# Patent

## Title
Full length human HCN1Ih channel subunit and variants

## Abstract

This invention relates to a DNA sequence encoding a member of the hyperpolarized activated ion channel family (HCN) and variants thereof, and the use of said sequences in assays for the measurement of gene expression. It also relates to assays for screening of Ih activators and blockers for clinical and therapeutic use in the management of human psychiatric and neurological dysfunction in the CNS, cardiovascular dysfunction of the heart, and reproductive dysfunction and/or contraception related to Ih function in testes and spermatozoa. Further, antibodies against the expressed DNA sequences and other compounds reactive with the expressed DNA sequence are also part of the invention.

## Background
FIELD OF THE INVENTION
This invention relates to a DNA sequence encoding a member of the hyperpolarised activated ion channel family (HCN) and variants thereof, and the use of said sequences in assays for the measurement of gene expression. It also relates to assays for screening of Ih activators and blockers for clinical and therapeutic use in the management of human psychiatric and neurological dysfunction in the CNS, cardiovascular dysfunction of the heart, and reproductive dysfunction and/or contraception related to Ih function in testes and spermatozoa. Further, antibodies against the expressed DNA sequences and other compounds reactive with the expressed DNA sequence are also part of the invention
BACKGROUND OF THE INVENTION
Voltage-gated ion channels play a critical role in shaping of electrical activity of neuronal and muscle cells, and in controlling the secretion of neurotransmitters and hormones through the gating of calcium ion entry. Large families of voltage gated sodium (Na+), potassium (K+) and calcium (Ca2+) ion channels have been defined using electrophysiological, pharmacological and molecular techniques [1, 18]; they are named according to their selective permeability for a particular cation with reference to their voltage dependence, kinetic behaviour or molecular identity. The importance of membrane voltage and ion permeability in the control of cell function ensures that modulation of ion channels will invariably have important consequences for cells and tissues, and such modulation can often be turned to therapeutic advantage. Major indication for ion channel modulators already include cardiac arrhythmia, hypertension, anxiety, epilepsy, pain, chemotherapy-induced nausea and diabetes as well as a range of drugs in development for important new indications such as neuroprotection and psychiatry.
A variety of bodily functions such as heart beat, sleep-wake cycles, secretion of hormones and control of behavioural state depend on the action of pacemakers, specialised cells that are able to generate rhythmic, spontaneously firing action potentials. The archetypal organ displaying autonomic rhythmicity is the heart.
Pacemaking in the heart is accomplished by the rhythmic discharge of the sino atrial node [8, 11, 12]. The firing rate of the sino atrial node is determined by the diastolic depolarisation phase of the action potential. During this phase the membrane potential is slowly depolarised to the threshold triggering the next action potential. The ionic conductance underlying the cardiac pacemaker depolarisation was identified in the late seventies and early eighties [10] and called If (f for ‘funny’) or Ih (h for hyperpolarisation activated). A similar current was subsequently discovered in neurones, first in photoreceptors, [2, 3, 6] and then in various central neurons eg hippocampal pyramidal cells [16] where is was called Iq (q for ‘queer’). This current was subsequently found in a wide variety of central and peripheral neurons [25].
Ih channels have several distinctive features. Unlike most voltage-gated channels, they open in response to negative-going voltage steps to potentials within the range of the normal resting potential. They conduct both K+ and Na+ ions with a three fold greater permeability to K+, yielding a reversal potential of −30 to −40 mV under physiological conditions. As a result, the opening of these channels near the resting potential ( ̃−60 mV) generates an inward, depolarising current that is largely carried by Na+ [25]. Another unusual property of these channels is their regulation by cyclic nucleotides [11], which speed up the rate of channel activation by binding to an intracellular site on the channel. In the heart, this in an important mechanism responsible for the acceleration of heart rate in response to sympathetic stimulation. Activation of β-adrenergic receptors leads to an activation of adenylyl cyclase with the resulting increase in intracellular cAMP directly activating the Ih channel. cAMP binding leads to a shift in the activation curve towards more positive voltages. This shift results in an increased inward current at a fixed membrane potential and therefore an acceleration of the diastolic depolarisation [9]. Muscarinic stimulation slows the heart rate, in part due to a decrease in cAMP level and a resulting reduction in the Ih current [13, 33].
In neurons, Ih channels have diverse functions. They were initially shown to be inward rectifiers; they are active near the resting potential and pass inward current more readily than outward current, thereby helping to control of resting potential and input resistance [25]. In photoreceptors, Ih channels help to damp the hyperpolarising effect of light; they are activated by hyperpolarisation, causing the voltage response to light to fade during the first 100-200 ms, thus producing sensory adaptation. In many CNS neurons, activation of Ih channels following post inhibitory post synaptic potentials contributes to a rebound afterdepolarisation (ADP), which can trigger an action potential. Ih is also generates or contributes to ‘pacemaker’ potentials that controls the rate of rhythmic oscillations, similar to its role in the heart. Ih has been found to regulate the rhythmic activity of thalamic relay neurons [23] and inferior olivary neurons [5] through interaction with a T-type calcium current. The oscillating single neurons are part of neuronal networks and are involved in the generation and modulation of rhythmic activity of these networks. A well studied example are spindle waves observed in the EEG during slow wave sleep, which are generated through interactions between thalamic reticular and relay neurons [4]. Regulation of Ih in these cells is important in the sleep-wake cycle. Although less well investigated, results suggest a similar role for Ih in the generation of oscillations in hippocampal neurons [21, 32] and respiratory neurons of the preBotzinger complex of the ventrolateral medulla [26]. Ih channels are also expressed in dendrites where they influence the cable properties of the dendrite and shape the time course of the EPSP as it is propagated to the soma [22]. A recent study has extended the role of Ih neurons by showing that these channels can alter neurotransmitter release from presynaptic terminals as Crayfish neuromuscular synapses. Presynaptic cAMP generation by via serotonin receptor activation directly modulates Ih in axons that produces an increase in synaptic strength which cannot be explained solely by depolarisation of the presynaptic membrane [7].
The genes encoding Ih channels were recently cloned from both mammals [19, 28, 29] and sea urchins [15]. These genes, called HCN1-4 in mammals, are members of the voltage gated K+ channel family. The encoded proteins contain six transmembrane segments, including a positively charged S4 voltage sensor and a pore-forming P region that includes the K+ channel signature sequence GYG. In addition, the C-terminus contains a 120-amino acid sequence that is homologous to the cAMP and cGMP binding domains of other proteins, and is therefore the likely site for cAMP regulation of channel opening [9,20]. All four mammalian genes are expressed in brain, with differing expression patterns [24]. While the first reported cloning of Ih was from sea urchin spermatozoa [15], the functional significance is poorly understood at present. The channel is expressed in sperm flagellum and it was postulated that it may be involved in the control of flagella beating. One of the cloned isoforms, HCN4, has been detected in testes suggesting that Ih may also have a function in mammalian testicular and sperm function [30].
These ion channels are the target for blocking molecules for therapeutic use in dysfunction in the CNS, cardiovascular dysfunction of the heart, and reproductive dysfunction and/or contraception related to Ih function in testes and spermatozoa. For instance, compounds have already been developed with the therapeutically interesting property of inducing bradycardia with minimal inotropic side effects [14, 17]. Unfortunately, there are no compounds yet described that distinguish cardiac from neuronal isoforms, and volunteers experienced optical hallucinations probably due to reduced functionality of Ih in photoreceptors. It is clear the ability to develop agents that are selective for the CNS vs heart or vice versa requires the availability of the cloned subunits to screen for compounds selective for subunits expressed in either neuronal or cardiac tissue.
BRIEF SUMMARY OF THE INVENTION
It is the object of the present invention to provide full-length functional human HCN1 channel subunits. Furthermore, variants of the full-length functional human HCN1 subunit are also provided. Human HCN1 channel subunits are preferred over subunits from other species since they are preferably used to select compounds that can be used to treat CNS disorders, cardiovascular dysfunction of the heart, and reproductive dysfunction and/or contraception related to Ih function in testes and spermatozoa in humans.


