GB2382576A - Voltage-gated ion channel - Google Patents

Abstract

The present invention provides an isolated voltage-gated ion channel polypeptide comprising <SL> <LI>(i) the amino acid sequence of SEQ ID NO: 2 or <LI>(ii) a variant thereof which is capable of forming part of a cation channel that can be activated by a change in membrane potential or <LI>(iii) a fragment of (i) or (ii) which is capable of forming part of a cation channel that can be activated by a change in membrane potential. </SL> Methods for the identification of substances which modulate the ion channel are also disclosed. Such modulators may be used in treating disorders responsive to voltage-gated ion channel modulation.

Description

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PROTEIN Field of the Invention The present invention relates to voltage-gated ion channel polypeptides.

Background of the Invention Ion channels are involved in a wide variety of neurological and other disorders in man. Voltage-gated calcium and sodium alpha-subunits have a conserved structural architecture, each consisting of four homologous repeats of six transmembrane alpha-helices. Thus each channel has a total of twenty four transmembrane alpha-helical domains. In contrast, voltage-gated potassium channels are formed through the association of four voltage-gated potassium channel subunit molecules in the cell membrane to form a functional channel pore. Each voltagegated potassium channel subunit has six transmembrane domains and thus is similar in structure to one of the four repeats of a voltage-gated calcium or sodium channel.

Thus functional voltage-gated potassium channels consist of four homologous subunits (each with six transmembrane domains), while sodium and calcium channels consist of a single subunit with four internally homologous repeats (each repeat containing six transmembrane domains).

The region of a voltage-gated calcium and sodium alpha-subunit that forms the pore of the channel is situated between the fifth and sixth transmembrane domains of each of the four repeats. These pore-loops contain the amino acids that are important in determining the ion selectivity of these channels. Sodium and calcium channels contain distinct selectivity residues at these positions with each of the four homologous repeats contributing one residue to the selectivity"signature"of the channel. The residues that form the distinct selectivity"signature"in voltagegated sodium channels are D-E-K-A (Catterall (1991) Curr. Opin Neurobiol. 1: 5; Marban et al. (1998) J. Physiol. 508: 647). In voltage-gated calcium channels the selectivity"signature"is E-E-E-E and in T-type calcium channels the selectivity "signature"is E-E-D-D. Thus the selectivity"signature"of sodium channels consists of one non-polar, one positively-charged and two negatively-charged amino acids, while the selectivity"signature"of the calcium channels consists of four negatively-

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charged amino acids.

The residues that are known to be important in the voltage sensitive activation of voltage-gated calcium and sodium channels reside within the fourth transmembrane alpha-helix of each repeat. These voltage-sensor helices have positively charged arginine or lysine residues at every third position, this spacing is consistent with these positive charges lining one side of the alpha-helix.

Summary of the Invention A novel voltage-gated ion channel, referred to herein as HIPHUM 39, is now provided. HIPHUM 39 is shown to be primarily expressed in medulla oblongata.

The novel voltage-gated ion channel is a screening target for the identification and development of novel pharmaceutical agents, including modulators of voltage-gated ion channel activity. These agents may be used in the treatment and/or prophylaxis of disorders such as irritable bowel syndrome (IBS), inflammatory pain, neuropathic pain, acute postoperative pain, headache, cluster headache, tension headache, migraine, glossopharyngeal neuralgia, tooth ache, ear ache, trigeminal neuralgia, cancer associated pain, psychogenic pain syndromes, intestinal pain, ileus, intestinal obstruction pain, abdominal pain, premenstrual syndrome, haemorrhoid pain, peptic ulcer pain, ulcerative colitis pain, medulloblastoma, hypertension, cardiac arrhythmias, congestive heart failure, Huchard's disease, acute bronchitis, acute respiratory failure in chronic obstructive pulmonary disease (COPD), adult respiratory distress syndrome, asthma, chronic obstructive pulmonary disease (COPD), emphysema, pulmonary hypertension, respiratory bronchiolitis-associated interstitial lung disease, respiratory distress syndrome, infant sudden death syndrome and allergic asthma.

