Patent Publication Number: US-2018030108-A1

Title: Novel voltage-dependent ion channel fusions and method of use thereof

Description:
FIELD OF THE INVENTION 
     The present invention relates to novel voltage-dependent ion channel fusion subunits, and to a functional bioluminescence resonance energy transfer (BRET) assay for screening in real time and characterizing candidate molecules or physical parameters for their ability to activate or inhibit voltage-dependent ion channels. The present invention also relates to a cell-based or cell-free composition comprising at least one voltage-dependent ion channel fusion subunit wherein a bioluminescent donor molecule and/or acceptor molecule are bound to either C-terminal, N-terminal, or to a loop of said channel subunit. The combination of the voltage-dependent ion channel fusion subunit bound to bioluminescent donor and/or fluorescent acceptor molecule enables bioluminescence resonance energy transfer (BRET) to be used to detect change of conformation of the voltage-dependent ion channel subunit, thereby indicating activation or inhibition of said channel. 
     BACKGROUND OF THE INVENTION 
     A large number of central processes in plant and animal cells are wholly or partially controlled via changes in the intracellular concentrations of certain ions, for example the proton concentration, and changes in the membrane potential and the ion gradients across the membrane. Virtually all animal cells are surrounded by a membrane composed of a lipid bilayer with proteins embedded in it. The membrane serves as both an insulator and a diffusion barrier to the movement of ions. Ion transporter/pump proteins actively push ions across the membrane to establish concentration gradients across the membrane, and ion channels allow ions to move across the membrane down those concentration gradients. Ion pumps and ion channels are electrically equivalent to a set of batteries and resistors inserted in the membrane, and therefore create a voltage difference between the two sides of the membrane. Ion channels participate in, and regulate, cellular processes as diverse as the generation and timing of action potentials, energy production, synaptic transmission, secretion of hormones and the contraction of muscles, etc. 
     Voltage-dependent ion channels are membrane proteins that conduct ions at high rates regulated by the voltage across the membrane. They play a fundamental role in the generation and propagation of the nerve impulse and in cell homeostasis. The voltage sensor is a region of the protein bearing charged amino acids that relocate upon changes in the membrane electric field. The movement of the sensor initiates a conformational change in the gate of the conducting pathway thus controlling the flow of ions. The first voltage-dependent ion channel that was isolated and purified was extracted from the eel electroplax where there is a large concentration of Na channels. Several years later, the sequence of the eel Na channel was deduced from its mRNA. The first K channel sequence was deduced from the Shaker mutant of  Drosophila melanogaster . Voltage-dependent ion channel proteins are selective for particular ions. Such ions include, for example, potassium, sodium, and calcium. 
     Voltage-dependent ion channels are present in every cell and are involved in generation of electrical activity and information processing. Accordingly, defect in their function can result in various conditions, such as heart arrhythmias, epilepsy, hypertension, etc. . . . . Many drugs exert their specific effects via modulation of ion channels. Examples include antiepileptic compounds which block voltage-dependent sodium channels in the brain, antihypertensive drugs which block voltage-dependent calcium channels in smooth muscle cells, and some stimulators of insulin release which block ATP-regulated potassium channels in the pancreas. Recent developments now allow ion channels, to be significantly more promising targets for drug discovery. Novel ion channels have been identified and are being targeted for the treatment of cancer, immune disorders and other diseases, thereby expanding the existing ion channel drug disease set well beyond CNS and cardiovascular disorders. 
     The amino acid sequence of a voltage-dependent ion channel protein across species is highly conserved. The proteins that function as voltage-gated ion channels have three remarkable properties that enable cells to conduct an electric impulse: (1) opening in response to changes in the membrane potential (voltage gating); (2) subsequent channel closing and inactivation; and (3) like all ion channels, exquisite specificity for those ions that will permeate and those that will not. Voltage-gated K +  channels, such as the Shaker protein from  Drosophila , are assembled from four similar subunits, each of which has six membrane-spanning a helices and a nonhelical P segment that lines the ion pore. The S4 α helix in each subunit acts as a voltage sensor. Voltage-gated Na +  and Ca 2+  channels are monomeric proteins containing four homologous domains each similar to a K + -channel subunit. The ion specificity of channel proteins is due mainly to coordination of the selected ion with specific residues in the P segments, thus lowering the activation energy for passage of the selected ion compared with other ions. Voltage-sensing a helices have a positively charged lysine or arginine every third or fourth residue. Their movement outward across the membrane, in response to a membrane depolarization of sufficient magnitude, causes opening of the channel. Voltage-gated K + , Na + , and Ca 2+  channel proteins contain one or more cytosolic domains that move into the open channel thereby inactivating it. It is postulated that voltage-gated channel proteins and possibly all K +  channels evolved from a common ancestral gene. 
     Among the voltage-dependent ion channels, the transient receptor-potential (TRP) ion channels, are garnering interest both as mediators of sensory signals and, more recently, as novel drug targets. There are six TRP subfamilies, the main ones being TRPM, TRPV and TRPC. The TRPM subfamily has been called the most novel of the TRP subfamilies, given the potential roles of TRPM channels in cell division, cell migration, and calcium signaling. For example, TRPM1 and TRPM8 show promise as oncology targets, while TRPM2 and TRPM4 are touted as novel targets for immune disorders. It is thought that most TRPs function as homotetramers. The formation of heteromultimeric channels between members of the same subfamily or different subfamilies has been described in several cases (such as between the TRPCs), and this could potentially create a wide variety of channels. A typical TRP protein contains six putative transmembrane segments (S1 to S6) with a pore-forming reentrant loop between S5 and S6. Intracellular amino and carboxyl termini are variable in length and consist of a variety of domains. The large intracellular domain can be seen as a ‘nested box’ structure: a ‘wire frame’ outer shell acts as a sensor for activators and modulators, and a globular inner chamber might modulate ion flow. Another feature in the amino termini of many TRPs is the presence of ankyrin repeats, 33-residue motifs consisting of pairs of antiparallel α-helices connected by β-hairpin motifs. The number of repeats in the ankyrin repeat domain (ARD) can vary between different TRPs: 3 to 4 in TRPCs, 6 in TRPVs, 14 to 15 in TRPAs and about 29 in TRPNs. Functionally, ARD seems to be connected with tetramerization of the channel and interactions with ligands and protein partners. 
     Amongst the other candidates, voltage-gated sodium channel is a protein embedded in the plasma membrane. This type of protein is found in the nerve and muscle cells and is used in the rapid electrical signaling found in these cells. The principle subunit of the voltage-gated sodium channel is a polypeptide chain of more than 1800 amino acids. When the amino acid sequence of any protein embedded in a membrane is examined, typically one or more segments of the polypeptide chain are found to be comprised largely of amino acids with non-polar side chains. Each of these segments coils is what is called a transmembrane domain, with a length approximately the width of the membrane. Moreover, within a transmembrane domain the side chains necessarily face outward where they readily interact with the lipids of the membrane. By contrast, the peptide bonds, which are quite polar, face inward, separated from the lipid environment of the membrane. In the case of the voltage-gated sodium channel, there are 24 such transmembrane domains in the polypeptide chain. Sodium channels comprise of one pore-forming a subunit, which may be associated with either one or two β subunits. α-Subunits consist of four homologous domains (I-IV), each containing six transmembrane segments (S1-56) and a pore-forming loop. The positively charged fourth transmembrane segment (S4) acts as a voltage sensor and is involved in channel gating. The crystal structure of the bacterial NavAb channel has revealed a number of novel structural features compared to earlier potassium channel structures including a short selectivity filter with ion selectivity determined by interactions with glutamate side chains. Interestingly, the pore region is penetrated by fatty acyl chains that extend into the central cavity which may allow the entry of small, hydrophobic pore-blocking drugs. Auxiliary β1, β2, β3 and β4 subunits consist of a large extracellular N-terminal domain, a single transmembrane segment and a shorter cytoplasmic domain. 
     Voltage-gated calcium (Ca 2+ ) channels are key transducers of membrane potential changes into intracellular Ca 2+  transients that initiate many physiological events. Ca 2+  channels purified from skeletal muscle transverse tubules are complexes of α1, α2, β, γ, and δ subunits. The α1 subunit is a protein of about 2000 amino acid residues in length with an amino acid sequence and predicted transmembrane structure like the previously characterized, pore-forming a subunit of voltage-gated sodium channels. The amino acid sequence is organized in four repeated domains (I-IV), which each contains six transmembrane segments (S1-S6) and a membrane-associated loop between transmembrane segments S5 and S6. The intracellular β subunit has predicted α helices but no transmembrane segments, whereas the γ subunit is a glycoprotein with four transmembrane segments, the cloned α2 subunit has many glycosylation sites and several hydrophobic sequences, whilst the δ subunit is encoded by the 3′ end of the coding sequence of the same gene as the α2 subunit, and the mature forms of these two subunits are produced by posttranslational proteolytic processing and disulfide linkage. 
     The sperm-specific CatSper channel controls the intracellular Ca 2+  concentration ([Ca 2+ ] i ) and, thereby, the swimming behaviour of sperm. These are putative 6TM, voltage-gated, calcium permeant channels that are presumed to assemble as a tetramer of a-like subunits and mediate the current. CatSper subunits are structurally most closely related to individual domains of voltage-activated calcium channels (Ca v ). CatSper1, CatSper2 and CatSpers 3 and 4, in common with a recently identified putative 2TM auxiliary CatSperβ protein and two putative 1TM associated CatSperγ and CatSperδ proteins, are restricted to the testis and localised to the principle piece of sperm tail. 
     Two-pore channel is a small family of 2 members putatively forming cation-selective ion channels. They are predicted to contain two K v -style six-transmembrane domains, suggesting they form a dimer in the membrane. These channels are closely related to CatSper channels and, more distantly, to TRP channels. 
     Cyclic nucleotide-regulated channels or cyclic nucleotide-gated channels (CNG) are responsible for signaling in the primary sensory cells of the vertebrate visual and olfactory systems. CNG channels are voltage-independent cation channels formed as tetramers. Each subunit has 6TM, with the pore-forming domain between TM5 and TM6. CNG channels were first found in rod photoreceptors, where light signals through rhodopsin and transducin to stimulate phosphodiesterase and reduce intracellular cGMP level. This results in a closure of CNG channels and a reduced ‘dark current’. Similar channels were found in the cilia of olfactory neurons and the pineal gland. The cyclic nucleotides bind to a domain in the C terminus of the subunit protein. Within this category, hyperpolarisation-activated, cyclic nucleotide-gated (HCN) channels are cation channels that are activated by hyperpolarisation at voltages negative to ˜−50 mV. The cyclic nucleotides cAMP and cGMP directly activate the channels and shift the activation curves of HCN channels to more positive voltages, thereby enhancing channel activity. HCN channels underlie pacemaker currents found in many excitable cells including cardiac cells and neurons. In native cells, these currents have a variety of names, such as I h , I q  and I f . The four known HCN channels have six transmembrane domains and form tetramers. It is believed that the channels can form heteromers with each other, as has been shown for HCN1 and HCN4. 
     Potassium channels function to conduct potassium ions down their electrochemical gradient, doing so both rapidly (up to the diffusion rate of K +  ions in bulk water) and selectively (excluding, most notably, sodium despite the sub-angstrom difference in ionic radius). Biologically, these channels act to set or reset the resting potential in many cells. In excitable cells, such as neurons, the delayed counterflow of potassium ions shapes the action potential. Potassium channels have a tetrameric structure in which four identical protein subunits associate to form a fourfold symmetric (C 4 ) complex arranged around a central ion conducting pore (i.e., a homotetramer). Alternatively four related but not identical protein subunits may associate to form heterotetrameric complexes with pseudo C 4  symmetry. All potassium channel subunits have a distinctive pore-loop structure that lines the top of the pore and is responsible for potassium selective permeability. Potassium ion channels remove the hydration shell from the ion when it enters the selectivity filter. The selectivity filter is formed by a five residue sequence, TVGYG, termed the signature sequence, within the P loop of each subunit. This signature sequence is highly conserved, with the exception that an isoleucine residue in eukaryotic potassium ion channels often is substituted with a valine residue in prokaryotic channels. This sequence in the P-loop adopts a unique structure, having their electro-negative carbonyl oxygen atoms aligned toward the centre of the filter pore and form a square anti-prism similar to a water-solvating shell around each potassium binding site. The hydrophobic region of the channel is used to neutralize the environment around the potassium ion so that it is not attracted to any charges. In turn, it speeds up the reaction. A central pore or a central cavity, 10 Å wide, is located near the center of the transmembrane channel, where the energy barrier is highest for the transversing ion due to the hydrophobity of the channel wall. The water-filled cavity and the polar C-terminus of the pore helices ease the energetic barrier for the ion. Repulsion by preceding multiple potassium ions is thought to aid the throughput of the ions. The presence of the cavity can be understood intuitively as one of the channel&#39;s mechanisms for overcoming the dielectric barrier, or repulsion by the low-dielectric membrane, by keeping the K +  ion in a watery, high-dielectric environment. 
     In 2006, Professor Okumura discovered voltage-gated hydrogen ion channels (proton channels), a voltage dependent membrane protein consisting of two modules: the voltage sensor and phosphatase. These ion channel molecules form conduction pathways for hydrogen ions in the cell membrane; however, they are not always open, opening only when the voltage sensor detects electric signals. The structure showed a ‘closed Wagasa (Japanese umbrella)’ shape with a long helix running through the cell membrane to the cytoplasm and featured a wide inner-accessible vestibule. Voltage sensing amino acids on the protein were located below the phenylalanine-containing “gascket” region in the center of membrane, indicating that structure was in resting state. 
     It follows naturally that detailed structural elucidation of ion channels and the development of high-throughput screening techniques will accelerate the discovery of effective and selective new ion channel drugs. Traditional screening methods (fluorescent dyes, radiometric flux, and binding assays) are indirect measures of ion channel activity. Electrophysiological techniques combined with pharmacology provide the most detailed and direct way to study ion channel function; however, conventional electrophysiological techniques do not meet the throughput demands of large compound library screening. New technologies, including automated electrophysiology and planar patch-clamp techniques, have also been tried and tested. However these methods are not yet performing at the high throughput speeds desired. 
     A major limitation of the patch clamp technique is its low throughput. Typically, a single, highly trained operator can test fewer than ten compounds per day using the patch clamp technique. Furthermore the technique is not easily amenable to automation, and produces complex results that require extensive analysis. 
     Førster resonance energy transfer (FRET), or simply resonance energy transfer (RET), is the non-radiative transfer of energy from an excited state donor molecule to a ground state acceptor molecule. Energy transfer efficiency is dependent on the distance between the donor and acceptor, the extent of the spectral overlap and the relative orientation of the acceptor and donor dipoles. In most cases both the fluorescent donor and acceptor are engineered variants of green fluorescent protein (GFP) from Aequoria victoria. The most widely used FRET pair is cyan fluorescent protein (CFP) as the donor alongside yellow fluorescent protein (YFP) as the acceptor and this FRET system has previously been used to quantify direct ligand binding by a number of GPCRs. 
     In bioluminescence resonance energy transfer (BRET), the donor fluorophore of FRET is replaced with a luciferase and the acceptor can be any suitable fluorophore. The use of a luciferase avoids the need for illumination as the addition of a substrate initiates bioluminescent emission and hence resonance energy transfer. FRET with odorant receptors has only previously been demonstrated for Class A (a2-adrenergic and parathyroid hormone), and Class B (secretin) GPCRs. 
     The main disadvantages of FRET, as opposed to BRET, are the consequences of the required excitation of the donor with an external light source. BRET assays show no photo bleaching or photoisomerization of the donor protein, no photodamage to cells, and no light scattering or autofluorescence from cells or microplates, which can be caused by incident excitation light. In addition one main advantage of BRET over FRET is the lack of emission arising from direct excitation of the acceptor. This reduction in background should permit detection of interacting proteins at much lower concentrations than it is possible for FRET. The present inventors have surprisingly found that bioluminescence resonance energy transfer (BRET) is particularly efficient to detect a target compound using voltage-dependent ion channel fusion subunits. 
     The present inventors have thus developed an advanced, non-destructive, cell-based or cell-free composition real time assay technology that is perfectly suited for voltage-dependent ion channel receptor research, with specific emphasis on drug screening and further mapping of signal transduction pathways. The bioluminescence resonance energy transfer (BRET) assay optimized by present inventors for voltage dependent ion channel receptor research, takes advantage of a naturally occurring phenomenon, namely, the Førster resonance energy transfer between a luminescent donor and a fluorescent acceptor. The transfer efficiency depends on the degree of the spectral overlap, the relative orientation, and the distance between the donor and acceptor. 
     Since the fluorescent energy transfer of the invention is based on stimulatory principles such as BRET, a biosensor as described herein based on FRET instead of BRET would also be expected to function well and is included within the scope of the present invention. 
     SUMMARY OF THE INVENTION 
     The present invention relates to novel nucleic acids encoding voltage-dependent ion channel fusion subunit comprising one or more bioluminescent donor molecule and/or one or more fluorescent acceptor molecules. According to the present invention, voltage-dependent ion channel subunit may be voltage-dependent cation channel or voltage-dependent anion channel. 
     The present invention also relates to expression vectors comprising nucleotide sequences encoding the voltage-dependent channel fusion subunits, recombinant cells comprising such expression vectors, process for the production of voltage-dependent ion channel subunit, as well as to the voltage-dependent fusion subunits per se. 
     This invention further relates to a method of identifying a compound or a candidate capable of binding to a target domain of a voltage-dependent ion channel by providing a nucleic acid comprising a nucleotide sequence encoding a voltage-dependent ion channel fusion subunit bound to a nucleotide sequence encoding at least one bioluminescent donor molecule and/or bound to a nucleotide sequence encoding at least one fluorescent acceptor molecule. Given the complex structure of these voltage-dependent ion channels which can comprise from 2 to 24 transmembrane domains, it was surprising that such a fusion subunit with BRET donor and acceptor tags would retain structural and functional integrity. In a preferred embodiment of the present invention, the voltage-dependent ion channel subunits may comprise subunit of a voltage-dependent anion channel. In a further preferred embodiment, the voltage-dependent ion channel subunits may comprise subunit of a voltage-dependent cation channel. These channels can be of any source as long as when expressed in a cell, the N-terminus and C-terminus are intracytoplasmic. Examples include, but are not limited to, mammalian receptors, or biologically active variants or fragments thereof. 
    
    
     
       BRIEF DESCRIPTION OF THE FIGURES 
         FIG. 1 : (A) is a schematic representation of a non-radiative transfer of energy between a donor and an acceptor.  FIG. 1(B)  is a schematic showing the overlapping of the wave lengths between donor and acceptor molecules.  FIG. 1(C)  is a schematic of the efficacy of the transfer function of the distance (r) between donor and acceptor molecules.  FIG. 1(C)  also presents the equation representing the efficacy of the transfer of energy E, wherein R 0  designates the distance of Førster for a specific donor-acceptor couple and is defined as being the distance allowing 50% of the maximal efficacy of transfer. 
         FIG. 2 : is a schematic representation of the intermolecular fluorescent probes (A) and intramolecular fluorescent probes (B). The donor molecule is represented as “D”, the acceptor is represented as “A” and the molecules of interest are “T1”, “T2”, or “T3”. 
         FIG. 3 : represents an example of use of an intramolecular BRET probe based on a voltage-dependent ion channel subunit according to the present invention. (A) Depending on the state of activation of the canal, the distance and/or the orientation between the N- and C-terminal ends of the voltage-dependent ion channel subunit were modified. (B) is a diagram of the spectra obtained in absence and in presence of BRET signal; Grey shadings within the graphs are representative of the light intensity of the acceptor on the right and the donor on the left. 
         FIG. 4 : represents the cloning strategy used to obtain expression plasmid of fusion channel subunit YFP-hTRPV1-rLuc. After amplification of the cDNA encoding hTRPV1 with appropriate primers, the amplicon is inserted in fusion in-between YFP and rLuc in the restriction site Age I of an existing expression vector where YFP and rLuc are already cloned in fusion (A). The resulting expression vector is represented in the  FIG. 4(B) . The same strategy was used to clone hTRPV3 fusion subunit with YFP and rLuc, except that the insertion was made in the restriction site EcoRI. 
         FIG. 5 : shows the evolution of the BRET signal over temperature in HEK293T cells transfected with YFP-hTRPV1-rLuc in control conditions (diamonds, n=10), in presence of 6 iodo-CAPS (squares, n=3) or with 2,2-Diphenyltetrahydrofuran (triangles, n=1). Δ BRET  represented the variation of BRET ratio over initial measure at 29.5° C. 
         FIG. 6 : shows the dose-response of capsaicin effect at 37° C. on BRET signal in HEK293T cells transfected with the probe YFP-hTRPV1-rLuc (n=3). 5 minutes before readings, cells were pre-incubated in presence of DMSO (as a control, square symbols), or either AMG517 (triangle symbols) or Capsazepine (CPZ) (diamond symbols), two well-known inhibitors of TRPV1. 
         FIG. 7 : shows the measure of Ca 2+  at 37° C. in presence of 1 μM of capsaicin injected after 100 sec (A) or 20 sec (B) in HEK293T cells transfected with hTRPV1 or with YFP-hTRPV1-rLuc (A) or with empty vector (B) and loaded with Fura 2. 
         FIG. 8 : shows the kinetic of evolution of the BRET signal in HEK293T cells transfected with YFP-hTRPV3-rLuc before and after injection of 2-aminoethyl diphenylborinate (2-APB) in the culture media. 
         FIGS. 9  (A) and (B): are schematic representations of the BRET tests used in the study with the YFP-TRPV1-Luc intramolecular BRET probe in A and the intermolecular BRET probe between TRPV1 and Calmodulin in B. YFP and Luc are fused to either N- or C-terminal of either TRPV1 or Calmodulin as indicated in the  FIG. 9 . Following activation of TRPV1, the distance d between Luc and YFP is expected to be modified. C and D, kinetic measurement of the effect of CAPS injection on cells expressing the YFP-TRPV1-Luc BRET probe (C) or the TRPV1-Luc/YFP-CaM constructs. The star indicates the time of CAPS injection. The time constant Tau is indicated. 
         FIGS. 10(A)  and (B): are dose-response curves of CAPS measured in HEK293T cells expressing the YFP-TRPV1-Luc BRET probe (A) or the TRPV1-Luc/YFP-CaM constructs (B). Before activation with CAPS, the cells were pre-incubated with either PBS, DMSO (vehicule), Capsazepine 1 μM (CPZ) or AMG517 1 μM.  FIG. 10(B)  shows the dose-response of capsaicin effect at 37° C. on BRET signal in HEK293T cells transfected with the probe YFP-hTRPV1-rLuc (n=3). 5 minutes before readings, cells were pre-incubated in presence of DMSO (as a control, square symbols), or either AMG517 (triangle symbols) or Capsazepine (CPZ) (diamond symbols), two well-known inhibitors of TRPV1. 
         FIGS. 11  (A)-(D): show the effect of temperature on the pharmacological properties of TRPV1 and its activation, measured by BRET. Efficacy and potency of CAPS on TRPV1 activation were measured by BRET when HEK293T cells expressing either the YFP-TRPV1-Luc BRET probe (A) or the TRPV1-Luc/YFP-CaM constructs (C), were incubated at 25° C., 31° C., 37° C. or 42° C. BRET signal was also recorded in real-time when the cell culture medium was heated from 25° C. to 50° C. ( FIGS. 11  B and D).  FIG. 11  (B) represents the evolution of the BRET signal in HEK293T cells expressing the YFP-TRPV1-Luc BRET probe in presence (empty diamonds), or absence (black circle) of 1 μM of CPZ (B). The evolution of the signal in cells expressing the TRPV1-Luc/YFP-CaM constructs is shown in (C, diamonds). In (C), the effects of calcium depletion in the extracellular medium (square) or the pre-incubation of cells with the TRPV1 inhibitor CPZ (circle) were also tested. 
         FIG. 12 : is a kinetic of the effect of 1 mM of either CARVACROL (circle) or 2-APB (square) on HEK293T cells expressing the YFP-TRPV3-Luc BRET probe. 
         FIGS. 13  (A) and (B): are dose response curves of the effect of CARVACROL (B) and 2-APB (A) on HEK293T cells expressing the TRPV3-Luc/YFP-CaM constructs. 
         FIGS. 14  (A) and (B): are kinetic (A) and a dose response curve (B) of the effect of the TRPV4 agonist GSK1016790A on HEK293T cells expressing the TRPV4-Luc/YFP-CaM constructs. In  FIG. 14(A)  cells were activated with 1 μM of agonist at the time indicated with an arrow. In  FIG. 14(B) , the test was done in presence (square) or absence (circle) of the TRPV inhibitor Ruthenium Red (10 μM). 
     
