modified fatty acid composition (WO 91/13972).
The seed treatment application of the active compound is carried out by spraying or by dusting the seeds before sowing of the plants and before emergence of the plants.
Compositions which are especially useful for seed treatment are e.g.:
A Soluble concentrates (SL, LS)
D Emulsions (EW, EO, ES)
E Suspensions (SC, OD, FS)
F Water-dispersible granules and water-soluble granules (WG, SG)
G Water-dispersible powders and water-soluble powders (WP, SP, WS)
H Gel-Formulations (GF)
I Dustable powders (DP, DS)
Conventional seed treatment formulations include for example flowable concentrates FS, solutions LS, powders for dry treatment DS, water dispersible powders for slurry treatment WS, water-soluble powders SS and emulsion ES and EC and gel formulation GF. These formulations can be applied to the seed diluted or undiluted. Application to the seeds is carried out before sowing, either directly on the seeds or after having pregerminated the latter
In a preferred embodiment a FS formulation is used for seed treatment. Typically, a FS formulation may comprise 1-800 g/l of active ingredient, 1-200 g/l Surfactant, 0 to 200 g/l antifreezing agent, 0 to 400 g/l of binder, 0 to 200 g/l of a pigment and up to 1 liter of a solvent, preferably water.
Especially preferred FS formulations of compounds identified according to the method of the invention for seed treatment usually comprise from 0.1 to 80% by weight (1 to 800 g/l) of the active ingredient, from 0.1 to 20% by weight (1 to 200 g/l) of at least one surfactant, e.g. 0.05 to 5% by weight of a wetter and from 0.5 to 15% by weight of a dispersing agent, up to 20% by weight, e.g. from 5 to 20% of an anti-freeze agent, from 0 to 15% by weight, e.g. 1 to 15% by weight of a pigment and/or a dye, from 0 to 40% by weight, e.g. 1 to 40% by weight of a binder (sticker/adhesion agent), optionally up to 5% by weight, e.g. from 0.1 to 5% by weight of a thickener, optionally from 0.1 to 2% of an anti-foam agent, and optionally a preservative such as a biocide, antioxidant or the like, e.g. in an amount from 0.01 to 1% by weight and a filler/vehicle up to 100% by weight.
Seed Treatment formulations may additionally also comprise binders and optionally colorants.
Binders can be added to improve the adhesion of the active materials on the seeds after treatment. Suitable binders are homo- and copolymers from alkylene oxides like ethylene oxide or propylene oxide, polyvinylacetate, polyvinylalcohols, polyvinylpyrrolidones, and copolymers thereof, ethylene-vinyl acetate copolymers, acrylic homo- and copolymers, polyethyleneamines, polyethyleneamides and polyethyleneimines, polysaccharides like celluloses, tylose and starch, polyolefin homo- and copolymers like olefin/maleic anhydride copolymers, polyurethanes, polyesters, polystyrene homo and copolymers
Optionally, also colorants can be included in the formulation. Suitable colorants or dyes for seed treatment formulations are Rhodamin B, C.I. Pigment Red 112, C.I. Solvent Red 1, pigment blue 15:4, pigment blue 15:3, pigment blue 15:2, pigment blue 15:1, pigment blue 80, pigment yellow 1, pigment yellow 13, pigment red 112, pigment red 48:2, pigment red 48:1, pigment red 57:1, pigment red 53:1, pigment orange 43, pigment orange 34, pigment orange 5, pigment green 36, pigment green 7, pigment white 6, pigment brown 25, basic violet 10, basic violet 49, acid red 51, acid red 52, acid red 14, acid blue 9, acid yellow 23, basic red 10, basic red 108.
Examples of a Gelling Agent is Carrageen (Satiagel®)
In the treatment of seed, the application rates of the compounds I are generally from 0.1 g to 10 kg per 100 kg of seed, preferably from 1 g to 5 kg per 100 kg of seed, more preferably from 1 g to 1000 g per 100 kg of seed and in particular from 1 g to 200 g per 100 kg of seed.
The invention therefore also relates to seed comprising a compound identified according to the method of the invention, or an agriculturally useful salt of I, as defined herein. The amount of the compound identified according to the method of the invention or the agriculturally useful salt thereof will in general vary from 0.1 g to 10 kg per 100 kg of seed, preferably from 1 g to 5 kg per 100 kg of seed, in particular from 1 g to 1000 g per 100 kg of seed. For specific crops such as lettuce the rate can be higher.
In one embodiment the invention relates to subject mater summarized as follows:
item a1. A method for identifying a insecticidally active compound that reduces the activity of an insect voltage-gated potassium channel ShaI (Shaker cognate I or Shaker-like) and/or its accessory protein KChIP (potassium channel-interacting protein) respectively which method comprises:

    
    
        a) assembling in a membrane a polypeptide with the activity of an insect voltage-gated potassium channel ShaI (Shaker cognate I or Shaker-like) and/or its accessory protein KChIP (potassium channel-interacting protein) respectively which is originally not present said membrane,
        b) applying at one side of the membrane the compound suspected of having the ability to inhibit the activity of said polypeptide which is originally not present said membrane,
        c) determining the activity of said polypeptide and
        d) identifying a compound applied in (b) that reduces the activity of said polypeptide.
    
    


item a2. A method according to item a1 whereby a gene coding for a polypeptide with the activity of an insect voltage-gated potassium channel ShaI (Shaker cognate I or Shaker-like) and/or its accessory protein KChIP (potassium channel-interacting protein) respectively is expressed in the membrane of a host cell.
item a3. A method of any one of the items 1 or 2 wherein the membrane comprises at least one polypeptide encoded by a nucleic acid molecule selected from the group consisting of:

    
    
        a) a nucleic acid molecule encoding the polypeptide shown in SEQ ID NO: 1, 5, 9, 13, 17, 21, 25 and/or 29;
        b) a nucleic acid molecule shown in SEQ ID NO: 1, 5, 9, 13, 17, 21, 25 and/or 29;
        c) a nucleic acid molecule, which, as a result of the degeneracy of the genetic code, can be derived from a polypeptide sequence according to SEQ ID NO: 2, 6, 10, 14, 18, 22, 26 and/or 30;
        d) a nucleic acid molecule having at least 50% identity with the nucleic acid molecule sequence of a polynucleotide comprising the nucleic acid molecule shown in SEQ ID NO: 1, 5, 9, 13, 17, 21, 25 and/or 29;
        e) a nucleic acid molecule encoding a polypeptide having at least 50% identity with the amino acid sequence of the polypeptide encoded by the nucleic acid molecule of (a) to (c) and having the activity of a voltage-gated potassium channel ShaI (Shaker cognate I or Shaker-like) and/or its accessory protein KChIP (potassium channel-interacting protein) respectively;
        f) nucleic acid molecule which hybridizes with a nucleic acid molecule of (a) to (c) under stringent hybridization conditions;
        g) a nucleic acid molecule encoding a polypeptide which can be isolated with the aid of monoclonal or polyclonal antibodies made against a polypeptide encoded by one of the nucleic acid molecules of (a) to (e) and having the activity of a voltage-gated potassium channel ShaI (Shaker cognate I or Shaker-like) and/or its accessory protein KChIP (potassium channel-interacting protein) respectively;
        h) a nucleic acid molecule encoding a polypeptide comprising the consensus sequence as shown in SEQ ID NO: 33 and/or 34 respectively or one or more motifs selected from the group consisting of SEQ ID NO: 35, 36, 37, 38, 39, 40, 41, 42, 43, 44, 45, 46, 47, 48, 49, 50, 51, 52, 53, 54 and/or 55, and/or 56, 57, 58, 59, 60, 61, 62, 63, 64, 65, 66, 67, 68, 69, 70 and/or 71 respectively;
        i) nucleic acid molecule which comprises a polynucleotide, which is obtained by amplifying a cDNA library or a genomic library using the primers in SEQ ID NO: 3, 4; 7, 8; 11, 12; 15, 16, 19, 20; 23, 24; 27, 28 and/or 31, 32 respectively; and
        j) a nucleic acid molecule which is obtainable by screening a suitable nucleic acid library under stringent hybridization conditions with a probe comprising a complementary sequence of a nucleic acid molecule of (a) or (b) or with a fragment thereof, having at least 15 nt, preferably 20 nt, 30 nt, 50 nt, 100 nt, 200 nt or 500 nt of a nucleic acid molecule complementary to a nucleic acid molecule sequence characterized in (a) to (e) and encoding a polypeptide and having the activity of a voltage-gated potassium channel ShaI (Shaker cognate I or Shaker-like) and/or its accessory protein KChIP (potassium channel-interacting protein) respectively.
    
    


item a4. A method of item a1 whereby the activity of said polypeptide with the activity of insect voltage-gated potassium channel ShaI (Shaker cognate I or Shaker-like) and/or its accessory protein KChIP (potassium channel-interacting protein) respectively is determinated electrophysiologically.
item a5. A method of item a4 whereby the activity of said polypeptide with the activity of insect voltage-gated potassium channel ShaI (Shaker cognate I or Shaker-like) and/or its accessory protein KChIP (potassium channel-interacting protein) respectively is determinated by patch clamp or in a HTS assay.
item a6. A method of item a2 whereby a gene coding for a polypeptide with the activity of an insect voltage-gated potassium channel ShaI (Shaker cognate I or Shaker-like) and/or its accessory protein KChIP (potassium channel-interacting protein) respectively is expressed in a mammalian cell.
item a7. A method of item a2 whereby a gene coding for a polypeptide with the activity of an insect voltage-gated potassium channel ShaI (Shaker cognate I or Shaker-like) and/or its accessory protein KChIP (potassium channel-interacting protein) respectively is expressed in a mammalian cell selected from the group consisting of: CHO-cells and HEK293.
item a8. A method of item a1 which comprises:

    
    
        a) e) applying to an insect, to a population of insects or to the location wherein said insect is to be controlled an insect-controlling amount a compound identified according to item aa1 d) and
        b) f) determining of the growth or the viability of said treated insect or population of insects or of insects or population of insects on said location and untreated insect, population of insects or location and
        c) g) selecting of compounds, which reduces the growth or the viability of said treated insect or population of insects or of insects or population of insects on said location following application of the compound of step e).
    
    


item a9. An assay system comprising a host organism, tissue, cells or a cell digest thereof or a membrane, which has embedded, assembled, intercalated or incorporated a nucleic acid molecule selected from the group consisting of the nucleic acid molecule as depicted in item a3 a) to 3 j) and, based on the expression of this nucleic acid molecule, a polypeptide having the biological activity of a insect voltage-gated potassium channel ShaI (Shaker cognate I or Shaker-like) and/or its accessory protein KChIP (potassium channel-interacting protein) respectively, for identifying insecticidally active compound that reduces the activity of an insect voltage-gated potassium channel ShaI (Shaker cognate I or Shaker-like) and/or its accessory protein KChIP (potassium channel-interacting protein) respectively.
item a10. The assay system of item a9 whereby the host organism is a stably transfected mammalian cell which expresses a nucleic acid molecule selected from the group consisting of the nucleic acid molecule as depicted in item a3 a) to 3 j).
item a11. The assay system of item a10 whereby the mammalian cell is selected from the group consisting of: CHO-cells, HEK293, COS, HeLa, NIH3T3, BAK21, Jurkat, CV-1, HepC-2-, Xenopus oocyte, Sf9, S2, Sf21, Hi5, Pc12 and U2OS.
item a12. A method for killing or inhibiting the growth or viability of an insect, comprising applying to the insect a compound identified according to the method of item a1.
item a13. A nucleic acid molecule selected from the group consisting of:

    
    
        a) a nucleic acid molecule encoding the polypeptide shown in 2, 6, 10, 14, 18, 22, 26 and/or 30;
        b) a nucleic acid molecule shown in SEQ ID NO: 1, 5, 9, 13, 17, 21, 25 and/or 29;
        c) a nucleic acid molecule, which, as a result of the degeneracy of the genetic code, can be derived from a polypeptide sequence according to SEQ ID NO: 2, 6, 10, 14, 18, 22, 26 and/or 30;
        d) a nucleic acid molecule having at least 50% identity with the nucleic acid molecule sequence of a polynucleotide comprising the nucleic acid molecule shown in SEQ ID NO: 1, 5, 9, 13, 17, 21, 25 and/or 29;
        e) a nucleic acid molecule encoding a polypeptide having at least 50% identity with the amino acid sequence of the polypeptide encoded by the nucleic acid molecule of (a) to (c) and having the activity of a voltage-gated potassium channel ShaI (Shaker cognate I or Shaker-like) and/or its accessory protein KChIP (potassium channel-interacting protein) respectively;
        f) nucleic acid molecule which hybridizes with a nucleic acid molecule of (a) to (c) under stringent hybridization conditions;
        g) a nucleic acid molecule encoding a polypeptide which can be isolated with the aid of monoclonal or polyclonal antibodies made against a polypeptide encoded by one of the nucleic acid molecules of (a) to (e) and having the activity of a voltage-gated potassium channel ShaI (Shaker cognate I or Shaker-like) and/or its accessory protein KChIP (potassium channel-interacting protein) respectively;
        h) a nucleic acid molecule encoding a polypeptide comprising the consensus sequence as shown in SEQ ID NO: 33 and/or 34 respectively or one or more motifs selected from the group consisting of SEQ ID NO: 35, 36, 37, 38, 39, 40, 41, 42, 43, 44, 45, 46, 47, 48, 49, 50, 51, 52, 53, 54 and/or 55, and/or 56, 57, 58, 59, 60, 61, 62, 63, 64, 65, 66, 67, 68, 69, 70 and/or 71 respectively;
        i) nucleic acid molecule which comprises a polynucleotide, which is obtained by amplifying a cDNA library or a genomic library using the primers in SEQ ID NO: 3, 4; 7, 8; 11, 12; 15, 16; 19, 20; 23, 24; 27, 28 and/or 31, 32 respectively; and
        j) a nucleic acid molecule which is obtainable by screening a suitable nucleic acid library under stringent hybridization conditions with a probe comprising a complementary sequence of a nucleic acid molecule of (a) or (b) or with a fragment thereof, having at least 15 nt, preferably 20 nt, 30 nt, 50 nt, 100 nt, 200 nt or 500 nt of a nucleic acid molecule complementary to a nucleic acid molecule sequence characterized in (a) to (e) and encoding a polypeptide and having the activity of a potassium channel ShaI (Shaker cognate I or Shaker-like) and/or its accessory protein KChIP (potassium channel-interacting protein) respectively.
    
    


item a14. A nucleic acid construct comprising a nucleic acid molecule according to item a13.
item a15. A vector comprising a nucleic acid construct according to item a14 or a nucleic acid molecule according to item a13.
item a16. A transgenic cell comprising a vector according to item a15, a nucleic acid construct according to item a14 or a nucleic acid molecule according to item a13.
item a17. A polypeptide encoded by a nucleic acid molecule according to item a13.
item a18. Use of a polypeptide with the activity of an insect voltage-gated potassium channel ShaI (Shaker cognate I or Shaker-like) and/or its accessory protein KChIP (potassium channel-interacting protein) respectively as insecticidal target.
item a19. Use of a polypeptide encoded by a nucleic acid molecule selected from the group consisting of the nucleic acid molecule as depicted in item a3 a) to 3 j) as insecticidal target.
item a20. A method for controlling a insecticidal pest comprising the application of a composition comprising as insecticidal active ingredient at least one compound as depicted in table I or a derivate thereof.
Item b1. A method for identifying a insecticidally active compound that reduces the activity of an insect Shaker channel and/or a Hyperkinetic beta subunit, preferably H-kv beta subunit A or C subtype respectively which method comprises:

    
    
        a) assembling in a membrane a polypeptide with the activity of an insect Shaker channel and/or a Hyperkinetic beta subunit, preferably H-kv beta subunit A or C subtype respectively which is originally not present said membrane,
        b) applying at one side of the membrane the compound suspected of having the ability to inhibit the activity of said polypeptide which is originally not present said membrane,
        c) determining the activity of said polypeptide and
        d) identifying a compound applied in (b) that reduces the activity of said polypeptide.
    
    


Item b2. A method according to item b1 whereby a gene coding for a polypeptide with the activity of an insect Shaker channel and/or a Hyperkinetic beta subunit, preferably H-kv beta subunit A or C subtype respectively is expressed in the membrane of a host cell.
Item b3. A method of any one of the items b1 or b2 wherein the membrane comprises at least one polypeptide encoded by a nucleic acid molecule selected from the group consisting of:
a) a nucleic acid molecule encoding a polypeptide comprising the polypeptide shown in SEQ ID NO: 73, 75, 77, 79, 81, 83, 85 and/or 87;
b) a nucleic acid molecule comprising a nucleic acid molecule shown in SEQ ID NO: 72, 74, 76, 78, 80, 82, 84 and/or 86;
c) a nucleic acid molecule, which, as a result of the degeneracy of the genetic code, can be derived from a polypeptide comprising a polypeptide sequence according to SEQ ID NO: 73, 75, 77, 79, 81, 83, 85 and/or 87;
d) a nucleic acid molecule having at least 50% identity with the nucleic acid molecule sequence of a polynucleotide comprising the nucleic acid molecule shown in SEQ ID NO: 72, 74, 76, 78, 80, 82, 84 and/or 86;
e) a nucleic acid molecule encoding a polypeptide having at least 50% identity with the amino acid sequence of the polypeptide encoded by the nucleic acid molecule of (a) to (c) and having the activity of a Shaker channel and/or a Hyperkinetic beta subunit, preferably H-kv beta subunit A or C subtype respectively;
f) nucleic acid molecule which hybridizes with a nucleic acid molecule of (a) to (c) under stringent hybridization conditions;
g) a nucleic acid molecule encoding a polypeptide which can be isolated with the aid of monoclonal or polyclonal antibodies made against a polypeptide encoded by one of the nucleic acid molecules of (a) to (e) and having the activity of a Shaker channel and/or a Hyperkinetic beta subunit, preferably H-kv beta subunit A or C subtype respectively;
h) a nucleic acid molecule encoding a polypeptide comprising the consensus sequence as shown in SEQ ID NO: 102 and/or 103 respectively or one or more motifs selected from the group consisting of SEQ ID NO: 104, 105, 106, 107, 108, 109, 110, 111, 112, 113, 114, 115, 116, 117, 118, 119, 120, 121, 122, 123, 124 and/or 125 and/or 126, 127 and/or 128 respectively;
i) nucleic acid molecule which comprises a polynucleotide, which is obtained by amplifying a cDNA library or a genomic library using the primers in SEQ ID NO: 88, 89; 90, 91; 92, 93; 94, 95; 96, 97; 98, 99 and/or 100, 101 respectively;
and
j) a nucleic acid molecule which is obtainable by screening a suitable nucleic acid library under stringent hybridization conditions with a probe comprising a complementary sequence of a nucleic acid molecule of (a) or (b) or with a fragment thereof, having at least 15 nt, preferably 20 nt, 30 nt, 50 nt, 100 nt, 200 nt or 500 nt of a nucleic acid molecule complementary to a nucleic acid molecule sequence characterized in (a) to (e) and encoding a polypeptide and having the activity of a Shaker channel and/or a Hyperkinetic beta subunit, preferably H-kv beta subunit A or C subtype respectively.
Item b4. A method of item b1 whereby the activity of said polypeptide with the activity of insect Shaker channel and/or a Hyperkinetic beta subunit, preferably H-kv beta subunit A or C subtype respectively is determinated electrophysiologically.
Item b5. A method of item b4 whereby the activity of said polypeptide with the activity of insect Shaker channel and/or a Hyperkinetic beta subunit, preferably H-kv beta subunit A or C subtype respectively is determinated by patch clamp or FLIPR.
Item b6. A method of item b2 whereby a gene coding for a polypeptide with the activity of an insect Shaker channel and/or a Hyperkinetic beta subunit, preferably H-kv beta subunit A or C subtype respectively is expressed in a mammalian cell.
Item b7. A method of item b2 whereby a gene coding for a polypeptide with the activity of an insect Shaker channel and/or a Hyperkinetic beta subunit, preferably H-kv beta subunit A or C subtype respectively is expressed in a mammalian cell selected from the group consisting of: CHO-cells, HEK293.
Item b8. A method of item b1 which comprises:
e) applying to an insect, to a population of insects or to the location wherein said insect is to be controlled an insect-controlling amount a compound identified according to item b1 d) and
f) determining of the growth or the viability of said treated insect or population of insects or of insects or population of insects on said location and untreated insect, population of insects or location and
g) selecting of compounds, which reduces the growth or the viability of said treated insect or population of insects or of insects or population of insects on said location following application of the compound of step e).
Item b9. An assay system comprising a host organism, tissue, cells or a cell digest thereof or a membrane, which has embedded, assembled, intercalated or incorporated a nucleic acid molecule selected from the group consisting of the nucleic acid molecule as depicted in item b3 a) to b3 j) and, based on the expression of this nucleic acid molecule, a polypeptide having the biological activity of a insect Shaker channel and/or a Hyperkinetic beta subunit, preferably H-kv beta subunit A or C subtype respectively, for identifying insecticidally active compound that reduces the activity of an insect Shaker channel and/or a Hyperkinetic beta subunit, preferably H-kv beta subunit A or C subtype respectively.
Item b10. The assay system of item b9 whereby the host organism is a stably transfected mammalian cell which expresses a nucleic acid molecule selected from the group consisting of the nucleic acid molecule as depicted in item b3 a) to b3 j).
Item b11. The assay system of item b10 whereby the mammalian cell is selected from the group consisting of: CHO-cells, HEK293, COS, HeLa, NIH3T3, BAK21, Jurkat, CV-1, HepC-2-, Xenopus oocyte, Sf9, S2, Sf21, Hi5, Pc12, U2O5.
Item b12. A method for killing or inhibiting the growth or viability of an insect, comprising applying to the insect a compound identified according to the method of item b1.
Item b13. A nucleic acid molecule selected from the group consisting of:
a) a nucleic acid molecule encoding a polypeptide comprising the polypeptide shown in SEQ ID NO: 73, 75, 77, 79, 81, 83, 85 and/or 87;
b) a nucleic acid molecule comprising a nucleic acid molecule shown in SEQ ID NO: 72, 74, 76, 78, 80, 82, 84 and/or 86;
c) a nucleic acid molecule, which, as a result of the degeneracy of the genetic code, can be derived from a polypeptide comprising a polypeptide sequence according to SEQ ID NO: 73, 75, 77, 79, 81, 83, 85 and/or 87;
d) a nucleic acid molecule having at least 50% identity with the nucleic acid molecule sequence of a polynucleotide comprising the nucleic acid molecule shown in SEQ ID NO: 72, 74, 76, 78, 80, 82, 84 and/or 86;
e) a nucleic acid molecule encoding a polypeptide having at least 50% identity with the amino acid sequence of the polypeptide encoded by the nucleic acid molecule of (a) to (c) and having the activity of a Shaker channel and/or a Hyperkinetic beta subunit, preferably H-kv beta subunit A or C subtype respectively;
f) nucleic acid molecule which hybridizes with a nucleic acid molecule of (a) to (c) under stringent hybridization conditions;
g) a nucleic acid molecule encoding a polypeptide which can be isolated with the aid of monoclonal or polyclonal antibodies made against a polypeptide encoded by one of the nucleic acid molecules of (a) to (e) and having the activity of a Shaker channel and/or a Hyperkinetic beta subunit, preferably H-kv beta subunit A or C subtype respectively;
h) a nucleic acid molecule encoding a polypeptide comprising the consensus sequence as shown in SEQ ID NO: 102 and/or 103 respectively or one or more motifs selected from the group consisting of SEQ ID NO: 104, 105, 106, 107, 108, 109, 110, 111, 112, 113, 114, 115, 116, 117, 118, 119, 120, 121, 122, 123, 124 and/or 125 and/or 126, 127 and/or 128 respectively;
i) nucleic acid molecule which comprises a polynucleotide, which is obtained by amplifying a cDNA library or a genomic library using the primers in SEQ ID NO: 88, 89; 90, 91; 92, 93; 94, 95; 96, 97; 98, 99 and/or 100, 101 respectively; and
j) a nucleic acid molecule which is obtainable by screening a suitable nucleic acid library under stringent hybridization conditions with a probe comprising a complementary sequence of a nucleic acid molecule of (a) or (b) or with a fragment thereof, having at least 15 nt, preferably 20 nt, 30 nt, 50 nt, 100 nt, 200 nt or 500 nt of a nucleic acid molecule complementary to a nucleic acid molecule sequence characterized in (a) to (e) and encoding a polypeptide and having the activity of a Shaker channel and a Hyperkinetic beta subunit, preferably H-kv beta subunit A or C subtype respectively.
Item b14. A nucleic acid construct comprising a nucleic acid molecule according to item b13.
Item b15. A vector comprising a nucleic acid construct according to item b14 or a nucleic acid molecule according to item b13.
Item b16. A transgenic cell comprising a vector according to item b15, a nucleic acid construct according to item b14 or a nucleic acid molecule according to item b13.
Item b17. A polypeptide encoded by a nucleic acid molecule according to item b13.
Item b18. Use of a polypeptide with the activity of an insect Shaker channel and a Hyperkinetic beta subunit, preferably H-kv beta subunit A or C subtype respectively as insecticidal target.
Item b19. Use of a polypeptide encoded by a nucleic acid molecule selected from the group consisting of the nucleic acid molecule as depicted in item b3 a) to b3 j) as insecticidal target.
Item c1. A method for identifying a insecticidal active compound that reduces the activity of an octopamine receptor selected from the group consisting of oa2, preferably from Drosophila melanogaster, Oamb, Oct-beta-2R and Oct-beta-3R which method comprises:

    
    
        a) assembling in a membrane a polypeptide with the activity of an octopamine receptor selected from the group consisting of oa2, preferably from Drosophila melanogaster, Oamb, Oct-beta-2R and Oct-beta-3R which is originally not present said membrane,
        b) applying at one side of the membrane the compound suspected of having the ability to inhibit the activity of said polypeptide which is originally not present said membrane,
        c) determining the activity of said polypeptide and
        d) identifying a compound applied in (b) that reduces the activity of said polypeptide.
    
    


Item c2. A method according to Item c1 whereby a gene coding for a polypeptide with the activity of an octopamine receptor selected from the group consisting of oa2, preferably from Drosophila melanogaster, Oamb, Oct-beta-2R and Oct-beta-3R is expressed in the membrane of a host cell.
Item c3. A method of any one of the Items c1 or c2 wherein the membrane comprises at least one polypeptide encoded by a nucleic acid molecule selected from the group consisting of:
a) a nucleic acid molecule encoding the polypeptide shown in SEQ ID NO: 130, 134, 138, 142, 146, 150, 154, 158, 162, 166, 170 and/or 174;
b) a nucleic acid molecule shown in SEQ ID NO: 129, 133, 137, 141, 145, 149, 153, 157, 161, 165, 169 and/or 173;
c) a nucleic acid molecule, which, as a result of the degeneracy of the genetic code, can be derived from a polypeptide sequence according to SEQ ID NO: 130, 134, 138, 142, 146, 150, 154, 158, 162, 166, 170 and/or 174
d) a nucleic acid molecule having at least 50% identity with the nucleic acid molecule sequence of a polynucleotide comprising the nucleic acid molecule shown in SEQ ID NO: 129, 133, 137, 141, 145, 149, 153, 157, 161, 165, 169 and/or 173;
e) a nucleic acid molecule encoding a polypeptide having at least 50% identity with the amino acid sequence of the polypeptide encoded by the nucleic acid molecule of (a) to (c) and having the activity of an octopamine receptor selected from the group consisting of oa2, preferably from Drosophila melanogaster, Oamb, Oct-beta-2R and Oct-beta-3R;
f) nucleic acid molecule which hybridizes with a nucleic acid molecule of (a) to (c) under stringent hybridization conditions;
g) a nucleic acid molecule encoding a polypeptide which can be isolated with the aid of monoclonal or polyclonal antibodies made against a polypeptide encoded by one of the nucleic acid molecules of (a) to (e) and having the activity of an octopamine receptor selected from the group consisting of oa2, preferably from Drosophila melanogaster, Oamb, Oct-beta-2R and Oct-beta-3R;
h) a nucleic acid molecule encoding a polypeptide comprising the consensus sequence as shown in SEQ ID NO: 177, 178 and/or 179 respectively or one or more motifs selected from the group consisting of SEQ ID NO: 180, 181, 182, 183, 184, 185, 186, 187, 188, 189 and/or 190, and/or 191, 192, 193, 194, 195, 196, 197, 198, 199, 200, 201, 202, 203, 204, 205, 206, 207 and/or 208, and/or 209, 210, 211, 212, 213, 214, 215, 216, 217, 218, 219, 220, 221, 222, 223, 224, 225 and/or 226 respectively;
i) nucleic acid molecule which comprises a polynucleotide, which is obtained by amplifying a cDNA library or a genomic library using the primers in SEQ ID NO: 131, 132; 135, 136; 139, 140; 143, 144; 147, 148; 151, 152; 155, 156; 159, 160; 163, 164; 167, 168; 171, 172 and/or 175, 176 respectively; and
j) a nucleic acid molecule which is obtainable by screening a suitable nucleic acid library under stringent hybridization conditions with a probe comprising a complementary sequence of a nucleic acid molecule of (a) or (b) or with a fragment thereof, having at least 15 nt, preferably 20 nt, 30 nt, 50 nt, 100 nt, 200 nt or 500 nt of a nucleic acid molecule complementary to a nucleic acid molecule sequence characterized in (a) to (e) and encoding a polypeptide and having the activity of an octopamine receptor selected from the group consisting of oa2, preferably from Drosophila melanogaster, Oamb, Oct-beta-2R and Oct-beta-3R.
Item c4. A method of Item c1 whereby the activity of said polypeptide with the activity of an octopamine receptor selected from the group consisting of oa2, preferably from Drosophila melanogaster, Oamb, Oct-beta-2R and Oct-beta-3R is determinated by fluorescence measurement.
Item c5. A method of Item c2 whereby the activity of said polypeptide with the activity of an octopamine receptor selected from the group consisting of oa2, preferably from Drosophila melanogaster, Oamb, Oct-beta-2R and Oct-beta-3R is determinated with Calcium concentration sensitive dyes.
Item c6. A method of Item c2 whereby a gene coding for a polypeptide with the activity of an octopamine receptor selected from the group consisting of oa2, preferably from Drosophila melanogaster, Oamb, Oct-beta-2R and Oct-beta-3R is expressed in a mammalian cell.
Item c7. A method of Item c2 whereby a gene coding for a polypeptide with the activity of an octopamine receptor selected from the group consisting of oa2, preferably from Drosophila melanogaster, Oamb, Oct-beta-2R and Oct-beta-3R is expressed in a mammalian cell selected from the group consisting of: CHO-cells, HEK293.
Item c8. A method of Item c1 which comprises:
e) applying to an insect, to a population of insects or to the location wherein said insect is to be controlled an insect-controlling amount a compound identified according to Item c1 d) and
f) determining of the growth or the viability of said treated insect or population of insects or of insects or population of insects on said location and untreated insect, population of insects or location and
g) selecting of compounds, which reduces the growth or the viability of said treated insect or population of insects or of insects or population of insects on said location following application of the compound of step e).
Item c9. An assay system comprising a host organism, tissue, cells or a cell digest thereof or a membrane, which has embedded, assembled, intercalated or incorporated a nucleic acid molecule selected from the group consisting of the nucleic acid molecule as depicted in Item c3 a) to c3 j) and, based on the expression of this nucleic acid molecule, a polypeptide having the biological activity of an octopamine receptor selected from the group consisting of oa2, preferably from Drosophila melanogaster, Oamb, Oct-beta-2R and Oct-beta-3R, for identifying insecticidally active compound that reduces the activity of an octopamine receptor selected from the group consisting of oa2, preferably from Drosophila melanogaster, Oamb, Oct-beta-2R and Oct-beta-3R.
Item c10. The assay system of Item c9 whereby the host organism is a stably transfected mammalian cell which expresses a nucleic acid molecule selected from the group consisting of the nucleic acid molecule as depicted in Item c3 a) to c3 j).
Item c11. The assay system of Item c10 whereby the mammalian cell is selected from the group consisting of: CHO-cells, HEK293, COS, HeLa, NIH3T3, BAK21, Jurkat, CV-1, HepC-2-, Xenopus oocyte, Sf9, S2, Sf21, Hi5, Pc12, U2O5.
Item c12. A method for killing or inhibiting the growth or viability of an insect, comprising applying to the insect a compound identified according to the method of Item c1.
Item c13. A nucleic acid molecule selected from the group consisting of:
a) a nucleic acid molecule encoding the polypeptide shown in 2, 6, 10, 14, 18, 22, 26, 30, 34, 38, 42 and/or 46;
b) a nucleic acid molecule shown in SEQ ID NO: 129, 133, 137, 141, 145, 149, 153, 157, 161, 165, 169 and/or 173;
c) a nucleic acid molecule, which, as a result of the degeneracy of the genetic code, can be derived from a polypeptide sequence according to SEQ ID NO: 130, 134, 138, 142, 146, 150, 154, 158, 162, 166, 170 and/or 174;
d) a nucleic acid molecule having at least 50% identity with the nucleic acid molecule sequence of a polynucleotide comprising the nucleic acid molecule shown in SEQ ID NO: 129, 133, 137, 141, 145, 149, 153, 157, 161, 165, 169 and/or 173;
e) a nucleic acid molecule encoding a polypeptide having at least 50% identity with the amino acid sequence of the polypeptide encoded by the nucleic acid molecule of (a) to (c) and having the activity of an octopamine receptor selected from the group consisting of oa2, preferably from Drosophila melanogaster, Oamb, Oct-beta-2R and Oct-beta-3R;
f) nucleic acid molecule which hybridizes with a nucleic acid molecule of (a) to (c) under stringent hybridization conditions;
g) a nucleic acid molecule encoding a polypeptide which can be isolated with the aid of monoclonal or polyclonal antibodies made against a polypeptide encoded by one of the nucleic acid molecules of (a) to (e) and having the activity of an octopamine receptor selected from the group consisting of oa2, preferably from Drosophila melanogaster, Oamb, Oct-beta-2R and Oct-beta-3R;
h) a nucleic acid molecule encoding a polypeptide comprising the consensus sequence as shown in SEQ ID NO: 177, 178 and/or 179 respectively or one or more motifs selected from the group consisting of SEQ ID NO: 180, 181, 182, 183, 184, 185, 186, 187, 188, 189 and/or 190, and/or 191, 192, 193, 194, 195, 196, 197, 198, 199, 200, 201, 202, 203, 204, 205, 206, 207 and/or 208, and/or 209, 210, 211, 212, 213, 214, 215, 216, 217, 218, 219, 220, 221, 222, 223, 224, 225 and/or 226 respectively;
i) nucleic acid molecule which comprises a polynucleotide, which is obtained by amplifying a cDNA library or a genomic library using the primers in SEQ ID NO: 131, 132; 135, 136; 139, 140; 143, 144; 147, 148; 151, 152; 155, 156; 159, 160; 163, 164; 167, 168; 171, 172 and/or 175, 176 respectively;
and
j) a nucleic acid molecule which is obtainable by screening a suitable nucleic acid library under stringent hybridization conditions with a probe comprising a complementary sequence of a nucleic acid molecule of (a) or (b) or with a fragment thereof, having at least 15 nt, preferably 20 nt, 30 nt, 50 nt, 100 nt, 200 nt or 500 nt of a nucleic acid molecule complementary to a nucleic acid molecule sequence characterized in (a) to (e) and encoding a polypeptide and having the activity of an octopamine receptor selected from the group consisting of oa2, preferably from Drosophila melanogaster, Oamb, Oct-beta-2R and Oct-beta-3R.
Item c14. A nucleic acid construct comprising a nucleic acid molecule according to Item c13.
Item c15. A vector comprising a nucleic acid construct according to Item c14 or a nucleic acid molecule according to Item c13.
Item c16. A transgenic cell comprising a vector according to Item c15, a nucleic acid construct according to Item c14 or a nucleic acid molecule according to Item c13.
Item c17. A polypeptide encoded by a nucleic acid molecule according to Item c13.
Item c18. Use of a polypeptide with the activity of an octopamine receptor selected from the group consisting of oa2, preferably from Drosophila melanogaster, Oamb, Oct-beta-2R and Oct-beta-3R as insecticidal target.
Item c19. Use of a polypeptide encoded by a nucleic acid molecule selected from the group consisting of the nucleic acid molecule as depicted in Item c3 a) to c3 j) as insecticidal target.
Item c20. Use of manserin and/or cyproheptadine as insecticidal active ingredients.
Item d1. A method for identifying a insecticidally active compound that reduces the activity of an insect small-conductance Ca2+-activated potassium channel which method comprises:

    
    
        a) assembling in a membrane a polypeptide with the activity of an insect small-conductance Ca2+-activated potassium channel which is originally not present said membrane,
        b) applying at one side of the membrane the compound suspected of having the ability to inhibit the activity of said polypeptide which is originally not present said membrane,
        c) determining the activity of said polypeptide and
        d) identifying a compound applied in (b) that reduces the activity of said polypeptide.
    
    