Accordingly, the present invention provides an isolated voltage-gated ion channel polypeptide comprising: (i) the amino acid sequence of SEQ ID NO: 2; (ii) a variant thereof which is capable of forming part of a cation channel that can be activated by a change in membrane potential; or (iii) a fragment of (i) or (ii) which is capable of forming part of a cation channel that can be activated by a change in membrane potential.

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According to another aspect of the invention there is provided a polynucleotide encoding a polypeptide of the invention which polynucleotide includes a sequence comprising: (a) the nucleic acid sequence of SEQ ID NO: 1 and/or a sequence complementary thereto; (b) a sequence which hybridises under stringent conditions to a sequence as defined in (a); (c) a sequence that is degenerate as a result of the genetic code to a sequence as defined in (a) or (b); or (d) a sequence having at least 60% identity to a sequence as defined in (a), (b) or (c).

The invention also provides: an expression vector which comprises a polynucleotide of the invention and which is capable of expressing a polypeptide of the invention; a host cell comprising an expression vector of the invention; a method of producing a polypeptide of the invention which method comprises maintaining a host cell of the invention under conditions suitable for obtaining expression of the polypeptide and isolating the said polypeptide; an antibody specific for a polypeptide of the invention; a method for identification of a substance that modulates voltage-gated ion channel activity and/or expression, which method comprises contacting a polypeptide, polynucleotide, expression vector or host cell of the invention with a test substance and determining the effect of the test substance on the activity and/or expression of the said polypeptide or the polypeptide encoded by the said polynucleotide, thereby to determine whether the test substance modulates voltage-gated ion channel activity and/or expression; a compound which stimulates or modulates voltage-gated ion channel activity and which is identifiable by the method referred to above; a method of treating a subject having a disorder that is responsive to voltage- gated ion channel stimulation or modulation, which method comprises administering to said subject an effective amount of substance of the invention; and

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use of a substance that stimulates or modulates voltage-gated ion channel activity in the manufacture of a medicament for the treatment or prophylaxis of a disorder that is responsive to stimulation or modulation of voltage-gated ion channel activity.

Preferably the disorder is selected from irritable bowel syndrome (IBS), inflammatory pain, neuropathic pain, acute postoperative pain, headache, cluster headache, tension headache, migraine, glossopharyngeal neuralgia, tooth ache, ear ache, trigeminal neuralgia, cancer associated pain, psychogenic pain syndromes, intestinal pain, ileus, intestinal obstruction pain, abdominal pain, premenstrual syndrome, haemorrhoid pain, peptic ulcer pain, ulcerative colitis pain, medulloblastoma, hypertension, cardiac arrhythmias, congestive heart failure, Huchard's disease, acute bronchitis, acute respiratory failure in chronic obstructive pulmonary disease (COPD), adult respiratory distress syndrome, asthma, chronic obstructive pulmonary disease (COPD), emphysema, pulmonary hypertension, respiratory bronchiolitis-associated interstitial lung disease, respiratory distress syndrome, sudden infant death syndrome and allergic asthma.

Brief Description of the Sequences SEQ ID NO: 1 shows the nucleotide and amino acid sequences of human protein HIPHUM 39.

SEQ ID NO: 2 shows the amino acid sequence alone of HIPHUM 39.

Detailed Description of the Invention Throughout the present specification and the accompanying claims the words "comprise"and"include"and variations such as"comprises","comprising", "includes"and"including"are to be interpreted inclusively. That is, these words are intended to convey the possible inclusion of other elements or integers not specifically recited, where the context allows.

The present invention relates to a human voltage-gated ion channel, referred to herein as HIPHUM 39, and variants thereof. Sequence information for HIPHUM 39 is provided in SEQ ID NO: 1 (nucleotide and amino acid) and in SEQ ID NO: 2.