    
    
     DETAILED DESCRIPTION OF THE INVENTION 
     Unless specifically defined otherwise, all technical and scientific terms used herein shall be taken to have the same meaning as commonly understood by one of ordinary skill in the art (e.g., in cell culture, donor, acceptor, voltage-dependent ion channel, fusion subunits). 
     Unless otherwise indicated, the recombinant protein, cell culture, and molecular cloning techniques utilized in the present invention are standard procedures, well known to those skilled in the art. Such techniques are described and explained throughout the literature in sources such as, J. Perbal, A Practical Guide to Molecular Cloning, John Wiley and Sons (1984), J. Sambrook et al., Molecular Cloning: A Laboratory Manual, Cold Spring Harbour Laboratory Press (1989), T. A. Brown (editor), Essential Molecular Biology: A Practical Approach, Volumes 1 and 2, IRL Press (1991), D. M. Glover and B. D. Hames (editors), DNA Cloning: A Practical Approach, Volumes 1-4, IRL Press (1995 and 1996), and F. M. Ausubel et al. (editors), Current Protocols in Molecular Biology, Greene Pub. Associates and Wiley-Interscience (1988, including all updates until present), Ed Harlow and David Lane (editors) Antibodies: A Laboratory Manual, Cold Spring Harbour Laboratory, (1988), and J. E. Coligan et al. (editors) Current Protocols in Immunology, John Wiley &amp; Sons (including all updates until present). 
     As used herein, the terms bioluminescent or fluorescent donor molecule refer to any molecule able to generate luminescence following either action on a suitable substrate, or its own excitation by an external source. 
     As used herein, the term substrate refers to any molecule that can be used in conjunction with a bioluminescent molecule to generate or absorb luminescence. 
     As used herein, the phrase allowing the bioluminescent or fluorescent donor molecule to modify the substrate refer to any enzymatic activity of the bioluminescent protein on the substrate that produces energy. 
     As used herein, the term acceptor molecule refers to any compound which can accept energy emitted as a result of the activity of a bioluminescent or fluorescent donor molecule, and re-emit it as light energy. 
     As used herein, bioluminescent resonance energy transfer (BRET) is a proximity assay based on the non-radiative transfer of energy between the bioluminescent donor molecule and the fluorescent acceptor molecule. As used herein, fluorescent resonance energy transfer (FRET) is a proximity assay based on the non-radiative transfer of energy between the fluorescent donor molecule and the fluorescent acceptor molecule. 
     As used herein, the term “modulate or modulation” or variations thereof refer to an alteration in the intensity and/or emission spectra of the bioluminescent donor and/or acceptor molecule. 
     As used herein, the term “spatial location” refers to the three dimensional positioning of the bioluminescent donor molecule relative to the acceptor molecule which changes as a result of the compound binding to the voltage-dependent ion channel or of the change in specific physical parameters. 
     As used herein, the term “dipole orientation” refers to the direction in three-dimensional space of the dipole moment associated either with the bioluminescent donor molecule and/or the acceptor molecule relative to their orientation in three-dimensional space. The dipole moment is a consequence of a variation in electrical charge over a molecule. 
     As used herein, the term “more sensitive” refers to a greater change in resonance energy transfer ratio between the ligand unbound form to the ligand bound form of one reporter system (for example, BRET) to another reporter system (for example, FRET). 
     As used herein, the term “contacting” refers to the addition of a candidate molecule and/or a sample capable of activating or inhibiting the voltage-dependent ion channel. The sample can be any substance or composition suspected of comprising a compound to be detected. 
     Voltage-dependent ion channels are a proven target for drug discovery, and many ion channel modulators are currently in clinical use for the treatment of pain, epilepsy, hypertension and other disease states. They comprise the molecular basis for essential physiological functions including fluid secretion, electrolyte balance, and bioenergetics and membrane excitability. Ion channels make good drug targets because they are physiologically essential, are pharmacologically accessible, are encoded by a variety of genes and usually operate as multimeric protein assemblies, resulting in a high degree of functional and anatomical specificity. Through molecular cloning, heterologous expression and electrophysiological characterization by patch-clamping, it is clear that the complexity of ion-channel biology also offers multiple opportunities for small-molecule drugs to achieve a specific, desired functional effect. For example, small molecules might influence a variety of biophysical properties of ion channels, such as voltage-dependence permeability, use-dependence, activation and inactivation. In contrast to simple blockers or openers, the discovery of modulatory compounds could allow the development of drugs that specifically act on cells or tissues exhibiting aberrant levels of ion-channel activity. 
     Modulators are any agents capable of altering the functional activity of voltage-dependent ion channel within a cell, and may represent a channel opener (functional agonist), a channel blocker (functional antagonist) or an agent that alters the level of expression of the channel at the cell membrane. An increase in expression will lead to greater numbers of voltage-dependent ion channels, which will lead to an increase in overall activity. Conversely, a decrease in expression will lead to a smaller overall activity. The major challenge in development of voltage-dependent ion channel modulators is to achieve selectivity for a specific target voltage-dependent ion channel over other related voltage-dependent ion channel subtypes, and for channels in the target tissue. 
     Voltage-dependent ion channels are known to exist in a number of different conformational states, referred to as gating states. For voltage-gated ion channels a channel can be viewed as residing in one of 3 gating states-closed (no ion permeation), open (ion flux occurs) and inactivated (no ion permeation; channel cannot be opened by depolarization), although it should be noted that some channels do not exhibit an inactivated state. Transition between gating states is voltage-dependent, and at any given time, equilibrium exists between these gating states, with the proportion of channels residing in each state depending upon the cellular membrane potential. 
     Many voltage-dependent ion channel modulators have been shown to bind preferentially to a specific gating state or states. For example, the voltage-gated sodium channel blocker Lamotrigine is thought to bind to the opened and inactivated states of the brain sodium channel protein. Preferential binding to a particular gating state may occur through an increase in channel affinity for the ion channel modulator, or simply through improved access of the drug to its binding site on the channel. 
     As used herein the terms “voltage-dependent ion channel fusion subunit” comprise a voltage-dependent ion channel subunit coupled or associated with at least a bioluminescent donor molecule and at least a fluorescent acceptor molecule. The bioluminescent donor molecule and the fluorescent acceptor molecule are preferably covalently attached, more preferably produced as a fusion protein with a subunit of the voltage-dependent ion channel or may be optionally coupled or fused to the subunit via a linker sequence. The bioluminescent donor molecule and the fluorescent acceptor molecule may be associated, either covalently attached or produced as a fusion protein to the same subunit or to different subunits of the same voltage-dependent ion channel. Alternatively, the bioluminescent donor molecule and the fluorescent acceptor molecule may be associated, either covalently attached or produced as a fusion protein of subunits of same or different subfamily of voltage-dependent ion channel. In addition, multiple combinations of bioluminescent donor molecule and the fluorescent acceptor molecule may be used to increase the sensitivity of the detection of tested compound candidates. 
     The bioluminescent donor molecule and the fluorescent acceptor molecule may completely replace or be inserted inside a specified region of the voltage-dependent ion channel. As the skilled person in the art will appreciate, the bioluminescent donor molecule and the fluorescent acceptor molecule are not inserted such that it makes the voltage-dependent ion channel inactive as result in a spatial change to the location and/or dipole orientation of the bioluminescent donor molecule relative to the fluorescent acceptor molecule. 
     By “substantially purified” or “purified” we mean a voltage-dependent channel subunit that has been separated from one or more lipids, nucleic acids, other polypeptides, or other contaminating molecules with which it is associated in its native state. It is preferred that the substantially purified polypeptide is at least 60% free, more preferably at least 75% free, and more preferably at least 90% free from other components with which it is naturally associated. However, at present there is no evidence that the polypeptides of the invention exist in nature. 
     The term “recombinant” in the context of a polypeptide refers to the polypeptide when produced by a cell, or in a cell-free expression system, in an altered amount or at an altered rate compared to its native state. In one embodiment, the cell is a cell that does not naturally produce the polypeptide. However, the cell may be a cell which comprises a non-endogenous gene that causes an altered, preferably increased, amount of the polypeptide to be produced. A recombinant polypeptide of the invention includes polypeptides which have not been separated from other components of the transgenic (recombinant) cell, or cell-free expression system, in which it is produced, and polypeptides produced in such cells or cell-free systems which are subsequently purified away from at least some other components. 
     The terms “polypeptide” and “protein” are generally used interchangeably and refer to a single polypeptide chain which may or may not be modified by addition of non-amino acid groups. It would be understood that such polypeptide chains may associate with other polypeptides or proteins or other molecules such as co-factors. The terms “proteins” and “polypeptides” as used herein also include variants, mutants, biologically active fragments, modifications, analogous and/or derivatives of the polypeptides described herein. 
     The % identity of a polypeptide is determined by GAP analysis (GCG program) with a gap creation penalty of 5, and a gap extension penalty of 0.3. The query sequence is at least 25 amino acids in length, and the GAP analysis aligns the two sequences over a region of at least 25 amino acids. More preferably, the query sequence is at least 50 amino acids in length, and the GAP analysis aligns the two sequences over a region of at least 50 amino acids. More preferably, the query sequence is at least 100 amino acids in length and the GAP analysis aligns the two sequences over a region of at least 100 amino acids. Even more preferably, the query sequence is at least 250 amino acids in length and the GAP analysis aligns the two sequences over a region of at least 250 amino acids. Even more preferably, the GAP analysis aligns the two sequences over their entire length. 
     As used herein, a “biologically active fragment” is a portion of a polypeptide as described herein, which maintains a defined activity of the full-length polypeptide. For example, a biologically active fragment of a voltage-dependent ion subunit must be capable of binding the target compound, resulting in a conformational change. Biologically active fragments can be any size as long as they maintain the defined activity. Preferably, biologically active fragments are at least 150, more preferably at least 250 amino acids in length. 
     As used herein, a “biologically active variant” is a molecule which differs from a naturally occurring and/or defined molecule by one or more amino acids but maintains a defined activity, such as defined above for biologically active fragments. Biologically active variants are typically least 50%, more preferably at least 80%, more preferably at least 90%, more preferably at least 95%, more preferably at least 97%, and even more preferably at least 99% identical to the naturally occurring and/or defined molecule. 
     With regard to a defined polypeptide or polynucleotide, it will be appreciated that % identity figures higher than those provided above will encompass preferred embodiments. Thus, where applicable, in light of the minimum % identity figures, it is preferred that the polypeptide or polynucleotide comprises an amino acid sequence which is at least 50%, more preferably at least 60%, more preferably at least 70%, more preferably at least 80%, more preferably at least 85%, more preferably at least 90%, more preferably at least 91%, more preferably at least 92%, more preferably at least 93%, more preferably at least 94%, more preferably at least 95%, more preferably at least 96%, more preferably at least 97%, more preferably at least 98%, more preferably at least 99%, more preferably at least 99.1%, more preferably at least 99.2%, more preferably at least 99.3%, more preferably at least 99.4%, more preferably at least 99.5%, more preferably at least 99.6%, more preferably at least 99.7%, more preferably at least 99.8%, and even more preferably at least 99.9% identical to the relevant nominated SEQ ID NO. 
     By an “isolated polynucleotide”, including DNA, RNA, or a combination of these, single or double stranded, in the sense or antisense orientation or a combination of both, we mean a polynucleotide which is at least partially separated from the polynucleotide sequences with which it is associated or linked in its native state. Preferably, the isolated polynucleotide is at least 60% free, preferably at least 75% free, and most preferably at least 90% free from other components with which they are naturally associated. 
     In recent years, bioluminescence resonance energy transfer approaches (BRET) have been increasingly used to study protein-protein interactions and drug discovery. Luminescence in general is a phenomenon in which energy is specifically channeled to a molecule to produce an excited state. Return to a lower energy state is accompanied by release of a photon. Luminescence includes fluorescence, phosphorescence, chemiluminescence and bioluminescence. Bioluminescence is the process by which living organisms emit light that is visible to other organisms. Where the luminescence is bioluminescence, creation of the excited state derives from an enzyme catalyzed reaction. Luminescence can be used in the analysis of biological interactions. The dependence of the energy transfer efficacy on the distance between energy donors and acceptors permits real time measurements that are both sensitive and specific to the labeling sites of the proteins thus allowing inference on the dynamic structural changes. 
     One technique for assessing protein-protein interaction is based on fluorescence resonance energy transfer (FRET). The theory of FRET has been described for the first time in 1948 by Théodore Førster. It is defined as a non-radiatif transfer of energy, i.e., a transfer of energy which occurs without emission of photons and which results in a dipole interaction between a donor of energy and an acceptor of energy ( FIG. 1 ). Therefore, in this process, one fluorophore donor transfers its excited-state energy to another fluorophore acceptor. The non-radiative transfer of energy from the donor to the acceptor results in the excitation of the acceptor. The acceptor returns at basal stage by emitting a photon at its own length wave ( FIG. 1A ). 
     According to Førster equation, FRET efficiency depends on five parameters: (i) the overlap—at least a partial overlap—between the absorption spectrum of the second fluorophore and the emission spectrum of the first fluorophore ( FIGS. 1A and 1B ), (ii) the relative orientation between the emission dipole of the donor and the absorption dipole of the acceptor, (iii) the distance between the fluorophores ( FIG. 1B ), (iv) the quantum yield of the donor and the acceptor, and (v) the extinction coefficient of the acceptor. 
     These donor and acceptor fluorophores may thus be used as molecular probes, either intramolecular probes ( FIG. 2 ,  FIG. 9A ) or intermolecular probes ( FIG. 9  B), 
     FRET has been used to assay protein-protein proximity in vitro and in vivo by chemically attaching fluorophores such as fluorescein and rhodamine to pairs of purified proteins and measuring fluorescence spectra of protein mixtures or cells that were microinjected with the labeled proteins. FRET, however, has several limitations. As with any fluorescence technique, photobleaching of the fluorophore and autofluorescence of the cells/tissue can significantly restrict the usefulness of FRET, and, in highly autofluorescent tissues, FRET is essentially unusable. Also, if the excitation light easily damages the tissue, the technique may be unable to give a value for healthy cells. Finally, if the cells/tissues to be tested are photoresponsive (e.g., retina), FRET may be impractical because as soon as a measurement is taken, the photoresponse may be triggered. 
     In order to overcome the above disadvantages and further taking advantage of multiple sites of energy donor and acceptor insertions in the protein-protein complex of interest, the present invention relies on the development of a BRET-based assay that directly monitors real-time interactions between voltage dependent ion channels and potential agonists and antagonists. 
     The BRET method is very similar to that of FRET wherein the donor is a bioluminescent molecule. No external source of excitation is thus required, thereby avoiding inconveniences of photobleaching and autofluorescence of the cells/tissues. The BRET is actually a natural phenomenon which is produced in living organisms such as  Renilla reniformis  and  Aequorea victoria . In those organisms, the luciferase enzyme, which is a bioluminescent protein, performs an oxidative degradation of the substrat ccelenterazin and at the same time emits some light with a peak at 480 nm. In these organisms, the proximity to the Green Fluorescent Protein (GFP) allows a non-radiative transfer of energy. A good acceptor of energy may be the Yellow Fluorescent Protein (YFP) which is excited at 512 nm and emits at 530 nm. The BRET signal is measured by the ratio between the light intensity at the peak of emission of the acceptor and the light intensity at the peak of emission of the donor ( FIG. 3 ). The BRET signal must be subtracted to the signal obtained in presence of luciferase alone in the same experimental conditions in order to obtain the net BRET signal. 
     The present invention recognizes for the first time a BRET analysis of voltage regulated ion channels. The present invention includes instrumentation and methods that provide for the accurate and reliable information generation. An object of the present invention is to provide a screening system that targets ion channels and has superior efficiency. The present invention provides a material for screening for compounds that act on a target ion channel, comprising cells which retain at least one nucleic acid encoding a voltage-dependent channel. 
     The invention also provides a useful tool to probe for conformational changes occurring in the voltage dependent ion channel complexes resulting from ligand binding. As a result, by multiplexing different BRET-biosensors of the voltage dependent ion channel subunits, the invention offers the possibility to set up pharmacological fingerprints that are specific to each receptor ligand, thus allowing differentiating the distinct signaling modes of different ligands toward the various signaling pathways engaged. 
     High-throughput screening for ion-channel function requires sensitive, simple assays and instrumentation that will report ion channel activity in living cells. 
     The present invention thus relates to a nucleic acid or polynucleotide comprising a nucleotide sequence encoding a voltage-dependent ion channel fusion subunit bound to a nucleotide sequence encoding at least one bioluminescent or fluorescent donor molecule and/or bound to a nucleotide sequence encoding at least one fluorescent acceptor molecule. 
     The voltage-dependent ion channel subunit is preferably a subunit of a voltage-dependent cation channel or voltage-dependent anion channel and comprises 2 to 24 transmembrane domains. Mammalian voltage dependent sodium channels have been identified and are well known in the art. According to the present invention, the voltage-dependent ion channels are described in the following Tables 1-11. These include also isoforms or active variants thereof. 
     In a preferred embodiment, voltage-dependent ion channel subunit is a subunit of a voltage-dependent cation channel or voltage-dependent anion channel and comprises 2 to 24 transmembrane domains. 
     
       
         
           
               
             
               
                 TABLE 1 
               
             
            
               
                   
               
               
                 Sodium channels, voltage-gated 
               
            
           
           
               
               
               
               
               
               
            
               
                 Approved 
                   
                 Tissue 
                   
                   
                   
               
               
                 Symbol 
                 Approved Name 
                 distribution 
                 Synonyms 
                 Chromosome 
                 Accession number 
               
               
                   
               
               
                 SCN1A 
                 sodium channel, 
                 CNS 
                 Nav1.1, 
                 2q24.3 
                 P35498, E9PG49 Q9C008, 
               
               
                   
                 voltage-gated, type  
                   
                 GEFSP2, 
                   
                 X03638 X65362, AF003372 
               
               
                   
                 I, alpha subunit 
                   
                 HBSCI, NAC1, 
                   
                   
               
               
                   
                   
                   
                 SMEI 
                   
                   
               
               
                 SCN1B 
                 sodium channel, 
                 CNS-brain, 
                   
                 19q13.12 
                 Q07699, Q5TZZ4, Q6TN97 
               
               
                   
                 voltage-gated, type 
                 skeletal muscle, 
                   
                   
                 M91808, L10338 
               
               
                   
                 I, beta subunit 
                 and heart 
                   
                   
                   
               
               
                 SCN2A 
                 sodium channel, 
                 CNS 
                 Nav1.2, 
                 2q24.3 
                 Q99250, A6NC14 A6NC14, 
               
               
                   
                 voltage-gated, type  
                   
                 HBSCII, HBSCI 
                   
                 X03639, X65361 M94055, X61149 
               
               
                   
                 II, alpha subunit 
                   
                   
                   
                   
               
               
                 SCN2B 
                 sodium channel, 
                 CNS 
                   
                 11q23.3 
                 O60939, O75302, Q9UNN3 
               
               
                   
                 voltage-gated, type  
                   
                   
                   
                 U37026, U37147, AF007783 
               
               
                   
                 II, beta subunit 
                   
                   
                   
                   
               
               
                 SCN3A 
                 sodium channel, 
                 CNS 
                 Nav1.3 
                 2q24 
                 Q9NY46, Q16142 Q9Y6P4, 
               
               
                   
                 voltage-gated, type 
                   
                   
                   
                 Y00766 
               
               
                   
                 III, alpha subunit 
                   
                   
                   
                   
               
               
                 SCN3B 
                 sodium channel, 
                 Heart 
                 HSA243396 
                 11q24.1 
                 Q06W27, A5H1I5, Q17RL3, 
               
               
                   
                 voltage-gated, type  
                   
                   
                   
                 Q9ULR2 
               
               
                   
                 III, beta subunit 
                   
                   
                   
                   
               
               
                 SCN4A 
                 sodium channel, 
                 skeletal 
                 Nav1.4, HYPP, 
                 17q23.3 
                 P35499, Q15478, Q16447, 
               
               
                   
                 voltage-gated, type 
                 muscle- 
                 SkM1 
                   
                 Q7Z6B1 M26643, M81758 
               
               
                   
                 IV, alpha subunit 
                 denervated and 
                   
                   
                   
               
               
                   
                   
                 innervated skeletal 
                   
                   
                   
               
               
                   
                   
                 muscle 
                   
                   
                   
               
               
                 SCN5A 
                 sodium channel, 
                 denervated 
                 Nav1.5, LQT3, 
                 3p21 
                 M27902, M77235, _Q86V90, 
               
               
                   
                 voltage-gated, type  
                 skeletal 
                 HB1, HBBD, 
                   
                 Q14524, A5H1P8, A5H1P8 
               
               
                   
                 V, alpha subunit 
                 muscle, heart 
                 PFHB1, IVF, 
                   
                   
               
               
                   
                   
                   
                 HB2, HH1, 
                   
                   
               
               
                   
                   
                   
                 SSS1, CDCD2, 
                   
                   
               
               
                   
                   
                   
                 CMPD2, ICCD 
                   
                   
               
               
                 SCN7A 
                 sodium channel, 
                 astrocytes PNS 
                 Nav2.1, Nav2.2, 
                 2q21-q23 
                 M96578 Y09164, Q01118 
               
               
                   
                 voltage-gated, type 
                 (DRG) 
                 NaG 
                   
                   
               
               
                   
                 VII, alpha subunit 
                   
                   
                   
                   
               
               
                 SCN8A 
                 sodium channel, 
                 CNS 
                 Nav1.6, NaCh6, 
                 12q13.1 
                 L39018 AF049239A 
               
               
                   
                 voltage gated, type  
                   
                 PN4, CerIII 
                   
                 F049240 U26707 
               
               
                   
                 VIII, alpha subunit 
                   
                   
                   
                 AF049617 AF050736 AF003373 
               
               
                   
                   
                   
                   
                   
                 Q9UQD0, Q9UPB2, B9VWG8 
               
               
                 SCN9A 
                 sodium channel, 
                 medullary 
                 Nav1.7, PN1, 
                 2q24 
                 Q15858 A1BUH5 Q8WWN4, 
               
               
                   
                 voltage-gated, type 
                 thyroid Ca 
                 NE-NA, NENA, 
                   
                 U79568 X82835 U35238 
               
               
                   
                 IX, alpha subunit 
                 Schwann 
                 ETHA 
                   
                   
               
               
                   
                   
                 Cells 
                   
                   
                   
               
               
                   
                   
                 PNS 
                   
                   
                   
               
               
                 SCN10A 
                 sodium channel, 
                 PNS 
                 Nav1.8, hPN3, 
                 3p22.2 
                 Q9Y5Y9, A6NDQ1, X92184 
               
               
                   
                 voltage-gated, type  
                   
                 SNS, PN3 
                   
                 U53833 Y09108 
               
               
                   
                 X, alpha subunit 
                   
                   
                   
                   
               
               
                 SCN11A 
                 sodium channel, 
                   
                 Nav1.9, NaN, 
                 3p22.2 
                 Q9UI33, A6NN05 Q9UHM0 
               
               
                   
                 voltage-gated, type 
                   
                 SNS-2 
                   
                   
               
               
                   
                 XI, alpha subunit 
               
               
                   
               
            
           
         
       
     
     
       
         
           
               
             
               
                 TABLE 2 
               
             
            
               
                   
               
               
                 Calcium channels, voltage-gated 
               
            
           
           
               
               
               
               
               
               
            
               
                 Approved 
                   
                   
                   
                   
                 Accession 
               
               
                 Symbol 
                 Approved Name 
                 Tissue distribution 
                 Synonyms 
                 Chromosome 
                 Number 
               
               
                   
               
               
                 CACNA1A 
                 calcium channel, 
                 Brain specific; mainly 
                 Cav2.1, EA2, 
                 19p13 
                 O00555, J3KP41, Q9UDC4 
               
               
                   
                 voltage-dependent, 
                 found in cerebellum, 
                 APCA, 
                   
                   
               
               
                   
                 P/Q type, alpha  
                 cerebral cortex, 
                 HPCA, FHM 
                   
                   
               
               
                   
                 1A subunit 
                 thalamus and 
                   
                   
                   
               
               
                   
                   
                 hypothalamus. 
                   
                   
                   
               
               
                   
                   
                 Expressed in the small 
                   
                   
                   
               
               
                   
                   
                 cell lung carcinoma 
                   
                   
                   
               
               
                   
                   
                 cell line SCC-9 
                   
                   
                   
               
               
                 CACNA1B 
                 calcium channel, 
                 Isoform Alpha-1b-1 
                 Cav2.2, 
                 9q34 
                 Q00975, B1AQK5 
               
               
                   
                 voltage-dependent, 
                 and isoform Alpha-1b- 
                 CACNN 
                   
                   
               
               
                   
                 N type, alpha 1B 
                 2 are expressed in the 
                   
                   
                   
               
               
                   
                 subunit 
                 central nervous system 
                   
                   
                   
               
               
                 CACNA1C 
                 calcium channel, 
                 Expressed in brain, 
                 Cav1.2, 
                 12p13.3 
                 Q13936, B2RUT3 
               
               
                   
                 voltage-dependent, 
                 heart, jejunum, ovary, 
                 CACH2, 
                   
                 Q99875 
               
               
                   
                 L type, alpha 1C 
                 pancreatic beta-cells 
                 CACN2, TS, 
                   
                   
               
               
                   
                 subunit 
                 and vascular smooth 
                 LQT8 
                   
                   
               
               
                   
                   
                 muscle. Overall 
                   
                   
                   
               
               
                   
                   
                 expression is reduced 
                   
                   
                   
               
               
                   
                   
                 in atherosclerotic 
                   
                   
                   
               
               
                   
                   
                 vascular smooth 
                   
                   
                   
               
               
                   
                   
                 muscle 
                   
                   
                   
               
               
                 CACNA1D 
                 calcium channel, 
                 Expressed in pancreatic 
                 Cav1.3, 
                 3p14.3 
                 Q01668, B0FYA3, Q9UDC3 
               
               
                   
                 voltage-dependent, 
                 islets and in brain, 
                 CACH3, 
                   
                   
               
               
                   
                 L type, alpha 1D 
                 where it has been seen 
                 CACN4 
                   
                   
               
               
                   
                 subunit 
                 in cerebral cortex, 
                   
                   
                   
               
               
                   
                   
                 hippocampus, basal 
                   
                   
                   
               
               
                   
                   
                 ganglia, habenula and 
                   
                   
                   
               
               
                   
                   
                 thalamus. Expressed in 
                   
                   
                   
               
               
                   
                   
                 the small cell lung 
                   
                   
                   
               
               
                   
                   
                 carcinoma cell line 
                   
                   
                   
               
               
                   
                   
                 SCC-9. 
                   
                   
                   
               
               
                 CACNA1E  
                 calcium channel, 
                 Expressed in neuronal 
                 Cav2.3, BII, 
                 1q25.3 
                 Q15878, Q14581, B1AM12 
               
               
                   
                 voltage-dependent, 
                 tissues and in kidney 
                 CACH6 
                   
                   
               
               
                   
                 R type, alpha 1E 
                   
                   
                   
                   
               
               
                   
                 subunit 
                   
                   
                   
                   
               
               
                 CACNA1F 
                 calcium channel, 
                 Expression in skeletal 
                 Cav1.4, JM8, 
                 Xp11.23 
                 O60840, A6NI29, Q9UHB1 
               
               
                   
                 voltage-dependent, 
                 muscle and retina 
                 JMC8, 
                   
                   
               
               
                   
                 L type, alpha 1F 
                   
                 CSNBX2, 
                   
                   
               
               
                   
                 subunit 
                   
                 CORDX3, 
                   
                   
               
               
                   
                   
                   
                 CSNB2A, 
                   
                   
               
               
                   
                   
                   
                 OA2 
                   
                   
               
               
                 CACNA1G 
                 calcium channel, 
                 Highly expressed in 
                 Cav3.1, 
                 17q22 
                 O43497, D6RA64, Q9Y5T3 
               
               
                   
                 voltage-dependent, 
                 brain, in particular in 
                 NBR13 
                   
                   
               
               
                   
                 T type, alpha 1G 
                 the amygdala, 
                   
                   
                   
               
               
                   
                 subunit 
                 subthalamic nuclei, 
                   
                   
                   
               
               
                   
                   
                 cerebellum and 
                   
                   
                   
               
               
                   
                   
                 thalamus. Moderate 
                   
                   
                   
               
               
                   
                   
                 expression in heart; 
                   
                   
                   
               
               
                   
                   
                 low expression in 
                   
                   
                   
               
               
                   
                   
                 placenta, kidney and 
                   
                   
                   
               
               
                   
                   
                 lung. Also expressed in  
                   
                   
                   
               
               
                   
                   
                 colon and bone marrow 
                   
                   
                   
               
               
                   
                   
                 and in tumoral cells to 
                   
                   
                   
               
               
                   
                   
                 a lesser extent. Highly 
                   
                   
                   
               
               
                   
                   
                 expressed in fetal brain, 
                   
                   
                   
               
               
                   
                   
                 but also in peripheral 
                   
                   
                   
               
               
                   
                   
                 fetal tissues as heart, 
                   
                   
                   
               
               
                   
                   
                 kidney and lung, 
                   
                   
                   
               
               
                   
                   
                 suggesting a 
                   
                   
                   
               
               
                   
                   
                 developmentally 
                   
                   
                   
               
               
                   
                   
                 regulated expression 
                   
                   
                   
               
               
                 CACNA1H 
                 calcium channel, 
                 Expressed in kidney, 
                 Cav3.2 
                 16p13.3 
                 O95180, B5ME00, Q9NYY5 
               
               
                   
                 voltage-dependent, 
                 liver, heart, brain. 
                   
                   
                   
               
               
                   
                 T type, alpha 1H 
                 Isoform 2 seems to be 
                   
                   
                   
               
               
                   
                 subunit 
                 testis-specific 
                   
                   
                   
               
               
                 CACNA1I 
                 calcium channel, 
                 Brain specific 
                 Cav3.3 
                 22q13.1 
                 Q9P0X4, B0QY12, Q9UNE6 
               
               
                   
                 voltage-dependent, 
                   
                   
                   
                   
               
               
                   
                 T type, alpha 1I 
                   
                   
                   
                   
               
               
                   
                 subunit 
                   
                   
                   
                   
               
               
                 CACNA1S 
                 calcium channel, 
                 Skeletal muscle 
                 Cav1.1, 
                 1q32 
                 Q13698, A4IF51, Q13934 
               
               
                   
                 voltage-dependent, 
                 specific. 
                 hypoPP 
                   
                   
               
               
                   
                 L type, alpha 1S 
                   
                   
                   
                   
               
               
                   
                 subunit 
               
               
                   
               
            
           
         
       
     
     
       
         
           
               
             
               
                 TABLE 3 
               
             
            
               
                   
               
               
                 Transient receptor potential cation channels 
               
            
           
           
               
               
               
               
               
               
            
               
                 Approved 
                   
                   
                   
                   
                 Accession  
               
               
                 Symbol 
                 Approved Name 
                 Tissue distribution 
                 Synonyms 
                 Chromosome 
                 number 
               
               
                   
               
               
                 TRPA1 
                 transient receptor 
                 Expressed at very low 
                   
                 8q13 
                 O75762, A6NIN6 
               
               
                   
                 potential cation 
                 level in human 
                   
                   
                   
               
               
                   
                 channel, subfamily 
                 fibroblasts and at a 
                   
                   
                   
               
               
                   
                 A, member 1 
                 moderate level in 
                   
                   
                   
               
               
                   
                   
                 liposarcoma cells 
                   
                   
                   
               
               
                 TRPC1 
                 transient receptor 
                 Seems to be ubiquitous 
                 HTRP-1 
                 3q23 
                 P48995, Q14CE4 
               
               
                   
                 potential cation 
                   
                   
                   
                   
               
               
                   
                 channel, subfamily 
                   
                   
                   
                   
               
               
                   
                 C, member 1 
                   
                   
                   
                   
               
               
                 TRPC2 
                 transient receptor 
                   
                   
                 11p15.4 
                 Q6ZNB5 
               
               
                   
                 potential cation 
                   
                   
                   
                   
               
               
                   
                 channel, subfamily 
                   
                   
                   
                   
               
               
                   
                 C, member 2, 
                   
                   
                   
                   
               
               
                   
                 pseudogene 
                   
                   
                   
                   
               
               
                 TRPC3 
                 transient receptor 
                 Expressed 
                   
                 4q27 
                 Q13507, A7VJS1, Q5G1L5 
               
               
                   
                 potential cation 
                 predominantly in brain 
                   
                   
                   
               
               
                   
                 channel, subfamily 
                 and at much lower 
                   
                   
                   
               
               
                   
                 C, member 3 
                 levels in ovary, colon, 
                   
                   
                   
               
               
                   
                   
                 small intestine, lung, 
                   
                   
                   
               
               
                   
                   
                 prostate, placenta and 
                   
                   
                   
               
               
                   
                   
                 testis 
                   
                   
                   
               
               
                 TRPC4 
                 transient receptor 
                 Strongly expressed in 
                 HTRP4 
                 13q13.3 
                 Q9UBN4, B1ALE0, Q9UIB2 
               
               
                   
                 potential cation 
                 placenta. Expressed at 
                 TRP4, 
                   
                   
               
               
                   
                 channel, subfamily 
                 lower levels in heart, 
                   
                   
                   
               
               
                   
                 C, member 4 
                 pancreas, kidney and 
                   
                   
                   
               
               
                   
                   
                 brain. Expressed in 
                   
                   
                   
               
               
                   
                   
                 endothelial cells. 
                   