Item d2. A method according to Item d1 whereby a gene coding for a polypeptide with the activity of an insect small-conductance Ca2+-activated potassium channel is expressed in the membrane of a host cell.
Item d3. A method of any one of the Items d1 or d2 wherein the membrane comprises at least one polypeptide encoded by a nucleic acid molecule selected from the group consisting of:
a) a nucleic acid molecule encoding the polypeptide shown in SEQ ID NO: 228, 230, 232;
b) a nucleic acid molecule shown in SEQ ID NO: 227, 229, 231;
c) a nucleic acid molecule, which, as a result of the degeneracy of the genetic code, can be derived from a polypeptide sequence according to SEQ ID NO: 228, 230, 232;
d) a nucleic acid molecule having at least 50% identity with the nucleic acid molecule sequence of a polynucleotide comprising the nucleic acid molecule shown in SEQ ID NO: 227, 229, 231;
e) a nucleic acid molecule encoding a polypeptide having at least 50% identity with the amino acid sequence of the polypeptide encoded by the nucleic acid molecule of (a) to (c) and having the activity of a small-conductance Ca2+-activated potassium channel;
f) nucleic acid molecule which hybridizes with a nucleic acid molecule of (a) to (c) under stringent hybridization conditions;
g) a nucleic acid molecule encoding a polypeptide which can be isolated with the aid of monoclonal or polyclonal antibodies made against a polypeptide encoded by one of the nucleic acid molecules of (a) to (e) and having the activity of a small-conductance Ca2+-activated potassium channel;
h) a nucleic acid molecule encoding a polypeptide comprising the consensus sequence as shown in SEQ ID NO: 239 or one or more motifs selected from the group consisting of SEQ ID NO: 240, 241, 242, 243, 244, 245, 246, 247, 248, 249, 250, 251, 252 and 253;
i) nucleic acid molecule which comprises a polynucleotide, which is obtained by amplifying a cDNA library or a genomic library using the primers in SEQ ID NO: 233, 234; 235, 236; 237, 238 respectively;
and
j) a nucleic acid molecule which is obtainable by screening a suitable nucleic acid library under stringent hybridization conditions with a probe comprising a complementary sequence of a nucleic acid molecule of (a) or (b) or with a fragment thereof, having at least 15 nt, preferably 20 nt, 30 nt, 50 nt, 100 nt, 200 nt or 500 nt of a nucleic acid molecule complementary to a nucleic acid molecule sequence characterized in (a) to (e) and encoding a polypeptide and having the activity of a small-conductance Ca2+-activated potassium channel.
Item d4. A method of Item d1 whereby the activity of said polypeptide with the activity of insect small-conductance Ca2+-activated potassium channel is determinated electrophysiologically.
Item d5. A method of Item d4 whereby the activity of said polypeptide with the activity of insect small-conductance Ca2+-activated potassium channel is determinated by patch clamp.
Item d6. A method of Item d2 whereby a gene coding for a polypeptide with the activity of an insect small-conductance Ca2+-activated potassium channel is expressed in a mammalian cell.
Item d7. A method of Item d2 whereby a gene coding for a polypeptide with the activity of an insect small-conductance Ca2+-activated potassium channel is expressed in a mammalian cell selected from the group consisting of: CHO-cells, HEK293.
Item d8. A method of Item d1 which comprises:
e) applying to an insect, to a population of insects or to the location wherein said insect is to be controlled an insect-controlling amount a compound identified according to Item d1 d) and
f) determining of the growth or the viability of said treated insect or population of insects or of insects or population of insects on said location and untreated insect, population of insects or location and
g) selecting of compounds, which reduces the growth or the viability of said treated insect or population of insects or of insects or population of insects on said location following application of the compound of step e).
Item d9. An assay system comprising a host organism, tissue, cells or a cell digest thereof or a membrane, which has embedded, assembled, intercalated or incorporated a nucleic acid molecule selected from the group consisting of the nucleic acid molecule as depicted in Item d3 a) to d3 j) and, based on the expression of this nucleic acid molecule, a polypeptide having the biological activity of a insect small-conductance Ca2+-activated potassium channel, for identifying insecticidally active compound that reduces the activity of an insect small-conductance Ca2+-activated potassium channel.
Item d10. The assay system of Item d9 whereby the host organism is a stably transfected mammalian cell which expresses a nucleic acid molecule selected from the group consisting of the nucleic acid molecule as depicted in Item d3 a) to d3 j).
Item d11. The assay system of Item d10 whereby the mammalian cell is selected from the group consisting of: CHO-cells, HEK293, COS, HeLa, NIH3T3, BAK21, Jurkat, CV-1, HepC-2-, Xenopus oocyte some more?. Sf9, S2, Sf21, Hi5, Pc12, U2O5.
Item d12. A method for killing or inhibiting the growth or viability of an insect, comprising applying to the insect a compound identified according to the method of Item d1.
Item d13. A nucleic acid molecule selected from the group consisting of:
a) a nucleic acid molecule encoding the polypeptide shown in SEQ ID NO: 230, 232;
b) a nucleic acid molecule shown in SEQ ID NO: 229, 231;
c) a nucleic acid molecule, which, as a result of the degeneracy of the genetic code, can be derived from a polypeptide sequence according to SEQ ID NO: 230, 232;
d) a nucleic acid molecule having at least 50% identity with the nucleic acid molecule sequence of a polynucleotide comprising the nucleic acid molecule shown in SEQ ID NO: 229, 231;
e) a nucleic acid molecule encoding a polypeptide having at least 50% identity with the amino acid sequence of the polypeptide encoded by the nucleic acid molecule of (a) to (c) and having the activity of a small-conductance Ca2+-activated potassium channel;
f) nucleic acid molecule which hybridizes with a nucleic acid molecule of (a) to (c) under stringent hybridization conditions;
g) a nucleic acid molecule encoding a polypeptide which can be isolated with the aid of monoclonal or polyclonal antibodies made against a polypeptide encoded by one of the nucleic acid molecules of (a) to (e) and having the activity of a small-conductance Ca2+-activated potassium channel;
h) a nucleic acid molecule encoding a polypeptide comprising the consensus sequence as shown in SEQ ID NO: 239 or one or more motifs selected from the group consisting of SEQ ID NO: 240, 241, 242, 243, 244, 245, 246, 247, 248, 249, 250, 251, 252 and 253;
i) nucleic acid molecule which comprises a polynucleotide, which is obtained by amplifying a cDNA library or a genomic library using the primers in SEQ ID NO: 233, 234; 235, 236; 237, 238 respectively;
and
j) a nucleic acid molecule which is obtainable by screening a suitable nucleic acid library under stringent hybridization conditions with a probe comprising a complementary sequence of a nucleic acid molecule of (a) or (b) or with a fragment thereof, having at least 15 nt, preferably 20 nt, 30 nt, 50 nt, 100 nt, 200 nt or 500 nt of a nucleic acid molecule complementary to a nucleic acid molecule sequence characterized in (a) to (e) and encoding a polypeptide and having the activity of a small-conductance Ca2+-activated potassium channel.
Item d14. A nucleic acid construct comprising a nucleic acid molecule according to Item d13.
Item d15. A vector comprising a nucleic acid construct according to Item d14 or a nucleic acid molecule according to Item d13.
Item d16. A transgenic cell comprising a vector according to Item d15, a nucleic acid construct according to Item d14 or a nucleic acid molecule according to Item d13.
Item d17. A polypeptide encoded by a nucleic acid molecule according to Item d13.
Item d18. Use of a polypeptide with the activity of an insect small-conductance Ca2+-activated potassium channel as insecticidal target.
Item d19. Use of a polypeptide encoded by a nucleic acid molecule selected from the group consisting of the nucleic acid molecule as depicted in Item d3 a) to d3 j) as insecticidal target.
EXAMPLES
Cell Biology
Molecular Biology: The full-length ShaI CDS (coding sequence) was subcloned into the pcDNA3/Neo(−) expression vector using the EcoR1 restriction site. The ShaI_delN mutant has an N-terminal deletion for the 2-40 aa coding region. The deletion mutation was inserted into the pcDNA3_ShaI construct using ShaI_del—2-4aa_fwd (5′ gca-gaattcgcccttgccaccatggagaagctcctga tcaacgtctccgg-3′) and ShaI_del—2-4aa_rev (5′-ccggagacgttgatcaggagcttctccatggtggcaagggc gaattctgc-3′) primers and the XL Site-Directed Mutagenesis Kit (Stratagene). A unique AscI restriction site was inserted before the ATG start codon in pcDNA_ShaI and pcDNA_ShaI_delN constructs by site-directed mutagenesis. The full length ShaI and ShaI_delN inserts were subcloned into the pcDNA3_AcGFPC1 vector (AscI and HindIII restriction sites) to obtain an AcGFP chimera (FIG. 1) The AcGFP is thus tagged to the N-terminus of both these constructs. All constructs were double-stranded sequenced for the ShaI coding region to confirm sequence integrity. The dominant-negative ShaI_DelNW362F mutant was made by changing the tryptophan (W) to phenylalanine (F) at position 362 in the pore region. KChIP, a ShaI accessory protein, was cloned from Drosophila and subcloned into the pcDNA3.1/Zeo vector.
FIG. 1: Primary ShaI clone. Vector NTI-generated map of the primary ShaI clone showing the major features of the construct. The ShaI coding region is 1473 bp in length
FIG. 2: Primary KChIP clone. Vector NTI-generated map of the primary KChIP clone showing the major features of the construct. The KChIP coding region is 621 bp in length. The expression construct, pcDNA3.1-Zeo-KChIP-intron1 (data not shown, notebook 1049294 #5) contains a  ̃700 bp intron to aid in cloning, and places the KChIP ORF downstream of a CMV promoter.
FIG. 3: ShaI and KChIP expression constructs. Maps of pcDNA3/AcGFP-ShaI and pcDNA3/AcGFP-ShaIdelN2-40aa and links to Vector NTI pcDNA3_ShaIDelN and pcDNA3_Zeo_KCHiP_intron1 constructs.
FIG. 4: Patch clamp data for full length and truncated constructs ShaI and ShaI_delN when coexpressed with AmCyan as well as AcGFP-tagged versions (top). Schematic representation of GFP-ShaI pore mutant and patch clamp data for wild type and pore mutant, showing that the measured currents indeed arise from overexpressed ShaI and not an upregulated endogenous current (bottom).
Expression in mammalian cells: Wild type and mutant ShaI channels were transiently expressed in CHO cells and tested for function at 48 hrs by whole-cell voltage clamp recordings. The untagged ShaI constructs were cotransfected with pcDNA3_AmCyan. The full length ShaI and ShaI_delN mutant mediated functional channel activity. The GFP tag at the amino terminus did not alter channel function. The ShaI_delN-W362F mutant did not form functional channels.
Stable cell line generation: Wild type CHO cells were transfected with AcGFP-ShaI and AcGFP-ShaI_delN DNA using FuGene Transfection Reagent (Roche). Stable clonal lines were generated by selecting the cells with G418 (900 ug/ml) at 24 hrs post transfection. 25 colonies were picked for each construct and evaluated for GFP expression using a fluorescence microscope. 70-75% of the clonal lines had very weak or no GFP expression. Based on the fluorescence rating, some of the high and medium GFP expressing clonal lines were tested for ShaI activity by patch clamp (Table 1). The ShaI_delN clones (Clone 10 & 15) had higher current density than the full length ShaI clones. Thus, the development of an assay was proceeded with these 2 clones.







TABLE 1







Summary of Shal clone screening showing relative fluorescent


response and current density.









Clone
Fluorescence rating
Current Density (pA/pF)





Shal C7
++
 54 (58; 50)


Shal C10
++
 86 (89; 83)


Shal C16
++
 41 (60; 21)


Shal C20
++
 36 (37; 35)


Shal C21
+++
 45 (41; 48)


Shal_DelN C5
++
n/a


Shal_DelN C8
++
no current


Shal_DelN C10
++
337 (216; 457)


Shal_DelN C11
++
no current


Shal_DelN C15
+++
107 (38; 118; 166)









ShaI_DelN C10 and C15 were tested for function on the FLIPR using the Molecular Devices blue membrane potential dye. The cells were seeded at 60K cells/well in 96-well Costar plates in complete media and assayed for function at 24 hrs. Depolarization with 15-60 mM KCl caused a 2-2.8 fold higher activity in the clonal line than the pcDNA control cell line (FIG. 5). Clone 10 had slightly higher activity than clone 15.
FIG. 5: ShaI_DelN clones tested on FLIPR with KCl depolarization. We also tested some known K-channel blockers on channel activity on the FLIPR (FIG. 6). Bay K8644, nicardipine, quinidine and niflumic acid showed dose dependent inhibition of KCl depolarization in both the ShaI_delN clonal lines and the control pcDNA3 cell line. Flecainide did not show any inhibition on the clones and control cell lines at the concentrations tested. The ShaI_delN clonal cell lines lost their GFP expression and assay window with cell passage. At passage 12, only 10-15% of the cell population was GFP positive.
FIG. 6: K channel blocker inhibition curves tested on the FLIPR. ShaI_delN C10 cells were subcloned by flow-sorting. The cells were flow-sorted into 96-well tissue culture plates to obtain GFP-positive single cells. Several clonal lines were expanded and tested for function using KCl depolarization on the FLIPR (FIG. 7).
FIG. 7: Screening ShaI_delN Clone10 subclones by KCl depolarization on FLIPR. Based on the FLIPR data, clones 10-2, 10-3 and 10-6 were further tested by patch clamp (FIG. 8). Clones 10-3 and 10-6 had high currents in the whole-cell patch clamp studies.
FIG. 8: Patch clamp data for clones 10-2, 10-3 and 10-6. IV curves showing the relationship of applied voltage to current.
A ShaI_delN clone 10-3 subclone maintained the GFP expression over several cell passages. At Passage12, about 80-85% of the cell population was GFP positive (FIG. 9).
FIG. 9: ShaI_DelN C10-3 cells at passage 12 showing stability of fluorescence over time. Left panel—fluorescence excitation, right panel—normal light.
Increase of Assay window:
The effects of barium (Ba2+) on ShaI_delN function were tested. Barium is known to inhibit some open rectifier and inward rectifier K-channel activities. Cells were dye loaded in the presence of BaCl2 for 1 hr at RT and assayed on the FLIPR using KCl depolarization for channel activation. Ba2+ at a concentration of 4-5 mM significantly reduced fluorescent responses in CHO_pcDNA3 control cells without any significant reduction in the ShaI_delN channel activity with KCl depolarization (FIG. 10).
FIG. 10: Effect of BaCl2 on ShaI channel activity on FLIPR.
Clone 10-3 had a higher assay window on FLIPR in the presence of Ba2+, than clone 10-6. ShaI outward currents were not inhibited by Ba2+ as shown by electrophysiology. The idea is that in pcDNA3 cells, Ba2+ inhibits the endogenous outward K+ currents leading to an accumulation in intracellular Potassium and therefore depolarization of the cells. Thus, with 0.5 mM KCl in the external solution and increasing Ba2+ concentration, the membrane potential is increased with complete collapse of the wild-type cell assay window at 5 mM BaCl2. In contrast the ShaI_delN clone 10-3 cell line still has significant ShaI outward K+ current activity at 5 mM Ba2+ resulting in an assay window. ShaI_delN clone 10-3 cells were tested for function on FLIPR up to passage 20 (P20, FIG. 11). The cells still had a significant assay window at passage window at P20. However, the size of the window is reduced with increase in cell passage.
FIG. 11: ShaI_delN C10-3 function on FLIPR at passage 20.
Increased Currents by Coexpression of an Accessory Protein: For some ion channels, currents can be increased by coexpression of accessory proteins, which is shown in FIG. 12.
FIG. 12: CHO-K1 cells stably expressing ShaI_delN were either mock transfected (red trace, lower line) or cotransfected with KChIP (blue trace, upper line).
Additional Electrophysiology
Chinese hamster ovary (CHO) cells transfected with (a) a Drosophila ShaI gene (b) a Drosophila ShaI pore mutant gene and (c) a Drosophila ShaI+KChIP gene, and were used for all measurements. Cells were plated in 35 mm Petri dishes 2-6 hours before the experiment.
Data were acquired and analyzed using pClamp software (version 9.0.1.16). The whole-cell configuration of the patch-clamp technique was used to voltage clamp cells at room temperature (22-25° C.). Pipettes were pulled from borosilicate glass capillaries (#8250, Garner Glass, Claremont, Calif.) using a DMZ Universal Puller (Zeitz, Munich, Germany) and had resistances of 1-2 MOhm when filled with pipette solution and measured in bath solution. The liquid junction potential between bath and pipette solution was always compensated before the formation of a gigaohm seal.
Membrane current was measured under whole-cell clamp, sampled at 2 kHz and filtered at 1 kHz by an Axoclamp 200B (Axon Instruments). Capacitance currents were electronically compensated at the beginning of each experiment. P/4 leak correction was applied.
To study ShaI currents on CHO cells, cells were held at −70 mV and a family of 500 ms test voltage pulses was applied starting from −70 to +70 mV, or +100 mV, in 10 mV increments. The amplitude, as measured for the current-voltage relationship, was defined as the maximal current at a given membrane potential.
Bath solution: NaCl (160 mM), KCl (2.5 mM), MgCl2 (1 mM), CaCl2 (2 mM), HEPES (10 mM) pH 7.4 (with NaOH)
Pipet solution: KCl (160 mM), MgCl2 (5 mM), EGTA (1 mM), CaCl2 (0.1 mM), NaGTP (0.1 mM), K2ATP (3 mM), HEPES (5 mM) pH 7.4 (with NaOH)
Additional Electrophysiology
By way of further assay validation, the presumptive ShaI blocker arachidonic acid (J. Neurosci. 1996, 16:2522) was tested on ShaI/KChIP 20 cells (FIG. 13).
FIG. 13: ShaI/KChIP inhibited by arachidonic acid. ShaI/KChIP 20 cells were perfused with a 100 uM [final] solution of arachidonic acid in bath solution. Pre- and post-compound currents were measured under whole-cell clamp. Left panel: Integrations of the areas under the curves (total current). Right panel: Peak current amplitudes.
Results and conclusions: Complete inhibition of total ShaI/KChIP current was obtained after incubation with arachidonic acid for five minutes, thus confirming a key indicator of this channel.
ShIP_GFP stable clones in CHO cell line, shown in FIG. 14.
FIG. 14: I/V curves for clones 18-13-6 and 18-13-20.
Buffer: KCNQ Int/Ext solution
Protocol: C:\FJB\patch clamp\Patch_parmeters\CHO cells_ShaI_IV
500 ms_to70 mV_Whole_cell.pro
Clone Generation and Selection
Transfection: ShaI_delN 10-3 cells were plated in a 35 mm 6-well plates at 2.5×105 cells/well and each well transfected 6 hours later with 1 ug pcDNA3.1/Zeo(+)KChIP DNA using FuGENE transfection reagent (Roche) and the manufacturer's recommendations. A mock transformation without KChIP DNA was also done to monitor antibiotic selection. For a “Neo/Zeo” control cell line, pcDNA3.1/Neo CHO cells at passage 44 were transfected with 1 ug pcDNA3.1/Zeo DNA. Cells were passaged 24 hr later to 175 cm2 flasks @750K cells/flask and placed under antibiotic selection (900 ug/ml G418; 1 mg/ml Zeocin). For the ShaI/KChIP transfection, once selection was complete (four days), cells were diluted to 12 cells/ml in complete culture medium and 250 ul was dispensed into each well of four 96-well culture plates. Wells containing a single colony were identified after one week's growth and picked for expansion and testing after an additional week. The Neo/Zeo control cells were propagated as a polyclonal pool.
Clone screening: 19 individual ShaI/KChIP clones were screened for function (FIG. 15). The amplitude and kinetic profile in response to depolarization with KCl was used to select clones for further evaluation.
FIG. 15: ShaI/KChIP clone screening and Neo/Zeo control line. ScreenWorks screenshots showing primary FLIPR data and a reduced data table for the 19 clones and the Neo/Zeo pool (middle panel, wells H4-6). Cells were plated at an estimated 5×104 cells/well into 96-well assay plates (TC-treated, BD Biosciences) and incubated overnight at 37° C./5% CO2. Culture medium was removed and replaced with 1× blue membrane potential dye in assay buffer and incubated for 0.5 hour at 25° C. The assay was read on a FLIPR Tetra by recording baseline fluorescence for 20 sec, and recording an additional 180 sec after activation with isometrically-substituted KCl (60 mM [final], columns 1, 2, 4 & 5). Columns 3 & 6 show the responses to normal assay buffer (0.5 mM KCl [final]). Subtract bias was set at 1 and negative control correction was OFF. For the data table, subtract bias was at 1 and negative control correction (from columns 3 & 6) was ON.
Results and conclusions: Seven ShaI/KChIP clones were identified for further testing (blue rows in data table). Clone 18 (left panel, row C) was ultimately selected for two rounds of subcloning in order to stabilize the response and reduce heterogeneity. Importantly, as the Neo/Zeo cell line was found to be essentially unresponsive to the activation method used (middle panel, wells H4 & 5), the requirement for it as a control line was effectively eliminated.
Intermediate subclone generation: ShaI/KChIP clones 17 & 18 were plated for subcloning as described previously. A combined total of 66 subclones were screened, and number 18-13 was selected as the source for final subclone generation (data not shown, notebook 1050115 #8).
Final subclone screening and selection: ShaI/KChIP subclone 18-13 was plated for final subcloning as described previously. Thirty-four subclones were evaluated for response to KCl depolarization (FIG. 16).
FIG. 16: ShaI/KChIP final subclone screening. Reduced FLIPR data from subclone screening resulting in the final cell line. Cells were plated at an estimated 5×104 cells/well into 96-well assay plates (TC-treated, BD Biosciences) and incubated overnight at 37° C./5% CO2. Culture medium was aspirated and replaced with 1× blue membrane potential dye in assay buffer and incubated for 0.5 hour at 25° C. The assay was read on a FLIPR Tetra by recording baseline fluorescence for 20 sec, and recording an additional 60 sec after activation with isometrically-substituted KCl (60 mM [final]). No data reductions were used during data export. Both the raw response (RFU, relative fluorescence units, blue bars) and ratioed responses (diamonds) are shown. The ratioed response is equal to the raw response divided by the signal test (i.e., baseline fluorescence)—this was done to compensate for variations in the number of cells plated for each clone. The green cells in the “ratio” column indicate clones with a raw/baseline ration >1. Clones were chosen for further evaluation by considering one and/or both reductions (indicated by marked lines).
Results and conclusions: Seven cell lines were chosen for further evaluation (marked in FIG. 15) on the FLIPR (data not shown) and in electrophysiology (see “Additional Electrophysiology” for examples). After testing clones via conventional patch clamp, it was decided to move forward with ShaI/KChIP 18-13-20. This cell line, known as ShaI/KChIP 20 (or ShIP 20), was used, unless indicated otherwise, in all subsequent HTS assay and screen development.
Assay & Screen Development
HTS Screening Strategy
This assay uses two fluid additions to permit the detection of activators and antagonists in a single experiment (FIG. 17). In screening, test compounds are added in the first addition and allowed to incubate for three minutes. An activating dose of KCl is then introduced in the second addition and the fluorescence read for an additional three minutes. Controls are run for both additions. A response (rise in fluorescence) significantly greater than that from buffer/DMSO in the first addition indicates that the compound may be an activator (green trace). A reduced response after the second addition indicates that the compound may be an antagonist (blue trace).
FIG. 17: ShaI/KChIP assay FLIPR response profiles. Sample control group averages from BioFocus compound screening showing first and second addition assay windows, and potential activator and antagonist profiles.
Basic Test Protocol
Cells were dispensed in a 50 μl volume containing 7,500 cells into 384-well TC clear/black assay plates (Greiner 781091) using a Multidrop 384 dispenser, incubated at ambient temperature for one hour (to reduce edge effects), and incubated overnight at 37° C./5% CO2. The cells were assayed 18-24 hours after seeding, at which point they were just approaching confluency in the wells. Culture medium was removed by flicking and tapping it out of the plate, and cells were loaded with 20 ul/well of 1× blue membrane potential dye in assay buffer for 30 minutes at 25° C. After 30 minutes the assay plate was placed into the FLIPR and run using a two-addition protocol. The first addition (5×, 5 ul) contained 2.5% DMSO in assay buffer. The pipetting height was set at 15 and the speed 20. The plate was read for three minutes after this addition. The second addition (2×, 25 ul) was either assay buffer or 120 mM isometrically-substituted KCl (i.e., KCl substituted for NaCl on a 1:1 molar basis). The height was 20 and the speed 25. Aspirate speeds were set at the lowest values and no hold or expel volumes applied. The plate was read for an additional three minutes after the second addition. The exported statistics were typically configured as stat1=average of 190-200 (the interval just before the first addition) and stat2=maximum of 260-maximum allowed. Subtract bias was set at 1 and negative control correction was OFF. Z′ statistics (an index of “screenability”, Z′>0.5=single-pass screen) were calculated using the following formula:

Z′=1−((3σmax+3σmin)/(Iμmax−μminI)).