DETAILED DESCRIPTION OF THE INVENTION
One aspect of the invention concerns the full length cDNA sequence of the human HCN1 channel subunit (SEQ ID NO: 1). The expression of this subunit is predominantly restricted to the CNS and has only been detected in some peripheral tissues to a very limited extend. This subunit is therefore a candidate for screening compounds with a selective effect in the CNS with a reduced propensity for undesirable side effects such as bradycardia via activity at Ih in the heart. In contrast with the prior art, the present invention provides the full-length sequence of human HCN1 and sequence variants whereas previous publications have only identified partial human HCN1 sequences. The major advantage of having a full-length sequence is that this allows functional expression in in vitro systems (ie in transfected cell lines) for the identification of compounds increasing (opening the channel) or decreasing (blocking the channel) ion flux through the expressed channel. While expression of partial sequences may be possible, these will not give rise to channels with the full functional characteristics of the full-length channel since they lack polypeptide domains that contribute to the functional characteristics of the channel. In addition, partial sequences may lack polypeptide domains with important target sites for compounds that modulate channel activity. Functional expression of partial sequences, particularly those lacking the 5′ end such as those reported for human HCN1 [1] is additionally problematic since they lack the sequence encoding the N-terminal signal peptide required for directing the nascent polypeptide to the endoplasmic reticulum where all nascent plasma membrane bound proteins are synthesised. The inherent advantage of an assay based on channel function, rather than, say a binding assay, is that a functional assay allows for the screening of compounds that interact with the channel as either blockers or openers. Binding assays give no such information, nor do they allow the identification of allosteric or use-dependent modulators of the channel.
By the term “full-length” as used herein we mean a DNA sequence that contains a complete open reading frame, beginning with a start codon at its 5′ end, preferably with a Kozak consensus, downstream to an in-frame stop codon.
The full-length DNA sequence for HCN1 (SEQ ID NO: 1) was obtained using a combined molecular biology and bioinformatics approach. Initial screening of a proprietary database with the published human HCN1 sequence [1] identified a single clone according to SEQ ID NO: 9 that matched at the 3′ end. Further analysis of the predicted amino acid sequence of this clone (SEQ ID NO: 10) confirmed that it encoded the 3′ end of human HCN1 with an in-frame stop codon. Comparison with the full-length murine HCN1 sequence indicated that the published human sequence lacks approximately 330 bases of 5′ sequence upstream to the translational initiation codon. Several approaches were adopted to attempt to obtain the missing 5′ end. These included cDNA library screening, genomic library screening and 5′ RACE-PCR. RACE-PCR consistently generated a further 153 bp (SEQ ID NO: 13) upstream of the published human HCN1 sequence. A search of genomic databases with this new 5′ end sequence identified a BAC clone (RP11-398G9) in the updated EMBL high throughput genomic (HTG) database (em62htgnew). Sequence analysis of this clone suggests that it contains an in-frame initiation codon and an open reading frame homologous to murine HCN1. The 5′ end of human HCN1 is extremely GC-rich (approximately 80%) thus explaining why conventional means of cloning this sequence proved fruitless.
Thus, sequence from a combination of newly identified genomic BAC clone, RACE-PCR, published sequence and the clone obtained from the proprietary database have been assembled to generate a full length cDNA encoding a full length human HCN1 Ih channel subunit (SEQ ID NO: 1).
In another aspect of the invention, the sequences of the present invention can be used to derive primers and probes for use in DNA amplification reactions in order to perform diagnostic procedures or to identify further, neighbouring genes which also may contribute to the expression of HCN1. Also, fragments may be generated with PCR procedures, as the sequences that are shown in SEQ ID NO: 13, SEQ ID NO: 14, SEQ ID NO: 15, SEQ ID NO: 18, SEQ ID NO: 19 and SEQ ID NO: 21. In this way mutation analysis may be performed.
It is known in the art that genes may vary within and among species with respect to their nucleotide sequence. The sequence of full length HCN1 cDNAs from other individuals as well as their gene sequence may now be readily identified using the above probes and primers.
Availability of the full-length sequence now allows to provide for assays that measure whether the functional characteristics of the full-length channel are altered. It is clear for the skilled person that now all kinds of functional equivalents may be genetically engineered. Therefore, the invention also comprises functional equivalents, which are characterised in that they are capable of hybridising to at least part of the HCN1 sequence shown in SEQ ID NO: 1, preferably under high stringency conditions.
The present invention also includes these functional variants. Examples of such variants are for example deletions, insertions, point substitutions (single nucleotide polymorphisms) and splice variants. Also included in this term are other sequence variants, for example splice variants of a sequence which may differ in size through inclusion or exclusion of different exons. Identification of sequence variants and single nucleotide polymorphisms in particular are important for two reasons: first by identifying the small differences that influence the type and severity of diseases contracted by individuals; and second, by being able to understand the reasons behind variation of response to medicines at the individual level. Understanding how these variations in the genetic code influence biological systems will be the key to discovering new medicines and enabling doctors to prescribe medicines to patients who are likely to respond.
The present invention provides such functional variants. SEQ ID NO: 3 which is a full-length variant that contains a 189 bp insertion at position 2114 in SEQ ID NO 1, indicative of a splicing variant compared to SEQ ID NO: 1. This insertion encodes a further 63 amino acids in SEQ ID NO 4 in a region predicted to encode a portion of the carboxy-terminal domain of the polypeptide (FIGS. 1 and 2). Two single nucleotide variants are also provided in SEQ ID NO 5. The first is a C (SEQ ID NO 1) to A (SEQ ID NO 5) transition at position 107 resulting in a change of amino acid from Pro (SEQ lD NO 2) to Thr (SEQ ID NO 6) at position 28. This occurs in a region predicted to encode the amino terminal domain of the polypeptide (FIGS. 1 and 2). The second single nucleotide variant is a C (SEQ ID NO 1) to T (SEQ ID NO 5) transition at position 2064 resulting in a change of amino acid from Ser (SEQ ID NO 2) to Phe (SEQ ID NO 6). This occurs in a region predicted to encode the carboxy-terminal domain of the polypeptide immediately upstream of the splice variant insertion site. Both single nucleotide variants may therefore occur in association with the splicing variant giving rise to SEQ ID NO 7 encoding the polypeptide SEQ ID NO 8. In addition, a functional variant may comprise only one of these single nucleotide differences in conjunction with the splicing isoforms.