                   
                   
               
               
                   
                   
                 Isoform alpha was 
                   
                   
                   
               
               
                   
                   
                 found to be the 
                   
                   
                   
               
               
                   
                   
                 predominant isoform. 
                   
                   
                   
               
               
                 TRPC5 
                 transient receptor 
                 Expressed in brain  
                 PPP1R159 
                 Xq23 
                 Q9UL62, B2RP53 
               
               
                   
                 potential cation 
                 with higher levels in  
                   
                   
                 Q9Y514 
               
               
                   
                 channel,  
                 fetal brain. Found  
                   
                   
                   
               
               
                   
                 subfamily C,  
                 in cerebellum and 
                   
                   
                   
               
               
                   
                 member 5 
                 occipital pole 
                   
                   
                   
               
               
                 TRPC6 
                 transient receptor 
                 Expressed primarily in 
                 TRP6 
                 11q22.1 
                 Q9Y210 Q52M59 Q9NQA9 
               
               
                   
                 potential cation 
                 placenta, lung, spleen, 
                   
                   
                   
               
               
                   
                 channel, subfamily 
                 ovary and small 
                   
                   
                   
               
               
                   
                 C, member 6 
                 intestine. Expressed in 
                   
                   
                   
               
               
                   
                   
                 podocytes and is a 
                   
                   
                   
               
               
                   
                   
                 component of the 
                   
                   
                   
               
               
                   
                   
                 glomerular slit 
                   
                   
                   
               
               
                   
                   
                 diaphragm 
                   
                   
                   
               
               
                 TRPC7 
                 transient receptor 
                 Highly expressed in 
                   
                 5q31.2 
                 O94759 D3DSL6 
               
               
                   
                 potential cation 
                 brain and peripheral 
                   
                   
                 Q96Q93_Q9HCX4 A1A4Z4 
               
               
                   
                 channel, subfamily 
                 blood cells, such as 
                   
                   
                 Q8IWP7 
               
               
                   
                 C, member 7 
                 neutrophils. Also 
                   
                   
                   
               
               
                   
                   
                 detected in bone 
                   
                   
                   
               
               
                   
                   
                 marrow, spleen, heart, 
                   
                   
                   
               
               
                   
                   
                 liver and lung. Isoform 
                   
                   
                   
               
               
                   
                   
                 2 is found in neutrophil 
                   
                   
                   
               
               
                   
                   
                 granulocytes 
                   
                   
                   
               
               
                 TRPM1 
                 transient receptor 
                 Expressed in the retina 
                 LTRPC1, 
                 15q13.3 
                 Q7Z4N2 D9IDV2 Q7Z4N5 
               
               
                   
                 potential cation 
                 where it localizes to  
                 CSNB1C 
                   
                   
               
               
                   
                 channel,  
                 the outer plexiform  
                   
                   
                   
               
               
                   
                 subfamily 
                 layer. Highly  
                   
                   
                   
               
               
                   
                 M, member 1 
                 expressed in 
                   
                   
                   
               
               
                   
                   
                 benign melanocytic 
                   
                   
                   
               
               
                   
                   
                 nevi and diffusely 
                   
                   
                   
               
               
                   
                   
                 expressed in various in 
                   
                   
                   
               
               
                   
                   
                 situ melanomas, but not 
                   
                   
                   
               
               
                   
                   
                 detected in melanoma 
                   
                   
                   
               
               
                   
                   
                 metastases. Also 
                   
                   
                   
               
               
                   
                   
                 expressed in 
                   
                   
                   
               
               
                   
                   
                 melanocytes and 
                   
                   
                   
               
               
                   
                   
                 pigmented metastatic 
                   
                   
                   
               
               
                   
                   
                 melanoma cell lines. In 
                   
                   
                   
               
               
                   
                   
                 melanocytes expression 
                   
                   
                   
               
               
                   
                   
                 appears to be regulated 
                   
                   
                   
               
               
                   
                   
                 at the level of 
                   
                   
                   
               
               
                   
                   
                 transcription and 
                   
                   
                   
               
               
                   
                   
                 mRNA processing 
                   
                   
                   
               
               
                 TRPM2 
                 transient receptor 
                 Detected in blood 
                 KNP3, 
                 21q22.3 
                 P10909 B2R9Q1 Q7Z5B9 
               
               
                   
                 potential cation 
                 plasma, cerebrospinal 
                 LTRPC2, 
                   
                   
               
               
                   
                 channel, subfamily 
                 fluid, milk, seminal 
                 NUDT9L1, 
                   
                   
               
               
                   
                 M, member 2 
                 plasma and colon 
                 NUDT9H, 
                   
                   
               
               
                   
                   
                 mucosa. Detected in 
                 EREG1 
                   
                   
               
               
                   
                   
                 the germinal center of 
                   
                   
                   
               
               
                   
                   
                 colon lymphoid 
                   
                   
                   
               
               
                   
                   
                 nodules and in colon 
                   
                   
                   
               
               
                   
                   
                 parasympathetic 
                   
                   
                   
               
               
                   
                   
                 ganglia of the 
                   
                   
                   
               
               
                   
                   
                 Auerbach plexus (at 
                   
                   
                   
               
               
                   
                   
                 protein level). 
                   
                   
                   
               
               
                   
                   
                 Ubiquitous. Detected in 
                   
                   
                   
               
               
                   
                   
                 brain, testis, ovary, 
                   
                   
                   
               
               
                   
                   
                 liver and pancreas, and 
                   
                   
                   
               
               
                   
                   
                 at lower levels in 
                   
                   
                   
               
               
                   
                   
                 kidney, heart, spleen 
                   
                   
                   
               
               
                   
                   
                 and lung 
                   
                   
                   
               
               
                 TRPM3 
                 transient receptor 
                   
                 KIAA1616, 
                 9q21.11 
                 Q504Y1 
               
               
                   
                 potential cation 
                   
                 LTRPC3, 
                   
                   
               
               
                   
                 channel, subfamily 
                   
                 GON-2 
                   
                   
               
               
                   
                 M, member 3 
                   
                   
                   
                   
               
               
                 TRPM4 
                 transient receptor 
                 Widely expressed with 
                 FLJ20041 
                 19q13.3 
                 Q8TD43 A2RU25 Q9NXV1 
               
               
                   
                 potential cation, 
                 a high expression in 
                   
                   
                   
               
               
                   
                 channel, subfamily 
                 intestine and prostate. 
                   
                   
                   
               
               
                   
                 M, member 4 
                 In brain, it is both 
                   
                   
                   
               
               
                   
                   
                 expressed in whole 
                   
                   
                   
               
               
                   
                   
                 cerebral arteries and 
                   
                   
                   
               
               
                   
                   
                 isolated vascular 
                   
                   
                   
               
               
                   
                   
                 smooth muscle cells. 
                   
                   
                   
               
               
                   
                   
                 Prominently expressed 
                   
                   
                   
               
               
                   
                   
                 in Purkinje fibers. 
                   
                   
                   
               
               
                   
                   
                 Expressed at higher 
                   
                   
                   
               
               
                   
                   
                 levels in T-helper 2 
                   
                   
                   
               
               
                   
                   
                 (Th2) cells as 
                   
                   
                   
               
               
                   
                   
                 compared to T-helper 1 
                   
                   
                   
               
               
                   
                   
                 (Th1) cells 
                   
                   
                   
               
               
                 TRPM5 
                 transient receptor 
                 Strongly expressed in 
                 LTRPC5, 
                 11p15.5 
                 Q9NZQ8 A6NHS0, Q52LU2, 
               
               
                   
                 potential cation 
                 fetal brain, liver and 
                 MTR1  
                   
                 Q9NY34 
               
               
                   
                 channel, subfamily 
                 kidney, and in adult 
                   
                   
                   
               
               
                   
                 M, member 5 
                 prostate, testis, ovary, 
                   
                   
                   
               
               
                   
                   
                 colon and peripheral 
                   
                   
                   
               
               
                   
                   
                 blood leukocytes. Also 
                   
                   
                   
               
               
                   
                   
                 expressed in a large 
                   
                   
                   
               
               
                   
                   
                 proportion of Wilms&#39; 
                   
                   
                   
               
               
                   
                   
                 tumors and 
                   
                   
                   
               
               
                   
                   
                 rhabdomyosarcomas. 
                   
                   
                   
               
               
                   
                   
                 In monochromosomal 
                   
                   
                   
               
               
                   
                   
                 cell lines shows 
                   
                   
                   
               
               
                   
                   
                 exclusive paternal 
                   
                   
                   
               
               
                   
                   
                 expression 
                   
                   
                   
               
               
                 TRPM6 
                 transient receptor 
                 Highly expressed in 
                 CHAK2, 
                 9q21.13 
                 Q9BX84 Q6VPR8 Q6VPS2 
               
               
                   
                 potential cation 
                 kidney and colon. 
                 FLJ22628 
                   
                   
               
               
                   
                 channel, subfamily 
                 Isoform TRPM6a and 
                   
                   
                   
               
               
                   
                 M, member 6 
                 isoform TRPM6b, are 
                   
                   
                   
               
               
                   
                   
                 coexpressed with 
                   
                   
                   
               
               
                   
                   
                 TRPM7 in kidney, and 
                   
                   
                   
               
               
                   
                   
                 testis, and are also 
                   
                   
                   
               
               
                   
                   
                 found in several cell 
                   
                   
                   
               
               
                   
                   
                 lines of lung origin. 
                   
                   
                   
               
               
                   
                   
                 Isoform TRPM6c is 
                   
                   
                   
               
               
                   
                   
                 detected only in testis 
                   
                   
                   
               
               
                   
                   
                 and in NCI-H510A 
                   
                   
                   
               
               
                   
                   
                 small cell lung 
                   
                   
                   
               
               
                   
                   
                 carcinoma cells. 
                   
                   
                   
               
               
                 TRPM7 
                 transient receptor 
                   
                 CHAK1, 
                 15q21 
                 Q96QT4 Q6ZMF5 Q9NXQ2 
               
               
                   
                 potential cation 
                   
                 LTRPC7, 
                   
                   
               
               
                   
                 channel, subfamily 
                   
                 TRP-PLIK 
                   
                   
               
               
                   
                 M, member 7 
                   
                   
                   
                   
               
               
                 TRPM8 
                 transient receptor 
                 Expressed in prostate. 
                   
                 2q37 
                 Q7Z2W7, A0AVG2, 
               
               
                   
                 potential cation 
                 Also expressed in 
                   
                   
                 Q9BVK1 
               
               
                   
                 channel, subfamily 
                 prostate tumors and in 
                   
                   
                   
               
               
                   
                 M, member 8 
                 non-prostatic primary 
                   
                   
                   
               
               
                   
                   
                 tumors such as colon, 
                   
                   
                   
               
               
                   
                   
                 lung, breast and skin 
                   
                   
                   
               
               
                   
                   
                 tumors 
                   
                   
                   
               
               
                 MCOLN1 
                 mucolipin 1 
                 Widely expressed in 
                 TRPML1, 
                 19p13.2 
                 Q9GZU1 D6W647 Q9H4B5 
               
               
                   
                   
                 adult and fetal tissues 
                 ML4, MLIV, 
                   
                   
               
               
                   
                   
                   
                 MST080, 
                   
                   
               
               
                   
                   
                   
                 MSTP080, 
                   
                   
               
               
                   
                   
                   
                 TRPM-L1 
                   
                   
               
               
                 MCOLN2 
                 mucolipin 2 
                   
                 TRPML2, 
                 1p22 
                 Q8IZK6 A6NI99 Q8N9R3 
               
               
                   
                   
                   
                 FLJ36691, 
                   
                   
               
               
                   
                   
                   
                 TRP-ML2 
                   
                   
               
               
                 MCOLN3 
                 mucolipin 3 
                   
                 TRPML3, 
                 1p22.3 
                 Q8TDD5 Q5T4H5, Q5T4H6, 
               
               
                   
                   
                   
                 FLJ11006, 
                   
                 Q9NV09 
               
               
                   
                   
                   
                 TRP-ML3 
                   
                   
               
               
                 PKD1 
                 polycystic kidney 
                   
                 PBP, Pc-1, 
                 16p13.3 
                 P98161 Q15140 Q15141 
               
               
                   
                 disease 1 
                   
                 TRPP1 
                   
                   
               
               
                   
                 (autosomal 
                   
                   
                   
                   
               
               
                   
                 dominant) 
                   
                   
                   
                   
               
               
                 PKD2 
                 polycystic kidney 
                 Strongly expressed in 
                 PKD4, PC2,  
                 4q22.1 
                 Q13563 O60441 Q2M1Q5 
               
               
                   
                 disease 2 
                 ovary, fetal and adult  
                 Pc-2, TRPP2 
                   
                   
               
               
                   
                 (autosomal 
                 kidney, testis, and 
                   
                   
                   
               
               
                   
                 dominant) 
                 small intestine. Not 
                   
                   
                   
               
               
                   
                   
                 detected in peripheral 
                   
                   
                   
               
               
                   
                   
                 leukocytes 
                   
                   
                   
               
               
                 PKD2L1 
                 polycystic kidney 
                 Expressed in adult 
                 PCL, TRPP3 
                 10q24.31 
                 Q9P0L9 O75972 Q9UPA2 
               
               
                   
                 disease 2-like 1 
                 heart, skeletal muscle, 
                   
                   
                   
               
               
                   
                   
                 brain, spleen, testis, 
                   
                   
                   
               
               
                   
                   
                 retina and liver. 
                   
                   
                   
               
               
                 PKD2L2 
                 polycystic kidney 
                 Testis, Brain and 
                 TRPP5 
                 5q31 
                 Q9NZM6 A6NK98 Q9UNJ0 
               
               
                   
                 disease 2-like 2 
                 Kidney 
                   
                   
                   
               
               
                 TRPV1 
                 transient receptor 
                 Widely expressed at 
                   
                 17p13.2 
                 Q8NER1 A2RUA9 Q9NY22 
               
               
                   
                 potential cation 
                 low levels. Expression 
                   
                   
                   
               
               
                   
                 channel, subfamily 
                 is elevated in dorsal 
                   
                   
                   
               
               
                   
                 V, member 1 
                 root ganglia. In skin, 
                   
                   
                   
               
               
                   
                   
                 expressed in cutaneous 
                   
                   
                   
               
               
                   
                   
                 sensory nerve fibers, 
                   
                   
                   
               
               
                   
                   
                 mast cells, epidermal 
                   
                   
                   
               
               
                   
                   
                 keratinocytes, dermal 
                   
                   
                   
               
               
                   
                   
                 blood vessels, the inner 
                   
                   
                   
               
               
                   
                   
                 root sheet and the 
                   
                   
                   
               
               
                   
                   
                 infundibulum of hair 
                   
                   
                   
               
               
                   
                   
                 follicles, differentiated 
                   
                   
                   
               
               
                   
                   
                 sebocytes, sweat gland 
                   
                   
                   
               
               
                   
                   
                 ducts, and the secretory 
                   
                   
                   
               
               
                   
                   
                 portion of eccrine 
                   
                   
                   
               
               
                   
                   
                 sweat glands (at protein 
                   
                   
                   
               
               
                   
                   
                 level). 
                   
                   
                   
               
               
                 TRPV2 
                 transient receptor 
                   
                 VRL, VRL-1, 
                 17p11.2 
                 Q9Y5S1, A6NML2, A8K0Z0, 
               
               
                   
                 potential cation 
                   
                 VRL1 
                   
                 Q9Y670 
               
               
                   
                 channel, subfamily 
                   
                   
                   
                   
               
               
                   
                 V, member 2 
                   
                   
                   
                   
               
               
                 TRPV3 
                 transient receptor 
                 Abundantly expressed 
                 VRL3 
                 17p13.3 
                 Q8NET8, Q8NDW7, 
               
               
                   
                 potential cation 
                 in CNS. Widely 
                   
                   
                 Q8NET9, Q8NFH2 
               
               
                   
                 channel, subfamily 
                 expressed at low levels. 
                   
                   
                   
               
               
                   
                 V, member 3 
                 Detected in dorsal root 
                   
                   
                   
               
               
                   
                   
                 ganglion (at protein 
                   
                   
                   
               
               
                   
                   
                 level). Expressed in the 
                   
                   
                   
               
               
                   
                   
                 keratinocyte layers of 
                   
                   
                   
               
               
                   
                   
                 the outer root sheath 
                   
                   
                   
               
               
                   
                   
                 and, to lesser extent, to 
                   
                   
                   
               
               
                   
                   
                 the matrix of the hair 
                   
                   
                   
               
               
                   
                   
                 follicles (at protein 
                   
                   
                   
               
               
                   
                   
                 level). 
                   
                   
                   
               
               
                 TRPV4 
                 transient receptor 
                 Found in the 
                 OTRPC4, 
                 12q24.1 
                 Q9HBA0, B7ZKQ6, Q9HBC0 
               
               
                   
                 potential cation 
                 synoviocytes from 
                 TRP12, 
                   
                   
               
               
                   
                 channel, subfamily 
                 patients with (RA) and 
                 VROAC, 
                   
                   
               
               
                   
                 V, member 4 
                 without (CTR) 
                 VRL-2, VR- 
                   
                   
               
               
                   
                   
                 rheumatoid arthritis (at 
                 OAC, 
                   
                   
               
               
                   
                   
                 protein level). 
                 CMT2C 
                   
                   
               
               
                 TRPV5 
                 transient receptor 
                 Expressed at high 
                 CaT2 
                 7q34 
                 Q9NQA5, A4D2H7, Q96PM6 
               
               
                   
                 potential cation 
                 levels in kidney, small 
                   
                   
                   
               
               
                   
                 channel, subfamily 
                 intestine and pancreas, 
                   
                   
                   
               
               
                   
                 V, member 5 
                 and at lower levels in 
                   
                   
                   
               
               
                   
                   
                 testis, prostate, 
                   
                   
                   
               
               
                   
                   
                 placenta, brain, colon 
                   
                   
                   
               
               
                   
                   
                 and rectum 
                   
                   
                   
               
               
                 TRPV6 
                 transient receptor 
                 Expressed at high 
                 CaT1 
                 7q34 
                 Q9H1D0, A4D2I8, Q9H296 
               
               
                   
                 potential cation 
                 levels in the 
                   
                   
                   
               
               
                   
                 channel, subfamily 
                 gastrointestinal tract, 
                   
                   
                   
               
               
                   
                 V, member 6 
                 including esophagus, 
                   
                   
                   
               
               
                   
                   
                 stomach, duodenum, 
                   
                   
                   
               
               
                   
                   
                 jejunum, ileum and 
                   
                   
                   
               
               
                   
                   
                 colon, and in pancreas, 
                   
                   
                   
               
               
                   
                   
                 placenta, prostate and 
                   
                   
                   
               
               
                   
                   
                 salivary gland. 
                   
                   
                   
               
               
                   
                   
                 Expressed at moderate 
                   
                   
                   
               
               
                   
                   
                 levels in liver, kidney 
                   
                   
                   
               
               
                   
                   
                 and testis. Expressed in 
                   
                   
                   
               
               
                   
                   
                 locally advanced 
                   
                   
                   
               
               
                   
                   
                 prostate cancer, 
                   
                   
                   
               
               
                   
                   
                 metastatic and 
                   
                   
                   
               
               
                   
                   
                 androgen-insensitive 
                   
                   
                   
               
               
                   
                   
                 prostatic lesions but not 
                   
                   
                   
               
               
                   
                   
                 detected in healthy 
                   
                   
                   
               
               
                   
                   
                 prostate tissue and 
                   
                   
                   
               
               
                   
                   
                 benign prostatic 
                   
                   
                   
               
               
                   
                   
                 hyperplasia 
               
               
                   
               
            
           
         
       
     
     
       
         
           
               
             
               
                 TABLE 4 
               
             
            
               
                   
               
               
                 CatSper channels 
               
            
           
           
               
               
               
               
               
               
            
               
                 Approved 
                   
                   
                   
                   
                   
               
               
                 Symbol 
                 Approved Name 
                 Tissue distribution 
                 Synonyms 
                 Chromosome 
                 Accession number 
               
               
                   
               
               
                 CATSPER1 
                 cation channel, 
                 Testis-specific 
                 CATSPER 
                 11q12.1 
                 Q8NEC5 Q96P76 
               
               
                   
                 sperm associated 1 
                   
                   
                   
                   
               
               
                 CATSPER2 
                 cation channel, 
                 Testis-specific 
                   
                 15q15.3 
                 Q96P56, Q8NHT9, Q96P54, 
               
               
                   
                 sperm associated 2 
                   
                   
                   
                 Q96P55 
               
               
                 CATSPER3 
                 cation channel, 
                 Testis-specific 
                 CACRC 
                  5q31.2 
                 Q86XQ3, Q86XS6 
               
               
                   
                 sperm associated 3 
                   
                   
                   
                   
               
               
                 CATSPER4 
                 cation channel, 
                 Testis-specific 
                   
                 1p35.3 
                 Q7RTX7, A1A4W6, Q5VY71 
               
               
                   
                 sperm associated 4 
               
               
                   
               
            
           
         
       
     
     
       
         
           
               
             
               
                 TABLE 5 
               
             
            
               
                   
               
               
                 Two-pore channels 
               
            
           
           
               
               
               
               
               
               
            
               
                 Approved 
                   
                   
                   
                   
                   
               
               
                 Symbol 
                 Approved Name 
                 Tissue distribution 
                 Synonyms 
                 Chromosome 
                 Accession number 
               
               
                   
               
               
                 TPCN1 
                 two pore segment 
                   
                 KIAA1169, 
                 12q24.21 
                 B7Z3R2 A5PKY2 H0YIK4 
               
               
                   
                 channel 1 
                   
                 FLJ20612, 
                   
                 F8VV93 
               
               
                   
                   
                   
                 TPC1 
                   
                   
               
               
                 TPCN2 
                 two pore segment 
                 Widely expressed. 
                 TPC2 
                 11q13.1 
                 Q8NHX9 Q9NT82_E7ETX0 
               
               
                   
                 channel 2 
                 Expressed at high level 
                   
                   
                   
               
               
                   
                   
                 in liver and kidney 
               
               
                   
               
            
           
         
       
     
     
       
         
           
               
             
               
                 TABLE 6 
               
             
            
               
                   
               
               
                 Cyclic nucleotide-regulated channels 
               
            
           
           
               
               
               
               
               
               
            
               
                 Approved 
                   
                 Tissue 
                   
                   
                   
               
               
                 Symbol 
                 Approved Name 
                 Distribution 
                 Synonyms 
                 Chromosome 
                 Accession number 
               
               
                   
               
               
                 CNGA1 
                 cyclic nucleotide gated 
                 Rod cells in the 
                 RCNC1, 
                 4p12 
                 P29973 A8K7K6 Q4W5E, 
               
               
                   
                 channel alpha 1 
                 retina 
                 RCNCa, 
                   
                 D6RCF1_D6R978, 
               
               
                   
                   
                   
                 CNG1, RP49 
                   
                 A0A024R9X3, Q14028, 
               
               
                   
                   
                   
                   
                   
                 H3BN09 Q9UMG2 
               
               
                 CNGA2 
                 cyclic nucleotide gated 
                   
                 CNG2, 
                 Xq27 
                 Q16280, A0AVD0, Q8IV77 
               
               
                   
                 channel alpha 2 
                   
                 OCNC1, 
                   
                   
               
               
                   
                   
                   
                 OCNCa, 
                   
                   
               
               
                   
                   
                   
                 OCNCALPHA, 
                   
                   
               
               
                   
                   
                   
                 OCNCalpha, 
                   
                   
               
               
                   
                   
                   
                 FLJ46312 
                   
                   
               
               
                 CNGA3 
                 cyclic nucleotide gated 
                 Prominently 
                 CCNC1, 
                 2q11.2 
                 Q16281, Q4VAP7, Q53RD2, 
               
               
                   
                 channel alpha 3 
                 expressed in retina 
                 CCNCa, CNG3 
                   
                 Q9UP64 
               
               
                 CNGA4 
                 cyclic nucleotide gated 
                   
                 OCNC2, 
                 11p15.4 
                 Q8IV77 
               
               
                   
                 channel alpha 4 
                   
                 OCNCb, CNG5 
                   
                   
               
               
                 CNGB1 
                 cyclic nucleotide gated 
                   
                 RCNC2, 
                 16q13 
                 Q16280, _Q16280 
               
               
                   
                 channel beta 1 
                   
                 RCNCb, 
                   
                   
               
               
                   
                   
                   
                 GARP, GAR1, 
                   
                   
               
               
                   
                   
                   
                 CNGB1B, 
                   
                   
               
               
                   
                   
                   
                 RP45 
                   
                   
               
               
                 CNGB3 
                 cyclic nucleotide gated 
                 Expressed 
                   
                 8q21.3 
                 Q9NQW8 C9JA51, Q9NRE9, 
               
               
                   
                 channel beta 3 
                 specifically in the 
                   
                   
                 H0YAZ4_B9EK43, _Q0QD47 
               
               
                   
                   
                 retina 
                   
                   
                   
               
               
                 HCN1 
                 hyperpolarization 
                 Detected in brain, 
                 BCNG-1, 
                 5p12 
                 O60741, Q86WJ6 
               
               
                   
                 activated cyclic 
                 in particular in 
                 HAC-2 
                   
                   
               
               
                   
                 nucleotide-gated 
                 amygdala and 
                   
                   
                   
               
               
                   
                 potassium channel 1 
                 hippocampus, 
                   
                   
                   
               
               
                   
                   
                 while expression in 
                   
                   
                   
               
               
                   
                   
                 caudate nucleus, 
                   
                   
                   
               
               
                   
                   
                 corpus callosum, 
                   
                   
                   
               
               
                   
                   
                 substantia nigra, 
                   
                   
                   
               
               
                   
                   
                 subthalamic 
                   
                   
                   
               
               
                   
                   
                 nucleus and 
                   
                   
                   
               
               
                   
                   
                 thalamus is very 
                   
                   
                   
               
               
                   
                   
                 low or not 
                   
                   
                   
               
               
                   
                   
                 detectable. 
                   
                   
                   
               
               
                   
                   
                 Detected at very 
                   
                   
                   
               
               
                   
                   
                 low levels in 
                   
                   
                   
               
               
                   
                   
                 muscle and 
                   
                   
                   
               
               
                   
                   
                 pancreas 
                   
                   
                   
               
               
                 HCN2 
                 hyperpolarization 
                 Highly expressed 
                 BCNG-2, 
                 19p13 
                 Q9UL51, O60742, Q9UBS2 
               
               
                   
                 activated cyclic 
                 throughout the 
                 HAC-1 
                   
                   
               
               
                   
                 nucleotide-gated 
                 brain. Detected at 
                   
                   
                   
               
               
                   
                 potassium channel 2 
                 low levels in heart. 
                   
                   
                   
               
               
                 HCN3 
                 hyperpolarization 
                 Detected in brain 
                 KIAA1535 
                 1q21.2 
                 Q9P1Z3, D3DV90, 
               
               
                   
                 activated cyclic 
                   
                   
                   
                 Q9BWQ2, Q2T9L6 Q86WJ5 
               
               
                   
                 nucleotide-gated 
                   
                   
                   
                   
               
               
                   
                 potassium channel 3 
                   
                   
                   
                   
               
               
                 HCN4 
                 hyperpolarization 
                 Highly expressed 
                   
                 15q24.1 
                 Q9Y3Q4 Q9UMQ7 
               
               
                   
                 activated cyclic 
                 in thalamus, testis 
                   
                   
                   
               
               
                   
                 nucleotide-gated 
                 and in heart, both 
                   
                   
                   
               
               
                   
                 potassium channel 4 
                 in ventricle and 
                   
                   
                   
               
               
                   
                   
                 atrium. Detected at 
                   
                   
                   
               
               
                   
                   
                 much lower levels 
                   
                   
                   
               
               
                   
                   
                 in amygdala, 
                   
                   
                   
               
               
                   
                   
                 substantia nigra, 
                   
                   
                   
               
               
                   
                   
                 cerebellum and 
                   
                   
                   
               
               
                   
                   
                 hippocampus 
               
               
                   
               
            
           
         
       
     
     
       
         
           
               
             
               
                 TABLE 7 
               
             
            
               
                   
               
               
                 Potassium channels, calcium-activated 
               
            
           
           
               
               
               
               
               
               
            
               
                 Approved 
                   
                 Tissue 
                   
                   
                   
               
               
                 Symbol 
                 Approved Name 
                 distribution 
                 Synonyms 
                 Chromosome 
                 Accession Number 
               
               
                   
               
               
                 KCNMA1 
                 potassium large 
                 Widely expressed. 
                 KCa1.1, mSLO1 
                 10q22 
                 Q12791 F8WA96 
               
               
                   
                 conductance calcium- 
                 Except in 
                   
                   
                 Q9UQK6 
               
               
                   
                 activated channel, 
                 myocytes, it is 
                   
                   
                   
               
               
                   
                 subfamily M, alpha 
                 almost ubiquitously 
                   
                   
                   
               
               
                   
                 member 1 
                 expressed 
                   
                   
                   
               
               
                 KCNN1 
                 potassium 
                   
                 KCa2.1, hSK1 
                 19p13.1 
                 Q92952 
               
               
                   
                 intermediate/small 
                   
                   
                   
                 Q5KR10, Q6DJU4 
               
               
                   
                 conductance calcium- 
                   
                   
                   
                   
               
               
                   
                 activated channel, 
                   
                   
                   
                   
               
               
                   
                 subfamily N, member 1 
                   
                   
                   
                   
               
               
                 KCNN2 
                 potassium 
                 Expressed in atrial 
                 KCa2.2, hSK2 
                 5q22.3 
                 Q9H2S1 A6NF94 
               
               
                   
                 intermediate/small 
                 myocytes (at 
                   
                   
                 Q6X2Y2 
               
               
                   
                 conductance calcium- 
                 protein level). 
                   