Use of Membrane Potential Dye in Assay
The ShaI cell line assay used the addition of membrane potential dye in the activation buffer to increase the size of the assay window (data not shown). After the addition of KChIP to the ShaI cell line, we examined whether this requirement for dye could be dropped in order to simplify the protocol and reduce the cost of the assay for both development and HTS (FIG. 18).
FIG. 18: Use of dye in activation buffer. ShaI/KChIP (ShIP), ShaI and Neo (ShaI control) cell lines were plated at 5×104 cells/well into 96-well assay plates (TC-treated, BD Biosciences) and incubated overnight at 37° C./5% CO2. Culture medium was aspirated and replaced with 50 ul 1× blue membrane potential dye in assay buffer and incubated for 0.5 hour at 25° C. The assay was read on a FLIPR Tetra by recording baseline fluorescence for 20 sec, and recording an additional 60 sec after addition of 50 ul isometrically-substituted KCl (60 mM [final]) with and without membrane potential dye. For the data export, subtract bias was set at 1. Error bars are +/−1 SD.
Results and conclusions: The removal of membrane potential dye from the activation buffer resulted in a significantly reduced fluorescence change upon KCl activation for all three cell lines. The Z′ statistic for ShaI/KChIP decreased from 0.77 to 0.59, which is still well above the single-pass screening cutoff of 0.5 (data not shown). As the addition of dye to the activation buffer would complicate the HTS protocol, and as the estimated potential cost savings over the course of development and screening was considerable, it was decided to progress without the use of dye in the activation buffer. The reduction in the Z′ statistic was largely recovered during subsequent development.
Assay Plate Selection
An experiment was undertaken to compare the assay's performance between our standard BD Falcon 353962 plates and the less-expensive Greiner 781091 384-well tissue culture-treated assay plates (FIG. 19) Note: Greiner is the manufacturer of the BD Falcon plates.
FIG. 19: Comparison of BD and Greiner 384-well assay plates: ShaI/KChIP 20 cells were plated at 7500 cells/well into BD and Greiner 384-well assay plates and incubated overnight at 37° C./5% CO2. Culture medium was flicked out and replaced with 20 ul 1× blue membrane potential dye in assay buffer and incubated for 0.5 hour at 25° C. The assay was read on a FLIPR Tetra. The first addition (5×, 5 ul) contained 2.5% DMSO in assay buffer, and was read for three minutes. The second addition (2×, 25 ul) consisted of isometrically-substituted KCl at dose, and was also read for three minutes. Each condition was measured from 2×48 replicates to allow the calculation of Z′s. The exported statistics were configured as stat1=average of 190-200 and stat2=maximum of 260-maximum allowed. Subtract bias was set at 1. Left panel: Window sizes (calculated as stat2-stat1) as a function of [KCl] for the two plate types. Right panel: Z′ statistics calculated as a function of [KCl] for the two plate types.
Results and conclusions: There was a small but consistent decrease ( ̃200 RFU) in the assay window size when using Greiner assay plates in this test. The Z′ statistics, however, were nearly identical due to slightly lower CVs on the Greiner plates. As the estimated potential cost savings over the course of development and screening was substantial, it was decided to continue development using the Greiner plates, and all subsequent work was done with them.
Assay Buffer Preparation Method Testing
While experimental results during early assay development indicated the use of freshly-prepared assay buffers, this requirement was eventually dropped during screen development (data not shown). The preparation method (additions of stock solutions of KCl, NaCl and DMSO to base buffer) remained in use however, and a volume-compensated buffer preparation calculator is included in this document.
Cell Density Optimization
Plating densities from 5,000 to 15,000 cells/well were examined for mean response and standard deviation, and Z′ statistics were calculated (FIG. 20).
FIG. 20: Cell density optimization. Reduced FLIPR data showing statistics for full plates at the indicated cell densities with resulting Z′ statistics. Platings below 5000 cells/well did not yield a monolayer the next day and were not tested. ShaI/KChIP cells were plated at the indicated densities incubated at 37° C./5% CO2 and assayed the following day. Cells were loaded with 20 ul 1× dye/well for 0.5 hour at 25° C. A two-addition protocol was used. The first addition (5×) was 5 ul 2.5% DMSO in assay buffer (0.5% [final]) read for 180 sec, and the second (2×) was 25 ul isometrically-substituted KCl in assay buffer (60 mM [final]) read for an additional 180 sec. The statistics were calculated using the average of reads 190-200 (minimum response) and the maximum of reads 260-end (maximum response). Spatial uniformity correction was ON. The bars show response to activation, and the line shows the trend in Z′ statistics
Results and conclusions: This assay tended to perform better at lower cell densities. It was decided to move development forward with 7500 cells/well (rather than 5000 cells/well, as would seem indicated) to avoid potential problems due to variations in cell counting and plating.
Dye Concentration
The performance of ShaI/KChIP intermediate subclones 18-13 and 18-28 was examined using 0.4× membrane potential dye (vs. 1×) as a possible cost saving measure (FIG. 21).
FIG. 21: Dye concentration evaluation. ShaI/KChIP clones 18-13 and 18-28 were plated at 5×104 cells/well into 96-well assay plates (TC-treated, BD Biosciences) and incubated overnight at 37° C./5% CO2. Culture medium was removed and replaced with 50 ul 1× or 0.4× blue membrane potential dye in assay buffer and incubated for 0.5 hour at 25° C. The assay was read on a FLIPR Tetra by recording baseline fluorescence for 20 sec, and recording an additional 60 sec after addition of 50 ul isometrically-substituted KCl (60 mM [final]). Maximum RFU values are shown. For the data export, subtract bias was set at 1 and negative control correction was ON. Error bars are +/−1 SD.
Results and conclusions: Dye used at 0.4× resulted in an assay window reduced to 70% the size of that obtained with full (1×) dye for both clones tested. To preserve the full assay window it was decided to carry out all subsequent development using 1× dye.
DMSO Tolerance
A cell-based assay's sensitivity to DMSO in the first addition factors into calculations made about the ability to screen compounds at desired levels from particular DMSO stock concentrations. The effects of first addition DMSO on assay window size and variability, and the resulting Z′ statistics were examined (FIG. 22).
FIG. 22: ShaI/KChIP assay window size, standard deviations and Z′ statistics. ShaI/KChIP18-13 cells were plated at 7500 cells/well into Greiner 384-well assay plates and incubated overnight at 37° C./5% CO2. Culture medium was flicked out and replaced with 20 ul 1× blue membrane potential dye in assay buffer and incubated for 0.5 hour at 25° C. The assay was run on a FLIPR Tetra using the following conditions: the first addition (5×, 5 ul) contained 5× [final] DMSO in assay buffer, and was read for three minutes. The second addition (2×, 25 ul) consisted of isometrically-substituted KCl (120 mM), and was read for an additional three minutes. Each condition was measured from 64 replicates to allow the calculation of Z′s. The exported statistics were configured as stat1=average of 180-200 and stat2=maximum of 260-380. Subtract bias was set at 1 and negative control correction was OFF. Blue bars (stat2-stat1) show response to activation, and the red line shows the trend in Z′ statistics. Error bars are +/−1 SD.
Results and conclusions: While standard deviations remained relatively constant, with the exception of 1% DMSO [final], window sizes decreased with increasing DMSO and, necessarily, there occurred a corresponding decrease in Z′ statistics. As a practical matter, given the concentrations of the compound DMSO stocks available to us (usually 2 or 10 mM), we can limit the cells' exposure to 0.5% DMSO [final]. In this experiment, that condition yielded an assay window of >2000 RFUs and a Z′ statistic of 0.73.
Dye Loading Time
The effect of progressively longer dye loading times on assay window size, standard deviation and resulting Z′ statistic was examined (FIG. 23).
FIG. 23: Effect of dye loading time on assay statistics. Filtered full-plate statistics showing the effect of dye loading times between 0.5 and 3 hours on window size, standard deviation and Z′ statistics. ShaI/KChIP18-13 cells were plated at 7500 cells/well in 384-well assay plates and tested the following day. Culture medium was removed by flicking the plates and cells loaded with 20 ul 1× membrane potential dye per well for 0.5-3 hours at 25° C. A two-addition protocol was used. The first addition (5×) was 5 ul 2.5% DMSO in assay buffer (0.5% [final]) read for 180 sec, and the second (2×) was 25 ul 120 mM isometrically-substituted KCl in assay buffer (60 mM [final]) read for an additional 180 sec. Statistics were calculated using the average of reads 190-200 (minimum response) and the maximum of reads 260-end (maximum response). Subtract bias was set at 1 and negative control correction was OFF, green bars (stat2-stat1) show response to activation, and the blue line shows the trend in Z′ statistics. Error bars are +/−1 SD. The data were minimally corrected for systematic artifacts.
Results and conclusions: Dye loading times between 0.5 and 3 hours all produced Z′ statistics above 0.7. There was an assay window maximum at 1.5 hours and a Z′ maximum at 2-2.5 hours. A visual inspection of the cells at three hours showed them to be in good condition. It was concluded that all dye loading times from 0.5-3 hours result in assays that perform well.
Cell Temperature for Dye Loading
A comparison was made regarding dye-loading temperature between cell plates allowed to cool at room temperature (25° C.) for one hour and those plates dye-loaded directly from 37° C. incubation (FIG. 24).
FIG. 24: The effects of pre-cooling cells to room temperature prior to dye-loading. ShaI/KChIP (ShIP), ShaI and Neo (ShaI control) cell lines were plated at 5×104 cells/well into 96-well assay plates (TC-treated, BD Biosciences) and incubated overnight at 37° C./5% CO2 for testing the following day. Culture medium was removed and replaced with 50 ul 1× blue membrane potential dye in assay buffer and incubated for 0.5 hour at 25° C. The assay was read on a FLIPR Tetra by recording baseline fluorescence for 20 sec, and recording an additional 60 sec after addition of 50 ul isometrically-substituted 120 mM KCl (60 mM [final]). For the data export, subtract bias was set at 1 and negative control correction was ON. Error bars are +/−1 SD. Results and conclusions: There was a statistically negligible difference between cool and warm cells for ShaI/KChIP, no difference for ShaI, and a significant reduction in response of the pre-cooled Neo control line. It was decided that subsequent development would be done by dye-loading assay plates that had been equilibrated at room temperature for one hour as the reduction of background response (as seen in the control line) was a desirable outcome. The requirement for pre-cooling assay plates places only a minor burden on the HTS protocol.
Activator EC50s
A dose response was established for ShaI/KChIP 18-13 by isometric KCl activation (FIG. 25).
FIG. 25: Group-averaged primary FLIPR data and preliminary EC50. ShaI/KChIP18-13 cells were plated at 7500 cells/well in 384-well assay plates and tested the following day. Culture medium was removed by flicking the plates and cells loaded with 20 ul 1× membrane potential dye per well for 0.5 hours at 25° C. A two-addition protocol was used. The first addition (5×) was 5 ul 2.5% DMSO in assay buffer (0.5% [final]) read for 180 sec, and the second (2×) was 25 ul 1 isometrically-substituted KCl at dose in assay buffer (0-60 mM [final]) read for an additional 180 sec. Statistics were calculated using the average of reads 180-200 and the maximum of reads 260-maximum allowed. Subtract bias was set at 1 and negative control correction was OFF. Top panel: ScreenWorks screenshot of reduced FLIPR data showing group average responses to KCl dosing. Bottom panel: Nonlinear regression/sigmoidal dose response showing calculated Hillslope and EC50.
Results and conclusions: The ShaI/KChIP cell line responded progressively and with low variability to activation by increasing levels of isometrically-substituted KCl. The highest achievable concentration of KCl using this method is 60 mM [final]. The EC50, using a fixed top response, was calculated to be 30 mM KCl in this experiment.
MDC vs Axygen FLIPR 384 Tips
A comparison was made between FLIPR 384 tips supplied by Molecular Devices Corporation (MDC) and those supplied by Axygen Scientific (FIG. 26). Note: Axygen is a former supplier of FLIPR 384 tips to MDC. The cost to us in RTP for Axygen tips is 2⁄3 that of MDC tips.
FIG. 26: Comparison of MDC and Axygen FLIPR 384 tips. GraphPad Prism scatter plots showing maximum and minimum responses with resultant statistics of the ShaI/KChIP assay. ShaI/KChIP18-13 cells were plated at 7500 cells/well in 384-well assay plates and tested the following day. Culture medium was removed by flicking the plates and cells loaded with 20 ul 1× membrane potential dye per well for 0.5 hours at 25° C. A two-addition protocol was used. The first addition (5×) was 5 ul 2.5% DMSO in assay buffer (0.5% [final]) read for 180 sec, and the second (2×) was 25 ul 120 mM isometrically-substituted KCl in assay buffer (60 mM [final]) read for an additional 180 sec. Statistics were exported using the average of reads 180-200 and the maximum of reads 260-maximum allowed. Subtract bias was set at 1 and negative control correction was OFF. Left panel: Molecular Devices FLIPR 384 tips. Right panel: Axygen FLIPR 384 tips.
Results and conclusions: The performance of the ShaI/KChIP assay was identical using either MDC or Axygen tips. As the cost savings over the course of assay development and HTS was calculated to be considerable, all subsequent protocols used Axygen Scientific FLIPR 384 tips.
Stability of Assay Buffer and Other Reagents
An assessment was made of the stability of the reagents used in the ShaI/KChIP assay, namely, the first addition assay buffer/DMSO, reconstituted dye and activation buffer (FIG. 27).
FIG. 27: Reagent stability. ShaI/KChIP 18-13 cells were plated at 7500 cells/well in 384-well assay plates and tested the following day. Culture medium was removed by flicking the plates and cells loaded with 20 ul 1× membrane potential dye per well for 0.5 hours at 25° C. A two-addition protocol was used. The first addition (5×) was 5 ul 2.5% DMSO in assay buffer (0.5% [final]) read for 180 sec, and the second (2×) was 25 ul 120 mM isometrically-substituted KCl in assay buffer (60 mM [final]) read for an additional 180 sec. Statistics were exported using the average of reads 180-200 and the maximum of reads 260-maximum allowed. Subtract bias was set at 1 and negative control correction was OFF. All reagents for the ShaI/KChIP assay were prepared the day prior (with the exception of the dye), early in the workday and freshly for each experiment. The assay was run at time points 0, 4 and 8 hrs and overnight (O/N) with both previously- and freshly-prepared reagents.
Results and conclusions: The performance of the ShaI/KChIP assay exhibited only minor fluctuations during the course of the day. It was concluded that the reagents were stable for at least one full day. Subsequently, it was found that the assay and activation buffers were stable over the course of at least one week (data not shown).
FLIPR Tetra Pipetting Optimization
A number of experiments (data not shown) were carried out to examine the effect of changes in pipetting heights, speeds, and hold and expel volumes on assay statistics (Table 2).
Results and conclusions: The following table shows the settings that consistently gave the best results, as measured by Z′ statistics:







TABLE 2







Pipetting optimization. First and second addition pipetting tip heights,


speeds, and expel volumes that consistently resulted in Z′ statistics of 0.5 or better.



















initial vol
1st add
vol
height
speed
tip up
expel
2nd add
vol
height
speed
tip up
expel





20 ul
5×
5 ul
15
20
6
0
2×
25 ul
20
25
6
0









The parameters were established using the basic test protocol. Note: No hold volumes were used during aspiration and no pauses or mixing applied to dispensing.
3-Day Minimum/Midpoint/Maximum Response
ShaI/KChIP 20 cells were examined for stability of response three times over the course of five days to assess the variability that could be expected during a screen (FIG. 28).
FIG. 28: Three-day minimum, midpoint and maximum statistics. GraphPad Prism scatterplots showing duplicate half-plate assay data for three days with resulting window, CV and Z′ statistics. ShaI/KChIP 20 cells were plated at 7500 cells/well in 384-well assay plates and tested the following day. Culture medium was removed by flicking the plates and the cells loaded with 20 ul 1× membrane potential dye per well for 0.5+ hours at 25° C. A two-addition protocol was used. The first addition (5×) was 5 ul 2.5% DMSO in assay buffer (0.5% [final]) read for 180 sec, and the second (2×) was 25 ul 60 mM (left-half of plate) or 120 mM (right-half of plate) isometrically-substituted KCl in assay buffer (30 and 60 mM [final], respectively) read for an additional 180 sec. Statistics were exported using the average of reads 180-200 and the maximum of reads 260-maximum allowed. Subtract bias was set at 1 and negative control correction was OFF. Statistics were compiled as follows: Min-left half of n001 stat1 & right half of n002 stat1; Mid-left half n001 stat2 & left half n002 stat2; Max-right half n001 stat2 & right half n002 stat2. The data were minimally corrected for systematic artifacts
Results and conclusions: This assay performed well over the course of five days with a maximum Z′ statistic range of 0.73 to 0.80, a mean of 0.77, and a standard deviation of 0.03. Note: Three-day single full-plate (three plates/day) experiments were also conducted (data not shown) with good results. The assay performed well over the course of six days with a maximum Z′ statistic range of 0.69 to 0.74, a mean of 0.72, and a standard deviation of 0.03. Taken together, this assay could be expected to perform well over the course of a screening campaign.
3-Day Activator Dose Response Curves & EC50s
The stability of response of ShaI/KChIP 20 to isometrically-substituted KCl dosing was measured three times across six days (FIG. 29).
FIG. 29: Three-day KCl dose response curves and EC50s. ShaI/KChIP 20 cells were plated at 7500 cells/well in 384-well assay plates and tested the following day. Culture medium was removed by flicking the plates and the cells loaded with 20 ul 1× membrane potential dye per well for 0.5 hours at 25° C. A two-addition protocol was used. The first addition (5×) was 5 ul 2.5% DMSO in assay buffer (0.5% [final]) read for 180 sec, and the second addition was 25 ul 2× isometrically-substituted KCl at dose in assay buffer (0-60 mM [final]) read for an additional 180 sec. Statistics were exported using the average of reads 180-200 and the maximum of reads 260-maximum allowed. Subtract bias was set at 1 and negative control correction was OFF. Analysis is nonlinear regression, sigmoidal dose-response, top constant. Data are from single plates with 48 replicates for each dose.
Results and conclusions: The assay responded reasonably consistently to KCl dosing over three days with an EC50 range of 32 to 42 mM KCl, a mean of 36 mM, and a standard deviation of 5 mM.
3-Day Antagonist Dose Response Curves & IC50s
The presumptive ShaI/KChIP antagonist amiloride, identified during BIOMOL compound screening at 25 uM, was used to generate duplicate dose responses over three consecutive days (FIG. 30).
FIG. 30: Three-day amiloride dose responses. ShaI/KChIP 20 cells were plated at 7500 cells/well in 384-well assay plates and tested the following day. Culture medium was removed by flicking the plates and the cells loaded with 20 ul 1× membrane potential dye per well for 0.5 hours at 25° C. A two-addition protocol was used. The first addition (5×) was 5 ul 2.5% DMSO in assay buffer (0.5% [final]) read for 180 sec, and the second addition was 25 ul 2× isometrically-substituted KCl at dose in assay buffer (0-60 mM [final]) read for an additional 180 sec. Statistics were exported using the average of reads 180-200 and the maximum of reads 260-maximum allowed. Subtract bias was set at 1 and negative control correction was OFF. Analysis is nonlinear regression, sigmoidal dose-response. The data were fixed to an extrapolated point at 55 mM KCl to compensate for first addition artifacts seen as inhibition approached 100%. Data are from duplicate plates each day with 48 replicates/plate for each dose. Results and conclusions: The assay responded consistently to inhibition by amiloride over three consecutive days with an IC50 range of 20 to 29 uM amiloride, a mean of 24 uM, and a standard deviation of 3 uM. It was concluded that this compound could reasonably be expected to perform well as an inhibition control for HTS.
Direct-to-Plate Assay
Experiments were undertaken to determine the feasibility of plating cells directly from liquid nitrogen storage to assay plates without any intervening cell culture (FIG. 31).
FIG. 31: Direct-to-plate assay. Frozen ShaI/KChIP 20 cells (2 ml×106/ml) were thawed, added to 82 ml complete culture medium, and plated in a 50 ul volume (1.2×104 cells/well) in 384-well plates for testing the following day. Culture medium was removed by flicking the plates and the cells loaded with 20 ul 1× membrane potential dye per well for 0.5 hours at 25° C. A two-addition protocol was used. The first addition (5×) was 5 ul 2.5% DMSO in assay buffer (0.5% [final]) read for 180 sec, and the second addition was 25 ul 60 mM or 120 mM isometrically-substituted KCl in assay buffer (30 mM or 60 mM [final], respectively) read for an additional 180 sec. Statistics were exported using the average of reads 180-200 and the maximum of reads 260-maximum allowed. Subtract bias was set at 1 and negative control correction was OFF.
This strategy is widely-employed throughout the lead discovery sector and often results in decreased assay variability, significant time savings, and reduced labor costs.
Results and conclusions: The ShaI/KChIP assay responded well when plated directly from liquid nitrogen storage. The Z′ achieved from the maximum response was 0.73. While this strategy will not be employed during the current campaign, it will be considered during future screen developments.
BIOMOL Compound Screening
An Ion Channel Ligand Library (BIOMOL #2805), comprising 71 activators and inhibitors covering the major ion channel types, was twice measured against ShaI/KChIP 20 activity at 25 uM in 384-well four-pont mode (FIGS. 32 & 33).
FIG. 32: BIOMOL compounds—primary screening data. ScreenWorks screenshots showing primary FLIPR data for BIOMOL compound screening. ShaI/KChIP 20 cells were plated at 7500 cells/well in 384-well assay plates and screened the following day. Culture medium was removed by flicking the plates and the cells were loaded with 20 ul 1× membrane potential dye per well for 0.5 hours at 25° C. A two-addition protocol was used. The first addition was 5 ul 5× control or compound in 2.5% DMSO in assay buffer (0.5% [final]) read for 180 sec, and the second addition was 25 ul 2× control or 120 mM isometrically-substituted KCl in assay buffer (0-60 mM [final]) read for an additional 180 sec. Statistics were exported using the average of reads 180-200 and the maximum of reads 260-maximum allowed. Subtract bias was set at 1 and negative control correction was OFF. Control columns 1⁄2 and 23/24 are described starting on pg. 53 of this document. Inset: Screenshot of group averages for the control and compound wells highlighted in the primary data screen.
FIG. 33: BIOMOL compounds—reduced screening data. Assay plate views showing percent inhibition of ShaI/KChIP response by BIOMOL compounds. Experimental conditions are described in the previous figure legend.
Results and conclusions: Using an arbitrary cutoff of 55% inhibition, three compounds showed reproducible inhibitory activity against the ShaI/KChIP response. Amiloride (B8) is a known calcium channel blocker. Both NS-1619 (E7) and flufenamic acid (F6) are known to stimulate KCa2+ channel activities. It was unlikely that these compounds would prove to be direct inhibitors of ShaI, and patch clamp measurements bore this out (data not shown). For screening purposes, amiloride proved to be consistent in its action, and so was used for subsequent BIOMOL and BioFocus validation screening, and is recommended for HTS.
BioFocus Compound Screening
FIG. 34: Compound preparation protocol for BioFocus screening. Compound preparation was carried out on the FLIPR Tetra. Library plates at 2 mM were diluted 40× in assay buffer and dispensed into quadrants for 384-well screening.
The 3222 unique members of the BioFocus SoftFocus Ion Channel Libraries #1-4 (#SF101-04) were screened in 384-well four-point mode against ShaI/KChIP at 10 uM in a combined activator and antagonist screen. The same FLIPR protocol was used as for the BIOMOL screening and was carried out over four non-consecutive days. The following compound preparation protocol was used to prepare screening plates: Briefly, ShaI-KChIP cells were seeded in 384-well plates at 7500 cells/well in 50 ul and incubated overnight at 37° C./5% CO2. The next day, culture medium was removed, 20 ul of 1×FMP dye was added, and the cell plate was incubated at 27° C. for 30 minutes. After incubation, the cell plate was placed into the Tetra and a two-addition protocol was used. The 1st addition was 5 μl of 5× compound (50 μM) with a three minute read. If the compound showed a response in the 1st addition, it was flagged as an activator. The 2nd addition was 25 ul of 2× activation buffer (120 mM isometric KCl substitution for NaCl) and read for an additional two minutes. A depression of the KCl response indicated the compound was an antagonist. All data were exported as statistics files. For the 1st addition, the average from reads 190-200 was used and for the 2nd addition, the maximum from 260—maximum allowed was used.
The activation and inhibition percentages were calculated by the following formulas and all results were calculated based on controls:

% Activation (1st addition statistic)=(test sample−μminA)/(μmaxA−μminA)×100 where μmaxA=mean 100% activation, and μminA=mean 0% activation.