In addition, the sequences of the present invention contain a number of other differences compared to the published sequence [1]. These include a further eleven point substitutions, corresponding to 5 amino acid changes, and divergent sequence at the 3′ end of the published sequence [1].
Two nucleic acid fragments are considered to have hybridisable sequences if they are capable to hybridising to one another under typical hybridisation and wash conditions, as described, for example in Maniatis, et al., pages 320-328, and 382-389, or using reduced stringency wash conditions that allow at most about 25-30% basepair mismatches, for example: 2×SSC, 0.1% SDS, room temperature twice, 30 minutes each, then 2×SSC, 0.1% SDS 37° C. once, 30 minutes; then 2×SSC, room temperature twice ten minutes each. Preferably, homologous nucleic acid strands contain 15-25% basepair mismatches, even more preferably 5-15% basepair mismatches. These degrees of homology can be selected by using wash conditions of appropriate stringency for identification of clones from gene libraries or other sources of genetic material, as is well known in the art.
Furthermore, to accommodate codon variability, the invention also includes sequences coding for the same amino acid sequences as the sequences disclosed herein. Also portions of the coding sequences coding for individual domains of the expressed protein are part of the invention as well as allelic and species variations thereof. Sometimes, a gene expresses different isoforms in a certain tissue which includes splicing variants, that may result in an altered 5′ or 3′ mRNA or in the inclusion of an additional exon sequence. Alternatively, the messenger might have an exon less as compared to its counterpart. These sequences as well as the proteins encoded by these sequences all are expected to perform the same or similar functions and form also part of the invention.
The sequence information as provided herein should not be so narrowly construed as to require inclusion of erroneously identified bases. The specific sequence disclosed herein can be used to isolate further genes which in turn can be subjected to further sequence analyses thereby identifying sequencing errors.
Thus, in one aspect, the present invention provides for a DNA sequence encoding a full length human hyperpolarised activated ion channel of the HCN 1 subtype or functional equivalents thereof.
The DNA according to the invention may be obtained from cDNA. Alternatively, the coding sequence might be obtained from genomic DNA, or prepared using DNA synthesis techniques. The polynucleotide may also be in the form of RNA. The polynucleotide may be in single stranded or double stranded form. The single strand might be the coding strand or the non-coding (anti-sense) strand.
The present invention further relates to polynucleotides which have at least 80%, preferably 90% and more preferably 95% and even more preferably at least 98% identity with SEQ ID NO:1, provided that such polynucleotides encode polypeptides which retain essentially the same biological function or activity as the natural, mature protein. Even more preferred is the full length sequence according to SEQ ID NO: 1, SEQ ID NO: 3, SEQ ID NO 5 or SEQ ID NO 7
The percentage of identity between two sequences can be determined with programs such as DNAMAN (Lynnon Biosoft, version 3.2). Using this program two sequences can be aligned using the optimal alignment algorithm of Smith and Waterman (1981, J. Mol. Biol, 147:195-197). After alignment of the two sequences the percentage identity can be calculated by dividing the number of identical nucleotides between the two sequences by the length of the aligned sequences minus the length of all gaps.
The DNA according to the invention will be very useful for in vivo or in vitro expression of the novel sequence according to the invention in sufficient quantities and in substantially pure form.
In another aspect of the invention, a full length human hyperpolarised activated ion channel of the HCN 1 subtype or functional equivalents thereof are provided. An example of such a polypeptide is a polypeptide sequence according to SEQ ID NO: 2. SEQ ID NO: 2 is encoded by the open reading frame of SEQ ID NO: 1 and consists of 827 amino acids.
Preferably, the polypeptides according to the invention comprise the amino acid sequence as shown in SEQ ID NO: 2.
Also functional equivalents, that is polypeptides homologous to SEQ ID NO: 2 or parts thereof having variations of the sequence while still maintaining functional characteristics, are included in the invention. Such variants are provided in SEQ ID NO: 4 which is a polypeptide encoded by the open reading frame of SEQ ID NO: 3, SEQ ID NO: 6 which is a polypeptide encoded by the open reading frame of SEQ ID NO: 5 and SEQ ID NO: 8 which is a polypeptide encoded by the open reading frame of SEQ ID NO: 7
The variations that can occur in a sequence may be demonstrated by (an) amino acid difference(s) in the overall sequence or by deletions, substitutions, insertions, inversions or additions of (an) amino acid(s) in said sequence. Amino acid substitutions that are expected not to essentially alter biological and immunological activities, have been described. Amino acid replacements between related amino acids or replacements which have occurred frequently in evolution are, inter alia Ser/Ala, Ser/Gly, Asp/Gly, Asp/Asn, Ile/Val (see Dayhof, M. D., Atlas of protein sequence and structure, Nat. Biomed. Res. Found., Washington D.C., 1978, vol. 5, suppl. 3). Based on this information Lipman and Pearson developed a method for rapid and sensitive protein comparison (Science, 1985, 227, 1435-1441) and determining the functional similarity between homologous polypeptides. It will be clear that also polynucleotides coding for such variants are part of the invention.
The polypeptides according to the present invention include the polypeptides comprising SEQ ID NO: 2 but also its isoforms, i.e. polypeptides with a similarity of 70%, preferably 85%, more preferably 90% even more preferably 95% or even 98%, provided these isoforms retain the same biological functions as the sequence shown in SEQ ID NO: 2, like the sequences shown in SEQ ID NO: 4, SEQ ID NO: 6 and SEQ ID NO: 8. Also fragments of such polypeptides still capable of conferring these biological effects are included. Especially portions which still bind to ligands form part of the invention. Such portions may be functional per se, e.g. in solubilized form or they might be linked to other polypeptides, either by known biotechnological ways or by chemical synthesis, to obtain chimeric proteins. Such proteins might be useful as therapeutic agent in that they may substitute the gene product in individuals with aberrant expression of the HCN1 gene. Polypeptides obtainable from alternative splice products are also included in this invention.
As an example of a functional equivalent SEQ ID NO: 4 is provided. This polypeptide corresponds to the open reading frame of SEQ ID NO: 3 and contains an insertion of 63 amino acids.