                   
                   
               
               
                   
                 activated channel, 
                 Widely expressed 
                   
                   
                   
               
               
                   
                 subfamily N, member 2 
                   
                   
                   
                   
               
               
                 KCNN3 
                 potassium 
                   
                 KCa2.3, hSK3, 
                 1q21.3 
                 Q9UGI6 B1ANX0 
               
               
                   
                 intermediate/small 
                   
                 SKCA3 
                   
                 Q8WXG7 
               
               
                   
                 conductance calcium- 
                   
                   
                   
                   
               
               
                   
                 activated channel, 
                   
                   
                   
                   
               
               
                   
                 subfamily N, member 3 
                   
                   
                   
                   
               
               
                 KCNN4 
                 potassium 
                 Widely expressed 
                 KCa3.1, hSK4, 
                 19q13.2 
                 O15554 Q53XR4 
               
               
                   
                 intermediate/small 
                 in non-excitable 
                 hKCa4, hIKCa1 
                   
                   
               
               
                   
                 conductance calcium- 
                 tissues 
                   
                   
                   
               
               
                   
                 activated channel, 
                   
                   
                   
                   
               
               
                   
                 subfamily N, member 4 
                   
                   
                   
                   
               
               
                 KCNT1 
                 potassium channel, 
                 Highest expression 
                 KCa4.1, 
                 9q34.3 
                 Q5JUK3 B3KXF7, 
               
               
                   
                 subfamily T, member 1 
                 in liver, brain and 
                 KIAA1422 
                   
                 Q9P2C5 
               
               
                   
                   
                 spinal cord. Lowest 
                   
                   
                   
               
               
                   
                   
                 expression in 
                   
                   
                   
               
               
                   
                   
                 skeletal muscle 
                   
                   
                   
               
               
                 KCNT2 
                 potassium channel, 
                   
                 KCa4.2, SLICK, 
                 1q31.3 
                 Q6UVM3, Q3SY59, 
               
               
                   
                 subfamily T, member 2 
                   
                 SLO2.1 
                   
                 Q5VTN1, Q6ZMT3 
               
               
                 KCNU1 
                 potassium channel, 
                 Testis-specific 
                 KCa5.1, Slo3, 
                 8p11.2 
                 A8MYU2 
               
               
                   
                 subfamily U, member 1 
                   
                 KCNMC1, 
                   
                   
               
               
                   
                   
                   
                 Kcnma3 
               
               
                   
               
            
           
         
       
     
     
       
         
           
               
             
               
                 TABLE 8 
               
             
            
               
                   
               
               
                 Potassium channels, voltage-gated 
               
            
           
           
               
               
               
               
               
               
            
               
                 Approved 
                   
                 Tissue 
                   
                   
                 Accession 
               
               
                 Symbol 
                 Approved Name 
                 distribution 
                 Synonyms 
                 Chromosome 
                 number 
               
               
                   
               
               
                 KCNA1 
                 potassium voltage-gated 
                   
                 Kv1.1, RBK1, 
                 12p13 
                 Q09470 A6NM83, 
               
               
                   
                 channel, shaker-related 
                   
                 HUK1, MBK1 
                   
                 Q3MIQ9 
               
               
                   
                 subfamily, member 1 
                   
                   
                   
                   
               
               
                   
                 (episodic ataxia with 
                   
                   
                   
                   
               
               
                   
                 myokymia) 
                   
                   
                   
                   
               
               
                 KCNA2 
                 potassium voltage-gated 
                   
                 Kv1.2, HK4 
                 1p13 
                 P16389 
               
               
                   
                 channel, shaker-related 
                   
                   
                   
                   
               
               
                   
                 subfamily, member 2 
                   
                   
                   
                 Q86XG6 
               
               
                 KCNA3 
                 potassium voltage-gated 
                   
                 Kv1.3, MK3, 
                 1p13.3 
                 P22001 Q5VWN2 
               
               
                   
                 channel, shaker-related 
                   
                 HLK3, HPCN3 
                   
                   
               
               
                   
                 subfamily, member 3 
                   
                   
                   
                   
               
               
                 KCNA4 
                 potassium voltage-gated 
                   
                 Kv1.4, HK1, 
                 11p14 
                 P22459 
               
               
                   
                 channel, shaker-related 
                   
                 HPCN2 
                   
                   
               
               
                   
                 subfamily, member 4 
                   
                   
                   
                   
               
               
                 KCNA5 
                 potassium voltage-gated 
                 Pancreatic islets 
                 Kv1.5, HK2, 
                 12p13 
                 P22460 Q4KKT8 
               
               
                   
                 channel, shaker-related 
                 and insulinoma 
                 HPCN1 
                   
                 Q9UDA4 
               
               
                   
                 subfamily, member 5 
                   
                   
                   
                   
               
               
                 KCNA6 
                 potassium voltage-gated 
                   
                 Kv1.6, HBK2, 
                 12p13 
                 P17658 
               
               
                   
                 channel, shaker-related 
                   
                 PPP1R96 
                   
                   
               
               
                   
                 subfamily, member 6 
                   
                   
                   
                   
               
               
                 KCNA7 
                 potassium voltage-gated 
                 Highly expressed 
                 Kv1.7, HAK6 
                 19q13.3 
                 Q96RP8, A1KYX7, 
               
               
                   
                 channel, shaker-related 
                 in skeletal 
                   
                   
                 Q9BYS4 
               
               
                   
                 subfamily, member 7 
                 muscle, heart and 
                   
                   
                   
               
               
                   
                   
                 kidney. 
                   
                   
                   
               
               
                 KCNA10 
                 potassium voltage-gated 
                 Detected in 
                 Kv1.8 
                 1p13.1 
                 Q16322 
               
               
                   
                 channel, shaker-related 
                 kidney, in 
                   
                   
                   
               
               
                   
                 subfamily, member 10 
                 proximal tubules, 
                   
                   
                   
               
               
                   
                   
                 glomerular 
                   
                   
                   
               
               
                   
                   
                 endothelium, in 
                   
                   
                   
               
               
                   
                   
                 vascular 
                   
                   
                   
               
               
                   
                   
                 endothelium and 
                   
                   
                   
               
               
                   
                   
                 in smooth muscle 
                   
                   
                   
               
               
                   
                   
                 cells 
                   
                   
                   
               
               
                 KCNB1 
                 potassium voltage-gated 
                   
                 Kv2.1 
                 20q13.2 
                 Q14721 Q14193 
               
               
                   
                 channel, Shab-related 
                   
                   
                   
                   
               
               
                   
                 subfamily, member 1 
                   
                   
                   
                   
               
               
                 KCNB2 
                 potassium voltage-gated 
                   
                 Kv2.2 
                 8q13.2 
                 Q92953, Q7Z7D0, 
               
               
                   
                 channel, Shab-related 
                   
                   
                   
                 Q9BXD3 
               
               
                   
                 subfamily, member 2 
                   
                   
                   
                   
               
               
                 KCNC1 
                 potassium voltage-gated 
                   
                 Kv3.1 
                 11p15 
                 P48547, K4DI87 
               
               
                   
                 channel, Shaw-related 
                   
                   
                   
                   
               
               
                   
                 subfamily, member 1 
                   
                   
                   
                   
               
               
                 KCNC2 
                 potassium voltage-gated 
                   
                 Kv3.2 
                 12q14.1 
                 Q96PR1, B7Z231, 
               
               
                   
                 channel, Shaw-related 
                   
                   
                   
                 Q96PR0 
               
               
                   
                 subfamily, member 2 
                   
                   
                   
                   
               
               
                 KCNC3 
                 potassium voltage-gated 
                   
                 Kv3.3 
                 19q13.33 
                 Q14003 
               
               
                   
                 channel, Shaw-related 
                   
                   
                   
                   
               
               
                   
                 subfamily, member 3 
                   
                   
                   
                   
               
               
                 KCNC4 
                 potassium voltage-gated 
                   
                 Kv3.4, 
                 1p21 
                 Q03721, Q3MIM4, 
               
               
                   
                 channel, Shaw-related 
                   
                 HKSHIIIC 
                   
                 Q5TBI6 
               
               
                   
                 subfamily, member 4 
                   
                   
                   
                   
               
               
                 KCND1 
                 potassium voltage-gated 
                 Widely 
                 Kv4.1 
                 Xp11.23 
                 Q9NSA2 B2RCG0, 
               
               
                   
                 channel, Shal-related 
                 expressed. 
                   
                   
                 O75671 
               
               
                   
                 subfamily, member 1 
                 Highly expressed 
                   
                   
                   
               
               
                   
                   
                 in brain, in 
                   
                   
                   
               
               
                   
                   
                 particular in 
                   
                   
                   
               
               
                   
                   
                 cerebellum and 
                   
                   
                   
               
               
                   
                   
                 thalamus; 
                   
                   
                   
               
               
                   
                   
                 detected at lower 
                   
                   
                   
               
               
                   
                   
                 levels in the other 
                   
                   
                   
               
               
                   
                   
                 parts of the brain 
                   
                   
                   
               
               
                 KCND2 
                 potassium voltage-gated 
                 Highly expressed 
                 Kv4.2, RK5, 
                 7q31 
                 Q9NZV8, O95012 
               
               
                   
                 channel, Shal-related 
                 throughout the 
                 KIAA1044 
                   
                 Q9UNH9 
               
               
                   
                 subfamily, member 2 
                 brain. Expression 
                   
                   
                   
               
               
                   
                   
                 is very low or 
                   
                   
                   
               
               
                   
                   
                 absent in other 
                   
                   
                   
               
               
                   
                   
                 tissues. 
                   
                   
                   
               
               
                 KCND3 
                 potassium voltage-gated 
                 Highly expressed 
                 Kv4.3, KSHIVB 
                 1p13.2 
                 Q9UK17, O60576, 
               
               
                   
                 channel, Shal-related 
                 in heart and 
                   
                   
                 Q9UK16 
               
               
                   
                 subfamily, member 3 
                 brain, in 
                   
                   
                   
               
               
                   
                   
                 particular in 
                   
                   
                   
               
               
                   
                   
                 cortex, 
                   
                   
                   
               
               
                   
                   
                 cerebellum, 
                   
                   
                   
               
               
                   
                   
                 amygdala and 
                   
                   
                   
               
               
                   
                   
                 caudate nucleus. 
                   
                   
                   
               
               
                   
                   
                 Detected at lower 
                   
                   
                   
               
               
                   
                   
                 levels in liver, 
                   
                   
                   
               
               
                   
                   
                 skeletal muscle, 
                   
                   
                   
               
               
                   
                   
                 kidney and 
                   
                   
                   
               
               
                   
                   
                 pancreas. Isoform 
                   
                   
                   
               
               
                   
                   
                 1 predominates in 
                   
                   
                   
               
               
                   
                   
                 most tissues. 
                   
                   
                   
               
               
                   
                   
                 Isoform 1 and 
                   
                   
                   
               
               
                   
                   
                 isoform 2 are 
                   
                   
                   
               
               
                   
                   
                 detected at 
                   
                   
                   
               
               
                   
                   
                 similar levels in 
                   
                   
                   
               
               
                   
                   
                 brain, skeletal 
                   
                   
                   
               
               
                   
                   
                 muscle and 
                   
                   
                   
               
               
                   
                   
                 pancreas 
                   
                   
                   
               
               
                 KCNF1 
                 potassium voltage-gated 
                 Detected in heart, 
                 Kv5.1, kH1, IK8 
                 2p25 
                 Q9H3M0, O43527, 
               
               
                   
                 channel, subfamily F, 
                 brain, liver, 
                   
                   
                 Q585L3 
               
               
                   
                 member 1 
                 skeletal muscle, 
                   
                   
                   
               
               
                   
                   
                 kidney and 
                   
                   
                   
               
               
                   
                   
                 pancreas. 
                   
                   
                   
               
               
                 KCNG1 
                 potassium voltage-gated 
                 Detected in brain 
                 Kv6.1, kH2, K13 
                 20q13 
                 Q9UIX4, A8K3S4, 
               
               
                   
                 channel, subfamily G, 
                 and placenta, and 
                   
                   
                 Q9BRC1 
               
               
                   
                 member 1 
                 at much lower 
                   
                   
                   
               
               
                   
                   
                 levels in kidney 
                   
                   
                   
               
               
                   
                   
                 and pancreas 
                   
                   
                   
               
               
                 KCNG2 
                 potassium voltage-gated 
                 Highly expressed 
                 Kv6.2, KCNF2 
                 18q23 
                 Q9UJ96 
               
               
                   
                 channel, subfamily G, 
                 in heart, liver, 
                   
                   
                   
               
               
                   
                 member 2 
                 skeletal muscle, 
                   
                   
                   
               
               
                   
                   
                 kidney and 
                   
                   
                   
               
               
                   
                   
                 pancreas. 
                   
                   
                   
               
               
                   
                   
                 Detected at low 
                   
                   
                   
               
               
                   
                   
                 levels in brain, 
                   
                   
                   
               
               
                   
                   
                 lung and placenta 
                   
                   
                   
               
               
                 KCNG3 
                 potassium voltage-gated 
                 Detected in many 
                 Kv6.3 
                 2p21 
                 Q8TAE7, Q53SC1 
               
               
                   
                 channel, subfamily G, 
                 parts of the brain 
                   
                   
                   
               
               
                   
                 member 3 
                 with the 
                   
                   
                   
               
               
                   
                   
                 exception of the 
                   
                   
                   
               
               
                   
                   
                 cerebellum, in 
                   
                   
                   
               
               
                   
                   
                 testis, pancreas, 
                   
                   
                   
               
               
                   
                   
                 lung, kidney, 
                   
                   
                   
               
               
                   
                   
                 ovary, small 
                   
                   
                   
               
               
                   
                   
                 intestine, colon, 
                   
                   
                   
               
               
                   
                   
                 thymus, adrenal 
                   
                   
                   
               
               
                   
                   
                 gland and spinal 
                   
                   
                   
               
               
                   
                   
                 cord 
                   
                   
                   
               
               
                 KCNG4 
                 potassium voltage-gated 
                 Highly expressed 
                 Kv6.4 
                 16q24.1 
                 Q8TDN1, Q96H24 
               
               
                   
                 channel, subfamily G, 
                 in brain, and at 
                   
                   
                   
               
               
                   
                 member 4 
                 lower levels in 
                   
                   
                   
               
               
                   
                   
                 liver, small 
                   
                   
                   
               
               
                   
                   
                 intestine and 
                   
                   
                   
               
               
                   
                   
                 colon. 
                   
                   
                   
               
               
                 KCNH1 
                 potassium voltage-gated 
                 Highly expressed 
                 Kv10.1, eag, h- 
                 1q32.2 
                 O95259 B1AQ26, 
               
               
                   
                 channel, subfamily H 
                 in brain and in 
                 eag, eag1 
                   
                 O76035, Q14CL3 
               
               
                   
                 (eag-related), member 1 
                 myoblasts at the 
                   
                   
                   
               
               
                   
                   
                 onset of fusion, 
                   
                   
                   
               
               
                   
                   
                 but not in other 
                   
                   
                   
               
               
                   
                   
                 tissues. Detected 
                   
                   
                   
               
               
                   
                   
                 in HeLa (cervical 
                   
                   
                   
               
               
                   
                   
                 carcinoma), SH- 
                   
                   
                   
               
               
                   
                   
                 SY5Y 
                   
                   
                   
               
               
                   
                   
                 (neuroblastoma) 
                   
                   
                   
               
               
                   
                   
                 and MCF-7 
                   
                   
                   
               
               
                   
                   
                 (epithelial tumor) 
                   
                   
                   
               
               
                   
                   
                 cells, but not in 
                   
                   
                   
               
               
                   
                   
                 normal epithelial 
                   
                   
                   
               
               
                   
                   
                 cells. 
                   
                   
                   
               
               
                 KCNH2 
                 potassium voltage-gated 
                 Highly expressed 
                 Kv11.1, HERG, 
                 7q36.1 
                 Q12809 A5H1P7 Q9H3P0 
               
               
                   
                 channel, subfamily H 
                 in heart and 
                 erg1 
                   
                   
               
               
                   
                 (eag-related), member 2 
                 brain. Isoforms 
                   
                   
                   
               
               
                   
                   
                 USO are 
                   
                   
                   
               
               
                   
                   
                 frequently 
                   
                   
                   
               
               
                   
                   
                 overexpressed in 
                   
                   
                   
               
               
                   
                   
                 cancer cells 
                   
                   
                   
               
               
                 KCNH3 
                 potassium voltage-gated 
                 Detected only in 
                 Kv12.2, BEC1, 
                 12q13 
                 Q9ULD8 
               
               
                   
                 channel, subfamily H 
                 brain, in 
                 elk2 
                   
                 Q9UQ06 
               
               
                   
                 (eag-related), member 3 
                 particular in the 
                   
                   
                   
               
               
                   
                   
                 telencephalon. 
                   
                   
                   
               
               
                   
                   
                 Detected in the 
                   
                   
                   
               
               
                   
                   
                 cerebral cortex, 
                   
                   
                   
               
               
                   
                   
                 occipital pole, 
                   
                   
                   
               
               
                   
                   
                 frontal and 
                   
                   
                   
               
               
                   
                   
                 temporal lobe, 
                   
                   
                   
               
               
                   
                   
                 putamen, 
                   
                   
                   
               
               
                   
                   
                 amygdala, 
                   
                   
                   
               
               
                   
                   
                 hippocampus and 
                   
                   
                   
               
               
                   
                   
                 caudate nucleus 
                   
                   
                   
               
               
                 KCNH4 
                 potassium voltage-gated 
                 Detected only in 
                 Kv12.3, elk1 
                 17q21 
                 Q9UQ05 
               
               
                   
                 channel, subfamily H 
                 brain, in 
                   
                   
                   
               
               
                   
                 (eag-related), member 4 
                 particular in the 
                   
                   
                   
               
               
                   
                   
                 telencephalon. 
                   
                   
                   
               
               
                   
                   
                 Detected in 
                   
                   
                   
               
               
                   
                   
                 putamen and 
                   
                   
                   
               
               
                   
                   
                 caudate nucleus, 
                   
                   
                   
               
               
                   
                   
                 and at lower 
                   
                   
                   
               
               
                   
                   
                 levels in cerebral 
                   
                   
                   
               
               
                   
                   
                 cortex, occipital 
                   
                   
                   
               
               
                   
                   
                 and hippocampus 
                   
                   
                   
               
               
                 KCNH5 
                 potassium voltage-gated 
                 Detected in brain, 
                 Kv10.2, H- 
                 14q23.1 
                 Q8NCM2, C9JP98 
               
               
                   
                 channel, subfamily H 
                 skeletal muscle, 
                 EAG2, eag2 
                   
                   
               
               
                   
                 (eag-related), member 5 
                 heart, placenta, 
                   
                   
                   
               
               
                   
                   
                 lung and liver, 
                   
                   
                   
               
               
                   
                   
                 and at low levels 
                   
                   
                   
               
               
                   
                   
                 in kidney 
                   
                   
                   
               
               
                 KCNH6 
                 potassium voltage-gated 
                 Expressed in 
                 Kv11.2, erg2, 
                 17q23.3 
                 Q9H252, Q9BRD7 
               
               
                   
                 channel, subfamily H 
                 prolactin- 
                 HERG2 
                   
                   
               
               
                   
                 (eag-related), member 6 
                 secreting 
                   
                   
                   
               
               
                   
                   
                 adenomas 
                   
                   
                   
               
               
                 KCNH7 
                 potassium voltage-gated 
                 Expressed in 
                 Kv11.3, HERG3, 
                 2q24.3 
                 Q9NS40, Q53QU4, 
               
               
                   
                 channel, subfamily H 
                 prolactin- 
                 erg3 
                   
                 Q8IV15 
               
               
                   
                 (eag-related), member 7 
                 secreting 
                   
                   
                   
               
               
                   
                   
                 adenomas 
                   
                   
                   
               
               
                 KCNH8 
                 potassium voltage-gated 
                 Nervous system 
                 Kv12.1, elk3 
                 3p24.3 
                 Q96L42, Q59GQ6 
               
               
                   
                 channel, subfamily H 
                   
                   
                   
                   
               
               
                   
                 (eag-related), member 8 
                   
                   
                   
                   
               
               
                 KCNQ1 
                 potassium voltage-gated 
                 Abundantly 
                 Kv7.1, KCNA8, 
                 11p15.5 
                 P51787, O00347 
               
               
                   
                 channel, KQT-like 
                 expressed in 
                 KVLQT1, 
                   
                 Q9UMN9 
               
               
                   
                 subfamily, member 1 
                 heart, pancreas, 
                 JLNS1, LQT1 
                   
                   
               
               
                   
                   
                 prostate, kidney, 
                   
                   
                   
               
               
                   
                   
                 small intestine 
                   
                   
                   
               
               
                   
                   
                 and peripheral 
                   
                   
                   
               
               
                   
                   
                 blood leukocytes. 
                   
                   
                   
               
               
                   
                   
                 Less abundant in 
                   
                   
                   
               
               
                   
                   
                 placenta, lung, 
                   
                   
                   
               
               
                   
                   
                 spleen, colon, 
                   
                   
                   
               
               
                   
                   
                 thymus, testis and 
                   
                   
                   
               
               
                   
                   
                 ovaries. 
                   
                   
                   
               
               
                 KCNQ2 
                 potassium voltage-gated 
                 In adult and fetal 
                 Kv7.2, ENB1, 
                 20q13.33 
                 O43526, O43796, Q99454 
               
               
                   
                 channel, KQT-like 
                 brain. Highly 
                 BFNC, KCNA11, 
                   
                   
               
               
                   
                 subfamily, member 2 
                 expressed in 
                 HNSPC 
                   
                   
               
               
                   
                   
                 areas containing 
                   
                   
                   
               
               
                   
                   
                 neuronal cell 
                   
                   
                   
               
               
                   
                   
                 bodies, low in 
                   
                   
                   
               
               
                   
                   
                 spinal chord and 
                   
                   
                   
               
               
                   
                   
                 corpus callosum. 
                   
                   
                   
               
               
                   
                   
                 Isoform 2 is 
                   
                   
                   
               
               
                   
                   
                 preferentially 
                   
                   
                   
               
               
                   
                   
                 expressed in 
                   
                   
                   
               
               
                   
                   
                 differentiated 
                   
                   
                   
               
               
                   
                   
                 neurons. Isoform 
                   
                   
                   
               
               
                   
                   
                 6 is prominent in 
                   
                   
                   
               
               
                   
                   
                 fetal brain, 
                   
                   
                   
               
               
                   
                   
                 undifferentiated 
                   
                   
                   
               
               
                   
                   
                 neuroblastoma 
                   
                   
                   
               
               
                   
                   
                 cells and brain 
                   
                   
                   
               
               
                   
                   
                 tumors 
                   
                   
                   
               
               
                 KCNQ3 
                 potassium voltage-gated 
                 Brain 
                 Kv7.3 
                 8q24 
                 O43525, A2VCT8, 
               
               
                   
                 channel, KQT-like 
                   
                   
                   
                 B4DJY4, E7EQ89 
               
               
                   
                 subfamily, member 3 
                   
                   
                   
                   
               
               
                 KCNQ4 
                 potassium voltage-gated 
                 Expressed in the 
                 Kv7.4 
                 1p34 
                 P56696, O96025 
               
               
                   
                 channel, KQT-like 
                 outer, but not the 
                   
                   
                   
               
               
                   
                 subfamily, member 4 
                 inner, sensory 
                   
                   
                   
               
               
                   
                   
                 hair cells of the 
                   
                   
                   
               
               
                   
                   
                 cochlea. Slightly 
                   
                   
                   
               
               
                   
                   
                 expressed in 
                   
                   
                   
               
               
                   
                   
                 heart, brain and 
                   
                   
                   
               
               
                   
                   
                 skeletal muscle. 
                   
                   
                   
               
               
                 KCNQ5 
                 potassium voltage-gated 
                 Strongly 
                 Kv7.5 
                 6q14 
                 Q9NR82, B4DS33. 
               
               
                   
                 channel, KQT-like 
                 expressed in 
                   
                   
                 Q9NYA6 
               
               
                   
                 subfamily, member 5 
                 brain and skeletal 
                   
                   
                   
               
               
                   
                   
                 muscle. In brain, 
                   
                   
                   
               
               
                   
                   
                 expressed in 
                   
                   
                   
               
               
                   
                   
                 cerebral cortex, 
                   
                   
                   
               
               
                   
                   
                 occipital pole, 
                   
                   
                   
               
               
                   
                   
                 frontal lobe and  
                   
                   
                   
               
               
                   
                   
                 temporal lobe. 
                   
                   
                   
               
               
                   
                   
                 Lower levels in 
                   
                   
                   
               
               
                   
                   
                 hippocampus and 
                   
                   
                   
               
               
                   
                   
                 putamen. Low to 
                   
                   
                   
               
               
                   
                   
                 undetectable 
                   
                   
                   
               
               
                   
                   
                 levels in medulla, 
                   
                   
                   
               
               
                   
                   
                 cerebellum and 
                   
                   
                   
               
               
                   
                   
                 thalamus. 
                   
                   
                   
               
               
                 KCNS1 
                 potassium voltage-gated 
                 Detected in all 
                 Kv9.1 
                 20q12 
                 A2RUL8, _Q96KK3, 
               
               
                   
                 channel, delayed- 
                 tissues tested 
                   
                   
                 A2RUL9, Q6DJU6, 
               
               
                   
                 rectifier, subfamily S, 
                 with the 
                   
                   
                 Q92953, Q7Z7D0, 
               
               
                   
                 member 1 
                 exception of 
                   
                   
                 Q9BXD3 
               
               
                   
                   
                 skeletal muscle. 
                   
                   
                   
               
               
                   
                   
                 Highly expressed 
                   
                   
                   
               
               
                   
                   
                 in adult and fetal 
                   
                   
                   
               
               
                   
                   
                 brain, fetal 
                   
                   
                   
               
               
                   
                   
                 kidney and lung, 
                   
                   
                   
               
               
                   
                   
                 and adult prostate 
                   
                   
                   
               
               
                   
                   
                 and testis. 
                   