% Inhibition (2nd addition statistic)=(μminI−test sample)/(μminI−μmaxI)×100 where μmaxI=mean 100% inhibition, and μminI=mean 0% inhibition.

The Z′ statistics for activation and inhibition were calculated using the following formula:

Z′=1−((3σmax+3σmin)/(Iμmax−σminI))

BioFocus SoftFocus® Ion Channel Focused Libraries #1-4: 1° Screening Activator Hits
The percent activity relative to control is plotted as a histogram for the BioFocus ion channel library activator screen (FIG. 35).
FIG. 35: ShaI/KChIP FLIPR BioFocus 1° screening—activation. Distribution of actives on ShaI/KChIP.
The activity was based on the mean and standard deviation of the compound set (minus controls and outliers, such as fluorescent compounds). Because the mean and standard deviation were so low, we chose to use a 6 σ cut-off of 12% activation to select compounds for follow-up. As shown in Table 3, there were no compounds that showed activation over the 6 σ cut-off of 12%.







TABLE 3







Summary of Shal/KChIP activation. Summary table showing


results from Shal/KChIP activator screening.











Shal-KChIP




Activation













Total compounds
3222



Mean of Data set
−0.3



SD of Data set
1.98



6-Sigma
  12%



Total Actives
0



% Hit Rate
0.00%









BioFocus SoftFocus® Ion Channel Focused Libraries #1-4: 1° Screening Antagonist Hits
The percent activity relative to control is plotted as a histogram for the BioFocus ion channel library antagonist screen. The activity was calculated based on the mean and standard deviation of the compound set (minus controls and outliers, such as fluorescent compounds). The distribution of the antagonist activity shows a normal distribution; however, it is centered at 10-15% inhibition, which we have seen previously with this compound set. We chose to use a 3 σ cut-off of 29% inhibition to select the compounds for follow-up (Table 4). Because the compounds were tested in quadruplicate in the FLIPR screen and did not show strong inhibition at 10 μM, and because the amount of compound available is limiting, IC50s will not be performed on the FLIPR. All follow-up for antagonist hits will be performed on the QPatch. Table I details the antagonist actives with inhibition >/=29%.







TABLE 4







Summary of Shal/KChIP inhibition. Summary table showing


results from Shal/KChIP inhibitor screening.









Shal-KChIP



Antagonist













Total compounds
3222



Mean of Data set
5.9



SD of Data set
825



3-Sigma
  29%



Total Actives
53



% Hit Rate
1.60%









Molecular Validation of ShaI/KChIP Cell Line
Experiments were undertaken to assess the integrity of the ShaI and KChIP coding sequences incorporated into the ShaI/KChIP stable cell line (Table A1). The strategy employed was to isolate genomic DNA (gDNA) from ShaI/KChIP 20 and control cell lines and to use these DNAs as templates for informative PCRs (polymerase chain reactions). Amplification primers were selected to generate products across the 5′ and 3′ ends of the ShaI and KChIP coding regions. Additional primers were chosen to verify the overall length of each coding region.







TABLE A1





Overview of primer pairs and PCR parameters used in molecular validation of Shal/KChIP cell line.
















Shal/KChIP: molecular validation














5′ primer
Tm
3′ primer
Tm
expected size





Shal



















1
Shal 5′ end
T7
48
Inside GFP +40 Reverse
52
180 
bp


2
Shal 3′ end
Shal 5′2
54
BGH rev
50
1.1 
kb


3
Shal full length
AcGFP_ATG_F
66
Shal_1451R
57
2.1 
kb












KChIP



















4
KChIP 5′ end
T7
48
KChIP-r
62
1.4 
kb


5
KChIP 3′ end
KCHiP-INTRON-2F
70
BGH rev
50
260 
bp













6
KChIP full length
KChIP-f
66
KChIP-r
62
1.3













Shal/KChIP gDNA
635 ng/ul
300 ulw/1.4 ug gDNA
2.2 ul





PCR
DNA/H2O
23 ul
(100 ng gDNA)



GoTaq 2×
25 ul




Primer1- 5′, 25 uM
 1 ul




Primer2- 3′, 25 uM
 1 ul





50 ul












Cycling parameters
Ends
Full-length














initial denature
94
2′
94
2′


denature
94
30′′
94
30′′


touchdown anneal
10× 65, 55; 20× 45
30′′
10× 60, 55; 20× 50
30′′


extension
72
30′′
72
3′


final extension
72
5′
72
5′


hold
4
end
4
end





PCR products were generated across the 5′ and 3′ ends as well as the full-length coding sequences of Shal and KChIP. Primer names, target regions, Tms (annealing temperatures) and expected fragment sizes are indicated. The Shal/KChIP 20 gDNA prep is described, as is the 50 ul reaction setup. The cycling parameters include a modified “touchdown” protocol to reduce the generation of non-specific products, and were designed to accommodate differing predicted optimal annealing temperatures.






Results and conclusions: All reactions yielded single products of the correct predicted size (FIG. 35A) with the exception of reaction #3 which produced a minor product of about 300 bp (FIG. 35A, lane 3). This product was also generated when amplifying control cell line gDNA (data not shown) and is presumed to be an artifact of the specific primer pair used. In conclusion, the coding sequences of ShaI and KChIP appear to be intact using this kind of molecular examination.
FIG. 35A: PCR products from the reactions described in Table A1. Each lane contains 20 ul of each 50 ul amplification reaction electrophoresed on a 1% agarose gel and stained with ethidium bromide. M1: BenchTop 1 kb DNA Ladder (Promega), M2: BenchTop PCR Markers (Promega), 1: ShaI 5′ end (180 bp), 2: ShaI 3′ end (1.1 kb), 3: ShaI full-length (2.1 kb), 4: KChIP 5′ end (1.4 kb), 5: KChIP 3′ end (260 bp), 6: KChIP full-length (1.3 kb).
The following examples are in connection with the shaker channel and/or a Hyperkinetic beta subunit:
Molecular Cloning and Vector Map
The pTriEx/Shaker plasmid we started with contains a Kozak sequence upstream 5′ additional N-terminal amino acids (MAISR). In order to clone the Shaker full-length cDNA into different expression vectors, the following strategy was performed:

    
    
        500 bp 5′-fragment amplification: two oligos have been designed for the amplification of the Shaker ATG codon together with a proper Kozak sequence without the additional 5′ Nterminal amino acids.
    
    











Oligo SH-UPP, 5′-CCGGTACCATGGCCGCCGTTGCC-3′



 



Oligo SH-LOW, 5′-CCGGTCTCCGTAGTCGGCCACC-3′






In bold KpnI restriction site; in bold, underlined the Kozak sequence, in underlined Shaker annealing sequence.
SH-LOW oligo is located downstream the unique SalI restriction site on Shaker sequence.
The PCR fragment has been cloned into pCR-Blunt vector and the sequence has been verified.
The pCR-Blunt/5′-PCR clone has been digested with KpnI (in SH-UPP oligo) and SalI (in Shaker sequence) and the 400 bp 5′-fragmet was purified.

    
    
        1770 bp 3′-fragment cloning: pTriEx/Shaker plasmid was digested with SalI and EcoR1 restriction enzymes and the 1770 bp fragment was purified.
        Shaker wt cloning into pExSelect and pIRES2EGFP expression vectors via pcDNA3.1(+) vector: the 400 bp KpnI-SalI 5′-fragment and the 1770 bp SalI-EcoRI 3′-fragment obtained as described above have been cloned into the pcDNA3.1(+) vector.
    
    


The NheI-EcoRI Shaker fragment of this construct has been cut and then cloned into the pExSelect and pIRES2EGFP previously digested with NheI and EcoRI. The VectorNTi map of the final pExSelect_Shaker construct is shown in FIG. 36.
Cell Culture Conditions and Transfection
CHO-K1 cells were maintained in Dulbecco's MEM/Nutrient Mix F12 (1:1) (DME-F12 Euroclone cat.#ECM0090L) supplemented with 1.6 mM Sodium Pyruvate, (100 mM solution, Euroclone cat.#ECM0542D), 13 mM Hepes (1M solution, Euroclone cat. #ECM0180D), 0.2% Sodium Bicarbonate (7.5% solution, Euroclone cat. #ECM0980D), 2 mM Ultraglutamine (BioWhittaker cat. #BE17-605E/U1), 10% FBS (Fetal Bovine Serum, Euroclone cat. #ECS0180L), and 1% Penicillin/Streptomycin (100× solution Euroclone cat. #B3001 D).
Propagation conditions consist of seeding about 6×105 cells/T75 flask twice a week.
Recovering
about 10-13×106 cells/T75 flask.
Transfection was performed by electroporating 1.0×106 cells in presence of 10 μg of DNA at 300 mV and 950 μF. Cells were then selected with medium containing 2 mg/ml G418 or 1 mg/ml Zeocin for 10-15 days. After antibiotic selection, resistant clones were maintained in 1 mg/ml G418 (Calbiochem cat. #345812) or 0.5 mg/ml Zeocin (InvivoGen cat. # ant-zn-5) medium.
All the cell lines are plated in complete medium without antibiotics for the FLIPR experiments.
Cellular Membrane Voltage Measurement by FLIPR
In order to detect the membrane depolarization elicited by KCl injection, the Membrane Potential sensitive dye was used both for FLIPR384 and FLIPRTETRA experiments. Cells were analyzed in 384 clear-bottom black MPTs (MATRIX cat. #4324) by seeding 5000, 7500, 10000 cells/well 24 hrs before experiment. Plates were incubated with 40 μl/w of 0.625× membrane potential dye for 45-60 minutes at 37° C. or room temperature and measured at FLIPR instrument by injecting 10 μl/w of 5× antagonist (in the presence of 0.5% DMSO, as indicated) followed by 3-5 minutes fluorescence reading; then a second injection of 25 μl/w of 3× KCl in Standard Tyrode solution) was performed and fluorescence measured for further 3-4 minutes. In experiments with a single injection (agonist), plates were incubated with 20 μl/w of 1× dye and injected with 20 μl/w of 2× KCl in Standard Tyrode solution.
Kinetic data obtained from different well replicates were analyzed with FLIPR, Excel and Spotfire software, by calculating the Integral or Maximum-Minimum RFU values after the injection; the obtained means and standard deviations were utilized to create sigmoidal dose-response fits by GraphPad PRISM® or Spotfire software and to calculate EC50-IC50 values and Z′ factors.
EC80 value was calculated according to the following formula:

ECx=(x/100−x)1/Hill Slope*EC50

For the calculation of the Z′ factor the following formula was used:




  
    Z
    ′
  
  =
  
    1
    -
    
      
        3
        *
        
          (
          
            
              
                ST
                .
                DEV
              
              ⁢
              
                
              
              ⁢
              agonist
            
            +
            
              
                ST
                .
                DEV
              
              ⁢
              
                
              
              ⁢
              Tyrode
            
          
        
      
      
        
          MEAN
          ⁢
          
            
          
          ⁢
          agonist
        
        -
        
          MEAN
          ⁢
          
            
          
          ⁢
          Tyrode
        
      
    
  




Current detection by Patch clamp
Electrophysiological Recordings
Standard whole-cell voltage-clamp experiments were performed at room temperature. For data acquisition and further analysis, we used the EPC10 digitally controlled amplifier in combination with PATCHMASTER software (HEKA Electronics, Lambrect, Germany). The EPC10 provides automatic subtraction of capacitance and leakage currents by mean of prepulse. The data were filtered at 66.7 KHz (−3 dB, 8-pole Bessel lowpass) and digitized at 5 μs per point. The input resistance of the patch pipettes was 2.0-4.0 MΩ and the capacitances of the cells were 15.3±2.1 pF (n=45); the residual series resistances (after up to 80% compensation) were 4.2±0.4 MΩ. Correction for liquid junction potential was routinely applied. Membrane potential was clamped at −100 mV and currents were elicited by 50 ms depolarization pulses (0.1 Hz) from −60 mV to +100 mV (or +60 mV).
Cell Culture
For electrophysiology experiments, CHO—K1/DmShaker cells have been treated and maintained in culture with the standard protocol described above. 24 hrs (or four to six hrs in the second limiting dilution tests) before experiments CHO—K1/DmShaker cells were seeded onto poly-D-lysine coated glasses (200000 cells each) and placed in six well plates in antibiotic free medium. Immediately before experiments coated glasses, with seeded cells, have been washed five times with patch clamp extracellular solution and then put into the recording chamber.
Solutions
The pipette solution contained (mM): KMeSO3 128, HEPES 10, EGTA 12, MgCl2 3, CaCl2 0.7, K2ATP 5, pH 7.2 with KOH.
The bath solution contained (mM): NaCl 145, KCl 5, MgCl2 1, CaCl2 2, HEPES 10, Glucose 10, pH 7.4 with NaOH.
Ligand Storage

    
    
        DMSO (Dimethyl sulfoxide SIGMA cat. #D-5879) was purchased from SIGMA®.
        KCl was purchased from SIGMA® and stock solution was prepared 3 M in water.
        TEA was purchased from SIGMA® and stock solution was prepared 1M in water.
    
    


Working solutions are prepared in the Standard Tyrode buffer (pH 7.4):
NaCl 130 mM, KCl 5 mM, CaCl2-2H2O 2 mM, MgCl2 1 mM, NaHCO3 5 mM, HEPES 20 mM.
Software
Data were analyzed using Excel, GraphPad Prism 4, FLIPR384 Control Software, ScreenWorks 1.2.0.
Stable Cell Line Generation
CHO—K1 cells have been stably transfected with pExSelect_Shaker or pExSelect vector alone (as mock control). 48 hrs after transfection cells have been cultured in complete medium supplemented with 2 mg/ml G418 to select a resistant pool. Antibiotic resistant DmShaker transfected cells (not FACSorted) have been further transfected with the H-kvβ subunit A or C subtype vectors received from BASF and 48 hrs later cells put on selection with 1 mg/ml Zeocin.
First Limiting Dilution Clone Selection
In order to obtain a pure Shaker and H-kvβ clone, a first limiting dilution step has been performed by diluting stable mock or Shaker transfected pools in 96 wp at a cell density of 1 cell/well (5×96 wp mock and 5×96 wp Shaker plus H-kvβ for each subunit). Confluent clones were replicated into clear bottom 384 wp and analyzed after 24 hrs at FLIPR384 by injecting 100 mM KCl. As shown in FIG. 37 the mock clones showed an hyperpolarization upon KCl injection while some of the Shaker and Hkvβ both A and C clones displayed a KCl response recorded as a sustained depolarization, as shown in FIG. 38.
FIG. 37. Mock I limiting dilution plate selection at FLIPR384: example of one plate
FIG. 38. Shaker+Hkvβ A or C subunit I limiting dilution clone selction at FLIPR384 Data were analyzed using the total integral RFU values after activator injection. On the basis of this data analysis, two mock clones and six clones each from both Shaker plus A or C subtype have been selected for re-test in a counted cells experiment: 10000 c/w have been plated and 24 hrs later a KCl dose-response at FLIPR384 has been performed. The resulting best clones are shown in FIG. 39.
FIG. 39. I limiting dilution clone re-test as counted cells at FLIPR384 
Protocol: 10000 c/w-24 h; MEM discarded; 45′ incubation at 37° C. with 40 μl of blue MP dye according to the described procedure; 20 μl/w KCl injection at FLIPR384 (3× Tyrode solution).
Clone Stability at Different Passages in Culture and After Freezing/Thawing
A-3A10 has been selected as the best responding clone and maintained in culture for more than two months. A-3A10 has been analyzed at FLIPR384 for KCl response stability at different cell passages and after freezing/thawing as shown in FIGS. 40 and 41
FIG. 40. A-3A10 signal stability at different cell passages: 10000 c/w-24 h; MEM discarded; 45′ incubation at 37° C. with 40 μl of blue MP dye according the described procedure; 10 μl/w 0.5% DMSO injection as 5× tyrode solutions; 3′ later, 25 μl/w KCl injection at FLIPR384 (3× tyrode solution) 1.5, 3.7, 7.5, 15, 30, 60, 90, 120 mM
FIG. 41. A-3A10 signal stability after freezing and thawing: 10000 c/w-24 h; MEM discarded; 45′ incubation. at 37° C. with 40 μl of blue MP dye according to the described procedure; 10 μl/w 0.5% DMSO injection 5× Tyrode solutions; 3′ later, 25 μl/w KCl injection at FLIPR384 (3× Tyrode solutions) 1.5, 3.7, 7.5, 15, 30, 60, 90, 120 mM
TEA Effect on A-3A10 Clone
TEA effect has been analyzed on A-3A10 clone by KCl stimulation upon 30, 60 or 90 mM TEA pre-injection by FLIPR 384. TEA blocking effect is visible in particular on 30 mM KCl stimulation as shown in FIG. 42.
FIG. 42. TEA effect on A-3A10 clone: 10000 c/w-24 h; MEM discarded; 45′ incubation at 37° C. with 40 μl of blue MP dye according the described procedure; 10 μl/w 0.5% DMSO inj. in the presence of 30, 60 or 90 mM TEA 5× Tyrode solutions; 3′ later, 25 μl/w KCl injection at FLIPR384 (3× tyrode solutions) 1.5, 3.7, 7.5, 15, 30, 60, 90, 120 mM
KCl Hypertonic or Isotonic Solution Analysis
Both KCl hypertonic or isotonic solutions have been analyzed on A-3A10 clone. Hypertonic solutions have been prepared starting from standard Tyrode buffer (135 mM NaCl+KCl) by adding the required volume of KCl stock solution (standard protocol). Isotonic solutions have been prepared starting from “Tyrode base” (0 M NaCl+KCl) and keeping NaCl+KCl salts concentration at 135 mM. No differences have been observed in the response of the tested clone both as RFU and as kinetic shape, when using the two solutions as shown in FIG. 43. So all validation experiments were performed in standard Tyrode.
FIG. 43. Hypertonic and isotonic solutions analysis: 10000 c/w-24 h; MEM discarded; 45′ inc. at 37° C. with 40 μl of blue MP dye according the described procedure; 10 μl/w 0.5% DMSO inj. 5× tyrode solutions; 3′ after, 25 μl/w KCl inj at FLIPR384 (3× tyrode solutions) 1.5, 3.7, 7.5, 15, 30, 60, 90, 120 mM
Second Limiting Dilution Clone Selection
The Shaker/H-kvβ A subunit best clone, A-3A10, has been put in second limiting dilution in 3×96 wp at a cell density of 1 cell/well. 30 clones have been picked-up and grown for testing. Ten clones have been tested first as counted cells using the standard loading and experimental protocols. KCl response of the best clones is shown in FIG. 44.
FIG. 44. A-3A10 II limiting dilution clone selection at FLIPR384
Example of best clones: 10000 c/w-24 h; MEM discarded; 45′ incubation at 37° C. with 40 μl of blue MP dye according the described procedure; 10 μl/w 0.5% DMSO injection 5× Tyrode solutions; 3′ later, 25 μl/w KCl inj at FLIPR384 (3× Tyrode solutions) 1.5, 3.7, 7.5, 15, 30, 60, 90, 120 mM
Two clones, n° 1 and n° 9, have been selected as the best responsive clones for further analysis at FLIPRTETRA. 10000, 7500, 5000 and 2500 c/w have been plated 24 hrs before experiment and KCl dose-response analysis has been performed. Data obtained are shown in FIG. 45
FIG. 45. n° 1 and n° 9 II limiting dilution clones analyzed at FLIPRTETRA: 10000 c/w-24 h; MEM discarded; 45′ incubation at 37° C. with 40 μl of blue MP dye according the described procedure; 10 μl/w 0.5% DMSO injection 5× Tyrode solutions; 3′ later, 25 μl/w KCl injection at FLIPRTETRA (3× Tyrode solutions) 1.5, 3.7, 7.5, 15, 30, 60, 90, 120 mM
Final Clone Optimization
The Shaker n° 1 clone has been chosen for further characterization and final clone optimization.
Cell Density Dependency
Shaker n° 1 clone has been seeded 10000, 7500 and 5000 c/w 24 h before experiment in order to determine the optimal cell density. The day of experiment a KCl dose-response has been injected at FLIPRTETRA and the EC50 has been calculated. The results are shown in FIG. 46.
FIG. 46. Cell density dependency, KCl dose-response: 10000, 7500, 5000 c/w-24 h; MEM discarded; 45′ incubation at RT with 40 μl of blue MP dye according the described procedure; 10 μl/w Tyrode injection; 3′ later, 25 μl/w KCl injection at FLIPRTETRA (3× Tyrode solutions) 1.5, 3.7, 7.5, 15, 30, 60, 90, 120 mM.
To draw the curve the Max-Min value after KCl injection has been considered.
DMSO Sensitivity
In order to determine if DMSO causes any effect on the activator response, clone n° 1 has been plated at the cell density of 10000, 7500 or 5000 c/w and analyzed at FLIPRTETRA 24 h later, by injecting KCl dose-response, after a instrument pre-injection of 0.5-1-1.5-3% DMSO.
As shown in FIG. 47-48-49 DMSO concentrations up to 1.5% have no significant effect on the KCl response; 3% DMSO gives rise to a decrease in KCl dependent response.
FIG. 47 DMSO sensitivity: 10000 c/w-24 h; MEM discarded; 45′ incubation at RT with 40 μl of blue MP dye according the described procedure; 10 μl/w 0.5, 1, 1.5, 3% DMSO injection 5× Tyrode solutions; 3′ after, 25 μl/w KCl inj at FLIPRTETRA (3× tyrode solutions) 1.5, 3.7, 7.5, 15, 30, 60, 90, 120 mM.
To draw the curve the Max-Min value after KCl injection has been considered.
FIG. 48 DMSO sensitivity: 7500 c/w-24 h; MEM discarded; 45′ incubation at RT with 40 μl of blue MP dye according the described procedure; 10 μl/w 0.5, 1, 1.5, 3% DMSO injection 5× Tyrode solutions; 3′ later, 25 μl/w KCl inj at FLIPRTETRA (3× Tyrode solutions) 1.5, 3.7, 7.5, 15, 30, 60, 90, 120 mM.
To draw the curve the Max-Min value after KCl injection has been considered.
FIG. 49 DMSO sensitivity: 5000 c/w-24 h; MEM discarded; 45′ incubation at RT with 40 μl of blue MP dye according the described procedure; 10 μl/w 0.5, 1, 1.5, 3% DMSO injection 5× Tyrode solutions; 3′ later, 25 μl/w KCl injection at FLPIRTETRA (3× Tyrode solutions) 1.5, 3.7, 7.5, 15, 30, 60, 90, 120 mM.
To draw the curve the Max-Min value after KCl injection has been considered.
Clone Stability After Freezing/Thawing
Clone n° 1 has been analyzed after culturing (p 6) and freezing/thawing: cells have been plated at the cell density of 10000, 7500 or 5000 c/w and analyzed at FLIPRTETRA after 24 h by injecting KCl dose-response in the presence of 0.5% DMSO.
The results are shown in FIG. 50.
FIG. 50 Clone stability in culture and after freezing/thawing: 10000, 7500, 5000 c/w-24 h; MEM discarded; 45′ incubation at RT with 40 μl of blue MP dye according the described procedure; 10 μl/w 0.5% DMSO injection 5× Tyrode solutions; 3′ later, 25 μl/w KCl inj at FLPIRTETRA (3× Tyrode solutions) 1.5, 3.7, 7.5, 15, 30, 60, 90, 120 mM.
To draw the curve the Max-Min value after KCl injection has been considered.
EC50 Stability on Three Separate Days
In order to verify the KCl response stability throughout different days of experiment, the final clone has been tested at FLIPR384 in three separate days by injecting a KCl dose-response. As shown in FIG. 51.
FIG. 51. EC50 reproducibility in three different days
Protocol: 10000 c/w-24 h; MEM discarded; 30′ incubation at 37° C. with 40 μl of blue MP dye according the described procedure; 10 μl/w Tyrode 0.5% DMSO injection at FLIPR384; read for 2′; 25 μl/w KCl injection at FLIPR384 (3× Tyrode solutions). Values in the graph refer to the Max-Min after KCl injection.
Patch Clamp Analysis
The two best clones coming from the first limiting dilution were tested through whole cell patch clamp technique. As shown (FIGS. 52a and b) the application of depolarization pulses to up +100 mV induced a Dm-Shaker current with a typical fast activation and fast inactivation profile. In some experiments, a TTX-sensitive inward current was present as shown in FIG. 52b. 
For each clone six experiments were performed and very promising results were obtained: 100% of the cells of clone 3a10 showed a Dm-Shaker current profile with a mean current density (at +60 mV) of 56.3±36 pA/pF, whereas five cells showed the potassium current for the clone 4d1 with a mean current densities of 13.4±18 pA/pF
As the best electrophysiology results were obtained from the Dm-Shaker A-subtype clone, it was decided to put into limiting dilution only the cell transfected with the Shaker channel plus the H-kvp A subunit. In the meanwhile the activity of the 3a10 clone was routinely checked every two weeks; moreover the clone expression stability after cells thawing was also checked.
In FIG. 53 we can see that pulses of depolarization up to +60 mV (b) or to +100 mV (a and c) induced the activation of Dm-Shaker. In particular the stability of the functional channel expression was still very high when cells were maintained in culture at least up to five-six weeks (FIG. 53a) and after cells thawing (FIG. 53c). In this experiments 83.3% and 100% of the cells respectively showed Dm-Shaker current (n=6 and 7).
After sub-culturing clone 3a10 for more than two months (passages 17-20), the expression of the channel was decreased and the cells showing Dm-Shaker current were 50% (n=8). In order to have kinetic informations about the voltage dependence of the activity of Dm-Shaker, the I-V relationship was plotted (FIG. 54). I-V plot was constructed by measuring peak currents and plotting the normalized values against membrane potential (n=8±SD).
Clone 1 coming from the second limiting dilution was then used for electrophysiology analysis and two compounds, were also tested on the selected clone. The two compounds affect the Dm-Shaker activity.
Screening Protocol
In the final assay adaptation for screening, several modifications of the protocols were done. The most substantial of these changes are:

    
    
        Implementation of an automated procedure for dye loading
        50 mM KCl: final concentration for screening (1:3 dilution of Activation buffer)
        Automated process
    
    


Where the activation buffer was prepared as follows:
150 mM KCl, 2 mM CaCl2*2H2O, 1 mM MgCl2, 5 mM NaHCO3, 20 mM HEPES, pH 7.4
The composition of the activation buffer represents a solution that is as close as possible to the physiological conditions in terms of osmolarity. That is the reason why it was decided to perform the screening adaptation and the full screening by using this buffer instead of standard tyrode.
Final FLIPR Protocol for Screening
The finalized FLIPR protocol has this setup:

    
    
        Source Plate 1: Compound dilution plate
        Source Plate 2: Activator plate
        Read Position: Assay plate
        Source Plate 3/Tips: not used (Tip box)
        Filters: Excitation: 510-545; Emission: 565-625
        Read settings (typical): Gain 60. Exp. Time 0.6; Excitation 60
        Wash Buffer A: water
        Wash Buffer B: water with 2.5% DMSO
    
    


The protocol (which is represented graphically above) includes these steps:

    
    
        Mix the compound dilution plate
        Transfer 10 μL from compound dilution plate to the assay plate
        Start read 3.0 minutes
        After 30 seconds, mix assay wells
        Wash tips (while reading)
        Transfer 25 μL from activator plate to the assay plate, mix immediately
        Read 4.0 minutes
        Wash tips (while reading)
    
    


The only substantial change in physical processing of the plate in the automated procedure versus the manual procedure is in the dye loading. In the manual process, this is accomplished by first dumping the medium out of the wells by “flicking” the liquid into a sink, and then aliquoting dye into the wells.
In the automated procedure, the dye loading is accomplished by the automated 384-well pipettor of our screening system. In order to remove the culture medium without disturbing the cell monolayer, we perform a cycle of aspiration/buffer dispense/aspiration in order to accomplish a “washing” of the well. In effect, our pipetting procedure resulted in a residual of 5% of the culture medium in the well with the dye. The final dye loading step involves removing the wash buffer, leaving 10 μL in the well, then adding 30 μL of 0.666× dye, for a final of 40 μL of 0.5× dye. We found that this resulted in an identical (or even better) performance of the assay as compared to manual emptying of the wells.
For the running of the automated process, an optimized schedule is compute to maximize plate throughput. An example of the optimized schedule for many plates is shown in FIG. 54. The throughput of this scheduled process is one plate each 13 minutes. This allows us to process 50-60 plates each day.
Overview of Automated Scheduled Process
The steps presented represent the processing of a single experiment. The scheduler system takes care of interlacing multiple assays in order to optimize throughout.
An overview of the automated scheduled process is depicted in FIG. 86.
The following examples are in connection with the G-protein coupled receptor:
Octopamine receptors can modulate their action through cyclic AMP production or intracellular calcium release, dependent on the receptor isoform. Although Oa2 endogenously signals though cAMP, we were able to force coupling to calcium via transfection of the receptor into a cell line expressing the promiscuous G-alpha-16-protein, which leads to calcium release upon its activation. The calcium release is measurable by fluorescent calcium sensing dyes (in this case Fluo-4). (FIG. 55A)
Cell Biology
Cell Line Construction
The OctR-pcDNA3.1(+)_zeo expression vector was created. This construct was used as a template for the creation of the fluorescently tagged pcDNA3.1-OctR-GFP plasmid. The pAcGFP sequence was inserted into the untagged OctR expression vector using a mega-primer strategy. The first step of this strategy was to use PCR to generate a gene fragment from the pAcGFP-N1-Asc1 plasmid, using primers specific for the pAcGFP vector with an additional overhang of approximately 20 base pairs specific to the OctR-pcDNA3.1 vector.
The primers used in this step were 6919-For (5′ ctgcatcccctgtacaccaacggcgcgcccatggtgagcaag 3′) and 6919-Rev2 (5′ ggatatctgcagaattcgcccttcacttgtacagctcatccatgc 3′). This 771 base-pair gene fragment was then purified from an agarose gel, concentration determined, and 125 ng of the purified PCR product was used as a mega-primer for a second PCR reaction, using the OctR-pcDNA3.1(+)_neo expression vector as the template and the QuickChange XL site-directed mutagenesis kit protocol. This PCR reaction generated pcDNA3.1-OctR-pAcGFP, which is the CG6919 dmOa2 octopamine receptor sequence with the AcGFP sequence attached to the C-terminus of the gene. The coding region of the construct was sequenced to confirm double-stranded sequence integrity. (FIG. 55B)
Transient Expression in Mammalian CHO Cells Coexpressing G 16
The untagged and GFP-tagged versions of OctR, which natively couples to cAMP, were transiently expressed in CHO cells stably expressing the G-alpha-16 promiscuous G protein, which signals through calcium. This allowed activation of the receptor to be measured by calcium-sensing fluorescent dyes.
In FIG. 55A, various amounts of untagged OctR-pcDNA3.1 DNA were transfected into CHO cells (6-well dish, 300,000 cells/well) and tested for function at 48 hours using Fluo-4 dye on the Discovery microscope. The graph shown below represents an average of all single-cell readings from cells expressing the cyan vector. It is assumed that the majority of cells expressing the cyan vector also express OctR. This experiment demonstrated that the OctR-pcDNA3.1 expression vector is fully functional, that the OctR receptor will couple to G-alpha16, and that a large assay window exists above control-transfected cells upon addition of a maximal dose of octopamine. In addition, it demonstrated that OctR is not toxic to the CHO cells, and increasing amounts of DNA yield greater expression and greater signals.
In FIG. 55B, a similar experiment is shown with the GFP-tagged OctR expression vector. Once again, the experiment demonstrates that the construct is fully functional and that the assay is capable of measuring calcium release specifically in response to stimulation with octopamine. FIG. 55C shows that the octopamine receptor is also activated by tyramine. Dose curves and competition studies were not performed, but preliminary data and published reports suggest that tyramine is only slightly less efficacious on the OctR than octopamine and has similar potency.
FIG. 55. Calcium-sensing Fluo-4 fluorescent dye response to octopamine or tyramine stimulation in CHO cells stably expressing G-alpha-16 and transiently expressing increasing amounts of untagged or GFP-tagged OctR-pcDNA3.1. Cells cotransfected with pAmCyan vector as a marker of transfected cells were stimulated with a maximal dose of octopamine or sub-maximal dose of tyramine. Well averages of single-cell response measured on the Discovery microscope are graphed above.
Stable Line Generation in Mammalian CHO Cells Coexpressing G-Alpha-16
A line of CHO cells stably co-expressing the pcDNA3(+)-OctR-AcGFP and G-alpha-16 was created. CHO cells stably expressing the G(16 construct (passage 20) were transiently transfected with 1.5 ug of pcDNA3(+)-OctR-AcGFP, the optimal amount of DNA as determined in FIG. 55. Forty-eight hours after transfection, the cells (already maintained under 4 ug/ml hygromycin selection) were put under zeocin selection (300 ug/ml). Cells were grown and passaged under selection for three weeks, then single-cell flow cytometry sorting by fluorescence was completed at Duke University to create monoclonal stable cell lines. FIG. 56 shows an experiment completed just prior to cell-sorting, demonstrating that about 10% of the stable pool population responded to octopamine. At the time of sorting, the G-alpha-16 CHO line was at passage 22.
FIG. 56. Following three weeks of zeocin selection, a stable pool of zeocin-resistant CHO-G-alpha-16 cells exists. Approximately 10% of the stable pool responded to octopamine at measurable levels. This pool was subsequently sorted into single-cell monoclonal colonies.
Monoclonal colonies were visually assessed for the expression of green fluorescent protein, and therefore by assumption, the fused OctR receptor. Colonies positive for green fluorescent protein expression were expanded and tested for response to octopamine.
Approximately 30 monoclonal lines positive for green fluorescent protein expression were tested for response to octopamine on the FLIPR Tetra (FIG. 57). All clones were stimulated by UTP as the positive control and a measure of the maximal calcium response, two doses of octopamine, and by buffer for negative control. Cells were seeded at a density of 20,000 cells/well. Of the approximately 30 lines, only 2 clonal lines (#13 and #30) demonstrated a measurable response to octopamine (FIG. 58). These two lines were selected for further testing.
FIG. 57. Screening of all GFP-positive monoclonal cell lines for response to octopamine. All lines responded to the positive control (10 μM UTP) and no response to the negative control (buffer addition), but only clones #13 and #30 had a measurable response to a maximal dose of octopamine
FIG. 58. Response of clones #13 and #30 on FLIPR Tetra. On each graph, top two lines are in response to maximal dose of UTP, followed in descending order by 10 uM octopamine, 50 nM octopamine, and buffer control.
Stable Lines #13 and #30 Show Best Response to Octopamine
After evaluation of approximately 30 GFP-positive monoclonal lines, clone #13 and #30 were selected for further analysis due to their measurable response to octopamine. The uniformity of each of these clonal lines was tested, by assessing single-cell response to octopamine. Clone #13 was assessed using Fura-2 fluorescent dye on the calcium imaging system, and Clone #30 was examined using Fluo-4 fluorescent dye on the Discovery microscope. FIG. 59 shows the results of these tests. Both clones appear to be comprised completely of cells expressing the OctR, as shown by their response to octopamine. However, a noticeable delineation exists between strong responders and weak responders. This may be due to a difference in receptor expression or in G 16 levels of expression from cell to cell. These experiments were performed following passage 7 after flow-sorting into monoclonal lines.
FIG. 59. Single-cell response to octopamine for clones #13 and #30. Clone #13 (A) was assessed by Fura-2 fluorescent dye on the calcium imaging system, and Clone #30 (B) was assessed by Fluo-4 fluorescent dye on the Discovery microscope.
Do to a shrinking of the signal over time in the FLIPR, further subcloning of both lines #13 and #30 were conducted. Single-cell flow cytometry sorting by fluorescence was completed at Duke University to create further subcloned monoclonal stable cell lines coexpressing the OctR-GFP and G(16. Single cells with medium or high expression of green fluorescent protein (and by assumption the fused OctR) were sorted into 96-well dishes and put under selection of hygromycin and zeocin.
Stable Cell Line Generation and Clone Selection
Over 50 subclones from the original #30 clonal line were evaluated on the FLIPR for several weeks looking for a maximum octopamine response above baseline. Clone 55 was chosen to move forward into assay development based on a good dynamic range in the FLIPR (FIG. 60: OCTR clone 55 selection) and over 82% homogeneity in the Discovery microscope (FIG. 61: Homogeneity of OCTR clone 55).
FIG. 60. OCTR clone 55 selection. OCTR clones were plated at 20K/well into black 96-well plates and assayed the next day. Culture medium was removed and cells were loaded with 50 ul of 2 μM Fluo-4 with probenecid per well for 1 hour at 25° C. Dye was removed and 50 μl of buffer with probenecid added. A single addition was performed in the Tetra. 50 μL of buffer or (2×) 200 μM Octopamine was added to cells while 10 μM UTP was used as a control for maximum intracellular calcium signal.
FIG. 61. Homogeneity of OCTR clone 55. OCTR cells were plated at 6K/well into a black 96-well plate and assayed the next day. Culture medium was removed and cells loaded with 50 ul Fluo-4 per well for 1 hour at 25° C. Dye was removed and 50 μl of buffer with probenecid added. 50 μl of 10 μM Octopamine or buffer was added and signal was recorded using the Discovery microscope. Well averages of single-cell response measured on the Discovery microscope are graphed above.
Pharmacology
Clone 55 was assessed for correct pharmacology and compared to published results based on a cAMP readout. As shown in FIGS. 62A&B, the agonists and antagonists showed the same rank order of potency as the published data, where in the case of the agonists, naphazoline>octopamine>amitraz>clonidine>tolazoline (Evans P, et al. 2005. Invertebrate Neuroscience 5: 111-118). In FIG. 62C, these same compounds had no effect on the parental cell line, showing the specificity of the ligands to the octopamine receptor.
FIG. 62. Cells were plated at 10K/well in 384 well plates and allowed to incubate overnight at 370 C. The cells were assayed 18-24 hours after seeding. Media was removed and cells were dye loaded for 60 minutes with 2 μM Fluo-4. After 1 hour, dye was removed and 20 μl of buffer added to the plate and placed into FLIPR. The 1st addition was antagonist dose response curves and the 2nd addition was agonist dose response curves. Antagonists were challenged with 100 nM Octopamine. In addition, these compounds were tested in the parental cell line CHO-Galpha16.
Assay Development
HTS Screening Strategy
This assay uses two fluid additions to permit the detection of activators and inhibitors in a single experiment. In screening, test compounds will be added in the first addition and allowed to incubate for three minutes. The activator octopamine will then be introduced in the second addition at an EC80 concentration and the fluorescence read for two minutes. Controls will be run for both additions. An increase in fluorescence above baseline in the first addition will indicate a possible activator and a reduced response or no increase in the second addition may indicate a possible inhibitor. FIG. 55C
Basic Test Protocol
Cells were plated at 10,000 cells/well in 50 μl into 384-well black TC plates and allowed to incubate overnight at 37° C./5% CO2. The cells were assayed 18-24 hours after seeding. Culture medium was removed by flicking and cells were dye loaded for 60 minutes with 20 μl of 2 μM Fluo-4 in assay buffer plus probenecid at 25° C. After one hour dye was removed by flicking and 20 μL of assay buffer was added, wait 5 min prior to placing into the FLIPR and run using a two-addition protocol. The first addition (20 ul, 2×) was 0.5% DMSO final in assay buffer. The second addition (20 ul, 3×) was either assay buffer or 100 nM octopamine final.
Cell Plating Method
Experiments were conducted to examine manual vs. multidrop dispensing of cells into assay plates (data not shown). The data showed that the multidrop plating was more reproducible than hand plating. There are two critical points to remember when plating cells with the multidrop:

    
    
        1. Cells need to stay in suspension during cell plating. We use a rotating plate or a rotomixer with a holder attached, where we can place a 500 ml conical bottom tube containing the cells.
        2. Do not leave the cells sitting in multidrop tubing between plates. The cells will settle in the tubing, which will result in streaking patterns in the data. If the cells sit, you will need to empty the tubing and reprime.
    