Further examples of functional variants are polypeptides derived from coding sequences with single nucleotide variations. These include SEQ ID NO: 6 and SEQ ID NO: 8 which correspond to the open reading frames of SEQ ID NO: 5, and SEQ ID NO: 7 respectively. Both polypeptides incorporate amino acid substitutions at position 28 (Pro/Thr) and 680 (Ser/Phe). It should be noted that either amino acid substitution may occur alone in the HCN1 polypeptide sequence such that any HCN1 polypeptide with either one or both of these substitutions are part of the invention.
The sequence according to SEQ ID NO: 1, SEQ ID NO: 3, SEQ ID NO: 5 or SEQ ID NO: 7 may also be used in the preparation of vector molecules for the expression of the encoded protein in suitable host cells. Also, fragments of these sequences may be cloned into an expression vector and expressed in order to determine individual functional sites along the cDNA molecule. The sequences may also be modified in that they are cloned into an expression vector either with our without the 5′ and/or 3′ non-coding regions. The coding region of a cDNA clone according to SEQ ID NO: 1 is shown in SEQ ID NO: 21. The coding region of a cDNA clone according to SEQ ID NO: 3 is shown in SEQ ID NO: 22. The coding region of a cDNA clone according to SEQ ID NO: 5 is shown in SEQ ID NO: 24. The coding region of a cDNA clone according to SEQ ID NO: 7 is shown in SEQ ID NO:25. The invention therefore also provides an expression vector comprising SEQ ID NO: 22, SEQ ID NO: 23, SEQ ID NO: 24 or SEQ ID NO: 25 or fragments thereof.
A wide variety of host cell and cloning vehicle combinations may be usefully employed in cloning the nucleic acid sequence coding for the HCN1 gene, the cDNA coding for HCN1 or parts thereof. For example, useful cloning vehicles may include chromosomal, non-chromosomal and synthetic DNA sequences such as various known bacterial plasmids and wider host range plasmids and vectors derived from combinations of plasmids and phage or virus DNA.
Vehicles for use in expression of the genes or a ligand-binding domain thereof of the present invention will further comprise control sequences operably linked to the nucleic acid sequence coding for a ligand-binding domain. Such control sequences generally comprise a promoter sequence and sequences which regulate and/or enhance expression levels. Of course control and other sequences can vary depending on the host cell selected.
Suitable expression vectors are for example bacterial or yeast plasmids, wide host range plasmids and vectors derived from combinations of plasmid and phage or virus DNA. Vectors derived from chromosomal DNA are also included. Furthermore an origin of replication and/or a dominant selection marker can be present in the vector according to the invention. The vectors according to the invention are suitable for transforming a host cell.
Recombinant expression vectors comprising the DNA of the invention as well as cells transformed with said DNA or said expression vector also form part of the present invention.
Suitable host cells according to the invention are bacterial host cells, yeast and other fungi, plant or animal host such as Chinese Hamster Ovary cells or monkey cells. Especially preferred cells are HEK 293. Thus, a host cell which comprises the DNA or expression vector according to the invention is also within the scope of the invention. The engineered host cells can be cultured in conventional nutrient media which can be modified e.g. for appropriate selection, amplification or induction of transcription. The culture conditions such as temperature, pH, nutrients etc. are well known to those ordinary skilled in the art.
The techniques for the preparation of the DNA or the vector according to the invention as well as the transformation or transfection of a host cell with said DNA or vector are standard and well known in the art, see for instance Sambrook et al., Molecular Cloning: A laboratory Manual. 2nd Ed., Cold Spring Harbor Laboratory, Cold Spring Harbor, N.Y., 1989.
The proteins according to the invention can be recovered and purified from recombinant cell cultures by common biochemical purification methods including ammonium sulfate precipitation, extraction, chromatography such as hydrophobic interaction chromatography, cation or anion exchange chromatography or affinity chromatography and high performance liquid chromatography. If necessary, also protein refolding steps can be included.
The gene products according to the present invention can be used for the in vivo or in vitro identification of novel ligands or analogs thereof. For this purpose binding studies can be performed with cells transformed with DNA according to the invention or an expression vector comprising DNA according to the invention, said cells expressing the HCN1 full length gene product according to the invention or fragments thereof.
Alternatively also the HCN1 gene products according to the invention as well as ligand-binding domains thereof can be used in an assay for the identification of functional ligands or analogs for the HCN1 gene products.
Methods to determine binding to expressed gene products as well as in vitro and in vivo assays to determine biological activity of gene products are well known. In general, expressed gene product is contacted with the compound to be tested and binding, stimulation or inhibition of a functional response is measured.
As a preferred way of detecting the binding of the ligand to the expressed protein, also signal transduction capacity may be measured.
The present invention thus provides for a quick and economic method to screen for therapeutic agents for the prevention and/or treatment of diseases related to CNS disorders such as human psychiatric and neurological dysfunction, cardiovascular dysfunction of the heart, and reproductive dysfunction and/or contraception related to Ih function in testes and spermatozoa. The method is especially suited to be used for the high throughput screening of numerous potential compounds.
Compounds which activate or inhibit the function of HCN1 gene product may be employed in therapeutic treatments to activate or inhibit the polypeptides of the present invention.
Also within the scope of the invention are antibodies, especially monoclonal antibodies raised against the polypeptide molecule according to the invention. Such antibodies can be used therapeutically to inhibit HCN1 gene product function and diagnostically to detect HCN1 gene products.
The invention furthermore relates to the use of the HCN1 gene products as part of a diagnostic assay for detecting clinical abnormalities or susceptibility to any of the above disorders or investigation of different clinical outcomes in response to medical treatment related to mutations in the nucleic acid sequences encoding the HCN1 gene. Two examples single nucleotide variants are provided in this invention. Such variants or mutations may e.g. be detected by using PCR (Saiki et al., 1986, Nature, 324, 163-166). Also the relative levels of RNA can be determined using e.g. hybridization or quantitative PCR technology. The presence and the levels of the HCN1 gene products themselves can be assayed by immunological technologies such as radioimmuno assays, Western blots and ELISA using specific antibodies raised against the gene products. Such techniques for measuring RNA and protein levels are well known to the skilled artisan.