                   
                   
               
               
                 KCNS2 
                 potassium voltage-gated 
                   
                 Kv9.2 
                 8q22 
                 Q9ULS6, A8KAN1, 
               
               
                   
                 channel, delayed- 
                   
                   
                   
                 Q92953, Q7Z7D0, 
               
               
                   
                 rectifier, subfamily S, 
                   
                   
                   
                 Q9BXD3, _Q14721, 
               
               
                   
                 member 2 
                   
                   
                   
                 Q14193 
               
               
                 KCNS3 
                 potassium voltage-gated 
                 Detected in 
                 Kv9.3 
                 2p24 
                 Q9BQ31, D6W520 
               
               
                   
                 channel, delayed- 
                 whole normal 
                   
                   
                 Q96B56, _C9J187, 
               
               
                   
                 rectifier, subfamily S, 
                 term placental 
                   
                   
                 L8E8W4, _Q92953, 
               
               
                   
                 member 3 
                 and placental 
                   
                   
                 Q7Z7D0, Q9BXD3 
               
               
                   
                   
                 chorionic plate 
                   
                   
                 Q14721, Q14193 
               
               
                   
                   
                 arteries and 
                   
                   
                   
               
               
                   
                   
                 veins. Detected in  
                   
                   
                   
               
               
                   
                   
                 syncytiotrophoblast 
                   
                   
                   
               
               
                   
                   
                 and in blood 
                   
                   
                   
               
               
                   
                   
                 vessel 
                   
                   
                   
               
               
                   
                   
                 endothelium 
                   
                   
                   
               
               
                   
                   
                 within 
                   
                   
                   
               
               
                   
                   
                 intermediate villi 
                   
                   
                   
               
               
                   
                   
                 and chorionic 
                   
                   
                   
               
               
                   
                   
                 plate (at protein 
                   
                   
                   
               
               
                   
                   
                 level). Detected 
                   
                   
                   
               
               
                   
                   
                 in most tissues, 
                   
                   
                   
               
               
                   
                   
                 but not in 
                   
                   
                   
               
               
                   
                   
                 peripheral blood 
                   
                   
                   
               
               
                   
                   
                 lymphocytes. The 
                   
                   
                   
               
               
                   
                   
                 highest levels of 
                   
                   
                   
               
               
                   
                   
                 expression are in 
                   
                   
                   
               
               
                   
                   
                 lung 
                   
                   
                   
               
               
                 KCNV1 
                 potassium channel, 
                 Detected in brain 
                 Kv8.1 
                 8q23.2 
                 Q6PIU1, Q9UHJ4, 
               
               
                   
                 subfamily V, member 1 
                   
                   
                   
                 B2R8Q7, _B4DMC1 
               
               
                   
                   
                   
                   
                   
                 Q76FP2 
               
               
                 KCNV2 
                 potassium channel, 
                 Detected in heart, 
                 Kv8.2 
                 9p24.2 
                 Q8TDN2, Q5T6X0 
               
               
                   
                 subfamily V, member 2 
                 brain, liver, 
                   
                   
                 P48547, K4DI87, _Q14721, 
               
               
                   
                   
                 skeletal muscle, 
                   
                   
                 Q14193, _Q9H3M0 
               
               
                   
                   
                 spleen, thymus, 
                   
                   
                 O43527, Q585L3 
               
               
                   
                   
                 prostate, testis, 
                   
                   
                   
               
               
                   
                   
                 ovary colon, 
                   
                   
                   
               
               
                   
                   
                 kidney and 
                   
                   
                   
               
               
                   
                   
                 pancreas 
               
               
                   
               
            
           
         
       
     
     
       
         
           
               
             
               
                 TABLE 9 
               
             
            
               
                   
               
               
                 Potassium channels, inwardly rectifying 
               
            
           
           
               
               
               
               
               
               
            
               
                 Approved 
                   
                   
                   
                   
                   
               
               
                 Symbol 
                 Approved Name 
                 Tissue distribution 
                 Synonyms 
                 Chromosome 
                 Accession number 
               
               
                   
               
               
                 KCNJ1 
                 potassium 
                 In the kidney and 
                 Kir1.1, 
                 11q24 
                 P48048, B2RMR4, 
               
               
                   
                 inwardly-rectifying 
                 pancreatic islets. Lower 
                 ROMK1 
                   
                 Q6LD67, A0A024R3K6 
               
               
                   
                 channel, subfamily 
                 levels in skeletal muscle,  
                   
                   
                   
               
               
                   
                 J, member 1 
                 pancreas, spleen, brain, 
                   
                   
                   
               
               
                   
                   
                 heart and liver 
                   
                   
                   
               
               
                 KCNJ2 
                 potassium 
                 Heart, brain, placenta, lung, 
                 Kir2.1, IRK1, 
                 17q24.3 
                 P63252, O15110, 
               
               
                   
                 inwardly-rectifying 
                 skeletal muscle, and 
                 LQT7  
                   
                 P48049, Q9NPI9 
               
               
                   
                 channel, subfamily 
                 kidney. Diffusely 
                   
                   
                   
               
               
                   
                 J, member 2 
                 distributed throughout the 
                   
                   
                   
               
               
                   
                   
                 brain. 
                   
                   
                   
               
               
                 KCNJ3 
                 potassium 
                   
                 Kir3.1, GIRK1, 
                 2q24.1 
                 P48549, B4DEW7, 
               
               
                   
                 inwardly-rectifying 
                   
                 KGA 
                   
                 Q8TBI0, D2XBF0, 
               
               
                   
                 channel, subfamily 
                   
                   
                   
                 D2X9V0 
               
               
                   
                 J, member 3 
                   
                   
                   
                   
               
               
                 KCNJ4 
                 potassium 
                 Heart, skeletal muscle, and 
                 Kir2.3, HIR, 
                 22q13.1 
                 P48050, Q14D44, 
               
               
                   
                 inwardly-rectifying 
                 several different brain 
                 HRK1, hIRK2, 
                   
                 A0A024R1L8, P63252, 
               
               
                   
                 channel, subfamily 
                 regions including the 
                 IRK3 
                   
                 O15110, P48049 
               
               
                   
                 J, member 4 
                 hippocampus 
                   
                   
                   
               
               
                 KCNJ5 
                 potassium 
                 Islets, exocrine pancreas 
                 Kir3.4, CIR, 
                 11q24 
                 Q4QRJ2, _P48544, 
               
               
                   
                 inwardly-rectifying 
                 and heart. Expressed in the 
                 KATP1, 
                   
                 B2R744 Q92807, 
               
               
                   
                 channel, subfamily 
                 adrenal cortex, particularly 
                 GIRK4, LQT13 
                   
                 H9A8K9, H7CGH0, 
               
               
                   
                 J, member 5 
                 the zona glomerulosa 
                   
                   
                 A0A024R3K7 
               
               
                 KCNJ6 
                 potassium 
                 Widely expressed. 
                 Kir3.2, GIRK2, 
                 21q22.1 
                 P48051 Q3MJ74, 
               
               
                   
                 inwardly-rectifying 
                 Expressed in cells of 
                 KATP2, BIR1, 
                   
                 Q53WW6, _B2RA12, 
               
               
                   
                 channel, subfamily 
                 hematopoietic origin (at 
                 hiGIRK2 
                   
                 Q96L92, Q32Q36 
               
               
                   
                 J, member 6 
                 protein level). Most 
                   
                   
                 Q9H3K8 
               
               
                   
                   
                 abundant in cerebellum, 
                   
                   
                   
               
               
                   
                   
                 and to a lesser degree in 
                   
                   
                   
               
               
                   
                   
                 islets and exocrine 
                   
                   
                   
               
               
                   
                   
                 pancreas 
                   
                   
                   
               
               
                 KCNJ8 
                 potassium 
                 Predominantly detected in 
                 Kir6.1 
                 12p12.1 
                 Q15842, O00657, 
               
               
                   
                 inwardly-rectifying 
                 fetal and adult heart 
                   
                   
                 F5GY12, 
               
               
                   
                 channel, subfamily 
                   
                   
                   
                 A0A024RAV6 
               
               
                   
                 J, member 8 
                   
                   
                   
                   
               
               
                 KCNJ9 
                 potassium 
                 Widely expressed. 
                 Kir3.3, GIRK3 
                 1q23.2 
                 Q92806, Q5JW75, 
               
               
                   
                 inwardly-rectifying 
                 Expressed in cells of 
                   
                   
                 Q96L92, Q32Q36, 
               
               
                   
                 channel, subfamily 
                 hematopoietic origin (at 
                   
                   
                 Q9H3K8 
               
               
                   
                 J, member 9 
                 protein level). 
                   
                   
                   
               
               
                 KCNJ10 
                 potassium 
                   
                 Kir4.1, Kir1.2 
                 1q23.2 
                 P78508, 
               
               
                   
                 inwardly-rectifying 
                   
                   
                   
                 A3KME7, Q92808, 
               
               
                   
                 channel, subfamily 
                   
                   
                   
                 Q547K1, Q9BXC5 
               
               
                   
                 J, member 10 
                   
                   
                   
                 Q9NPI9 
               
               
                 KCNJ11 
                 potassium 
                   
                 Kir6.2, BIR 
                 11p15.1 
                 Q14654, 
               
               
                   
                 inwardly-rectifying 
                   
                   
                   
                 B4DWI4, Q8IW96 
               
               
                   
                 channel, subfamily 
                   
                   
                   
                 E9PPF1, H0YES9, 
               
               
                   
                 J, member 11 
                   
                   
                   
                 D2K1F9 
               
               
                 KCNJ12 
                 potassium 
                 Heart, skeletal muscle, and 
                 Kir2.2, Kir2.2v, 
                 17p11.1 
                 Q14500, O43401, 
               
               
                   
                 inwardly-rectifying 
                 several different brain 
                 IRK2, hIRK1 
                   
                 Q15756, Q8NG63, 
               
               
                   
                 channel, subfamily 
                 regions including the 
                   
                   
                 B7U540, Q9HAP6, 
               
               
                   
                 J, member 12 
                 hippocampus 
                   
                   
                 P48050, Q14D44 
               
               
                 KCNJ13 
                 potassium 
                 Predominantly expressed in 
                 Kir7.1, Kir1.4, 
                 2q37 
                 O60928 A0PGH1 
               
               
                   
                 inwardly-rectifying 
                 small intestine. Expression 
                 LCA16 
                   
                 Q8N3Y4_C9JWD6 
               
               
                   
                 channel, subfamily 
                 is also detected in stomach, 
                   
                   
                 H7C4D1 
               
               
                   
                 J, member 13 
                 kidney, and all central 
                   
                   
                   
               
               
                   
                   
                 nervous system regions 
                   
                   
                   
               
               
                   
                   
                 tested with the exception of 
                   
                   
                   
               
               
                   
                   
                 spinal cord 
                   
                   
                   
               
               
                 KCNJ14 
                 potassium 
                 Expressed preferentially in 
                 Kir2.4, IRK4 
                 19q13 
                 Q9UNX9 Q99712 
               
               
                   
                 channel, subfamily 
                 retina 
                   
                   
                 D3DSH5 Q99446 
               
               
                   
                 J, member 14 
                   
                   
                   
                   
               
               
                 KCNJ15 
                 potassium 
                   
                 Kir4.2, Kir1.3, 
                 21q22.2 
                 Q99712 D3DSH5 
               
               
                   
                 inwardly-rectifying 
                   
                 IRKK 
                   
                 Q99446, A8K9U7 
               
               
                   
                 channel, subfamily 
                   
                   
                   
                   
               
               
                   
                 J, member 15 
                   
                   
                   
                   
               
               
                 KCNJ16 
                 potassium 
                 Highly expressed in 
                 Kir5.1, BIR9 
                 17q24.3 
                 Q9NPI9 
               
               
                   
                 inwardly-rectifying 
                 kidney, pancreas and 
                   
                   
                   
               
               
                   
                 channel, subfamily 
                 thyroid gland 
                   
                   
                   
               
               
                   
                 J, member 16 
                   
                   
                   
                   
               
               
                 KCNJ18 
                 potassium 
                 Specifically expressed in 
                 KIR2.6, TTPP2 
                 17p11.2 
                 B7U540 
               
               
                   
                 inwardly-rectifying 
                 skeletal muscle 
                   
                   
                   
               
               
                   
                 channel, subfamily 
                   
                   
                   
                   
               
               
                   
                 J, member 18 
               
               
                   
               
            
           
         
       
     
     
       
         
           
               
             
               
                 TABLE 10 
               
             
            
               
                   
               
               
                 Potassium channels, two-P 
               
            
           
           
               
               
               
               
               
               
            
               
                 Approved 
                   
                   
                   
                   
                   
               
               
                 Symbol 
                 Approved Name 
                 Tissue distribution 
                 Synonyms 
                 Chromosome 
                 Accession number 
               
               
                   
               
               
                 KCNK1 
                 potassium channel, 
                 Widely expressed with 
                 K2p1.1, DPK, 
                 1q42-q43 
                 O00180, Q13307, 
               
               
                   
                 subfamily K, 
                 high levels in heart and 
                 TWIK-1 
                   
                 Q5T5E8 
               
               
                   
                 member 1 
                 brain and lower levels in 
                   
                   
                   
               
               
                   
                   
                 placenta, lung, liver and 
                   
                   
                   
               
               
                   
                   
                 kidney 
                   
                   
                   
               
               
                 KCNK2 
                 potassium channel, 
                   
                 K2p2.1, TREK- 
                 1q41 
                 Q6ZW95, Q13303, 
               
               
                   
                 subfamily K, 
                   
                 1 
                   
                 A0AVM9, Q99411 
               
               
                   
                 member 2 
                   
                   
                   
                   
               
               
                 KCNK3 
                 potassium channel, 
                 Widespread expression in 
                 K2p3.1, TASK, 
                 2p23 
                 O14649, Q535U2, 
               
               
                   
                 subfamily K, 
                 adult. Strongest expression 
                 TASK-1 
                   
                 B9EIJ4 
               
               
                   
                 member 3 
                 in pancreas and placenta. 
                   
                   
                   
               
               
                   
                   
                 Lower expression in brain, 
                   
                   
                   
               
               
                   
                   
                 lung, prostate, heart, 
                   
                   
                   
               
               
                   
                   
                 kidney, uterus, small 
                   
                   
                   
               
               
                   
                   
                 intestine and colon 
                   
                   
                   
               
               
                 KCNK4 
                 potassium channel, 
                   
                 K2p4.1, 
                 11q13 
                 Q2YDA1 
               
               
                   
                 subfamily K, 
                   
                 TRAAK 
                   
                   
               
               
                   
                 member 4 
                   
                   
                   
                   
               
               
                 KCNK5 
                 potassium channel, 
                 Abundant expression in 
                 K2p5.1, TASK- 
                 6p21 
                 O95279 B2RAQ6, 
               
               
                   
                 subfamily K, 
                 kidney, also detected in 
                 2 
                   
                 B5TJL2, Q5VV76 
               
               
                   
                 member 5 
                 liver, placenta and small 
                   
                   
                   
               
               
                   
                   
                 intestine. In the kidney, 
                   
                   
                   
               
               
                   
                   
                 expression is restricted to 
                   
                   
                   
               
               
                   
                   
                 the distal tubules and 
                   
                   
                   
               
               
                   
                   
                 collecting ducts. 
                   
                   
                   
               
               
                 KCNK6 
                 potassium channel, 
                 Widespread expression, 
                 K2p6.1, TWIK- 
                 19q13.1 
                 Q9Y257 Q9HB47 
               
               
                   
                 subfamily K, 
                 detected in all tissues tested 
                 2 
                   
                   
               
               
                   
                 member 6 
                 except for skeletal muscle. 
                   
                   
                   
               
               
                   
                   
                 Strongest expression in 
                   
                   
                   
               
               
                   
                   
                 placenta, pancreas, heart, 
                   
                   
                   
               
               
                   
                   
                 colon and spleen, lower 
                   
                   
                   
               
               
                   
                   
                 levels detected in 
                   
                   
                   
               
               
                   
                   
                 peripheral blood 
                   
                   
                   
               
               
                   
                   
                 leukocytes, lung, liver, 
                   
                   
                   
               
               
                   
                   
                 kidney and thymus. Lowest 
                   
                   
                   
               
               
                   
                   
                 expression detected in 
                   
                   
                   
               
               
                   
                   
                 brain 
                   
                   
                   
               
               
                 KCNK7 
                 potassium channel, 
                   
                 K2p7.1 
                 11q13 
                 Q3MI97 
               
               
                   
                 subfamily K, 
                   
                   
                   
                   
               
               
                   
                 member 7 
                   
                   
                   
                   
               
               
                 KCNK9 
                 potassium channel, 
                 Mainly found in the 
                 K2p9.1, 
                 8q24.3 
                 Q9NPC2, Q2M290, 
               
               
                   
                 subfamily K, 
                 cerebellum. Also found in 
                 TASK3, 
                   
                 Q540F2 
               
               
                   
                 member 9 
                 adrenal gland, kidney and 
                 TASK-3 
                   
                   
               
               
                   
                   
                 lung 
                   
                   
                   
               
               
                 KCNK10 
                 potassium channel, 
                 Abundantly expressed in 
                 K2p10.1, 
                 14q31 
                 P57789, B2R8T4, 
               
               
                   
                 subfamily K, 
                 pancreas and kidney and to 
                 TREK-2, 
                   
                 Q9HB59 
               
               
                   
                 member 10 
                 a lower level in brain, 
                 TREK2, 
                   
                   
               
               
                   
                   
                 testis, colon, and small 
                 PPP1R97 
                   
                   
               
               
                   
                   
                 intestine. Isoform b is 
                   
                   
                   
               
               
                   
                   
                 strongly expressed in 
                   
                   
                   
               
               
                   
                   
                 kidney (primarily in the 
                   
                   
                   
               
               
                   
                   
                 proximal tubule) and 
                   
                   
                   
               
               
                   
                   
                 pancreas, whereas isoform 
                   
                   
                   
               
               
                   
                   
                 c is abundantly expressed 
                   
                   
                   
               
               
                   
                   
                 in brain 
                   
                   
                   
               
               
                 KCNK12 
                 potassium channel, 
                   
                 THIK-2, 
                 2p16.3 
                 Q9HB15 
               
               
                   
                 subfamily K, 
                   
                 THIK2, 
                   
                   
               
               
                   
                 member 12 
                   
                 K2p12.1 
                   
                   
               
               
                 KCNK13 
                 potassium channel, 
                   
                 K2p13.1, 
                 14q32.11 
                 Q9HB14, B5TJL8, 
               
               
                   
                 subfamily K, 
                   
                 THIK-1, 
                   
                 Q96E79 
               
               
                   
                 member 13 
                   
                 THIK1 
                   
                   
               
               
                 KCNK15 
                 potassium channel, 
                 Detected in pancreas, heart, 
                 K2p15.1, 
                 20q13.12 
                 Q9H427, Q52LL3, 
               
               
                   
                 subfamily K, 
                 placenta, lung, liver, 
                 dJ781B1.1, 
                   
                 Q9HBC8 
               
               
                   
                 member 15 
                 kidney, ovary, testis, 
                 KT3.3, 
                   
                   
               
               
                   
                   
                 skeletal muscle and adrenal 
                 KIAA0237, 
                   
                   
               
               
                   
                   
                 gland, and at lower levels 
                 TASK5, 
                   
                   
               
               
                   
                   
                 in prostate, spleen and 
                 TASK-5 
                   
                   
               
               
                   
                   
                 thyroid gland 
                   
                   
                   
               
               
                 KCNK16 
                 potassium channel, 
                 Highly expressed in 
                 K2p16.1, 
                 6p21.2-p21.1 
                 Q96T55, B5TJL9, 
               
               
                   
                 subfamily K, 
                 pancreas. 
                 TALK-1, 
                   
                 Q9H591 
               
               
                   
                 member 16 
                   
                 TALK1 
                   
                   
               
               
                 KCNK17 
                 potassium channel, 
                   
                 K2p17.1, 
                 6p21 
                 Q96T54, E9PB46, 
               
               
                   
                 subfamily K, 
                   
                 TALK-2, 
                   
                 Q9H592, B2RCT9 
               
               
                   
                 member 17 
                   
                 TALK2, 
                   
                   
               
               
                   
                   
                   
                 TASK4, 
                   
                   
               
               
                   
                   
                   
                 TASK-4 
                   
                   
               
               
                 KCNK18 
                 potassium channel, 
                 Expressed specifically in 
                 K2p18.1, 
                 10q26.11 
                 Q7Z418, Q5SQQ8 
               
               
                   
                 subfamily K, 
                 dorsal root ganglion and 
                 TRESK-2, 
                   
                   
               
               
                   
                 member 18 
                 trigeminal ganglion 
                 TRESK2, 
                   
                   
               
               
                   
                   
                 neurons. Detected at low 
                 TRESK, TRIK 
                   
                   
               
               
                   
                   
                 levels in spinal cord. 
               
               
                   
               
            
           
         
       
     
     
       
         
           
               
             
               
                 TABLE 11 
               
             
            
               
                   
               
               
                 Hydrogen voltage-gated ion channels 
               
            
           
           
               
               
               
               
               
               
            
               
                 Approved 
                   
                   
                   
                   
                   
               
               
                 Symbol 
                 Approved Name 
                 Tissue distribution 
                 Synonyms 
                 Chromosome 
                 Accession number 
               
               
                   
               
               
                 HVCN1 
                 hydrogen voltage- 
                 Enriched in immune 
                 MGC15619, 
                 12q24.11 
                 Q96D96, A8MQ37, 
               
               
                   
                 gated channel 1 
                 tissues, such as lymph 
                 Hv1, VSOP 
                   
                 Q96IS5 
               
               
                   
                   
                 nodes, B-lymphocytes, 
                   
                   
                   
               
               
                   
                   
                 monocytes and spleen 
               
               
                   
               
            
           
         
       
     
     The nucleic acid according to the current invention can encode a portion or entire fragment of voltage-dependent cation channel subunit as elaborated in Tables 1-11 or specifically selected from Calcium-activated potassium channels including subfamily M, alpha member 1, subfamily N, member 1, subfamily N, member 2, subfamily N, member 3, subfamily N, member 4, potassium channel, subfamily T, member 1, potassium channel, subfamily T, member 2, potassium channel, subfamily U, member 1, CatSper (1 to 4) and Two-Pore channels, Cyclic nucleotide-regulated channels including channel alpha 1, channel alpha 2, channel alpha 3, channel alpha 4, channel beta 1, channel beta 3, hyperpolarization activated cyclic nucleotide-gated potassium channel 1 to 4, potassium channels, Two-P including subfamily K, member 1-18, potassium channels including inwardly rectifying potassium channels like subfamily J, member 1-18, Voltage-gated calcium channels, Voltage-gated potassium channels including shaker-related subfamily, member 1-10, Shab-related subfamily, member 1 and 2, Shaw-related subfamily, member 1 to 4, Shal-related subfamily, member 1-3, subfamily F, member 1, subfamily G, member 1, subfamily G, member 2, subfamily G, member 3, subfamily G, member 4, subfamily H (eag-related), member 1-8, KQT-like subfamily, member 1-5, delayed-rectifier, subfamily S member 1-3, subfamily V, member 1-2, Voltage-gated sodium channels including type I, alpha subunit, type I, beta subunit, type II, alpha subunit, type II, beta subunit, type III, alpha subunit, type III, beta subunit, type IV, alpha subunit, type V, alpha subunit, type VII, alpha subunit, type VIII, alpha subunit, type IX, alpha subunit, type X, alpha subunit, type XI, alpha subunit, Calcium channel, voltage-dependent including P/Q type, alpha 1A subunit, N type, alpha 1B subunit, L type, alpha 1C subunit, L type, alpha 1D subunit, R type, alpha lE subunit, L type, alpha 1F subunit, T type, alpha 1G subunit, T type, alpha 1H subunit, T type, alpha 1I subunit, L type, alpha 1S subunit, Transient Receptor Potential channels like Transient receptor potential cation channels including subfamily A, member 1, subfamily C, member 1, subfamily C, member 2, pseudogene, subfamily C, member 3, subfamily C, member 4, subfamily C, member 5, subfamily C, member 6, subfamily C, member 7, subfamily M, member 1, subfamily M, member 2, subfamily M, member 3, subfamily M, member 4, subfamily M, member 5, subfamily M, member 6, subfamily M, member 7, subfamily M, member 8, mucolipin, subfamily V, member 1, subfamily V, member 2, subfamily V, member 3, subfamily V, member 4, subfamily V, member 5, subfamily V, member 6, Hydrogen voltage-gated ion channels and the like. 
     In preferred embodiments, the voltage-dependent cation channel subunit is a subunit of channel comprising 6 transmembrane domains, 2 intracellular loops, 1 transmembrane loop, and intracellular N- and C-termini, such as transient receptor potential (TRP) channel. Such TRP may be TRPC (canonical) channel, TRPV (vanilloid), TRPM (melastatin), TRPA (ankyrin), TRPP (polycystin), or TRPML (mucolipin). 
     In particularly preferred embodiments, TRPV channel is TRPV1, TRPV2, TRPV3, TRPV4, TRPV5, or TRPV6, wherein said TRPC channel is TRPC1, TRPC2, TRPC3, TRPC4, TRPC5, TRPC6, or TRPC7, wherein said TRPM channel is TRPM1, TRPM2, TRPM3, TRPM4, TRPM5, TRPM6, TRPM7, or TRPM8, wherein said TRPP channel is TRPP1, TRPP2, TRPP3, or TRPP4, or wherein TRPA is TRPA1. 
     In particularly preferred embodiments, TRPV channel is TRPV1, TRPV2, TRPV3, TRPV4, or TRPA1. 
     The present invention relates to a novel nucleic acids encoding voltage-dependent ion channel fusion subunit comprising one or more bioluminescent donor molecule and/or one or more fluorescent acceptor molecules. 
     The present invention relates to a nucleic acid comprising a nucleotide sequence encoding a voltage-dependent ion channel fusion subunit comprising a voltage-dependent cation channel subunit bound to at least one bioluminescent donor molecule and/or bound to at least one fluorescent acceptor molecule, wherein said voltage-dependent cation channel subunit is a subunit of a transient receptor potential (TRP) channel. According to the invention, bioluminescent donor molecule overlaps with the absorbance spectrum of the acceptor molecule, so that the light energy delivered by the bioluminescent donor molecule is at a wavelength that is able to excite the acceptor molecule. The intramolecular BRET probes according to the present invention are thus useful to monitor TRP channel structural conformational changes and TRP channel activation in living cells. 
     According to a preferred embodiment, the present invention relates to a nucleic acid comprising a nucleotide sequence encoding a voltage-dependent ion channel fusion subunit comprising a voltage-dependent cation channel subunit bound to at least one bioluminescent donor molecule and bound to at least one fluorescent acceptor molecule, wherein said voltage-dependent cation channel subunit is a subunit of a transient receptor potential (TRP) channel, and wherein said bioluminescent donor molecule and acceptor molecule are selected so that the emission spectrum of the bioluminescent donor molecule overlaps with the absorbance spectrum of the acceptor molecule, so the light energy delivered by the bioluminescent donor molecule is at a wavelength that is able to excite the acceptor molecule. Also, said voltage-dependent cation channel subunit is preferably a subunit of channel comprising 6 transmembrane domains, 2 intracellular loops, 1 transmembrane loop, and intracellular N- and C-termini as described herein above. Preferred subunits belong to a member of TRPV subfamily, such as without any limitations TRPV1, TRPV3 and TRPV4. 
     Embodiments of this disclosure include bioluminescence resonance energy transfer (BRET) systems, methods of detecting a protein-protein interaction, methods to determine efficacy of a test compound, BRET vectors, kits relating to each of the above. In general, BRET systems involve the non-radiative transfer of energy between a bioluminescence donor molecule and a fluorescent acceptor molecule by the FORSTER mechanism. The energy transfer primarily depends on: (i) an overlap between the emission and excitation spectra of the donor and acceptor molecules, respectively and (ii) the proximity of about 100 Angstroms (A) between the donor and acceptor molecules. The donor molecule in BRET produces light via chemiluminescence, so it is amenable to small animal imaging. In addition, the BRET system does not use an external light excitation source, which provides potentially greater sensitivity in living subjects because of the low signal to noise ratio. 
     An embodiment of a BRET system, among others, includes: nucleic acid comprising a nucleotide sequence encoding a voltage-dependent ion channel fusion subunit bound to a nucleotide sequence encoding at least one bioluminescent donor molecule and/or bound to a nucleotide sequence encoding at least one fluorescent acceptor molecule. 
     Preferably, the nucleic acid according to the present invention comprises a nucleotide sequence encoding a voltage-dependent ion channel fusion subunit as described above bound, optionally via a linker sequence, to at least one bioluminescent donor molecule and also bound, optionally via a linker, to a nucleotide sequence encoding at least one fluorescent acceptor molecule. More precisely, said bioluminescent donor molecule and acceptor molecule may be bound to either C-terminal, N-terminal, or to a loop of said channel subunit, optionally via a linker sequence. Possible coupling according to the present invention may be as follows:
         (i) the bioluminescent donor molecule is bound to C-terminal of channel subunit and acceptor molecule is bound to N-terminal of said channel subunit,   (ii) the bioluminescent donor molecule is bound to N-terminal of channel subunit and acceptor molecule is bound to C-terminal of said channel subunit,   (iii) the bioluminescent donor molecule is bound to C-terminal of channel subunit and acceptor molecule forms part of the first or the second intracellular loop,   (iv) the bioluminescent donor molecule is bound to N-terminal of channel subunit and acceptor molecule forms part of the first or the second intracellular loop,   (v) said acceptor molecule is bound to C-terminal of channel subunit and the bioluminescent donor molecule forms part of the first or second intracellular loop,   (vi) said acceptor molecule is bound to N-terminal of channel subunit and the bioluminescent donor molecule forms part of the first or second intracellular loop,   (vii) the bioluminescent donor molecule forms part of the first intracellular loop and the acceptor molecule forms part of the second intracellular loop,   (viii) the bioluminescent donor molecule forms part of the second intracellular loop and the acceptor molecule forms part of the first intracellular loop.       