    


DMSO Tolerance Study
Full octopamine dose response curves were performed in the 1st and 2nd additions to test the sensitivity of this assay to varying concentrations of DMSO. As shown in FIG. 63A, there was a significant shift in the EC50 for octopamine at 2% DMSO (final). For the 2nd addition (FIG. 63B), final DMSO concentrations greater than 0.67% showed a significant shift in the EC50. For this assay, we will be using final concentrations of 0.5% DMSO in the 1st addition and 0.33% in the second addition, which will have a minimal effect on the octopamine EC50.
FIG. 63: Octopamine EC50 curves in different DMSO concentrations in 1st and 2nd additions. OctR cells were plated at 10K cells/well and assayed the following day. Culture medium was removed and cells loaded with 20 ul of 2 μM Fluo-4 plus probenecid per well for 1 hour at 25° C. After one hour, dye was removed and 20 μl of buffer plus probenecid was added to plate and placed in Tetra. A two-addition protocol was used. The first addition (2×) was either 20 ul indicated % DMSO+octopamine DRC (FIG. 63A) or 20 ul indicated % DMSO in assay buffer (FIG. 63B) then read for 180 sec. The second addition, for FIG. 63B, was (2×) 20 μl of octopamine DRC and read for an additional 180 sec. Statistics were calculated using the maximum of reads 20-180 (1st addition) and the maximum of reads 200-360 (2nd addition).
Cell Density Optimization
Plating densities at 7K, 10K, and 14K per well were examined for variability and Z′ statistics and data are shown in FIG. 64. All densities tested performed resulted in excellent Z′ statistics. It was decided to move forward with 10K/well based on the low variability and cost reduction.
FIG. 64: Cell density optimization. FLIPR data showing maximum and minimum statistics for full plates at the indicated densities with resulting CV and Z′ statistics. Platings below 4K cells/well did not yield a confluent monolayer the next day and were not tested. OCTR cells were plated by-hand at the appropriate densities and assayed the following day. Culture medium was removed and cells loaded with 20 ul/well of 2 μM Fluo-4 plus probenecid and incubated for 1 hour at 25° C. After one hour, dye was removed and 20 μl/well of buffer plus probenecid was added and placed in Tetra. A two-addition protocol was used. The first addition (2×) was 20 ul 1% DMSO in assay buffer (0.5% [final]) read for 180 sec, and the second (3×) was 20 ul 300 nM Octopamine in assay buffer (100 nM [final]) read for an additional 180 sec. The statistics were calculated using the maximum of reads 20-180 (minimum response) and the maximum of reads 200-360 (maximum response).
Dye Concentration
An experiment comparing the response of cells loaded for one hour with 1 μM, 2 μM and 4 μM Fluo-4 dye (Notebook 1052125 #1 p. 102) is shown in FIG. 65. All concentrations of Fluo-4 tested resulted in acceptable window size and Z′ statistics. It was decided to move forward with 2 μM Fluo-4 based on the good dynamic range and significant cost reduction.
FIG. 65: Dye concentration. Maximum statistics showing the effect of dye concentration on window size. OCTR cells were plated at 10K cells/well and assayed the following day. Culture medium was removed and cells loaded with 20 ul of 1 μM, 2 μM or 4 μM Fluo-4 per well for 1 hour at 25° C. After one hour, dye was removed and 20 μl of buffer was added to plate and placed in Tetra. A two-addition protocol was used. The first addition (2×) was 20 ul 1% DMSO in assay buffer (0.5% [final]) read for 180 sec, and the second (3×) was 20 ul 300 nM octopamine in assay buffer (100 nM [final]) read for an additional 180 sec. The statistics were calculated using the maximum of reads 20-180 (minimum response) and the maximum of reads 200-360 (maximum response). (not shown Z′ for 1 μM Fluo-4=0.73, 2 μM=0.70, and 4 μM=0.80)
Dye Loading Time
The effect of dye loading time on variability and Z′ statistics was examined in FIG. 66. In addition, we compared MDC tips versus Axygen Tetra tips. The results showed that dye loading times between 1.0 and 3.0 hours had no significant effect on EC50, window sizes or, as a consequence, Z′ statistics. There was no difference between the MDC or the Axygen tips.
FIG. 66: Dye loading time. OctR cells were loaded for 1 hr, 2 hr and 3 hrs to determine if there would be a shift in the octopamine EC50 with increasing dye loading time. In addition, we compared MDC tips with Axygen tips over the same amount of time. OctR cells were plated at 10K cells/well and assayed the following day. Culture medium was removed and cells loaded with 20 ul of 2 μM Fluo-4/probenecid per well at 25° C. After one two, and three hours, dye was removed and 20 μl of buffer was added to plate and placed in Tetra. A two-addition protocol was used. The first addition (2×) was 20 μl DMSO (0.5% final) in assay buffer read for 180 sec, and the second (3×) was 20 μl of octopamine at given dose in assay buffer read for an additional 180 sec. Statistics were calculated using the maximum of reads 20-180 (minimum response) and the maximum of reads 200-360 (maximum response).
Dye Loading Temperature
The OctR cells loaded well at room temperature, so 37° C. was not examined.
Assay Buffer pH Optimization
Standard HBSS buffer at pH 7.4 was used throughout assay development.
Stability of Assay Buffer
Assay buffer stored at 4° C. was tested out to one week (data not shown) with no significant difference in performance.
Stability of Octopamine
Octopamine was tested comparing freshly diluted versus a 24-hour solution (data not shown). The response obtained with the 24-hour solution stored at room temperature was identical to that of the freshly diluted solution. We have repeatedly freeze-thawed 4° C. DMSO stocks of 10 mM octopamine with no reduction in activity.
Pipette Washing and Octopamine Carryover Assessment
In order to reduce the cost of the screen, tip washing experiments were performed to evaluate the ability to remove octopamine from the tips, so that the tips could be used multiple times. Octopamine is extremely sticky and is easily carried over on the tips to the next plate. The following DMSO wash protocol was evaluated:
4 cycles times 2 strokes @ 28 μL in 2.5% DMSO
Mix 10 strokes @ 25 μL in 100% DMSO
1 cycle times 2 strokes @ 28 μL in 2.5% DMSO
1 cycle times 2 strokes @ 28 μL in water
Based on the data shown in FIGS. 67A and B, the DMSO wash protocol did not eliminate octopamine from the tips and resulted in significant carryover into the 2nd plate (FIG. 67B). In addition, it should be noted that the length of the assay increased to over 11 minutes, which significantly impacted the number of plates that could be run per day (Notebook 1052125 #1 p124). Acetone was also tested once and worked very well to eliminate carryover (data not shown), but due to the low flash point, it would be a safety hazard to use acetone in the Tetra instrument. For this screen, tips will be changed in between plates.
FIG. 67: Tip washing assessment. FLIPR data showing maximum and minimum statistics for full plates with resulting CV and Z′ statistics. OCTR cells were plated at 10K/well and assayed the following day. Culture medium was removed and cells loaded with 20 ul of 2 μM Fluo-4 plus probenecid per well for 1 hour at 25° C. After one hour, dye was removed and 20 μl of buffer plus probenecid was added to plate and placed in Tetra. A two-addition wash protocol was used. The first addition (2×) was 20 ul 1% DMSO in assay buffer (0.5% [final]) read for 180 sec, and the second (3×) was 20 ul 300 nM Octopamine in assay buffer (100 nM [final]) read for an additional 180 sec. The statistics were calculated using the maximum of reads 20-180 (minimum response) and the maximum of reads 200-360 (maximum response).
3-Day Variability Assessment
Three independent assays were performed on three different days to validate the appropriate concentrations of agonist (octopamine) or antagonist (mianserin) to be used during screening, and to validate the inter and intra plate variability of the assay. On each day, duplicate plates were run for each condition. The assay protocol was as follows: OctR cells were plated at 10K/well in 50 μl media and incubated at 370 C/5% CO2 for 18-24 hours prior to running in the Tetra. On the day of the assay, cells were dye loaded with 2 μl Fluo-4 for 1 hour at 250 C, dye removed, 20 μl buffer added to the plate, incubated at RT for 5 minutes, then run in the Tetra.
For the 3-day variability assessment, the following concentrations of agonist and antagonist were used:
100% Agonist=100 nM (day 1) and 1 μM (day 2 and 3)
80% Agonist=40 nM (day 1) and 100 nM (day 2 and 3)
50% Agonist=5 nM (all days)
50% Antagonist=40 nM (all days)
Time Cuts:
Read 1=20-180 max stat
Read 2=200-360 max stat

    
    
        1) Buffer+50% agonist+100% agonist. The following scatter plots show 3 days of duplicate plates in order to validate the ability of the assay to identify agonists. The 50% dose of octopamine was achieved on days 2 and 3
        2) Buffer+80% agonist+100% agonist. The following scatter plots show 3 days of duplicate plates in order to validate the 80% dose of octopamine, which will be used in the 2nd addition. As shown, the 80% dose was achieved on all three days.
        3) Buffer+50% antagonist+80% agonist. The following scatter plots show 3 days of duplicate plates in order to validate the ability of the assay to identify antagonists. Unfortunately, the incorrect dose of mianserin was used for all three days; however, the correct dose was achieved during the twenty-plate DMSO test run (FIG. 69B).
    
    


3-Day Agonist and Antagonist Dose Response Curves
Octopamine and mianserin dose response curves were run on three separate days to analyze EC50 shifts through time. The data in FIGS. 68A and B show that the EC50 for octopamine and the IC50 for mianserin were very reproducible over three days. FIG. 68. OCTR cells were plated at 10K/well and assayed the following day. Culture medium was removed and cells loaded with 20 ul of 2 μM Fluo-4 plus probenecid per well for 1 hour at 25° C. After one hour, dye was removed and 20 μl of buffer plus probenecid was added to plate and placed in Tetra. A two-addition wash protocol was used. The first addition (2×) was 20 ul DMSO or mianserin at given dose in assay buffer read for 180 sec, and the second (3×) was 20 μl of octopamine at given dose (FIG. 55A) or 100 nM final octopamine (FIG. 55B) and read for an additional 180 sec. Statistics were calculated using the maximum of reads 20-180 (minimum response) and the maximum of reads 200-360 (maximum response).
20-Plate DMSO Test Run
A 20 plate DMSO test run was performed in order to test the following:
Compound dilution scheme (page 30)
Reagent preparation volumes for 20-plate run (SOP page 30-31)
Dynamics of running: timing, equipment issues etc.
Table 5 shows the Z′ for all 20 plates. All plates have acceptable Z′, except for plate 6, which had a multidrop pipetting error that resulted in variable volumes of liquid being added across the plate. Plate 2 had one control well in the 1st addition (activation) that was low. Removing that point from the calculation resulted in the Z′ activation=0.60.










TABLE 5






Plate
z′ Activation
z′ Inhibition








Plate01
0.60
0.79



Plate02
0.47
0.65



Plate03
0.60
0.59



Plate04
0.64
0.81



Plate05
0.59
0.72



Plate06
0.20
0.29



Plate07
0.70
0.78



Plate08
0.66
0.76



Plate09
0.65
0.68



Plate10
0.68
0.69



Plate11
0.57
0.70



Plate12
0.64
0.74



Plate13
0.61
0.72



Plate14
0.71
0.76



Plate15
0.63
0.74



Plate16
0.63
0.74



Plate17
0.64
0.76



Plate18
0.60
0.73



Plate19
0.66
0.78



Plate20
0.53
0.78









FIG. 69A is a scatter plot of plate #7, showing the controls and test wells for the agonist portion of the screen. The green squares are the 100% (1 μM) octopamine controls, the blue squares are the 0% or buffer controls, and the black diamonds are the test samples, in this case 0.5% final DMSO.
FIG. 69B is a scatter plot of plate #7, showing the controls and test wells for the antagonist portion of the screen. The green squares are the 80% (100 nM) octopamine controls, the blue squares are the 0% or buffer controls, and the black diamonds are the test samples, in this case 100 nM octpamine+0.33% final DMSO. The black diamonds with larger values than the green squares are a 100% dose of octopamine (1 μM). These wells are not used in data calculation, but are included in the assay to validate the 80% dose of octopamine. The black diamonds with lower values than the green squares are the 100% controls from the 1st addition that have decreased over time. They are not included in any data calculations. The red squared are the 50% antagonist control (267 nM mianserin) and are included to ensure that the cells are responding properly.
DATA CALCULATIONS
The % activation and % inhibition were calculated by the following formulas:

% Activation=((Test sample−ave 0% act)/(ave 100% act−ave 0% act))×100

% Inhibition=((Test sample−ave 0% inhib)/(ave 80% inhib−ave 0% inhib))×100

The Z′ statistic for activation were calculated using the following formula:

1−((3×ave. STDEV min+3×ave STDEV max)/(abs ave 100%−ave 0%))

    
    
        where min=0% activation and Max=100% activation
    
    


The Z′ statistic for activation were calculated using the following formula:

1−((3×ave. STDEV min+3×ave STDEV max)/(abs ave 80%−ave 0%))

    
    
        where min=0% activation and Max=80% activation
    
    


HTS SOP
1) Materials













Vendor
Catalog #



















Base media type





Ham's F12
Mediatech
010-080-cv



FBS
Hyclone
Sh30071.02



trypsin-EDTA (high Conc.)
Mediatech
15400-054



hygromycin (per 50 ml bottle)
Calbiochem
400052



zeocin (per 1 g-10 ml-8 tubes/box)
Invitrogen
46-0509



Pen/Strep (P/S/antimycotic)
Mediatech
bw17602e



PBS
Mediatech
bw17512q



Consumables (Plastics) Tissue





Culture Treated





Flasks (T175)
Nunc
159910



Plates (black/clear, 384 well)
bd falcon
353962



compound plates
NUNC
264573



Tetra tips
axygen









Assay Buffer (HBSS):
20 mM Hepes; 11.1 mM Glucose; 1.8 mM CaCl2; 1 mM MgCl12; 125 mM NaCl; 2.5 mM KCl; 5 mM Probenecid; pH to 7.4 Osm 290
To make 1 L of HBSS, add 20 mls of 1M Hepes, 11.1 mls of 1M glucose, 1.8 mls of 1M CaCl2, 31.25 mls of 4M NaCl, 1 ml of 1M MgCl2 and 0.83 mls of 3M KCl to 935 mls of dH20. pH to 7.4 with NaOH. Store at 4° C.
Probenecid:
MW=285.4; ICN Biomedical (156370)
Add 14.2 grams of powder to 100 mls of 1M NaOH=500 mM
Add 10 ml of 500 mM stock to 1 L of HBSS and pH to 7.4 with HCL. Store at RT.
Fluo-4AM:
MW=1096.95; Molecular Probes (F-14202)
Add 912 μl of 100% DMSO to 1 mg vial=1 mM stock
20% Pluronic F-127:
MW-12500; Invitrogen (P3000MP)
20% solution in DMSO
Octopamine:
MW=189.64; Sigma (O-0250)
10 mM stock prepared in 100% DMSO
Mianserin:
MW=300.8; Sigma (M-2525)
10 mM stock prepared in 100% DMSO
2) Cell Culture
A) Cell Culture Protocol
This assay uses one cell line: OCTR clone 55
Complete culture medium: Prepared in biosafety cabinet/laminar flow hood.
1. 500 ml of Dulbecco's Modified Eagle's Medium/Ham's Nutrient Mixture F-12.
2. Add 50 ml ml fetal bovine serum.
3. Add 5 ml penicillin-streptomycin solution.
4. Add 2.2 ml Hygromycin
5. Add 1.5 ml Zeocin
6. Sterile filter through 0.2 um filter and store at 4° C. Thawing frozen cells: It is best at first to thaw cells early in the day on Monday so that the cell growth can be monitored and numbers adjusted during the week. The cell numbers listed below can only be considered rough guides as the cells may grow at a different rate in your culture medium.
1. Remove a vial from liquid nitrogen storage and rapidly thaw the cells by immediately placing it in a 37° C. water bath and applying gentle agitation. Keep the O-ring and cap out of the water to reduce the possibility of contamination.
2. When the contents are nearly thawed, remove the vial from water bath, mix by inverting, and decontaminate by spraying with 70% ethanol. All operations from this point on are carried out under aseptic conditions.
3. Transfer the 1 ml of thawed cell suspension into a 50 ml conical bottom centrifuge tube containing 30 ml of complete medium.
4. Centrifuge the cells for 5-7 minutes at 150 g.
5. After centrifugation, remove medium by aspiration and resuspend the pellet in 5 ml of complete medium.
6. Transfer the 5 ml of resuspended cells into a 225 cm2 flask containing 25 ml of complete medium.
7. Inspect the culture flask the following morning and split for expansion or replace medium for further growth.
B) Subculture of Cells/Harvest Protocol
1. Remove flasks from incubator.
2. Aseptically aspirate old media from the flask in the hood.
3. Add 5-10 ml PBS per flask.
4. Rinse flask with the PBS briefly
5. Aspirate PBS
6. Add 3-5 ml RT trypsin
7. Wash trypsin over cells briefly.
8. Aspirate trypsin.
9. Allow cells to detach for 3 minutes and tap flask to loosen cells.
10. Add 8 ml media to the flask.
11. Pipette several times to break up any clumps and to wash cells from the bottom of the flask. Stand flask on end while next steps are completed.
12. If harvesting multiple flasks, then combine cells from all flasks into one flask and mix thoroughly.
13. Remove 20 ul cell suspension from the flask and place into a tube for counting.
14. Proceed to step C below.
C) Performing Cell and Viability Counts/Seeding of Flasks and Plates:
1. After harvesting cells, prepare cells for counting by diluting in trypan blue and PBS at a 1:1 dilution: 20 μl cell suspension+20 μl of 0.4% trypan blue solution NOTE: Trypan blue is available as a 0.4% solution and is used at a working concentration of 0.02%-0.04%, but works well at 0.2%. After being stained with trypan blue, the cells are counted within 3 minutes; after that time viable cells will begin to take up the dye.
2. Using a pipette, withdraw a small amount of the stained cell suspension and place the tip of the pipette onto the slot of a clean hemacytometer with the coverslip. The cell suspension will pass under the coverslip by capillary action. Fill the opposite chamber. Do not overfill. The cell distribution should be homogeneous in both chambers.
3. Place the hemacytometer on the stage of microscope and view one of the four large corner squares ruled by three lines. Viable cells will be slightly opalescent, round and pale with a darker outline. Nonviable cells will be dark, opaque blue.
4. Count the viable cells in the four squares. Count the cells that overlap outside borders of squares but not those overlapping inside borders. Calculate the average number of viable cells per square (total viable cells in the four squares, divided by four).
5. Record the cell counts from both chambers. If the counts differ by more than 20%, prepare a third sample to verify the count.
6. The viable cell number is calculated using the formula:

Viable cell number/ml=average number of viable cells×104×dilution factor % viability=number of viable cell counted/total number of cells×100

For seeding flasks, calculate the cells/ml of suspension and volume of cells needed.
For example,

    
    
        Seed 10 flasks at 8.5e6 cells/flask using a cell suspension at 2.5e6 cells/ml
        Pipette 25 ml of complete media into each flask.
        Calculate the volume of cell suspension that needs to be added to each flask.
        8.5e6/2.5e6=3.4 ml of cells added to each flask.
        Pipette the cells into each flask.
        Rock flasks gently to evenly coat the flask with cells and media.
        Place flasks into 370 C/5% CO2 incubator.
        The following table shows the seeding schedule and densities for T175 flasks.
    
    














Cell Culture Schedule for 20 plates/day 4 days/week













# of vessels
Mon.
Tues.
Wed.
Thurs.
Fri.






available
13
13
13
13
0
# of flasks need


for plating








to make
27
0
0
0
20
# of flasks need


more vessels








total
40
13
13
13
20
Total # of flasks










Splitting schedule









overnight
10e6 c/f



2 days
8.5e6 c/f
Cells need to be refed on second day ie. RF Th


3 days
5e6 c/f



4 days
2-2.5e6 c/f









For seeding plates, calculate the number of cells per plate needed.
For example,

    
    
        Seed 20 plates at 10K cells/well using a cell suspension at 2.5e6 cells/ml
        Calculate the volume of cell suspension you will need.
        10,000 cells/well×384 wells/plates=3.84e6 cells/plate
        3.84e6 cells/plate×23 plates=8.832e7 cells total
        (Note: 23 plates is used in the calculation so that there is a dead volume for the multidrop)
        8.83e7 cells total/2.5e6 cells/ml=35.3 ml of cell suspension
        Calculate the total volume that you will add the cells suspension to:
        50 μl/well×384 wells/plates=19.2 ml/plate
        19.2 ml/plate×23 plates=442 ml total
        To the 35.3 ml cell suspension, add 406.3 ml media.
        Mix thoroughly and take a sample to count.
        The cell count should be 5e5 cells/ml or 2.5e4 cells/50 μl
        Use the multidrop to dispense cells into the plates:
        
            Put multidrop head into the dispenser
            Flush the head with 50 ml 70% EtOH
            Flush with 70 ml sterile PBS
            Place the cell suspension in a 500 ml conical bottom tube on the rotomix table and place the multidrop tubing into the cell suspension.
            Prime the head with the cell suspension.
            Dispense 50 μl/well into each plate. Make certain to keep the cells in suspension while dispensing and that the cells do not sit in the tubing. Both will result in patterns in the data.
            Leave plates in a single layer at room temperature for at least 30 minutes, prior to placing into 370 C/5% CO2 incubator.
            Make certain that plates are in a single layer in the incubator.
            Clean multidrop tubing by flushing with 70 ml of 70% EtOH followed by 70 ml of dH2O.
        
        
    
    


3) HTS 20 Plate Screening Assay
A) One Day Prior to Assay:

    
    
        Harvest cells from flasks. Seed 20× 384-well plates at 10 Kcells/well/50 μl per well. Leave plates at RT for at least 30 min to reduce edge effects (see page 29 for details).
        Incubate at 37(C/5% CO2 overnight (16-24 hours, stacking no more than 1 plate high).
    
    


B) Day of the Assay:

    
    
        1. Prepare Tripos Read 1:
        Place the 45 μL 2 mM stock plate into source position #1 on Tetra
        Place a new 384-well plate with 45 μl of HBSS (no DMSO) in columns 3-22 in source position #2
        Place a new 384-well plate with 90 μl of HBSS (no DMSO) in columns 3-22 in read position
        Run Tripos compound dilution control file
    
    


The Tripos compound dilution control file is depicted in FIG. 87.
Or,

    
    
        1. Prepare Divpick Read 1:
        Using multidrop, add 45 μl of HBSS to stock plate containing 5 μl of compound for a final stock of 200 μM.
        Place the 50 μL 200 μM stock plate into source position #2 in Tetra
        Place a new 384-well plate with 90 μl of HBSS (no DMSO) in columns 3-22 in read position
        Run Divpick compound dilution control file
    
    


The Divpick compound dilution control file is depicted in FIG. 88.