The determination of expression levels of the HCN1 gene products and variants thereof in individual patients may lead to fine tuning of treatment protocols.
Also, transgenic animals may be prepared in which the expression of the HCN1 gene is altered or abolished.
EXAMPLE 1
Full-length Sequence Identification
Proprietary databases were screened by BLAST2 for the presence of related human cDNA sequences using parts of the DNA sequence of the human HCN1 channel (Accession No AF064876) [29] and mouse HCN1 (Accession number AJ225123). A single cDNA clone (SEQ ID NO: 9) was identified from a brain cDNA library, obtained and sequenced using an ABI Prism 310 Genetic analyser (PE Biosystems). Sequencing reactions were performed using ABI Prism BigDye Terminator cycle sequencing Ready reaction kit (PE Biosystems). Each sequencing reaction contained 300 ng cDNA clone, 3.2 pmol sequencing primer, and PE Biosystems Terminator Ready reaction mix in a final volume of 20 ul. Reactions were cycled as follows: 25 cycles of 96° C. for 10 sec, 50° C. for 5 sec and 60° C. for 4 min in a PE Biosystems GeneAmp PCR system 9700. Following cycling, the extension products were precipitated by adding 2 ul 3M NaOAc (pH 4.6) and 50 ul 95% ethanol. Products were precipitated at RT for 15 min and collected by centrifugation at 14000 rpm for 20 min. Pellets were washed 2× with 70% ethanol prior to resuspension in 20 ul
The published human HCN1 sequence and the 5′ sequence obtained by RACE-PCR was used to design primers to PCR the region of human HCN1 flanked by the RACE-PCR product (SEQ ID NO: 13) and SEQ ID NO: 9 and covered by the published sequence. Each PCR reaction contained 1× PCR buffer (Expand High Fidelity buffer), 1.5 mM MgCl2, 2□1 whole brain Marathon-Ready cDNA(Clontech), 400 nM primer 1 (HCN1PCRA 5′-TGCTGCAGCCCGGGGTCAACAAAT-3′ (SEQ ID NO:37)), 400 nM primer 2 (HCN1PCRB 5′-GAGGCGGTGGGGGAGGCATAGTGG-3′ (SEQ ID NO:38)), 400□M dATP, 400 μM dCTP, 400 μM dGTP, 400□M dTTP, 5% DMSO and 2.625 units Expand High Fidelity PCR enzyme mix in a total volume of 50 μl. Reactions were cycled in a MJ Research PTC-200 Thermal Cycler using the following conditions: 94° C., 2 min and 35 cycles of 24° C. for 30 sec, 60° C. for 30 sec, 72° C. for 90 sec, followed by an extension of 72° C. for 5 min. A PCR product of approximately 1.9 kb was identified, purified and sequenced as previously described (SEQ ID NO: 19). Some independently derived clones were also found to contain a single nucleotide variation (a C to T transition) indicated in SEQ ID NO: 20.
The full length sequence of HCN1 indicates that the cDNA consists of 2484 bp open reading frame (SEQ ID NO: 22) encoding an 827 amino acid channel. The functional equivalent sequence shown in SEQ ID NO: 3 comprises an 2673 bp open reading frame (SEQ ID NO: 23) encoding a 890 amino acid channel. Two single nucleotide variants were found which altered the amino acid sequence, and these are indicated in conjunction with the splice variant in SEQ ID NO: 24 and SEQ ID NO: 25. To date, four members of the hyperpolarisation-activated cation channel have been identified and cloned from mouse (HCN1-4). Full length clones of human HCN-2 and HCN-4 (Accession NOs AF065164 and AJ238850) have also been obtained whereas only a single partial sequence exists for human HCN-1. These channels share close overall homology. Analysis of the channel sequence have shown that these channels have the classic K+ channel structure of 6 transmembrane domains and an ion conducting pore loop. Additionally, they possess a cyclic nucleotide binding domain. Within the transmembrane, pore and CNBD regions there is very high conservation among the channel subtypes. Alignment of the HCN1 sequences according to the invention with the other members of the HCN channel family suggest that this sequence does encode an hyperpolarisation activated ion channel. Regions of conservation between the human HCN channels are denoted by asterixes in FIG. 1. The transmembrane domains, pore regions and CNBD are underlined. Alignment of HCN1 polypeptides shows that the novel sequences do encode HCN1 channels (FIG. 2). The single amino acid variants are indicated in bold and are underscored with a # in the alignment in FIG. 2.
The cloned full length mouse HCN1 channel contains a polyglutamine repeat in the C-terminal region [29]. Expansions of polyglutamine repeats have been implicated in several neurodegenerative diseases [27]. Although no polyglutamine repeat appears to be present in the novel sequence the possibility arises of allelic variation and expansion of a polyglutamine tract resulting in increased propensity to neurodegenerative diseases. The 189 bp deletion identified in the new splice variant spans the corresponding region in the mouse HCN1 channel containing the polyglutamine repeat.
EXAMPLE 2
Tissue Distribution of SEQ ID NO: 1 and SEQ ID NO: 3
In order to analyze the expression of the ion channels comprising SEQ ID NO:1 and SEQ ID NO: 3 in different human tissues, Northern blot analysis was performed. A human Multiple Tissue Expression (MTE) Array was obtained from Clontech, which contains poly A+ RNA from a variety of human tissues including nervous system, cardiovascular, digestive, and immune tissue. Prehybridization was performed in Clontech's ExpressHyb supplemented with 100 μg/ml denatured sheared salmon DNA for 2 hr at 65° C. For RNA detection, 32P labeled DNA fragments were generated with an Oligolabeling kit (High Prime, Boehringer Mannheim) using a 627 bp fragment generated from SEQ ID NO: 9. This fragment starts at a Smal site 717 bp from the 5′ end of the coding sequence and stops at the Notl cloning site (SEQ ID NO:21) and is common to both SEQ ID NO: 1 and SEQ ID NO: 3. The 32P labeled probes were hybridized to the array for 16 hours at 65° C. Subsequently, the array was washed with 2×SSC/1% SDS at 65° C. (4×20 min) followed by 2×20 min washes at 50° C. in 0.1×SSC/0.5% SDS. Filters were exposed to a phosphorscreen for 20 hr and analysed using Molecular Dynamics Storm Scan phosphorimage package.
From the results it can be concluded that human SEQ ID NO: 1 and SEQ ID NO: 3 are expressed predominantly in nervous tissue. Positive signals were detected in a variety of brain regions including cerebral cortex, cerebellum, occipital lobe, temporal lobe and nucleus accumbens, Weaker expression was observed in fetal brain. Peripherally, only weak expression was detected in atrial tissue of the heart.
EXAMPLE 3
Tissue Distribution of SEQ ID: 1 and 3 in Nervous Tissue
In order to further analyze the expression of the ion channels comprising SEQ ID NO:1 and SEQ ID NO: 3 in nervous tissue, Northern blot analysis was performed. A human Multiple Tissue Northern was obtained from Clontech (Human Brain MTN Blot II), which contains poly A+ RNA from a variety of brain regions. Prehybridization was performed in Clontech's ExpressHyb for 2 hr at 65° C. For RNA detection, 32P labeled DNA fragments were generated as described previously for MTE array. The 32P labeled probes were hybridized to the filters for 16 hours at 65° C. Subsequently, the filters were washed with 2×SSC/0.05% SDS at room temperature (2×40 min) followed by 2×20 min washes at 50° C. in 0.1×SSC/0.1% SDS. Filters were exposed to a phosphorimager screen for 20 hr and analysed using Molecular Dynamics Storm Scan phosphorimage package.