     In preferred aspects of the invention, said bioluminescent donor molecule and acceptor molecule are bound to the N and C termini of the channel subunit (items (i) and (ii) above). In highly preferred aspects of the invention, the bioluminescent donor molecule is bound to the C terminus of the channel subunit and the acceptor molecule is bound to the N terminus of the channel subunit. 
     According to another embodiment, the bioluminescent donor molecule and acceptor molecule may be bound to a different subunit of a transient receptor potential (TRP) channel, either C-terminal, N-terminal, or to a loop of two distinct channel subunits belonging to the same TRP channel or two distinct TRP channels of a different family or subfamily of TRP channel. The nucleic acid according to this embodiment comprises a first nucleotide sequence encoding a voltage-dependent ion channel fusion subunit as described above bound, optionally via a linker sequence, to at least a nucleotide sequence encoding at least one bioluminescent donor molecule or one fluorescent acceptor molecule, and a second nucleotide sequence encoding a voltage-dependent ion channel fusion subunit as described above bound, optionally via a linker sequence, to at least a nucleotide sequence encoding at least one bioluminescent donor molecule or one fluorescent acceptor molecule. If the first subunit is bound to at least one bioluminescent donor molecule, then the second subunit is bound to at least one fluorescent acceptor molecule and vice versa. The various possible coupling may have the same configurations as that described above under (i) to (viii) with the difference that the bioluminescent donor molecule and acceptor molecule are coupled to distinct subunit of the same or of distinct TRP channel. 
     At least one bioluminescent donor molecule or at least one acceptor molecule may be bound to a subunit of a transient receptor potential (TRP) channel, either C-terminal, N-terminal, or to a loop of said subunit and two distinct channel subunits belonging to the same TRP channel. 
     According to still another embodiment, the present invention comprises a nucleotide sequence encoding a voltage-dependent ion channel fusion subunit as described above bound to at least one bioluminescent donor molecule or to at least one fluorescent acceptor molecule. The bioluminescent donor molecule or fluorescent acceptor molecule may be bound, optionally via a linker sequence, to either C-terminal, N-terminal, or to a loop of said channel subunit. One or the other of the donor or the acceptor molecule is bound to the subunit. The other partner of the BRET probe is bound to either in C-terminal or in N-terminal to a modulator or effector of the voltage-dependent cation channel subunit. Preferably, the voltage-dependent cation channel subunit is a subunit of a transient receptor potential (TRP) channel. Therefore, according to this embodiment, if a bioluminescent donor molecule is coupled to a TRP subunit (optionally via a linker, either in C-terminal, N-terminal, or to a loop of the subunit), then the fluorescent acceptor molecule is coupled in C-terminal or N-terminal of effector or modulator of the TRP channel to which the TPR subunit fusion belongs, and vice versa. According to this particular embodiment, the modulator protein may be for example calmodulin ( FIG. 9B ), which is known to binds to TRPV channel in a calcium dependent manner. Indeed, desensitization of the TPRV channel depends on the presence of intracellular calcium, and calmodulin acts as a sensor transducing the changes in calcium concentration into changes in channel conformational changes and behavior. The intermolecular BRET probes according to this particular embodiment of the present invention are thus useful to monitor TRP channel activation in living cells resulting both from structural conformational changes and in the recruitment of intracellular effector proteins such as calmodulin. 
     Modulators, effector proteins and/or ligands have been defined above and have been well characterized for the TRP channels. As indicated above, TRPV1 is a non-selective cation channel and is activated by noxious stimuli, heat, protons, low pH. TRPV1 may be activated by endogenous vanilloid agonists, a class of compounds referred to as endovanilloids; by conjugates of biogenic amines, e.g., N-arachidonylethanolamine, N-arachidonoyldopamine, N-oleoylethanolamine N-arachidonolylserine, and various N-acyltaurines and N-acylsalsolinols; oxygenated eicosatetraenoic acids, such as the lipoxygenase products 5-, 12-, and 15-hydroperoxyeicosatetraenoic acids, prostaglandins, and leukotriene B 4 ; and further by adenosine, ATP, and polyamines (such as spermine, spermidine, and putrescine). TRPV1 may be also activated by exogenous agonists, such as capsaicinoids and capsinoids, piperine, eugenol, gingerol and by synthetic agonists such as lidocaine. 
     As TRPV1, TRPV2 is activated by noxious heat as well as by several exogenous chemical ligands such as for example phenylborate, DPBA, Cannabidiol, and cannabinol. It is blocked by diuretic amiloride, tetraethylammonium, 4-amino-pyridine, and 1-(2-(trifluoromethyl)phenyl) imidazole, and the monoterpene aldehyde citral. 
     TRPV3 is activated by a number of natural and synthetic exogenous ligands which include for example thymol, eugenol, cresol, cembrane diterpenoid incensole acetate, carvacrol, 2-aminoethoxydiphenylboronate (2-APB), drofenine, diphenylboronic anhydride (DPBA), and 2,2-diphenyltetrahydrofuran (DPTHF), etc. . . . . 
     TRPV4 is activated by physical stimuli, such as cell swelling, innocuous warmth, by endogenous chemical ligands such as endocannabinoids and arachidonic acid metabolites, and by small molecules such as bisandrographolide, α-phorbol esters, dimeric diterpenoid bisandrographolide A, and GSK1016790A (See Thorneloe K S et al., J Pharmacol Exp Ther 326: 432-442). Antagonists of TRPV4 include for example RN-1734 and RN-9893. 
     TRPV5 and TRPV6 are activated by 1,25-dihydroxy vitamine D3, the Ca 2+  binding protein S100A10, and annexin II. Antagonists thereof include for example the antifungal azole econazole, etc. . . . . 
     Said bioluminescent donor molecule and acceptor molecule are selected so that the emission spectrum of the bioluminescent donor molecule overlaps with the absorbance spectrum of the acceptor protein, so the light energy delivered by the bioluminescent donor molecule is at a wavelength that is able to excite the acceptor molecule. 
     Bioluminescent donor refers to any moiety capable of acting on a suitable substrate to transform chemical energy into light energy. For example, it may refer to an enzyme which converts a substrate into an activated product which then releases energy as it relaxes. The activated product (generated by the activity of the bioluminescent protein on the substrate) is the source of the bioluminescent protein-generated luminescence that is transferred to the acceptor molecule. 
     Given the size of bioluminescent molecules it was surprising that functional voltage-dependent ion channel subunits according to the present invention could be produced. There are a number of different bioluminescent donor molecules that can be employed in the present invention. Light-emitting systems have been known and isolated from many luminescent organisms including bacteria, protozoa, coelenterates, molluscs, fish, millipedes, flies, fungi, worms, crustaceans, and beetles, particularly click beetles of genus  Pyrophorus  and the fireflies of the genera  Photinus, Photuris , and  Luciola . Additional organisms displaying bioluminescence are listed in international publications WO 00/024878 and WO 99/049019. 
     One well characterized example is the class of proteins known as luciferases. Luciferases proteins catalyze an energy-yielding chemical reaction in which a specific biochemical substance, a luciferin (a naturally occurring substrate), is oxidized by an enzyme having a luciferase activity. Both prokaryotic and eukaryotic organisms including species of bacteria, algae, fungi, insects, fish and other marine forms can emit light energy in this manner and each has specific luciferase activities and luciferins, which are chemically distinct from those of other organisms. Luciferin/luciferase systems are very diverse in form, chemistry and function. For example, there are luciferase activities which facilitate continuous chemiluminescence, as exhibited by some bacteria and mushrooms, and those which are adapted to facilitate sporadic, or stimuli induced, emissions, as in the case of dinoflagellate algae. As a phenomenon, which entails the transformation of chemical energy into light energy, bioluminescence is not restricted to living organisms, nor does it require the presence of living organisms. It is simply a type of chemiluminescent reaction that requires a luciferase activity, which at one stage or another had its origins from a biological catalyst. Hence the preservation or construction of the essential activities and chemicals suffices to have the means to give rise to bioluminescent phenomena. 
     Examples of bioluminescent proteins with luciferase activity may be found in U.S. Pat. Nos. 5,229,285, 5,219,737, 5,843,746, 5,196,524, and 5,670,356. Two of the most widely used luciferases are: (i)  Renilla  luciferase (from  R. reniformis ), a 35 kDa protein, which uses coelenterazine as a substrate and emits light at 480 nm; and (ii) Firefly luciferase (from  Photinus pyralis ), a 61 kDa protein, which uses luciferin as a substrate and emits light at 560 nm. In preferred embodiments bioluminescent donor molecule is a protein chosen among luciferase, chosen among  Renilla  luciferase, Firefly luciferase, Coelenterate luciferase, North American glow worm luciferase, click beetle luciferase, a railroad worm luciferase,  Gaussia  luciferase, Aequorin, Arachnocampa luciferase, or a biologically active variant or fragment of any one.  Gaussia  luciferase (from  Gaussia princeps ) is a 20 kDa protein that oxidises coelenterazine in a rapid reaction resulting in a bright light emission at 470 nm. Luciferases useful for the present invention have also been characterized from  Anachnocampa  sp (WO 2007/019634). These enzymes are about 59 kDa in size and are ATP-dependent luciferases that catalyze luminescence reactions with emission spectra within the blue portion of the spectrum. 
     Alternative, non-luciferase, bioluminescent molecules may be any enzymes which can act on suitable substrates to generate a luminescent signal. Specific examples of such enzymes are β-galactosidase, lactamase, horseradish peroxydase, alkaline phosphatase, β-glucuronidase, or β-glucosidase. Synthetic luminescent substrates for these enzymes are well known in the art and are commercially available from companies, such as Tropix Inc. (Bedford, Mass., USA). 
     By way of examples, several bioluminescent donor molecules have been listed in the following Table 12. 
     
       
         
           
               
             
               
                 TABLE 12 
               
             
            
               
                   
               
               
                 Examples of bioluminescent molecules 
               
            
           
           
               
               
               
               
               
               
            
               
                   
                   
                   
                 MW 
                 Emission 
                   
               
               
                 Species 
                 Name 
                 Organism 
                 kDa × 10 −3   
                 (nm) 
                 Substrate 
               
               
                   
               
            
           
           
               
               
               
               
               
               
            
               
                 Insect 
                 FFluc 
                 
                   Photinus pyralis 
                 
                 ~61 
                 560 
                 D-(−)-2-(6′-hydroxybenzo- 
               
               
                   
                   
                 (North American Firefly) 
                   
                   
                 thiazolyl)-Δ 2 -thiazoline-4- 
               
               
                   
                   
                   
                   
                   
                 carboxylic acid, HBTTCA, 
               
               
                   
                   
                   
                   
                   
                 (C 11 H 8 N 2 O 3 S 2 )(Luciferin) 
               
               
                 Insect 
                 FF′luc 
                 
                   Luciola cruciate 
                 
                   
                 560-590 (many 
                 Luciferin 
               
               
                   
                   
                 (Japanese Firefly) 
                   
                 mutants) 
                   
               
               
                 Insect 
                   
                 Phengodid beetles(railroad 
                   
                   
                   
               
               
                   
                   
                 worms) 
                   
                   
                   
               
               
                 Insect 
                   
                   Arachnocampa  sp. 
                   
                   
                 Luciferin 
               
               
                 Insect 
                   
                 
                   Orphelia fultoni 
                 
                   
                   
                   
               
               
                   
                   
                 (North American glow 
                   
                   
                   
               
               
                   
                   
                 worm) 
                   
                   
                   
               
               
                 Insect 
                 Clluc 
                 
                   Pyrophorus 
                 
                   
                 546, 560 578 
                 Luciferin 
               
               
                   
                   
                 
                   plagiophthalamus 
                 
                   
                 and 593 
                   
               
               
                 Jelly Fish 
                 Aequorea 
                 
                   Aequorea 
                 
                 44.9 
                 460-470 
                 Coelenterazine 
               
               
                 Sea Pansy 
                 Rluc 
                 
                   Renilla Reniformis 
                 
                 36 
                 480 
                 Coelenterazine 
               
               
                 Sea 
                 Rluc8 
                 
                   Renilla 
                 
                 36 
                 487(peak) 
                 Coelenterazine/Deep Blue C 
               
               
                 Pansy(modified) 
                   
                   Reniformis (modified) 
                   
                   
                   
               
               
                 Sea Pansy 
                 Rmluc 
                 
                   Renilla mullerei 
                 
                 36.1 
                 ~480 
                 Coelenterazine 
               
               
                 Sea Pansy 
                   
                 
                   Renilla kollikeri 
                 
                   
                   
                   
               
               
                 Crustacea(shrimp) 
                 Vluc 
                 
                   Vargula hilgendoifii 
                 
                 ~62 
                 ~460 
                 coelenterazine *  
               
               
                 Crustacea 
                   
                   Cypridina (sea firefly) 
                 75 
                 460 
                 coelenterazine ** 
               
               
                 Dinofagellate 
                   
                 
                   Gonyaulax polyedra 
                 
                 130 
                 ~475 
                 Tetrapyrrole 
               
               
                 (marine algae) 
                   
                   
                   
                   
                   
               
               
                 Mollusc 
                   
                   Latia  (Freshwater limpet) 
                 170 
                 500 
                 Enol formate, 
               
               
                   
                   
                   
                   
                   
                 terpene,aldehyde. 
               
               
                 Hydroid 
                   
                 Obelia  biscuspidata   
                 ~20 
                 ~470 
                 Coelenterazine 
               
               
                 Shrimp 
                   
                 
                   Oplophorus gracilorostris 
                 
                 31 
                 462 
                 Coelenterazine 
               
               
                 Others 
                 Ptluc 
                 
                   Ptilosarcus 
                 
                   
                 ~490 
                 Coelenterazine 
               
               
                   
                 Glue 
                 
                   Gaussia 
                 
                 ~20 
                 ~475 
                 Coelenterazine 
               
               
                   
                 Plluc 
                 
                   Pleuromamma 
                 
                 22.6 
                 ~475 
                 Coelenterazine 
               
               
                   
               
            
           
         
       
     