    
    
        2. Prepare Read plate 1 controls.
        Make up HBSS 1% DMSO buffer by adding 750 μl of 100% DMSO to 75 mls of HBSS. Add 60 μl to appropriate wells according to READ 1 plate map below.
        Make up 100% Octopamine by adding 5 μL of 10 mM stock to 25 ml of HBSS for a 2 μM (2×) concentration. Add 60 μl to appropriate wells according to READ 1 plate map below.
        Make up 50% Mianserin by adding 50 μL of 100 μM stock to 6.25 ml of HBSS for a 800 nM (2×) concentration. Add 60 μl to appropriate wells according to READ 1 plate map below.
    
    


The Read 1 plate map is depicted in FIG. 89.

    
    
        3. Prepare Read 2 Plates
        Make up 80% octopamine by adding 165 μL of 1 mM stock to 550 ml of HBSS for a 300 nM (3×) concentration. Add 60 μls to appropriate wells according to READ 2 plate map below.
        Make up 100% octopamine by adding 37.5 μL of 1 mM stock to 12.5 ml of HBSS for a 3 μM (3×) concentration. Add 60 μls to appropriate wells according to READ 2 plate map below.
        Add 60 μLs of HBSS (no DMSO) to appropriate wells according to READ 2 plate map below.
    
    


The Read 2 plate map is depicted in FIG. 90.
4. Dye Load

    
    
        Prepare dye by adding 0.5 ml of 1 mM Fluo-4 together with 0.5 ml of 20% pluronic into a tube, mix, then add mixture into 250 mls of HBSS/probenecid.
        Remove media from cell plates by flicking plates into sink containing Clorox, tap gently on kimwipes to remove excess, and add 20 μl/well of dye using multidrop.
        Place in RT incubator for 60 min.
    
    


5. Run the Assay

    
    
        Remove dye from cell plate by flicking into sink containing bleach; tap gently on Kimwipes to remove excess dye.
        Add 20 μl of HBSS using multidrop to cell plate; place in read position in FLIPR and wait 5 minutes prior to running in FLIPR. (***Failure to wait 5 minutes will increase the variability in the plate)
        Place Read 1 compound plate into source #1
        Place Read 2 compound plate into source #2
        Run protocol: OCTR HTS screen on Tetra
        FLIPR read settings:
    
    


1st addition=180 reads at 1 sec interval with 20 μl addition after 10 baseline reads
2nd addition=120 reads at 1 sec interval with 20 μl addition after 10 baseline reads
7. Data Export
After 20 plates have been run, manually batch export maximum statistics from read 1-180 as stat1 and reads 200-300 as stat2. These statistics are then copied into an excel spreadsheet to calculate the percent control for inhibition and activation.
A screenshot of the Batch Export statistics interface is depicted in FIG. 91.
The following examples are in connection with the SK-channel:
1.
The potassium channel is responsible for the survival of Dm. In transient RNAi experiments it was demonstrated that reduced viability and lethality effects are induced in Dm. In comparison to buffer control as-well-as injection of a known nonlethal RNAi, RNAi produced from SEQ ID NO: 227, shows measurable reduced viability in Dm.



















Construct
injected eggs
developing eggs
larvae hatched
dev E to L rate
Pupae
L to P rate
Adults
P to A rate
survival devE to A







Buffer only
82
71
50
70.42%
40
80.00%
37
92.50%
52.11%


Nonlethal Control
99
92
79
85.87%
74
93.67%
63
85.14%
68.48%


Seq No: 1 RNAi
87
74
50
67.57%
33
66.00%
31
93.94%
41.89%


Lethal Control
82
72
28
38.89%
13
46.43%
11
84.62%
15.28%









2. Expression of Drosophila SK Gene in CHO Cells
Gene name small conductance calcium-activated potassium channel
Synonyms CG10706
Species Drosophila melanogaster 
Final clone name pCRII-SK2+4D (coding sequence only) FIG. 70
Base vector pCRII-TOPO (Invitrogen)
Primers









(SEQ ID NO: 233)



Forward: SK 5′1 5′ATGAAAACACCTTCCATTGC 3′



 



(SEQ ID NO: 224)



Reverse: SK 3′2 5′TCAGCTGCCGTATTTGTTGG 3′






Cloning strategy: Coding sequence from mixed fs-cDNA (head and 2nd instar) was PCR amplified using the above primers, and cloned into pCRII-TOPO vector. 2 independent clones were chosen for sequencing, 2 and 4D, and a perfect sequence was created by subcloning the AvrI/NdeI 498 bp fragment from 2 into the AvrI/NdeI 5233 bp vector fragment of 4D.
Expression Construct
pTriEx3 Neo SK was created by ligating the 1.8 kb EcoRV/BamHI fragment from pCRII-SK2+4D to the EcoRV and BamHI sites of pTriEx3-Neo. The resulting construct contains the SK CDS downstream of the CMV promoter and adds nine codons to the 5′ end as a result being in-frame with the Kozak translation start consensus. FIG. 71 Transfection: CHO—K1 cells were plated in 35 mm 6-well plates at 2.5×104 cells/well and transfected the following day with 1 ug pTriEx3 Neo SK DNA from each of three different bacterial clones using FuGENE 6 transfection reagent (Roche Diagnostics Corporation) and the manufacturer's recommendations. Cells were passaged 24 hr later into 75 cm2 flasks and placed under antibiotic selection (400 ug/ml G-418, Calbiochem).
3. Testing Functionality
Basic test protocol: Cells were plated at 5×104/well into 96-well poly-D-lysine assay plates (BD Biosciences) and placed at 37 C/5% CO2 the day before testing. Culture medium was aspirated and replaced with 0.4× blue membrane potential dye (Molecular Devices Corporation) in assay buffer and incubated for 1 hour at RT. The assay was run by reading baseline fluorescence for 20 sec, and reading an additional 60 or 180 sec after activation.
Pool testing: Once selection was complete (about 10 days) the cells were passaged again into assay plates and culture vessels for expansion. The three transfection pools were tested for function using 1 uM [final] ionomycin (a Ca2+ ionophore) as an activator. Pool 3 (clone SK3) showed the expected hyperpolarization in response to presumptive Ca2+ influx. FIG. 72
4. Assay Protocol
Assay Buffer (Prepared Fresh Daily):














Stock solution
Final concentration









1M KCl
1 mM



1M CaCl2
2.3 mM  



0.5M NaHCO3
5 mM



1M MgCl2
1 mM



5M NaCl
154 mM 



1M D(+) glucose
5.5 mM  



1M HEPES pH 7.4
20 mM 










Stock solutions can be sterile-filtered and stored indefinitely at room temperature, with the exception of glucose, which is stored at 4° C. The assay buffer is prepared by starting with 0.8 volume of high-quality water; adding stock components to the final concentration, and adjusting the volume. The solution is sterile-filtered and the pH adjusted to 7.4 with 1N NaOH.
A 10 mM ionomycin (MW=709) stock is prepared by dissolving the powder in DMSO to a final concentration of 7.1 mg/ml. This solution is stored at 4° C. and is stable for one year.
Activation solution (5×) is prepared by adding 10 mM ionomycin to assay buffer to a final concentration of 5 uM (1:2000 dilution). This solution should be prepared fresh daily. We have heard reports of ionomycin sticking to certain types of polypropylene, but have not seen this with the tips, tubes and compound plates we are using, with the exception of the black FLIPR Tetra 96-well tips.
This assay was developed using the Molecular Devices blue membrane potential dye at 0.4× the normal concentration. It is important to vortex the dye upon hydration and rinse the vial repeatedly to ensure that all the dye has been recovered and that it is in solution. The dye can be stored during the day at 4°. Dye loading is carried out by flipping the culture medium from the 96-well poly-D-lysine plate, gently tapping the plate on Kimwipes to remove excess medium, replacing it with 180 ul 0.4× dye per well, and incubating in the dark at room temperature (25° C.-28° C.) for 3-5 hr. The assay plate is now ready for testing which comprises a 20 ul (10×) first addition and 50 ul (5×) second addition. 50 uM propafenone [final] is used as a control inhibitor. The assay is read for three minutes after activation to allow maximum window development relative to controls.
Special reagents: Ionomycin, free acid (Calbiochem #407950)

    
    
        Propafenone hydrochloride (Sigma #P4670)
    
    


5. Cloning:
Cells from pool 3 were diluted to 12 cells/ml and 250 ul was dispensed into each well of two 96-well culture plates. Wells containing a single clone were identified after one week's growth and picked for expansion and testing the following week.
Clone screening: Fifteen individual clones were screened for function using the same conditions as pool testing. The time and extent of maximum hyperpolarization as well as a measurement of the sustainability of the hyperpolarization were used to select the best performing clones for further evaluation.
6. Functional Validation
An experiment was undertaken to examine the response to elimination of external Ca2+. If the channel is indeed activated by the import of Ca2+ by ionomycin, then removing Ca2+ from the outside of the cell should reduce the differential response. FIG. 73
Conclusion: Elimination of Ca2+ from the assay buffer reduced maximum hyperpolarization to within 20% of wildtype and abolished the differential response altogether by timepoint 120 sec. Therefore, the SK-expressing cells require Ca2+ for full response, indicating a Ca2+-dependent activation.
7. Activation Optimization/EC50
Tests were conducted to determine the optimal concentration of ionomycin in the activation buffer needed to produce a robust signal with a large response window relative to controls. The EC50 for ionomycin on SK3 was calculated in three separate experiments, yielding a value of 200 nM (SD=10). There was no increase in the size of the response window relative to controls above 800 nM ionomycin. These results correspond to literature values of 1 uM for full activation of Ca2+-dependent channels (Terstappen et al., 2001. Neuropharmacology 40).
Sample data: FIG. 74
pTX-CHO is a mock transfected cell line, i.e. a cell line transfected with vector that does not contain the gene of interest to show any activity in screening is due to the gene of interest and not the vector.
8. Variability in Relation to Dye Loading Time
Tests were conducted to examine the assay window size relative to dye loading time. 24 wells each SK3-9 and pTx-CHO were loaded at room temperature for the times indicated on the following chart, up to nearly five hours: FIG. 75
The window size stabilized at maximum after 3 hours and variability among wells was reduced (data not shown). Based on these results, a dye load time of 3-5 hours is recommended for screening.
9. DMSO Tolerance
Cells responded predictably to increasing levels of DMSO in the first (10×) addition with increased instability and a narrowing of the assay window. Additions resulting in DMSO levels above 0.2% showed severe perturbations after ionomycin activation. FIG. 76
10. Ionomycin Stability
An experiment was conducted (data not shown) to examine the stability of ionomycin in solution. Ionomycin was diluted to 5 uM in assay buffer, loaded into a compound plate and compared with freshly-prepared ionomycin for SK activation the following day. The freshly-prepared ionomycin yielded a statistically significant 6% increase in assay window size, so our recommendation is for freshly-prepared ionomycin.
11. Statistical Tests
The following half-plate data (FIG. 77) was used to calculate two statistical parameters from timepoint 260 sec using zero baseline with a lag time of 80 sec.
t-test: The two-tailed P value is less than 0.0001. Therefore, the probability of the differential response occurring by chance is essentially zero.
Z′-factor data table (units are -K RFUs, n=24):




















SK3-9
220
205
217
218
215
208




239
227
223
224
217
224




250
236
238
228
224
237




250
240
266
243
233
239




AVE
230
STD
14





pTx-CHO
10
−5
9
2
15
−21




32
20
25
15
20
5




32
28
27
18
26
38




50
34
38
36
31
41




AVE
22
STD
16










Z′-factor
0.56









This statistic places the test in the “an excellent assay” category (Zhang et al., 1999. Journal of Biomolecular Screening 4. A Simple Statistical Parameter for Use in Evaluation and Validation of High Throughput Screening Assays).
12. Ion Channel Library Testing
The DmSK3 clone was tested at 10 uM on a set of 71 compounds comprised of known activators, agonists and inhibitors across the major ion channel types (BIOMOL #2805, Ion Channel Ligand Library). Cells were incubated with compound for 1 min before activation by 1 uM ionomycin.
Summary of Results
Seventeen of the 71 compounds (24%) had a clear effect on the response of the cells to ionomycin. Of these, nine (13%) appeared to be inhibitory to hyperpolarization and eight (11%) had an activator/agonist effect on response. In general terms, DmSK3 tended to respond to compounds involved in Ca2+ movements and were relatively insensitive to K+ and Na+ channel modulators (with some exceptions).
Selected Results (from SK3 Testing)
BAY K-8644 L-type Ca2+ channel agonist FIG. 78
Propafenone Efficacious potassium channel blocker. FIG. 79
TEA (SK3) (Tetraethylammonium) FIG. 80
4-AP (SK3-4) (4-aminopyridine) FIG. 81
propafenone (SK3) FIG. 82
13. Apamin Sensitivity
Mammalian SK channels are characterized by their sensitivity to the peptide toxin apamin, from bee venom. We found DmSK to be apamin-insensitive, even at high dose (10 uM, data not shown).
14. SK Expression in CHO Cells Tested with the Patch-Clamp Technique
Materials and Methods
Cells:
Chinese hamster ovary (CHO) cells stably transfected with a Drosophila SK gene were used for all measurements. Cells were plated in 35 mm Petri dishes 18-22 hours before the experiment.
Electrophysiology:
Data were acquired and analyzed using pClamp software (version 9.0.1.16). The whole-cell configuration of the patch-clamp technique was used to voltage clamp cells at room temperature (22-25° C.). Pipettes were pulled from borosilicate glass capillaries (8250, Garner Glass, Claremont, Calif.) using a DMZ Universal Puller (Zeitz, Munich, Germany) and had resistances of 2-3 MOhm when filled with pipette solution and measured in bath solution. The liquid junction potential between bath and pipette solution was always compensated before the formation of a gigaohm seal.
Membrane current was measured under whole-cell clamp, sampled at 2 kHz and filtered at 1 kHz by an Axoclamp 200B (Axon Instruments). Capacitance currents were electronically compensated at the beginning of each experiment. Due to the linear nature of the IV-curve, leak correction was not applied.
To study SK currents on CHO cells, cells were held at −40 mV and a family of 200 ms test voltage pulses were applied starting from −100 to +130 mV in 10 mV increments every 2 sec. The amplitude, as measured for the current-voltage relationship, was defined as the maximal outward current at a given depolarizing potential.
Inhibitors were added directly to the bath solution.
















Bath

pipette




solution:

solution:











mM

mM
















KCl
30
KCl
140



NaCl
110



MgCl2
1
MgCl2
1



CaCl2
1
CaCl2
0.1



HEPES
10
HEPES
10












pH 7.2

pH 7.2




(with NaOH)

(with KOH)



295 mOsm

280 mOsm










Results
Experiment: Test of DmSK expression in CHO cells (Clone SK) FIG. 83
Conclusion:
CHO cells transfected with the DmSK gene (clone SK) show functional expression of the channels.
Experiment: Effect of Propafenone on DmSK expressing CHO cells (Clone SK)
Purpose: To confirm the results obtained with the plate-based FlexStation II and FLIPR experimental procedure, cells were subjected to 70 uM Propafenone, under whole-cell patch clamp conditions. FIG. 85 Conclusion:
The DmSK-A2 channels are completely blocked by the addition of 70 uM Propafenone.
15. Xenopus laevis Oocyte Expression of the DmSK Potassium Channel
Purpose: To utilize the oocyte two-electrode voltage clamp expression system to assay functional expression of the voltage-gated Drosophila melanogaster small-conductance calcium-activated potassium channel (SK) within Xenopus laevis oocytes.
Ovarian lobes freshly harvested from a Xenopus laevis frog were ordered from NASCO (Fort Atkinson, Wis.).
Oocytes were isolated with 1.5 mg/ml collagenase Type 1A in 20 ml of calcium-free OR-2 oocyte buffer at room temperature.
Stage V-VI oocytes were injected with 50 nl of in vitro transcribed DmSK RNA (1 ug/ul) using a Drummond Nanoject Injector (pCRII-SK2+4D plasmid linearized with HindIII and transcribed using the Ambion T7 mMessage mMachine transcription kit).
Oocytes were incubated at 18° C. for 2 days in supplemented ND-96.
Channel expression was assayed using two-electrode voltage clamp (borosilicate glass 1.5 mm×1.12 mm filled with 3M KCl) through a Turbo Tec 10C Amplifier (NPI Instruments) connected to an Apple PowerMac G3 via an ADInstruments PowerLab system.
Injected oocytes with resting potentials greater than −30 mV were clamped at +5 mV and perfused with ND-96 bath solution (96 mM NaCl, 2 mM KCl, 1 mM MgCl2, 0.3 mM CaCl2, 5 mM HEPES at pH 7.5 and 230 mOsm) Currents measured were filtered at 2 kHz and with a 50/60 Hz HumBug noise eliminator.
SK channels were activated with intracellular injection of 100 mM cadmium chloride (9.2 nl) using the nanoject while clamped. Measured currents were compared to published results of SK activation within oocytes (FIG. 85).
Results:
Initial evaluation of the functional expression of the DmSK Channel within the oocyte expression system produced outward currents activated by at least three 9.2 nl injections of cadmium chloride inside the oocytes. Compared to the results examined using expression of rat SK (ref. 4), this insect SK channel appears to require more calcium-like activation (28 nl of 100 mM CdCl2).
A reference graph of activity of rat SK channel in oocyte expression system from Grunnet et al. Journal of Neuroscience Methods 2004 is reproduced in FIG. 92.

## Claims

1. A method for identifying an insecticidal active compound that reduces the activity of an insect voltage-gated potassium channel Sha1 (Shaker cognate 1 or Shaker-like) and its accessory protein KChIP (potassium channel-interacting protein) which method comprises:
a) assembling in a membrane polypeptides, originally not present in said membrane, having the activity of an insect voltage-gated potassium channel Sha1 (Shaker cognate 1 or Shaker-like) and its accessory protein KChIP (potassium channel-interacting protein),
b) applying at one side of the membrane the compound suspected of having the ability to inhibit the activity of said polypeptides which are originally not present in said membrane,
c) determining the activity of said polypeptides,
d) identifying the compound applied in (b) that reduces the activity of said polypeptides as an insecticidal active compound; and
e) selecting the compound identified in step d) as the insecticidal active compound;

wherein said polypeptides having the activity of Sha1 are encoded by one or more nucleic acid molecules selected from the group consisting of:
a) a nucleic acid molecule encoding the polypeptide shown in SEQ ID NO: 2, or a polypeptide of a ShaI_delN mutant which is a polypeptide with an N-terminal deletion of amino acids 2-40 of the polypeptide shown in SEQ ID NO: 2;
b) a nucleic acid molecule shown in SEQ ID NO: 1 or a nucleic acid molecule which is a nucleic acid molecule with a deletion of the coding region in SEQ ID NO: 1 that encodes amino acids 2-40 of the polypeptide shown in SEQ ID NO: 2;
c) a nucleic acid molecule encoding a polypeptide having at least 80% identity with the amino acid sequence of the polypeptide encoded by the nucleic acid molecule of (a) to (b) and having the activity of a voltage-gated potassium channel Sha1 (Shaker cognate 1 or Shaker-like); and

wherein said polypeptides having the activity of KChIP are encoded by one or more nucleic acid molecules selected from the group consisting of:
a) a nucleic acid molecule encoding a polypeptide with KChIP activity as shown in SEQ ID NO: 26;
b) a nucleic acid molecule shown in SEQ ID NO: 25;
(c) a nucleic acid molecule encoding a polypeptide having at least 80% identity with the amino acid sequence of the polypeptide encoded by the nucleic acid molecule of (a) to (b) and having the activity of a voltage-gated potassium channel accessory protein KChIP (potassium channel-interacting protein).

2. A method according to claim 1 wherein the polypeptides with the activity of an insect voltage-gated potassium channel Sha1 (Shaker cognate 1 or Shaker-like) and its accessory protein KChIP (potassium channel-interacting protein) are co-expressed in the membrane of a host cell.

3. A method of claim 1 whereby the activity of said polypeptides with the activity of insect voltage-gated potassium channel Sha1 (Shaker cognate 1 or Shaker-like) and its accessory protein KChIP (potassium channel-interacting protein) respectively is determinated electrophysiologically by patch clamp or in a HTS assay.

4. A method of claim 1 wherein the polypeptides with the activity of an insect voltage-gated potassium channel Sha1 (Shaker cognate 1 or Shaker-like) and its accessory protein KChIP (potassium channel-interacting protein) are expressed in a mammalian cell said mammalian cell selected from the group consisting of: CHO-cells and HEK293 cells.

5. A method of claim 1 which comprises:
f) applying to an insect, to a population of insects or to the location wherein said insect is to be controlled an insect-controlling amount a compound identified according to the method of claim 1 and
g) determining of the growth or the viability of said treated insect or population of insects or of insects or population of insects on said location and untreated insect, population of insects or location and
h) selecting of compounds, which reduces the growth or the viability of said treated insect or population of insects or of insects or population of insects on said location following application of the compound of step f).