From the results it can be concluded that human SEQ ID NO: 1 and SEQ ID NO: 3 are expressed in a variety of nervous tissue. Multiple transcripts were detected in a cerebral cortex, cerebellum, occipital lobe, temporal lobe and frontal lobe. Weaker expression was observed in putamen and medulla, with no expression in spinal cord.
EXAMPLE 4
Functional Assay for Detecting HCN1 Channel Currents
Human HCN1 channel currents were detected under voltage-clamp using the whole-cell configuration of the patch-clamp technique. Appropriate cells (HEK293) expressing human HCN1 channel subunits were placed in a recording chamber containing extracellular solution (Sodium chloride 135 mM; Potassium chloride 5 mM; Calcium chloride 1.8 mM; Magnesium chloride 0.5 mM; HEPES 5 mM) at a temperature of 20-25 degrees centigrade. In some experiments the extracellular solution contained 30 mM Potassium chloride, in which cases the Sodium chloride concentration was reduced to 110 mM. The glass pipettes used for recording contained pipette solution (Sodium chloride 10 mM; Potassium chloride 130 mM; Magnesium chloride 0.5 mM; HEPES 5 mM; EGTA 1 mM) Using standard equipment and methodology, a cell was voltage-clamped at a holding potential of −40 mV (not allowing for liquid junction potentials). The membrane current was recorded throughout the experiment. To determine the current-voltage relationship of human HCN1 channels, every 8 seconds the membrane potential was stepped sequentially to the following values (in mV) for 2 or 3 seconds each: −20, −40, −60, −80, −100, −120, −140 (FIGS. 5 and 6). To determine the effects of caesium chloride (5 mM) or ZD 7288 (100 micromolar) on human HCN1 channel currents, t every 8 or 10 seconds the cell potential was hyperpolarised to a potential of −120 mV (not allowing for liquid junction potentials) for 1-5 seconds and the potential then returned to the holding potential of −40 mV. The membrane current was measured just after (1-10 milliseconds) the start of the hyperpolarising step and just before (10-100 milliseconds) its end. The difference in these two values of membrane current was taken as the amplitude of the current due to the human HCN1 channel subunits. For each cell, the current amplitude was determined first in normal extracellular solution and then in extracellular solution containing caesium chloride (FIG. 3) or ZD 7288 (FIGS. 4 and 7). Alternatively, the effect of caesium chloride (5 mM) on human HCN1 channel currents was determined by measuring the current-voltage relationship in normal extracellular solution and also in extracellular solution containing caesium chloride (FIG. 6). Human HCN1 channel currents were reduced by caesium chloride (5 mM) and by ZD 7288 (100 micromolar) [17, 25].
A clone which has the following characteristics is considered to be an Ih ion channel:
Template suppression reagent (PE Biosystems) for sequencing. This clone encoded the 3′ end of human HCN-1. The sequence is shown in SEQ ID NOs: 9.
The presence of a second splice variant was confirmed by PCR using primers designed against the published sequence flanking either side of the 189 bp deletion identified in SEQ ID NO: 9 compared to the published sequence. Each PCR reaction contained 1×PCR buffer (Expand High Fidelity buffer), 1.5 mM MgCl2, 2□1 whole brain Marathon-Ready cDNA (Clontech), 400 nM primer 1 and 2 (HCN1SPLICEA 5′-TGCTGCAGCCCGGGGTCAACAAAT-3′ (SEQ ID NO:26) and HCN1SPLICE B 5′-CTCCTGCCCCCTGCCTGAAG-3′ (SEQ ID NO:27), or HCN1SPLICEC 5′-TCTACTACGACCCCGACCTC-3′ (SEQ ID NO:28) and HCN1SPLICED 5′-TGGCTCCCGACGACATCT-3′ (SEQ ID NO:29)), 400□M dATP, 400 μM dCTP, 400□M dGTP, 400□M dTTP, 5% DMSO and 2.625 units Expand High Fidelity PCR enzyme mix in a total volume of 50□1. Reactions were cycled in a MJ Research PTC-200 Thermal Cycler using the following conditions: 94° C., 2 mm and 35 cycles of 24° C. for 30 sec, 60° C. for 30 sec, 72° C. for 90 sec, followed by an extension of 72° C. for 5 mm. PCR products of approximately 560 bp (HCN1SPLICEA and HCN1SPLICEB) and 600 bp (HCN1SPLICEC and HCN1SPLICED) were identified, purified and sequenced as previously described (SEQ ID NO: 17). This sequence had no deletion compared to the published sequence. Several independently derived clones were observed to contain a single nucleotide variant (a C to T transition) shown in SEQ ID NO: 11 which results in a change in amino acid (Ser to Phe) in SEQ ID NO: 12.
Primers were designed using the known human HCN1 sequence to attempt to obtain the 5′ end of HCN1 by RACE-PCR using Clontech SMART RACE cDNA amplification kit. SMART 5′-RACE-ready cDNA was synthesised as follows, 1 μg of hippocampal polyA+ RNA was mixed with 2 μM 5′-RACE cDNA synthesis primer (Clontech 5′-(T)25N−1N-3′) and 2 M SMART II oligonucleotide (Clontech 5′-AAGCAGTGGTAACAACGCAGAGTACGCGGG-3′ (SEQ ID NO: 30)) in a total volume of 5 μl. Tubes were incubated at 70° C. for 2 min and cooled on ice for a further 2 min. The following was then added, 1× First strand buffer (50 mM Tris-HCl pH8.3, 75 mM KCl and 6 mM MgCl2), 2 mM DTT, 1 mM dATP, 1 mM dCTP, 1 mM dGTP, 1 mM dTTP and 200 u MMLV reverse transcriptase. The reactions were incubated at 42° C. for 1.5 hr in an air incubator. The reaction product was diluted with 250 ul Tricine-EDTA buffer and heated at 72° C. for 7 min prior to storage at 20° C.
Each RACE-PCR reaction contained 1×PCR buffer (Clontech Advantage-GC 2 PCR buffer, 40 mM Tricine-KOH pH9.2, 15 mM KOAc, 3.5 mM Mg(OAc)2, 5% DMSO, 187.5 ng BSA, 0.005% Nonidet P-40 and 0.005% Tween-20), 200 uM dATP, 200 uM dCTP, 200 uM dGTP, 200 uM dTTP, 2.5 ul RACE-ready cDNA, Clontech Universal primer mix (20 nM long primer 5′-CTAATACGACTCACTATAGGGCAAGCAGTGGTAACAACGCAGAGT-3′ (SEQ ID NO:31), 100 nM short primer 5′-AAGCAGTGGTAACAACGCAGACT-3′SEQ ID NO:32), 200 nM Gene Specific Primer (BCNG1R2, 5′-CTGGCTGTCTFfGTAAACTFITCAGATCCATFF-3′ (SEQ ID NO: 33), BCNG1R3 5′-CTGTAAGGGTGGATAATCCAGAAGCCTGC-3′ (SEQ ID NO: 34), BCNG R4B, 5′-TTCTGGCTCCCAAACATGCGGAGG-3′ (SEQ ID NO: 35)), 1× Advantage-GC 2 polymerase mix (1% glycerol, 0.3 mM Tris-HCl pH 8.0, 1.5 mM KCl, 1 uM EDTA) and 0.5M GC-melt (Clontech) in a total volume of 50 ul. Reactions were cycled in a MJ Research PTC-200 Thermal Cycler using the following conditions: 95° C., 3 min and 40 cycles of 94° C. for 20 sec, 65° C. for 20 sec, 72° C. for 90 sec, followed by an extension of 72° C. for 5 min. A PCR product of approximately 300 bp was identified, purified and sequenced as previously described. RACE-PCR generated novel sequence information of 153 bp upstream of published human HCN1 sequence SEQ ID NO: 13.
SEQ ID NO: 13 was used to screen by BLAST2 human public EST and genomic databases available from EMBL [31]. A human genomic bacterial artificial chromosome (BAC) clone (RP11 -398G9: accession number AC013384) that contained sequence similarity was identified from the high throughput genomic division of the EMBL release 62. The region corresponding to human HCN-1 was sequenced as described previously. This clone appeared to encode the 5′ end of human HCN-1 including the translation initiation codon. The sequences are shown in SEQ ID NO: 14. Several independently derived clones were found to have a single nucleotide variant (a C to A transition) indicated in SEQ ID NO: 15 resulting in a change of amino acid from Pro to Thr.