     The choice of the substrate of the bioluminescent donor molecule can impact on the wavelength and the intensity of the light generated by the bioluminescent protein. A widely known substrate is coelenterazine which occurs in cnidarians, copepods, chaetognaths, ctenophores, decapod shrimps, mysid shrimps, radiolarians and some fish taxa. For  Renilla  luciferase for example, coelenterazine analogues/derivatives are available that result in light emission between 418 and 512 nm. A coelenterazine analogue/derivative (400A, DeepBlueC) has been described emitting light at 400 nm with  Renilla  luciferase (WO 01/46691). Other examples of coelenterazine analogues/derivatives are EnduRen and ViviRen. 
     Luciferin relates to a class of light-emitting biological pigments found in organisms capable of bioluminescence, which are oxidised in the presence of the enzyme luciferase to produce oxyluciferin and energy in the form of light. Luciferin, or 2-(6-hydroxybenzothiazol-2-yl)-2-thiazoline-4-carboxylic acid, was first isolated from the firefly  Photinus pyralis . Since then, various forms of luciferin have been discovered and studied from various different organisms, mainly from the ocean, for example fish and squid, however, many have been identified for example in worms, beetles and various other insects. 
     There are at least five general types of luciferin, which are each chemically different and catalysed by chemically and structurally different luciferases that employ a wide range of different cofactors. First, is firefly luciferin, the substrate of firefly luciferase, which requires ATP for catalysis (EC 1.13.12.7). Second, is bacterial luciferin, also found in some squid and fish, that consists of a long chain aldehyde and a reduced riboflavin phosphate. Bacterial luciferase is FMNH-dependent. Third, is dinoflagellate luciferin, a tetrapyrrolic chlorophyll derivative found in dinoflagellates (marine plankton), the organisms responsible for night-time ocean phosphorescence. Dinoflagellate luciferase catalyses the oxidation of dinoflagellate luciferin and consists of three identical and catalytically active domains. Fourth, is the imidazolopyrazine vargulin, which is found in certain ostracods and deep-sea fish, for example, Porichthys. Last, is coelanterazine (an imidazolpyrazine), the light-emitter of the protein aequorin, found in radiolarians, ctenophores, cnidarians, squid, copepods, chaetognaths, fish and shrimp. 
     There are a number of different acceptor molecules that can be employed in this invention. The acceptor molecule may be a protein or non-proteinaceous. 
     Representative acceptor proteins can include, but are not limited to, green fluorescent protein (GFP), variant of green fluorescent protein (such as GFP10), blue fluorescent protein (BFP), cyan fluorescent protein (CFP), yellow fluorescent protein (YFP), enhanced GFP (EGFP), enhanced CFP (ECFP), enhanced YFP (EYFP), GFPS65T, mAmetrine, LSS-mOrange, LSS-mKate, Emerald, Topaz, GFPuv, destabilised EGFP (dEGFP), destabilised ECFP (dECFP), destabilised EYFP (dEYFP), HcRed, t-HcRed, DsRed, DsRed2, mRFPl, pocilloporin,  Renilla  GFP, Monster GFP, paGFP, Kaede protein or a Phycobili protein, or a biologically active variant or fragment of any one thereof. 
     The most frequently used bioluminescent or fluorophore is the green fluorescent protein from the jellyfish  Aequorea victoria  and numerous other variants (GFPs) obtained for example mutagenesis and chimeric protein technologies. GFPs are classified based on the distinctive component of their chromophores, each class having distinct excitation and emission wavelengths: class 1, wild-type mixture of neutral phenol and anionic phenolate: class 2, phenolate anion: class 3, neutral phenol: class 4, phenolate anion with stacked s-electron system: class 5, indole: class 6, imidazole: and class 7, phenyl. One example of an engineered system which is suitable for BRET is a  Renilla  luciferase and enhanced yellow mutant of GFP (EYFP) pairing which do not directly interact to a significant degree with one another alone in the absence of a mediating protein(s) (in this case, the G protein coupled receptor). 
     Examples of non-proteinaceous acceptor molecules are Alexa™ (Molecular Probes), fluor dye, Bodipy Dye™ (Life technologies), Cy Dye™ (Life technologies), fluorescein, dansyl, umbelliferone (7-hydroxycoumarin), fluorescent microsphere, luminescent nanocrystal, Marina Blue™ (Life technologies), Cascade Blue™ (Life technologies), Cascade Yellow™ (Life technologies), Pacific Blue™ (Life technologies), Oregon Green™ (Life technologies), Tetramethylrhodamine, Rhodamine, Texas Red™ (Life technologies), rare earth element chelates, or any combination or derivatives thereof. 
     Other representative acceptor molecules can include, but are not limited to sgGFP, sgBFP, BFP blue shifted GFP (Y66H), Cyan GFP, DsRed, monomeric RFP, EBFP, ECFP, GFP (S65T), GFP red shifted (rsGFP), non-UV excitation (wtGFP), UV excitation (wtGFP), GFPuv, HcRed, rsGFP, Sapphire GFP, sgBFP™, sgBFP™ (super glow BFP), sgGFP™, sgGFP™ (super glow GFP), Yellow GFP, semiconductor nanoparticles (e.g., raman nanoparticles), 1,5 IAEDANS; 1,8-ANS; 4-Methylumbelliferone; 5-carboxy-2,7-dichlorofluorescein; 5-Carboxyfluorescein (5-FAM); 5-Carboxynapthofluorescein; 5-Carboxytetramethylrhodamine (5-TAMRA); 5-FAM (5-Carboxyfluorescein); 5-HAT (Hydroxy Tryptamine); 5-Hydroxy Tryptamine (HAT); 5-ROX (carboxy-X-rhodamine); 5-TAMRA (5-Carboxytetramethylrhodamine); 6-Carboxyrhodamine 6G; 6-CR 6G; 6-JOE; 7-Amino-4-methylcoumarin; 7-Aminoactinomycin D (7-AAD); 7-Hydroxy-4-methylcoumarin; 9-Amino-6-chloro-2-methoxyacridine; AB Q; Acid Fuchsin; ACMA (9-Amino-6-chloro-2-methoxyacridine); Acridine Orange; Acridine Red; Acridine Yellow; Acriflavin; Acriflavin Feulgen SITSA; Aequorin (Photoprotein); AFPs (AutoFluorescent Protein; Quantum Biotechnologies); Alexa Fluor 350™; Alexa Fluor 430™; Alexa Fluor 488™; Alexa Fluor 532™; Alexa Fluor 546™; Alexa Fluor 568™; Alexa Fluor 594™; Alexa Fluor 633™; Alexa Fluor 647™; Alexa Fluor 660™; Alexa Fluor 680™; Alizarin Complexon; Alizarin Red; Allophycocyanin (APC); AMC, AMCA-S; AMCA (Aminomethylcoumarin); AMCA-X; Aminoactinomycin D; Aminocoumarin; Aminomethylcoumarin (AMCA); Anilin Blue; Anthrocyl stearate; APC (Allophycocyanin); APC-Cy7; APTRA-BTC; APTS; Astrazon Brilliant Red 4G; Astrazon Orange R; Astrazon Red 6B; Astrazon Yellow 7 GLL; Atabrine; ATTO-TAG™ CBQCA; ATTO-TAG™ FQ; Auramine; Aurophosphine G; Aurophosphine; BAO 9 (Bisaminophenyloxadiazole); BCECF (high pH); BCECF (low pH); Berberine Sulphate; Beta Lactamase; Bimane; Bisbenzamide; Bisbenzimide (Hoechst); bis-BTC; Blancophor FFG; BlancophorSV; BOBO™-1; BOBO™-3; Bodipy 492/515; Bodipy 493/503; Bodipy 500/510; Bodipy 505/515; Bodipy 530/550; Bodipy 542/563; Bodipy 558/568; Bodipy 564/570; Bodipy 576/589; Bodipy 581/591; Bodipy 630/650-X; Bodipy 650/665-X; Bodipy 665/676; Bodipy Fl; Bodipy FL ATP; Bodipy Fl-Ceramide; Bodipy R6G SE; Bodipy TMR; Bodipy TMR-X conjugate; Bodipy TMR-X, SE; Bodipy TR; Bodipy TR ATP; Bodipy TR-X SE; BO-PRO™-1; BO-PRO™-3; Brilliant Sulphoflavin FF; BTC; BTC-5N; Calcein; Calcein Blue; Calcium Crimson™; Calcium Green; Calcium Green-1 Ca 2+  Dye; Calcium Green-2 Ca 2+ ; Calcium Green-5N Ca 2+ ; Calcium Green-C18 Ca 2+ ; Calcium Orange; Calcofluor White; Carboxy-X-rhodamine (5-ROX); Cascade Blue™; Cascade Yellow; Catecholamine; CCF2 (GeneBlazer); CFDA; Chlorophyll; Chromomycin A; Chromomycin A; CL-NERF; CMFDA; Coumarin Phalloidin; C-phycocyanine; CPM Methylcoumarin; CTC; CTC Formazan; Cy2™; Cy3.18; Cy3.5™; Cy3™; Cy5.18; Cy5.5™; Cy5™; Cy7™; cyclic AMP Fluorosensor (FiCRhR); Dabcyl; Dansyl; Dansyl Amine; Dansyl Cadaverine; Dansyl Chloride; Dansyl DHPE; Dansyl fluoride; DAPI; Dapoxyl; Dapoxyl 2; Dapoxyl 3′ DCFDA; DCFH (Dichlorodihydrofluorescein Diacetate); DDAO; DHR (Dihydorhodamine 123); Di-4-ANEPPS; Di-8-ANEPPS (non-ratio); DiA (4-Di-16-ASP); Dichlorodihydrofluorescein Diacetate (DCFH); DiD-Lipophilic Tracer; DiD (Di1C18(5)); DIDS; Dihydorhodamine 123 (DHR); Dil (DilC18(3)); Dinitrophenol; DiO (DiOC18(3)); DiR; DiR (Di1C18(7)); DM-NERF (high pH); DNP; Dopamine; DTAF; DY-630-NHS; DY-635-NHS; ELF 97; Eosin; Erythrosin; Erythrosin ITC; Ethidium Bromide; Ethidium homodimer-1 (EthD-1); Euchrysin; EukoLight; Europium (III) chloride; EYFP; Fast Blue; FDA; Feulgen (Pararosaniline); FIF (Formaldehyd Induced Fluorescence); FITC; Flazo Orange; Fluo-3; Fluo-4; Fluorescein (FITC); Fluorescein Diacetate; Fluoro-Emerald; Fluoro-Gold (Hydroxystilbamidine); Fluor-Ruby; FluorX; FM 1-43™; FM 4-46; Fura Red™ (high pH); Fura Red™/Fluo-3; Fura-2; Fura-2/BCECF; Genacryl Brilliant Red B; Genacryl Brilliant Yellow 10GF; Genacryl Pink 3G; Genacryl Yellow SGF; GeneBlazer (CCF2); Gloxalic Acid; Granular blue; Haematoporphyrin; Hoechst 33258; Hoechst 33342; Hoechst 34580; HPTS; Hydroxycoumarin; Hydroxystilbamidine (FluoroGold); Hydroxytryptamine; Indo-1, high calcium; Indo-1, low calcium; Indodicarbocyanine (DiD); Indotricarbocyanine (DiR); Intrawhite Cf; JC-1; JO-JO-1; JO-PRO-1; LaserPro; Laurodan; LDS 751 (DNA); LDS 751 (RNA); Leucophor PAF; Leucophor SF; Leucophor WS; Lissamine Rhodamine; Lissamine Rhodamine B; Calcein/Ethidium homodimer; LOLO-1; LO-PRO-1; Lucifer Yellow; Lyso Tracker Blue; Lyso Tracker Blue-White; Lyso Tracker Green; Lyso Tracker Red; Lyso Tracker Yellow; LysoSensor Blue; LysoSensor Green; LysoSensor Yellow/Blue; Mag Green; Magdala Red (Phloxin B); Mag-Fura Red; Mag-Fura-2; Mag-Fura-5; Mag-Indo-1; Magnesium Green; Magnesium Orange; Malachite Green; Marina Blue; Maxilon Brilliant Flavin 10 GFF; Maxilon Brilliant Flavin 8 GFF; Merocyanin; Methoxycoumarin; Mitotracker Green FM; Mitotracker Orange; Mitotracker Red; Mitramycin; Monobromobimane; Monobromobimane (mBBr-GSH); Monochlorobimane; MPS (Methyl Green Pyronine Stilbene); NBD; NBD Amine; Nile Red; Nitrobenzoxadidole; Noradrenaline; Nuclear Fast Red; Nuclear Yellow; Nylosan Brilliant lavin EBG; Oregon Green; Oregon Green 488-X; Oregon Green™; Oregon Green™ 488; Oregon Green™ 500; Oregon Green™ 514; Pacific Blue; Pararosaniline (Feulgen); PBFI; PE-Cy5; PE-Cy7; PerCP; PerCP-Cy5.5; PE-TexasRed [Red 613]; Phloxin B (Magdala Red); Phorwite AR; Phorwite BKL; Phorwite Rev; Phorwite RPA; Phosphine 3R; PhotoResist; Phycoerythrin B [PE]; Phycoerythrin R [PE]; PKH26 (Sigma); PKH67; PMIA; Pontochrome Blue Black; POPO-1; POPO-3; PO-PRO-1; PO-PRO-3; Primuline; Procion Yellow; Propidium lodid (PI); PyMPO; Pyrene; Pyronine; Pyronine B; Pyrozal Brilliant Flavin 7GF; QSY 7; Quinacrine Mustard; Red 613 [PE-TexasRed]; Resorufin; RH 414; Rhod-2; Rhodamine; Rhodamine 110; Rhodamine 123; Rhodamine 5 GLD; Rhodamine 6G; Rhodamine B; Rhodamine B 200; Rhodamine B extra; Rhodamine BB; Rhodamine BG; Rhodamine Green; Rhodamine Phallicidine; Rhodamine Phalloidine; Rhodamine Red; Rhodamine WT; Rose Bengal; R-phycocyanine; R-phycoerythrin (PE); S65A; S65C; S65L; S65T; SBFI; Serotonin; Sevron Brilliant Red 2B; Sevron Brilliant Red 4G; Sevron Brilliant Red B; Sevron Orange; Sevron Yellow L; SITS; SITS (Primuline); SITS (Stilbene Isothiosulphonic Acid); SNAFL calcein; SNAFL-1; SNAFL-2; SNARF calcein; SNARF1; Sodium Green; SpectrumAqua; SpectrumGreen; SpectrumOrange; Spectrum Red; SPQ (6-methoxy-N-(3-sulfopropyl)quinolinium); Stilbene; Sulphorhodamine B can C; Sulphorhodamine Extra; SYTO 11; SYTO 12; SYTO 13; SYTO 14; SYTO 15; SYTO 16; SYTO 17; SYTO 18; SYTO 20; SYTO 21; SYTO 22; SYTO 23; SYTO 24; SYTO 25; SYTO 40; SYTO 41; SYTO 42; SYTO 43; SYTO 44; SYTO 45; SYTO 59; SYTO 60; SYTO 61; SYTO 62; SYTO 63; SYTO 64; SYTO 80; SYTO 81; SYTO 82; SYTO 83; SYTO 84; SYTO 85; SYTOX Blue; SYTOX Green; SYTOX Orange; Tetracycline; Tetramethylrhodamine (TRITC); Texas Red™; Texas Red-X™ conjugate; Thiadicarbocyanine (DiSC3); Thiazine Red R; Thiazole Orange; Thioflavin 5; Thioflavin S; Thioflavin TCN; Thiolyte; Thiozole Orange; Tinopol CBS (Calcofluor White); TMR; TO-PRO-1; TO-PRO-3; TO-PRO-5; TOTO-1; TOTO-3; TriColor (PE-Cy5); TRITC TetramethylRodaminelsoThioCyanate; True Blue; TruRed; Ultralite; Uranine B; Uvitex SFC; WW 781; X-Rhodamine; XRITC; Xylene Orange; Y66F; Y66H; Y66W; YO-PRO-1; YO-PRO-3; YOYO-1; YOYO-3, Sybr Green, Thiazole orange (interchelating dyes), or combinations thereof. 
     Alternatively, the acceptor molecule may be a fluorescent nanocrystal. Nanocrystals, have several advantages over organic molecules as fluorescent labels, including resistance to photodegradation, improved brightness, non-toxicity, and size dependent, narrow emission spectra that enables the monitoring of several processes simultaneously. Additionally, the absorption spectrum of nanocrystals is continuous above the first peak, enabling all sizes, and hence all colors, to be excited with a single excitation wavelength. 
     Fluorescent nanocrystals may be attached, or “bioconjugated”, to proteins in a variety of ways. For example, the surface cap of a “quantum dot” may be negatively charged with carboxylate groups from either dihydrolipoic acid (DHLA) or an amphiphilic polymer. Proteins can be conjugated to the DHLA-nanocrystals electrostatically, either directly or via a bridge consisting of a positively charged leucine zipper peptide fused to recombinant protein. The latter binds to a primary antibody with specificity for the intended target. Alternatively, antibodies, streptavidin, or other proteins are coupled covalently to the polyacrylate cap of the nanocrystal with conventional carbodiimide chemistry. 
     There are colloidal methods to produce nanocrystals, including cadmium selenide, cadmium sulfide, indium arsenide, and indium phosphide. These quantum dots can contain as few as 100 to 100,000 atoms within the quantum dot volume, with a diameter of 10 to 50 atoms. Some quantum dots are small regions of one material buried in another with a larger band gap. These can be so-called core-shell structures, for example, with CdSe in the core and ZnS in the shell or from special forms of silica called ormosil. Conversely, smaller dots emit bluer (higher energy) light. The coloration is directly related to the energy levels of the quantum dot. Quantitatively speaking, the bandgap energy that determines the energy (and hence color) of the fluoresced light is inversely proportional to the square of the size of the quantum dot. Larger quantum dots have more energy levels which are more closely spaced. This allows the quantum dot to absorb photons containing less energy, i.e. those closer to the red end of the spectrum. 
     The acceptor molecule may also be a fluorescent microsphere. These are typically made from polymers, and contain fluorescent molecules (for example fluorescein GFP or YFP) incorporated into the polymer matrix, which can be conjugated to a variety of reagents. Fluorescent microspheres may be labeled internally or on the surface. Internal labeling produces very bright and stable particles with typically narrow fluorescent emission spectra. With internal labeling, surface groups remain available for conjugating ligands (for example, proteins) to the surface of the bead. Internally-labeled beads are used extensively in imaging applications, as they display a greater resistance to photobleaching. 
     Carboxylate-modified fluorescent microspheres are suitable for covalent coupling of proteins using water-soluble carbodiimide reagents such as 1-ethyl-3-(3-dimethylaminopropyl) carbodiimide hydrochloride (EDAC). Sulfate fluorescent microspheres are relatively hydrophobic and will passively and nearly irreversibly adsorb almost any protein. Aldehyde-sulfate fluorescent microspheres are sulfate microspheres that have been modified to add surface aldehyde groups, and react with proteins. 
     Finally, the acceptor molecule may be a luminescent microsphere. These are typically made from polymers, which contain luminescent molecules (for example complexes of europium or platinum) incorporated into the polymer matrix, which can be conjugated to a variety of reagents. 
     Criteria which should be considered in determining suitable pairings for BRET is the relative emission/fluorescence spectrum of the acceptor molecule compared to that of the bioluminescent donor molecule. The emission spectrum of the bioluminescent protein should overlap with the absorbance spectrum of the acceptor molecule such that the light energy from the bioluminescent protein luminescence emission is at a wavelength that is able to excite the acceptor molecule and thereby promote acceptor molecule fluorescence when the two molecules are in a proper proximity and orientation with respect to one another. For example, it has been demonstrated that an  Renilla  luciferase/EGFP pairing is not as good as an  Renilla  luciferase/EYEF pairing based on observable emission spectral peaks. 
     The bioluminescent protein emission can be manipulated by modifications to the substrate. In the case of luciferases the substrate is coelenterazine. The rationale behind altering the bioluminescent protein emission is to improve the resolution between donor emission and acceptor emission. The original BRET system uses the  Renilla  luciferase as donor, EYFP (or Topaz) as the acceptor and coelenterazine derivative as the substrate. These components when combined in a BRET assay generate maximal light in the 475-485 nm range for the bioluminescent protein and the 525-535 nm range for the acceptor molecule, giving a spectral resolution of 40-60 nm. 
       Renilla  luciferase generates a broad emission peak overlapping substantially the GFP emission, which in turn contributes to decrease the signal to noise of the system. Various coelenterazine derivatives are known in the art, including coe1400a, that generate light at various wavelengths (distinct from that generated by the wild type coelenterazine) as a result of  Renilla  luciferase activity. A skilled person in the art would appreciate that because the light emission peak of the donor has changed, it is necessary to select an acceptor molecule which will absorb light at this wavelength and thereby permit efficient energy transfer. Spectral overlapping between light emission of the donor and the light absorption peak of the acceptor is one condition among others for an efficient energy transfer. Class 3 and 1 GFPs are known to absorb light at 400 nm and re-emit between 505-511 nm. This results in a wavelength difference between donor and acceptor emissions of approximately 111 nm. 
     Bioluminescent donor and acceptor molecule pairs are well known in the art and are well within the reach of a person of skill in art. Based on the knowledge regarding the ability of donor to generate luminescence and the ability of an acceptor to accept energy emitted as a result of the activity of a bioluminescent donor protein, and re-emit it as light energy or based on transfer of excited-state energy between such pairs of molecules, spectral overlap, the relative orientation, and the distance between the donor and acceptor many bioluminescent proteins and acceptor molecule pairs can be selected by a person of skill in art. 
     The present invention also relates to an expression vector comprising an acid nucleic encoding a voltage-dependent ion channel fusion subunit as described above. The nucleic acid or polynucleotide of the present invention may be inserted into several commercially available expression vectors. Non-limiting examples include prokaryotic plasmid vectors, such as the pUC-series, pBluescript (Stratagene), the pET-series of expression vectors (Novagen) or pCRTOPO (Invitrogen) and vectors compatible with an expression in mammalian cells like pREP (Invitrogen), pcDNA3 (Invitrogen), pCEP4 (Invitrogen), pMC1neo (Stratagene), pXT1 (Stratagene), pSG5 (Stratagene), EBO-pSV2neo, pBPV-1, pdBPVMMTneo, pRSVgpt, pRSVneo, pSV2-dhfr, pIZD35, pLXIN, pSIR (Clontech), pIRES-EGFP (Clontech), pEAK-10 (Edge Biosystems) pTriEx-Hygro (Novagen) and pCINeo (Promega). Examples for plasmid vectors suitable for  Pichia pastoris  comprise e.g. the plasmids pAO815, pPIC9K and pPIC3.5K (all Invitrogen). The nucleic acid or polynucleotide of the present invention referred to above may also be inserted into vectors such that a translational fusion with another polynucleotide is generated. The other polynucleotide may encode a protein which may e.g. increase the solubility and/or facilitate the purification of the fusion protein. Non-limiting examples include pET32, pET41, pET43. The vectors may also contain an additional expressible polynucleotide coding for one or more chaperones to facilitate correct protein folding. Suitable bacterial expression hosts comprise e.g. strains derived from BL21 (such as BL21(DE3), BL21(DE3)PlysS, BL21(DE3)RIL, BL21(DE3)PRARE) or Rosetta®. In preferred embodiments, expression vector according to the invention is a DNA or RNA vector, capable of transforming eukaryotic host cells and effecting stable or transient expression of said TRP channel fusion subunit, and wherein said vector is a plasmid, a virus like adenovirus, adeno associated virus (AVV), lentiviral, Epstein-Barr, Herpes Simplex, Papilloma, Polyoma, Retro, SV40, Vaccinia, any retroviral vector, influenza viral vector and other non-viral vectors including naked DNA, or liposomes. 
     Generally, vectors can contain one or more origin of replication (ori) and inheritance systems for cloning or expression, one or more markers for selection in the host, e.g., antibiotic resistance, and one or more expression cassettes. Suitable origins of replication (ori) include, for example, the Col E1, the SV40 viral and the M 13 origins of replication. The coding sequences inserted in the vector can e.g. be synthesized by standard methods, or isolated from natural sources. Ligation of the coding sequences to transcriptional regulatory elements and/or to other amino acid encoding sequences can be carried out using established methods. Transcriptional regulatory elements (parts of an expression cassette) ensuring expression in prokaryotes or eukaryotic cells are well known to those skilled in the art. These elements comprise regulatory sequences ensuring the initiation of the transcription (e.g., translation initiation codon, promoters, enhancers, and/or insulators), internal ribosomal entry sites (IRES) and optionally poly-A signals ensuring termination of transcription and stabilization of the transcript. Additional regulatory elements may include transcriptional as well as translational enhancers, and/or naturally-associated or heterologous promoter regions. Preferably, the polynucleotide of the invention is operatively linked to such expression control sequences allowing expression in prokaryotes or eukaryotic cells. The vector may further comprise nucleotide sequences encoding secretion signals as further regulatory elements. Such sequences are well known to the person skilled in the art. Furthermore, depending on the expression system used, leader sequences capable of directing the expressed polypeptide to a cellular compartment may be added to the coding sequence of the polynucleotide of the invention. Such leader sequences are well known in the art. 
     Possible examples for regulatory elements ensuring the initiation of transcription comprise the cytomegalovirus (CMV) promoter, SV40-promoter, RSV-promoter (Rous sarcome virus), the lacZ promoter, human elongation factor 1α-promoter, CMV enhancer, CaM-kinase promoter, the  Autographa californica  multiple nuclear polyhedrosis virus (AcMNPV) polyhedral promoter or the SV40-enhancer. For the expression in prokaryotes, a multitude of promoters including, for example, the tac-lac-promoter, the lacUV5 or the trp promoter, has been described. Examples for further regulatory elements in prokaryotes and eukaryotic cells comprise transcription termination signals, such as SV40-poly-A site or the tk-poly-A site or the SV40, lacZ and AcMNPV polyhedral polyadenylation signals, downstream of the polynucleotide. 
     According to the present invention, nucleotide sequence encoding the channel fusion subunit may be operably linked to a promoter and optionally to an enhancer. Preferred promoters may be selected from but are not limited to promoter is CMV promoter, RSV promoter, SV40 promoter, adenovirus promoter, adenovirus E1A, Heat Shock protein promoter, a promoter from Mycobacteria genes and RNA,  Mycobacterium bovis  MPB70, MPB59, or MPB64 antigen promoter, P1 promoter from bacteriophage Lambda, a tac promoter, a trp promoter, a lac promoter, a lacUV5 promoter, an Ipp promoter, a P L λ promoter, a P R λ promoter, a racy promoter, a β-lactamase, a recA promoter, a SP6 promoter, a T7 promoter, a, a metallothionine promoter, a growth hormone promoter, a hybrid promoter between a eukaryotic promoter and a prokaryotic promoter, ubiquitin promoter, E2F, CEA, MUC1/DF3, α-fetoprotein, erb-B2, surfactant, tyrosinase, PSA, TK, p21, hTERT, hKLK2, probasin or a cyclin gene derived promoter and wherein said enhancer is selected from immediate early enhancer, β-actin, or an adenovirus inverted terminal repeats (ITR). Enhancers may be selected from immediate early enhancer, b-actin, Adenovirus inverted terminal repeats (ITR) and the like. 
     Preferred vectors of the present invention are expression vectors comprising a selectable marker. Examples of selectable markers include neomycin, ampicillin, and hygromycine, kanamycine resistance and the like. Specifically-designed vectors allow the shuttling of DNA between different hosts, such as bacteria-fungal cells or bacteria-animal cells (e.g. the Gateway® system available at Invitrogen). 
     In a particularly preferred embodiment, the subunit of a voltage-dependent cation channel or voltage-dependent anion channel comprises 2 to 24 transmembrane domains. The bioluminescent protein can form part of any of the transmembrane or non-transmembrane loops (domains) or the C-terminus or the N-terminus of the nucleic acid of the present invention. The acceptor molecule also can form part of any of the transmembrane or non-transmembrane loops (domains) or the C-terminus or the N-terminus of the nucleic acid of the present invention. The acceptor molecule cannot be in the same region as the donor molecule when part of the same nucleic acid construct, however, the acceptor molecule can be in the equivalent region as the donor molecule. For example, the donor can form part of the C-terminus whilst the acceptor molecule can form part of the N-terminus and vice versa. In another embodiment, the donor forms part of the transmembrane loop of the subunit, and the acceptor molecule forms part of another transmembrane loop within the same subunit. In yet another embodiment, the acceptor or donor molecule forms part of transmembrane loop of the subunit whilst the other occupies N or C terminal portion. In yet another embodiment, the acceptor or acceptor molecule forms part of any of the intracellular loop of the subunit whilst the other occupies N- or C-terminal portion. Donor and acceptor can also bind to different intracellular loops within the same unit. In further embodiment, acceptor or donor molecule forms part of any of the transmembrane loops while the other member occupies any of the intracellular loops. 
     A person of skill in the art can readily determine the N-terminal end, C-terminal end, transmembrane domains, non-transmembrane loops (domains) and intracellular domains within the test nucleic acid. For example, a variety of bioinformatics approaches may be used to determine the location and topology of transmembrane domains, non-transmembrane domains and intracellular domains in a nucleic acid or an amino acid, based on its sequence and similarity with known domain of voltage dependent receptor sequences. Alignments and nucleic acid and/or amino acid sequence comparisons are routinely performed in the art, for example, by using the BLAST program or the clustalw programs. Based on alignments with known transmembrane domain or intracellular domain-containing nucleic acids and/or proteins, it is possible for one skilled in the art to predict the location of such domains. Furthermore, the 3 dimensional structures of many such receptors are known and catalogued in public information sources. Based on analysis and comparisons with such 3D structures, it may be possible to predict the location and topology of transmembrane domains in other such nucleic acid sequences encoding subunits of voltage dependent receptors. There are also many programs available for predicting the location and topology of transmembrane domains in proteins. For example, one may use one or a combination of the TMpred which predicts membrane spanning segments: TopPred which predicts the topology of membrane proteins; PRFDATOR, which predicts secondary structure from single and multiple sequences; TMAP, which predicts transmembrane regions of proteins from multiply aligned sequences; and numerous other programs which predicts transmembrane regions from single sequences. 
     The present invention further relates to a cell, genetically engineered with the nucleic acid or polynucleotide of the present invention or the vector of the present invention. Said cell may be in cell culture or part of a host. Said cell or said host may be produced by introducing said polynucleotide or vector(s) into a cell or host which upon its/their presence mediates the expression of the polypeptide, i.e., fusion subunit encoded by said nucleic acid as described herein above. The cell or host may be any prokaryote or eukaryotic cell. The host may be any prokaryote or eukaryotic cell. Suitable eukaryotic host may be a mammalian cell, an amphibian cell, a fish cell, an insect cell, a fungal cell or a plant cell. Eukaryotic cell may be an insect cell such as a  Spodoptera frugiperda  cell, a yeast cell such as a  Saccharomyces cerevisiae  or  Pichia pastoris  cell, a fungal cell such as an  Aspergillus  cell or a vertebrate cell. Suitable prokaryotes may be  E. coli  (e.g.,  E coli  strains HB101, DH5a, XL1 Blue, Y1090 and JM101),  Salmonella typhimurium, Serratia marcescens, Burkholderia glumae, Pseudomonas putida, Pseudomonas fluorescens, Pseudomonas stutzeri, Streptomyces lividans, Lactococcus lactis, Mycobacterium smegmatis  or  Bacillus subtilis.    
     The present invention is thus also directed to a recombinant host cell containing an expression vector for expression of the voltage-dependent ion channel subunit fusion as described above, wherein said vector contains a polynucleotide comprising a nucleic acid sequence encoding the voltage-dependent ion channel subunit or a functionally equivalent active fragment thereof as described above. The expressed voltage-dependent ion channel fusion subunit can be in the form of a fusion protein, for example a fusion to a bioluminescent donor molecule and a bioluminescent acceptor molecule. 
     Methods of producing polynucleotides and vectors (e.g., viral and non-viral) are well known in the art. Preferably, the said vector is a plasmid, cosmid, virus, bacteriophage or another vector used conventionally in genetic engineering. 
     Recombinant host cells according to the present invention thus comprise an expression vector wherein said channel fusion subunit is expressed and is able to co-assemble with other homomeric or heteromeric channel subunits in vitro and in vivo to form a functional fusion channel. The present invention embodies a process for the production of said channel fusion subunits as described above comprising culturing said recombinant cell according to the invention, and expressing said channel fusion subunit. 
     Recombinant host cells according to the present invention preferably present as a stable line. 
     The present invention also provides a fusion subunit encoded by the nucleic acid according to the present invention, wherein said N-terminal extremity, C-terminal extremity or a loop is bound to at least one bioluminescent donor protein and/or at least one acceptor protein. It is also within the skill of a person of ordinary skill in art, to incorporate the donor and acceptor on parts of two different subunits, instead of on a single subunit. 
     The current invention further relates to cell free composition and live cell assays which are more sensitive and existing chemical detection systems such as fluorescence and a gas chromatographic technique with flame ionization detection (GC-FID). The present invention relates to methods and nucleic acids for detecting a compound in a sample. In particular, the present invention relates to the use of a cell-free or whole cell composition comprising at least one voltage-dependent ion channel subunit in combination or coupled or bound to a nucleotide sequence encoding at least one bioluminescent donor molecule and/or bound to a nucleotide sequence encoding at least one fluorescent acceptor molecule. 
     According to the present invention, the cell free composition comprises the voltage-dependent ion channel fusion subunit or a functional fusion channel as described above, wherein said fusion subunit is embedded in a lipid bilayer, preferably in a bilayer of a liposome. Such lipid bilayer comprises two layers of, typically amphiphilic, with a common hydrophobic bilayer interior and two hydrophilic surfaces. The lipid bilayer can be naturally occurring or artificial. Such lipid bilayer may be a cellular or bio-membrane for example from mammalian or yeast cell membrane, into which voltage-dependent ion channel fusion subunit is inserted. 
     Methods for preparing cell-free compositions from cells are well-known in the art and consist in obtaining recombinant cells as described above and disrupting the membrane of the cells. These methods generally include repeated cycles of freezing and thawing, grinding, treatment of cells with ultrasound in a sonicator device, homogenization, and use of a French press, the addition of detergent and/or enzymes, glass-bead lysis, differential centrifugation, and several density gradient procedures using a variety of gradient media. These techniques are inter alia described in details in “Current Protocols in Protein Science”; John E. Caligan; Ben M. Dunn; Hidde L. Ploegh; David W. Speicher; Paul T. Wingfield; Wiley and Sons). For isolating or preparing cell membrane extracts, a combination of these methods is usually employed. Generally, cells are lysed either by mechanical means, or using detergents, and the membrane fractions isolated via differential centritugation. Liposomes containing recombinant cell as per the invention may be created by disrupting the phospholipid membrane of cells expressing the protein in water, for example by sonication. The phospholipids would reassemble into liposomal spheres containing a core of aqueous solution. Low shear rates would create multilamellar liposomes, which have many layers. Continued high-shear sonication would form smaller unilamellar liposomes, more suited to the application of the present invention. 
     Assay for BRET 
     In a preferred embodiment, the energy transfer occurring between the bioluminescent protein and acceptor molecule is presented as calculated ratios from the emissions measured using optical filters (one for the acceptor molecule emission and the other for the bioluminescent protein emission) that select specific wavelengths (see equation 1). 
       Equation 1: 
         Ea/Ed =BRET ratio  (1)
 
     where Ea is defined as the acceptor molecule emission intensity (emission light is selected using a specific filter adapted for the emission of the acceptor) and Ed is defined as the bioluminescent protein emission intensity (emission light is selected using a specific filter adapted for the emission of the bioluminescent protein). 
     It should be readily appreciated by those skilled in the art that the optical filters may be any type of filter that permits wavelength discrimination suitable for BRET. For example, optical filters used in accordance with the present invention can be interference filters, long pass filters, short pass filters, etc. Intensities (usually in counts per second (CPS) or relative luminescence units (RLU)) of the wavelengths passing through filters can be quantified using either a photo-multiplier tube (PMT) or a CCD camera. The quantified signals are subsequently used to calculate BRET ratios and represent energy transfer efficiency. The BRET ratio increases with increasing intensity of the acceptor emission. 
     Generally, a ratio of the acceptor emission intensity over the donor emission intensity is determined (see equation 1), which is a number expressed in arbitrary units that reflects energy transfer efficiency. The ratio increases with an increase of energy transfer efficiency. 
     Energy transfer efficiencies can also be represented using the inverse ratio of donor emission intensity over acceptor emission intensity (see equation 2). In this case, ratios decrease with increasing energy transfer efficiency. Prior to performing this calculation the emission intensities are corrected for the presence of background light and auto-luminescence of the substrate. This correction is generally made by subtracting the emission intensity, measured at the appropriate wavelength, from a control sample containing the substrate but no bioluminescent protein, acceptor molecule or polypeptide of the invention. 
       Equation 2: 
         Ed/Ea =BRET ratio  (2)
 
     where Ea and Ed are as defined above. 
     The light intensity of the bioluminescent protein and acceptor molecule emission can also be quantified using a monochromator-based instrument such as a spectrofluorometer, a charged coupled device (CCD) camera or a diode array detector. Using a spectrofluorometer, the emission scan is performed such that both bioluminescent protein and acceptor molecule emission peaks are detected upon addition of the substrate. The areas under the peaks represent the relative light intensities and are used to calculate the ratios, as outlined above. Any instrument capable of measuring lights for the bioluminescent protein and acceptor molecule from the same sample can be used to monitor the BRET system of the present invention. 
     In an alternative embodiment, the energy transfer efficiency is represented using a ratiometric measurement which only requires one optical filter for the measurement. In this case, light intensity for the donor or the acceptor is determined using the appropriate optical filter and another measurement of the samples is made without the use of any filter (intensity of the open spectrum). In this latter measurement, total light output (for all wavelengths) is quantified. Ratio calculations are then made using either equation 3 or 4. For the equation 3, only the optical filter for the acceptor is required. For the equation 4, only the optical filter for the donor is required. 
       Equation 3: 
         Ea/Eo−Ea =BRET ratio or = Eo−Ea/Ea   (3)
 
       Equation 4: 
         Eo−Ed/Ed =BRET ratio or = Ed/Eo−Ed   (4)
 
     where Ea and Ed are as defined above and Eo is defined as the emission intensity for all wavelengths combined (open spectrum). 
     It should be readily apparent to one skilled in the art that further equations can be derived from equations 1 through 4. For example, one such derivative involves correcting for background light present at the emission wavelength for bioluminescent protein and/or acceptor molecule. 
     In performing a BRET assay, light emissions can be determined from each well using any appropriate instrument known by one of ordinary skill in the art. BRET measurement can be performed for instance using readers that can detect at least the acceptor molecule emission and preferably two wavelengths (for the acceptor molecule and the bioluminescent protein) or more. 
     In an embodiment of the invention, BRET is detected using a microfluidics device. Microfluidics devices conveniently require only an aliquot of the sample, generally not more than about 50 μL, to be transferred to the sample reservoir of the micro fluidics device. This is performed either manually or by pneumatic injection via a syringe, capillary or the like. 
     An automated luminescence biochip device using microfluidics may be used to perform all the necessary BRET reaction steps. Automating BRET reactions in a microfluidic biochip platform is desirable as this avoids multiple manual handling steps and reduces human time and effort in performing experiments. The microfluidics device may contain a self-contained disposable biochip with patterned microchannels and compartments having storage means for storing a plurality of samples, reagents, and substrates. The steps of transferring sequentially at least one of the samples, or reagents, and then luminescent substrate from compartments through microchannels to the reaction sites could be automated. The luminescent substrates would then react with the donor molecules resulting in luminescence, which would be detected by an optical detector. An example of a microfluidics device for detecting luminescence is described in U.S. Pat. No. 6,949,377. 
     The present invention further relates to a process of screening in real time agonist or antagonist of the voltage-dependent ion channel. 
     Recombinant DNA techniques are used to isolate a nucleic acid encoding a voltage-dependent ion channel subunit, for example, any of the channels listed in Tables 1-11. Standard recombinant DNA protocols are used to clone the nucleic acids encoding the nucleic acids into any plasmid that facilitates the expression of an in frame fusion of the nucleic acid bound to a nucleotide sequence encoding at least one bioluminescent or fluorescent donor molecule and/or bound to a nucleotide sequence encoding at least one fluorescent acceptor molecule. Any means now known or discovered in the future are used to co-introduce the expression constructs into a host cell. The host cells are propagated using known culturing methods, and fusion proteins are co-expressed in the recombinant host cells. 
     To detect agonist activity, samples of the transfected cells can be added to a 96-well plate (Costar 3912). Various concentrations of known agonists or test compounds are added to the wells with the cell samples. Following a short incubation period, for example about 20 minutes, the BRET reaction is started by submitting cell sample to a stimulus appropriate for triggering the BRET. The stimulus may be selected for instance from the addition of a substrate, an increase of temperature, and an irradiation with radiofrequences. One of ordinary skill in the art is able to adapt the stimulus to the voltage-dependent ion channel, the donor molecule and/or the fluorescent acceptor molecule. The emission of light at various wavelengths is measured on a Mithras LB940 plate reader (Berthold, Germany) or instrument with similar capabilities. For example, BRET optimized filters (Berthold, Germany) are used to detect luciferase emission at 395 nm and fluorescent emission at 510 nm wavelength. The emissions are measured for one second each. Data from the BRET assays are fit to the equation R=D+(A−D)/(1+(x/c)), where A=minimum response, D=maximum response and c=EC50 (R=response, x=concentration of ligand) using the Prism 4.0 software (GraphPad Software, Dan Diego, Calif., USA). The readings from three wells are used to generate each data point. The data points for cells treated with test compounds are compared to the data points for cells not treated with known agonists or antagonists. A comparison of the BRET signals to untreated cells, or cells treated with known agonists or antagonists, is used to characterize the test compound as a ligand, as well as if the ligand is an agonist, antagonist, or inverse agonist of the voltage-dependent ion channel subunit. 
     To detect antagonist activity, samples of the transfected cells are added to a 96 well plate. A known agonist is added to the cells, and the cells are incubated for a period of time, such as for 10 minutes. Following incubation with the known agonist, various concentrations of a known antagonist or test compound are added to the cell samples. Cells are incubated with the known antagonist or test compound, for example for 10 minutes. Following a short incubation period, for example about 20 minutes, the BRET reaction is started by submitting to the cell sample to a stimulus appropriate for triggering the BRET. The stimulus may be selected for instance from the addition of a substrate, an increase of temperature, and an irradiation with radiofrequences. One of ordinary skill in the art is able to adapt the stimulus to the voltage-dependent ion channel, the donor molecule and/or the fluorescent acceptor molecule. The emission of light at various wavelengths is measured on a Mithras LB940 plate reader (Berthold, Germany) or instrument with similar capabilities. For example, BRET optimized filters (Berthold, Germany) are used to detect luciferase emission at 395 nm and fluorescent emission at 510 nm wavelength. The emissions are measured for one second each. Data from the BRT BRET assays are fit to the equation R=D+(A−D)/(1+(x/c)), where A=minimum response, D=maximum response and c=EC50 (R=response, x=concentration of ligand) using the Prism 4.0 software (GraphPad Software, Dan Diego, Calif., USA). The readings from three wells are used to generate each data point. The data points for cells treated with test compounds are compared to the data points for cells not treated with known agonists. A comparison of the BRET signals to untreated cells, or cells treated with known agonists, is used to characterize the test compound as a ligand, as well as if the ligand is an antagonist of the voltage-dependent ion channel subunit or more specifically TRP channel. 
     The current invention provides a method of screening in real time a compound candidate capable of activating or inhibiting channel, comprising: (i) contacting the candidate with the recombinant host cell according to the invention, or a cell free composition according to the invention, (ii) providing a substrate of the bioluminescent donor molecule; (iii) measuring the variation of BRET signal. 
     In a further aspect, the present invention provides a kit for screening agonist or inhibitor compound candidate of voltage-dependent ion channel comprising a nucleic acid or a polynucleotide of the invention, a vector of the invention, a recombinant cell or a host cell of the invention, or a cell-free composition or a composition of the invention, and/or a biosensor of the invention. 
     The present invention can be used to detect a wide variety of compounds which may act as agonists or antagonists to the voltage-dependent ion channels. To that effect the present invention also provides a biosensor or a device for the detection of an analyte that combines a biological component with a physicochemical detector component. It typically consists of three parts, firstly at least one nucleotide subunit encoding a voltage-dependent ion channel subunit. Second, a transducer or detector element, which works in a physicochemical way (eg. optical, electrochemical) that transforms the signal resulting from the interaction of the compound with the test substance into another signal (ice transducers) that can be more easily measured and quantified. Third, an associated electronic or signal processor which then displays the results of the interaction in a user-friendly way. The present invention provides a method of assessing whether a test compound functions as a ligand for a voltage-dependent ion channel, the method comprising: (i) providing a cell comprising a nucleotide sequence encoding a voltage-dependent ion channel fusion subunit bound to a nucleotide sequence encoding at least one bioluminescent donor molecule and/or bound to a nucleotide sequence encoding at least one acceptor molecule; (ii) contacting said cell with a test compound; and (iii) determining the resultant reaction or interaction and output. The current invention also provides biosensor comprising a nucleotide sequence encoding a voltage-dependent ion channel fusion subunit bound to a nucleotide sequence encoding at least one bioluminescent donor molecule and/or bound to a nucleotide sequence encoding at least one acceptor molecule. Still further the invention provides bioluminescence resonance energy transfer system comprising a nucleotide sequence encoding a voltage-dependent ion channel fusion subunit bound to a nucleotide sequence encoding at least one bioluminescent donor molecule and/or bound to a nucleotide sequence encoding at least one acceptor molecule. 
     In another embodiment, the methods of screening according to the current invention are used for drug discovery and/or development. Also contemplated within the scope of invention is a method of making a pharmaceutical composition comprising (i) performing the method according to the current invention (ii) identifying a test compound that interacts with voltage-dependent ion channel; and (iii) combining said test compound with a pharmaceutically acceptable carrier. 
     The fusion subunits of the present invention may be further useful for studying the effect of other stimuli than the action of a modulator (inhibitor or activator) of the channel, such as the effect of temperature and/or radiofrequences on said channel. Actually, the conformational change of the fusion channel subunit(s) that triggers the BRET emission may be obtained by contacting said channel with a modulator thereof, or by submitting said channel to another stimulus, such as an increase in temperature or an irradiation with radiofrequences. 
     Throughout this application, various references are referred to and disclosures of these publications in their entireties are hereby incorporated by reference into this application to more fully describe the state of the art to which this invention pertains. 
     EXAMPLES 
     Example 1 
     Example 1.1 Methods of Analyzing in Real Time the TRP Channel Activation on Live Cells 
     Voltage-dependent ion channel such as TRPV1 and TRPV3 were used to study the effects of specific agonist and temperature increase on TRP channel activation. Pharmacology of TRPV1 for example, has been well characterized and various specific antagonists thereof are commercially available. On the other hand, TRPV1 has been studied and is known to open at 43° C. 
     Two methods were used. The first method used was a direct method of assessing the activation of channels such as TRP. This method consists in measuring BRET signals from voltage-dependent ion channels, TRPV1, TRPV3 or TRPV4, which N- and C-termini have been fused to YFP and to luciferase, respectively ( FIG. 3 ). The activation of the channels following for example agonist exposure or cell culture temperature increase results in a change of the conformation of the monomer subunits which result in a modification of either or both the distance and the orientation of the donor and acceptor molecules in N- and C-terminal ends of the channels. Another method consist in measuring BRET signals from two voltage-dependent ion channels subunits, each ones being fused to either the YFP or the luciferase at one of each monomer extremity. 
     Example 1.2: Construction of Expression Vectors 
     Expression vectors were constructed using intramolecular probes YFP-hTRPV1-RLuc and YFP-hTRPV3-RLuc, wherein protein sequences of human channel subunits TRPV1 et TRPV3 (hTRPV1, hTRPV3) are inserted between  Renilla  Luciferase, in 3′, and YFP, in 5′, according to the strategy presented in  FIG. 4 . 
     Constructs YFP-hTRPV1-RLuc and YFP-hTRPV3-RLuc have been obtained by amplification of hTRPV1 et hTRPV3 cDNA via PCR, using specific primers allowing fusion of amplicons between sequences of YFP and RLuc in a pcDNA3.1(+) YFP-RLuc vector ( FIG. 4 ). cDNA encoding hTRPV1 et hTRPV3 were used as matrices, and were provided from database PlasmID (Harvard Medical School, U.S.A). Sense and antisense primer sequences that were used to amplify by PCR hTRPV1 and hTRPV3 are as follows: 
     