    1. It opens through hyperpolarisation and closes at positive voltage values (Vm-10 mV;
    2. Whose activation and deactivation proceeds with a relatively slow time course;
    3. Which conducts not only K+ ions but also Na+ ions;
    4. Which are blocked preferentially by extracellular caesium ions rather than by extracellular barium ions.
    5. Which are directly modulated by cyclic nucleotides, particularly cAMP and cGMP


BRIEF DESCRIPTION OF THE DRAWING
FIG. 1: CLUSTAL W Multiple Sequence Alignments of SEQ ID NO: 2 SEQ ID NO: 4, SEQ ID NO: 6 and SEQ ID NO: 8 with human HCN1 (SEQ ID NO:36), human HCN2 (SEQ ID NO:39) and human HCN4 (SEQ ID NO: 40).
FIG. 2: CLUSTAL W Multiple Sequence Alignments of SEQ ID NO: 2, SEQ ID NO: 4, SEQ ID NO: 6 and SEQ ID NO: 8 with rat (SEQ ID NO: 41), mouse (SEQ ID NO: 42) and partial human HCN1 (SEQ ID NO: 43) channels.
FIG. 3: Effect of caesium chloride (5 mM) on hHCN1 (full-length) currents. Whole-cell patch-clamp recording from a HEK 293F cell transfected with an expression vector containing SEQ ID NO 7. Panel A: each data point represents a measurement from one data sweep. Caesium chloride was added to the bathing medium for the time represented by the bar above the data points. Panel B: representative data sweeps (corresponding letters in panels A & B identify the times at which the sweeps in panel B were taken). Inward membrane currents in panel B are plotted as negative quantities (following convention), but the amplitude of hHCN1 current in panel A is plotted as a positive value.
FIG. 4: Effect of ZD 7288 (100 micromolar) on hHCN1 (full-length) currents. Whole-cell patch-clamp recording from a HEK 293F cell transfected with an expression vector containing SEQ ID NO 7. Panel A: each data point represents a measurement from one data sweep. ZD 7288 was added to the bathing medium for the time represented by the bar above the data points. Panel B: representative data sweeps (corresponding letters in panels A & B identify the times at which the sweeps in panel B were taken). Inward membrane currents in panel B are plotted as negative quantities (following convention), but the amplitude of hHCN1 current in panel A is plotted as a positive value.
FIG. 5: Current-voltage relationships of whole-cell patch-clamp recordings from HEK 293F cells transfected with DNA for hHCN1. Panel A: SEQ ID NO 5. Panel B: SEQ ID NO 7. For each panel, the upper traces display the currents evoked by the voltage steps shown in the lower traces.
FIG. 6: Current-voltage relationship and block by caesium chloride of hHCN1 current. Whole-cell patch-clamp recording from a HEK 293F cell transfected with DNA expressing SEQ ID NO:3. Each panel shows the currents evoked by voltage commands (voltage traces not shown). Panel A: normal extracellular solution. Panel B: extracellular solution containing 5 mM caesium chloride. Panel C: return to normal extracellular solution. All panels from the same cell.
FIG. 7: Effect of ZD 7288 (100 micromolar) on hHCN1 current. Whole-cell patch-clamp recording from a HEK 293 cell transfected with KDNA expressing SEQ ID NO:3. Panel A: each data point represents a measurement from one data sweep. ZD 7288 was added to the bathing medium for the time represented by the bar above the data points. Panel B: representative data sweeps (corresponding letters in panels A & B identify the times at which the sweeps in panel B were taken). Note that inward membrane currents in panel B are plotted as negative quantities (following convention), but that the amplitude of hHCN1 current in panel A is plotted as a positive value.
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## Claims

1. An isolated DNA sequence encoding a full length human hyperpolarized activated ion channel of the HCN 1 subtype, comprising:
SEQ ID: 5.

2. An isolated DNA sequence comprising SEQ ID NO: 22 or SEQ ID NO: 24.

3. An isolated DNA sequence comprising SEQ ID NO: 23 or SEQ ID NO: 25.

4. An isolated DNA sequence comprising SEQ ID NO: 3 or SEQ ID NO: 7.

5. An isolated, full length human hyperpolarized activated ion channel of the HCN1 subtype, comprising an amino acid sequence selected from the group consisting of SEQ ID NO: 2, SEQ ID NO: 4, SEQ ID NO: 6 and SEQ ID NO: 8.

6. The isolated, full length human hyperpolarized activated ion channel of the HCN 1 subtype of claim 5, comprising:
SEQ ID NO: 6.

7. The isolated, full length hyperpolarized activated ion channel of the HCN1 subtype of claim 5, comprising SEQ ID NO:8.

8. An in vitro screening assay for the selection of compounds that bind the human hyperpolarised activated ion channel of the HCN 1 subtype, comprising:
providing cells expressing the full length human hyperpolarized activated ion channel of the HCN 1 subtype according to claim 5,
contacting the compounds with the full length human hyperpolarized activated ion channel of the HCN 1 subtype, and
determining whether the compounds bind specifically to the full length human hyperpolarized activated ion channel of HCN 1 subtype.

9. A method to screen for compounds that influence a function of the human hyperpolarised activated ion channel of the HCN 1 subtype, comprising:
assaying compounds with the full length human hyperpolarized activated ion channel of the HCN 1 subtype according to claim 5 to determine whether the compounds activate or inhibit said ion channel.

10. An isolated expression vector comprising SEQ ID NO: 22, SEQ ID NO: 24, SEQ ID NO: 23, SEQ ID NO: 25.