       
         
           
               
            
               
                 hTRPV1 sense (S) 
               
               
                 SEQ ID NO: 1 
               
               
                 5′TGTGTACCGGTGAATTCTGGTGGAGGCGGATCTATGAAGAAATGGAGC 
               
               
                 AGCACAGACT-3′ 
               
               
                   
               
               
                 hTRPV1 anti-sense (AS) 
               
               
                 SEQ ID NO: 2 
               
               
                 5′CACCAGAATTCACCGGTACCTTCTCCCCGGAAGCGGCAGGACTC-3′ 
               
               
                   
               
               
                 hTRPV3 sense (S) 
               
               
                 SEQ ID NO: 3 
               
               
                 5′TGTGTACCGGTGAATTCTGGTGGAGGCGGATCTATGAAAGCCCACCCC 
               
               
                 AAGGAGATGG-3′ 
               
               
                   
               
               
                 hTRPV3 anti-sense 
               
               
                 SEQ ID NO: 4 
               
               
                 5′CACCAGAATTCACCGGTACCACCGAGGTTTCCGGGAATTCCTCG-3′ 
               
               
                   
               
               
                 hTRPM2 Fus AS 
               
               
                 SEQ ID NO: 5 
               
               
                 5′ CACCAGAATTCACCGGTACGTAGTGAGCCCCGAACTCAGCGGC-3′ 
               
               
                   
               
               
                 Fusion hTRPM2 S 
               
               
                 SEQ ID NO: 6 
               
               
                 5′TGTGTACCGGTGAATTCTGGTGGAGGCGGATCTATGGAGCCCTCAGCC 
               
               
                 CTGAGGAAAGC-3′ 
               
               
                   
               
               
                 hTRPV4_Fus_AS 
               
               
                 SEQ ID NO: 7 
               
               
                 5′CACCAGAATTCACCGGTACGAGCGGGGCGTCATCAGTCCTCCACTTG 
               
               
                 CG-3′ 
               
               
                   
               
               
                 Fusion_hTRPV4_S: 
               
               
                 SEQ ID NO: 8 
               
               
                 5′TGTGTACCGGTGAATTCTGGTGGAGGCGGATCTATGGCGGATTCCAGC 
               
               
                 GAAGGCCCCCG-3′ 
               
               
                   
               
               
                 YFP sense 
               
               
                 SEQ ID NO: 9 
               
               
                 5′-TGTCTAAGCTTGGATCCGCCACCATGGTGAGCAAGGGCGAGGAGCTG 
               
               
                 TTCACC-3′ 
               
            
           
         
       
     
     Using the above primers, the amino acid sequence of the region linking YFP to hTRPV1/3 in N-terminal and linking hTRPV1/3 to RLuc in C-terminal of the fusion channel subunit, was VPVNSGGGS (SEQ ID NO: 10). 
     The PCR reaction was performed in a volume of 50 μl in a buffer comprising 1 μM of each primers, 250 μM dNTP, 5 μl PCR buffer Phusion HF II 10× (Thermoscientific), 5% DMSO, 1 U Phusion enzyme HF II 10× (Thermoscientific), and 25 ng matrix DNA. The PCR was performed in a thermocyclor (Rotor Gene Q, Qiagen) according to program listed in the following Table 13. 
     
       
         
           
               
             
               
                 TABLE 13 
               
             
            
               
                   
               
               
                 program of PCR amplication of hTRPV1 and hTRPV3 
               
            
           
           
               
               
               
               
               
            
               
                   
                   
                   
                   
                 Number of 
               
               
                   
                 Stages 
                 Temp (° C.) 
                 Time 
                 cycles 
               
               
                   
                   
               
            
           
           
               
               
               
               
               
            
               
                   
                 Initial denaturation 
                 95 
                 1 min 
                 1 
               
               
                   
                 Denaturation 
                 95 
                 45 sec 
                 25 
               
               
                   
                 Hybridation 
                 55 
                 45 sec 
                   
               
               
                   
                 Elongation 
                 72 
                 2 min 
                   
               
               
                   
                 Final elongation 
                 72 
                 4 min 
                 1 
               
               
                   
                   
               
            
           
         
       
     
     PCR products were then purified on agarose gel using the QIAquick Gel Extraction kit (Qiagen). PCR products and vector pcDNA3.1(+) YFP-RLuc were then digested either by the Age I enzyme for the cloning of hTRPV1, or by EcoRI enzyme in the case of hTRPV3 cloning. The vector pcDNA3.1(+) YFP-RLuc was also dephosphorylated using a phosphatase alcaline enzyme (Thermoscientific). DNAs were then prepared and purified on a column using QIAquick PCR purification Kit (Qiagen), and further quantified on gel. Ligation of the amplicons of PCR at the restriction site within the opened vector pcDNA3.1(+) YFP-RLuc was then performed by incubating 50 ng of opened vectors with 20 to 100 ng insert (molar ratio greater or equal to 3:1) in 12 μl of ligation buffer 1× (Thermoscientific) comprising 1.2 mM ATP and 1 U of T4 DNA ligase (Thermoscientific), at 23° C. for 2 hours. The ligation mix was then used to transform  Escherichia coli  DH5α by thermal choc. 100 μl of competent DH5α bacteria were incubated on ice in presence of 12 μl of the ligation mix during 1 hour. Bacteria were then put at 42° C. for 20 sec before being transferred back to ice for 2 minutes. They were then placed under agitation at 200 RPM, at 37° C. for 1 hour after addition of 500 μl Luria-Bertani medium (LB) (Fischer Scientific), and spread on LB-Agar box containing 100 μg/ml of ampicillin allowing to select successfully transformed bacteria. 
     The insertion of the sequence hTRPV1/3 in between YFP and hRLuc was not orientated. It was thus necessary to further screen colonies to select those that contained inserts hTRPV1/3 in the correct orientation. This was performed using the PCR technique using the &lt;&lt;sense&gt;&gt; primer which was capable of hybridizing in 5′ of the coding sequence of YFP and using the antisense primer hybridizing in 5′ of the non-coding sequence of hTRPV1/3. This analytical PCR protocol was similar to that of above described PCR except for the Dream Taq polymerase (thermoscientific) which was used during 30 cycles of PCR with an initial denaturation step of 5 min at 95° C. allowing to destroy bacteria and releasing plasmid DNA. Colonies which contained cDNA of hTRPV1/3 in phase with YFP allowed amplication only. A positive clone is thus cultured in 100 ml of LB medium (16 hours, 37° C.). Plasmid DNA is prepared by alcaline lysis of bacteria and purification using the midiprep kit (Qiagen). DNA so obtained was then double checked by sequencing. 
     The same protocol was successfully used to construct expression vectors respectively comprising the hTRPV4 and hTRPM2 sequences. 
     Example 1.3: Cell Culture and Transfection 
     Human cell line HEK293T which originated from kidney embryonic cells, had been selected for this experiment. These cells were cultivated in DMEM medium (Dulbecco&#39;s Modified Eagle&#39;s Medium) containing GLUTAmax (Invitrogen), 10% fetal calf serum, 100 units/ml of penicillin and streptomycin (Invitrogen), 1 mM of sodium pyruvate (Invitrogen). These cells were placed in culture flasks T25 or T75 up to 70-90% confluence on the day of transfection. Transient transfections were performed using polyethylenimin (linear PEI, Polysciences, Inc., Warrington, USA) in Opti-MEM medium (Invitrogen) with a ratio PEI:DNA of 4:1. Transfection of the cells cultured in a T75 flask required 15 μg of total DNA placed in 700 μl of opti-MEM medium (5 minutes, RT) wherein 60 μl of 1 μg/μl PEI are added. The mixture was vortexed 5 sec and incubated at RT during 20 min before being added to the cell culture. The next day, cells were collected using trypsin-EDTA 1% and cultured for 24 hours either in 96 well plaques (1·10 5  cells/well) for dose-response experiments, or in 24 well plaques containing glass slides of 12 mm de diameter (1·10 6  cells/well) for all other experiments. Both types of support had been treated with L-polylysine (Sigma). 
     Example 1.4: Measure of Intracellular Calcium by Fura 2-AM 
     HEK293T Cells were transfected with DNA encoding wild-type TRPV1 or YFP-hTRPV1-rLuc. After 24 hours, 150 000 cells are plated in a 96 wells-plates in DMEM medium. Further an additional 24 hours, the medium is replaced by HBSS buffer (140 mM NaCl, 4.2 mM KCl, 0.4 mM Na 2 HPO 4 , 0.5 mM NaH 2 PO 4 , 0.3 mM MgCl 2 , 0.4 mM MgSO 4 , 1 mM CaCl 2 , 20 mM Hepes, 5 mM glucose, pH 7.4) comprising 2 μM Fura 2-AM (Tocris) and 0.02% of pluronic acid, and cells were incubated at 37° C. for 1 hour without any light stimulation. Reading of the fluorescent signal was performed with multifonction Flexstation III plaque reader (Molecular device) preheated at 37° C. The light signal has been integrated at 505 nm for 1 second after having sequentially excited the probe at 340 nm and 380 nm. After 100 seconds of reading, 1 μM of capsaicin was added to the cellular medium. The results presented on  FIG. 7A  present the ratio of light intensity as obtained at 505 nm during the excitation at 340 nm and at 380 nm (I 340 /I 380 ). 
     The same protocol was successfully used to measure intracellular calcium in cells similarly transfected with wild-type TRPV3 or YFP-hTRPV3-rLuc. 
     Example 1.5: Measure of BRET Signal 
     Readings of BRET signals in experiments of dose-response were realized in of 96 opaque wells-plates with a multifonction Flexstation III plaque reader (Molecular device) preheated at 37° C. 36-48 hours after the transfection, the cellular medium was replaced with PBS containing 0.2% BSA and various concentrations of capsaicin ligand (Tocris). After 5 minutes of incubation at 37° C., the substrate ccelenterazine H (Nanolight Technology) was added to a final concentration of 5 μM and the light signal was integrated for 1 second sequentially at 480 nm and 530 nm, e.g., the wavelengths of emission of luciferase and YFP, respectively. A third reading was performed at 300 nm in order to measure the background signal of the device. The raw BRET signal was calculated by the following equation: 
       BRET=( I   530   −I   300 /( I   480   −I   300 ), 
     wherein I is the light intensity as obtained at the given wavelength. 
     The BRET signal was then corrected by removing the BRET signal obtained during the light emission of luciferase when the protein TRPV1-rLuc was expressed in absence of YFP. 
     Example 1.6: Analysis of the Data 
     The results of BRET obtained with variations of temperatures and with the Fura 2-AM probe have been analyzed on Excel (Microsoft). The results obtained in dose-response experiments have been analyzed with the GraphPad Prism software v6.00 (GraphPad Software Inc, La Jolla, Calif., USA). 
     Example 2: Direct Measuring Methods of Activation/Inhibition of a Voltage-Dependent Cation Channel Using Intramolecular BRET Probes 
     In order to study the activation/inhibition of the voltage-dependent cation channel fusion subunit, for example of the family TRPV (vanilloid) channel, such as TRPV1, by the temperature or a chemical modulator, expression vectors were first constructed encoding fusion channel subunit wherein sequences of TRPV1 were taken in sandwich between N-terminal YFP fused subunit and C-terminal RLuc fused subunit. 
     Example 2.1: Characterization of Intramolecular TRPV1 BRET Probe 
     In order to study the behaviour of the intramolecular BRET probe, i.e., YFP-hTRPV1-rLuc over temperature, HEK293T cells were transfected using the expression vector encoding the fusion subunits. 24 hours after transfection, cells were progressively heated from 29 to 50° C. with a Peltier thermostat, and the BRET signals were measured using known techniques. 
     The analysis of the results showed that the BRET ratio did not change between 29 and 34° C., and slightly increased between 34° C. and 43° C. Over 43° C., an important decrease of the BRET value was observed ( FIG. 5 ). Temperature of 43° C. corresponded to the threshold of opening of the TRPV1 channel (Clapham, 2003  Nature  426, 517-524). 
     The same cells were then pre-incubated with two antagonists of TRPV1, namely 6 iodo-CAPS and 2,2-Diphenyltetrahydrofuran which modify the initial value of net BRET of the probe YFP-hTRPV1-Luc. The ratio of net BRET as initially measured at 29.5° C. was of 0.43 in control conditions, and of 0.28 in presence of 6 iodo-CAPS and of 0.93 in presence 2,2-Diphenyltetrahydrofuran. These important changes of the initial BRET level could have reflected a transition of the conformation state of the TRPV1 channels to a state having a lesser thermosensitivity. According to this hypothesis, a completely different behavior of the probe was observed with variations of temperature in presence of 6 iodo-CAPS or 2,2-Diphenyltetrahydrofuran. In both cases, BRET signal increased between 32 and 37° C., and plateaued at 52° C. in the case of 2,2-Diphenyltetrahydrofuran, and only decreased as from 48° C. for 6 iodo-CAPS ( FIG. 5 ). 
     The sensitivity of the probe YFP-hTRPV1-rLuc to chemical activators was also assessed. The BRET signal in HEK293T cells expressing the YFP-hTRPV1-rLuc fusion protein was measured after incubation of the cells in presence of different concentrations of capsaicin. The BRET signal was function of the concentration of capsaicin, thereby showing a significant increase of the ratio with an EC50 of 650±33 nM ( FIG. 6 ) showing that the effect of capsaicin on to the probe. In this case, the ratio of initial BRET signal was about 0.28 but could not be compared to the values obtained in the preceeding experiments, since it was performed with a different device system. As expected, known hTRPV1 antagonists like AMG517 or Capsazepine induce a right-shift of the capsaicin dose-response curve, which is in agreement with the notion that these molecules decrease TRPV1 activation threshold by capsaicin ( FIG. 6 ). 
     The functionality of the fused subunit channels YFP-hTRPV1-rLuc was compared to that of wild-type channels. The flux of calcium was measured in HEK293T cells expressing either the probe BRET YFP-hTRPV1-rLuc, or the wild-type TRPV1, after the addition of 1 μM agonist capsaicin. We have used a radiometric probe Fura 2-AM which has spectral properties compatible with excitation spectrum of YFP. The addition of capsaicin resulted in a rapid and important increase of intracellular calcium in cells expressing wild-type TRPV1 as well as in cells expressing YFP-hTRPV1-rLuc with similar kinetic and amplitude ( FIG. 7A ). No calcium increase has been measured in HEK293T cells which are transfected with an empty vector after the addition of capsaicin ( FIG. 7B ), thereby confirming that the signal as measured in  FIG. 7A  is dependent of the ectopic expression of TRPV1. 
     Example 2.2 Characterization of the Intramolecular TRPV3 BRET Probe 
     The sensitivity of the probe YFP-hTRPV3-rLuc to chemical activators was assessed. The kinetic of the evolution of the BRET signal in HEK293T cells expressing the YFP-hTRPV3-rLuc fusion protein was measured at 33° C. At a defined time, a TRPV3 activator, such as 2-Aminoethyl diphenylborinate (2-APB), was added in the culture media producing an increase in the BRET value within seconds ( FIG. 8 ). 
     Example 3: Direct Measuring Methods of Activation/Inhibition of a Voltage-Dependent Cation Channel Using Intermolecular BRET Probe 
     Example 3.1: Characterization of Intermolecular TRPV1 BRET Probe 
     An intermolecular TRPV1 BRET probe was used wherein the RLuc is fused to the C-terminal part with of the TRPV1 subunit and the YFP was fused to Calmodulin which is known to bind to TRPV channel in a calcium dependent manner ( FIG. 9B ). Such intermolecular probe thus allowed measuring Calmodulin docking on TRPV1 intracellular part following activation and calcium entry inside the cell. TRPV1 channel activation was then monitored using this intermolecular BRET probe as well as the intramolecular TRPV1 BRET probe described in Example 2.1 ( FIG. 9A ). 
     HEK293T cells expressing either YFP-TRPV1-Luc or TRPV1-Luc and YFP-CaM were processed for BRET analysis. In both case, a basal BRET signals was observed most likely reflecting either the different mode of energy transfer between Rluc and YFP inside the tetrameric organisation of the channel or some constitutive interaction between TRPV1 and Calmodulin ( FIGS. 9C and 9D ). 
     Using both types of probes according to the present invention, exposure to capsaicin induced a robust increase of BRET over the basal signal within seconds following activation albeit with a different time constant (the time constant was equal to 2 sec for the YFP-TRPV1-Luc probe, while it was equal to 27 seconds with the TRPV1-Luc/YFP-CaM BRET test). The increase in BRET signal following capsaicin (CAPS) activation underscored the potential of these BRET tests to study TRPV1 channel activation in living cells. 
     Dose-responses of capsaicin (CAPS) on BRET signal were also measured with intramolecular and intermolecular probes, in HEK293T cells pre-incubated in presence of DMSO, or with TRPV1 inhibitors, e.g., either AMG517 or Capsazepine (CPZ).  FIGS. 10(A)  and (B) showed a right shift of the CAPS dose response curve, thereby confirming the decrease of the activation threshold by CAPS ( FIG. 10 ). This pharmacological competitive test thus confirmed that both intra- and intermolecular types of BRET probes according to the present invention were suitable for high-throughput screening of potential TRPV1 inhibitors or activators. 
     Further, the effect of temperatures was assessed on both intermolecular and intramolecular TRPV1 BRET probes according to the present invention. HEK293T cells expressing either the intramolecular YFP-TRPV1-Luc BRET probe ( FIG. 11A ) or the intermolecular TRPV1-Luc probe/YFP-CaM construct ( FIG. 11C ) were incubated at progressively greater temperatures of 25° C., 31° C., 37° C. or 42° C. using a peltier heating system. BRET signals were recorded in real time using an optical fiber coupled to a spectrometer when the HEK293T cell culture medium was heated from 25° C. to 50° C. 
     Considering the BRET signal measured from cells expressing the YFP-TRPV1-Luc probe, a complex profile where the basal BRET signal remained stable between 25° C. and 30° C. and increased slightly from 30° C. to 37° where it reached a plateau that was maintained until 43° C. From 43° C. a major decrease of the signal was observed until 50° C. ( FIG. 11  B). 
     Knowing that 43° C. is considered to be the temperature-threshold of TRPV1 pore opening, such a decrease of the signal could be indicative of some intramolecular and/or intermolecular conformational change experimented inside the TRPV1 tetrameric structure following temperature activation. To verify this hypothesis, the experiment was repeated in the presence of a TRPV1 channel blocker: CPZ. In presence of CPZ, the signal evolved completely differently with a major increase from 37° C. up to 47° C., where it finally started to decrease ( FIG. 11B ). This indicated that the conformational changes occurring in TRPV1 in response to the temperature increase were completely modified in presence of the CPZ inhibitor. Nonetheless, the fact that CPZ was able to modify the conformational change of the probe indicated that the YFP-TRPV1-Luc BRET probe reproduced the temperature activation behavior of the native TRPV1 channel. In addition, the emission spectra shape of both YFP and luciferase was not affected by the change of temperature between 25° C. and 50° C. 
     A similar experiment was conducted using our second BRET test, measuring the variation of the BRET signal between TRPV1-Luc and YFP-CaM when the temperature increased from 25° C. to 50° C. ( FIG. 11D ). Using this BRET pair, a weak BRET signal was measured and remained stable between 25° C. and 37° C. From 37° C., the BRET signal started to increase with the temperature until 45° C. where it reached a plateau. If the same assay was performed in a buffer without calcium or if the cells were preincubated with AMG517 (a potent and selective TRPV1 inhibitor), no significant BRET increase could be observed. The fact that the signal remained stable between 43° C. and 50° C. also excluded the possibility of a bias in our measure due to the denaturation of the Luciferase or the YFP groups with the increasing of the temperature. 
     Knowing that the temperature increase could potentiate TRPV1 channel by lowering the activation threshold, it was checked whether such an effect using our BRET tests could be detected. Dose response curves of TRPV1 activation by capsaicin were therefore obtained at 25° C., 31° C., 37° C. and 42° C. using both the intramolecular ( FIG. 11A ) and intermolecular ( FIG. 11C ) BRET probes. In both tests, the temperature rise from 25° C. to 42° C. resulted in a leftward shift of the power of capsaicin while the efficacy remained unchanged ( FIGS. 11A &amp; 11C , and Table 13 below). These results indicated that both TRPV1 BRET probes did mimic the temperature sensitivity of the native TRPV1 channel. 
     
       
         
           
               
             
               
                 TABLE 13 
               
             
            
               
                   
               
               
                 Efficacy of CAPS as a function of the temperature, measured in  
               
               
                 HEK293T cells expressing either the YFP-TRPV1-Luc probe or the  
               
               
                 TRPV1-Luc/YFP-CaM constructs. 
               
            
           
           
               
               
            
               
                   
                 Temperature (° C.) 
               
            
           
           
               
               
               
               
               
            
               
                   
                 25° C. 
                 31° C. 
                 37° C. 
                 42° C. 
               
               
                   
               
            
           
           
               
               
               
               
               
            
               
                 Log EC50 (M) 
                  −5.4 ±  
                 −5.57 ±  
                 −6.08 ± 0.25 
                 −6.3 ± 0.3 
               
               
                 (YFP-TRPV1-Luc) 
                 0.39 
                 0.31 
                   
                   
               
               
                 Log EC50 (M) 
                 −5.45 ±  
                  −5.8 ±  
                 −6.43 ± 0.17 
                 −6.9 ± 0.2 
               
               
                 (TRPV1-Luc/YFP-CaM) 
                 0.15 
                 0.11 
               
               
                   
               
            
           
         
       
     
     Example 3.2: Characterization of Intramolecular TRPV3 BRET Probe 
     The functionality and sensitivity of the intramolecular YFP-hTRPV3-rLuc BRET probe was confirmed in HEK293T cells expressing the YFP-hTRPV3-rLuc fusion protein and treated with two agonists of TRPV3, namely 2-aminoethyl diphenylborinate (2-APB) and carvacrol.  FIG. 12  shows a kinetic of BRET signal and the effect of carvacrol and 2-APB on HEK293T cells expressing the YFP-TRPV3-Luc BRET probe, thereby confirming the functionality of the fusion protein. 
     In the same manner as that described for TRPV1 in Example 3.1, an intermolecular TRPV3 BRET probe was constructed wherein the RLuc is fused to the C-terminal part of TRPV3 and the YFP was fused to the N-terminus part of Calmodulin. Also, dose response curves of TRPV3 activation by carvacrol and 2-APB were obtained in HEK293T cells expressing the TRPV3-Luc/YFP-CaM constructs ( FIGS. 13A and 13B ), thereby also showing the functionality of the intermolecular TRPV3-Luc BRET probe. 
     Example 3.3 Characterization of the Intermolecular TRPV4 BRET Probe 
     An intermolecular TRPV4 BRET probe where the RLuc was fused to the TRPV4 in C-terminal and YFP was fused to the N-terminus part of Calmodulin. Kinetic ( FIG. 14A ) and dose response curve ( FIG. 14B ) of the effect of the TRPV4 agonist GSK1016790A on HEK293T cells expressing the TRPV4-Luc/YFP-CaM constructs were obtained. In  FIG. 14A , cells were activated with 1 μM of agonist at the time indicated with an arrow. In  FIG. 14B , the test was done in presence (square) or absence of the TRPV inhibitor Ruthenium Red (10 μM). It was found that Log EC50=−4.89±0.12 M for the control condition. No BRET increase could be measured in presence of Ruthenium Red. 
     A similar strategy as that of Examples 2 and 3 may be used to study activation or inhibition of other voltage-dependent cation channels belonging to TRPC (canonical) channel, TRPV (Vaniloid), TRPM (melastatin) channel, TRPA (ankyrin) channel, TRPP (polycystin) channel, or TRPML (mucolipin) channel, or sodium gated channel (SCN channel), calcium gated channel (CACNA channel), CATPSER channel, CNG channel, voltage dependent potassium channel (KCN channel), and/or hydrogen voltage-gated ion channel.