A polypeptide of the invention thus consists essentially of the amino acid sequence

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of SEQ ID NO: 2 or of a variant of that sequence, or of a fragment of either thereof.

Polypeptides of the invention may be in a substantially isolated form. It will be understood that the polypeptide may be mixed with carriers or diluents which will not interfere with the intended purpose of the polypeptide and still be regarded as substantially isolated. A polypeptide of the invention may also be in a substantially purified form, in which case it will generally comprise the polypeptide in a preparation in which more than 50%, e. g. more than 80%, 90%, 95% or 99%, by weight of the polypeptide in the preparation is a polypeptide of the invention.

Routine methods, can be employed to purify and/or synthesise the proteins according to the invention. Such methods are well understood by persons skilled in the art, and include techniques such as those disclosed in Sambrook et al, Molecular Cloning: a Laboratory Manual, 2nd Edition, CSH Laboratory Press, 1989, the disclosure of which is included herein in its entirety by way of reference.

The term"variant"refers to a polypeptide which has a same essential character or basic biological functionality as HIPHUM 39. The essential character of HIPHUM 39 can be defined as follows: HIPHUM 39 is a voltage-gated ion channel.

Preferably the polypeptide is a subunit of a cation channel that can be activated by a change in cell membrane potential. Preferably a variant polypeptide is one which binds to the same cation channel subunits as HIPHUM 39. Preferably the cation channel is formed by the association of four subunits. HIPHUM 39 may form homotetrameric channels or may associate with one, two or more other cation channel subunits to form a heterotetrameric channel. A channel comprising HIPHUM 39 may be permeable to calcium and/or sodium. Preferably a channel comprising HIPHUM 39 is a calcium channel. A homotetramer formed by variant polypeptide preferably has a selectivity"signature"consisting of four negatively charged amino acids. More preferably the selectivity"signature"consists of four aspartate residues.

A polypeptide having the same essential character as HIPHUM 39 may be identified by monitoring for a function of the voltage-gated ion channel selected from changes in intracellular cation concentrations, for example [Ca2+] or [Na+], or changes in current flowing across the cell membrane following a change in the cell membrane potential in cells expressing the variant.

In another aspect of the invention, a variant is one which does not show the

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same activity as HIPHUM 39 but is one which inhibits a basic function of HIPHUM 39. For example, a variant polypeptide is one which inhibits cation channel activity of HIPHUM 39, for example by binding to HIPHUM 39 or another cation channel subunit to prevent activity mediated by homooligomerisation of HIPHUM 39 or by binding of HIPHUM 39 to another cation channel subunit.

Typically, polypeptides with more than about 65% identity preferably at least 80% or at least 90% and particularly preferably at least 95% at least 97% or at least 99% identity, with the amino acid sequences of SEQ ID NO: 2, are considered as variants of the proteins. Such variants may include allelic variants and the deletion, modification or addition of single amino acids or groups of amino acids within the protein sequence, as long as the peptide maintains a basic biological functionality of HIPHUM 39.

Amino acid substitutions may be made, for example from 1,2 or 3 to 10,20 or 30 substitutions. The modified polypeptide generally retains activity as a voltagegated ion channel. Conservative substitutions may be made, for example according to the following Table. Amino acids in the same block in the second column and preferably in the same line in the third column may be substituted for each other.

ALIPHATIC Non-polar GAP IL Polar-uncharged C S T M NQ Polar-charged DE KR AROMATIC HFWY

Shorter polypeptide sequences are within the scope of the invention. For example, a peptide of at least 20 amino acids or up to 50,60, 70,80, 100,150, 200,

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300,400 or 500 amino acids in length is considered to fall within the scope of the invention as long as it demonstrates a basic biological functionality of HIPHUM 39.

In particular, but not exclusively, this aspect of the invention encompasses the situation when the protein is a fragment of the complete protein sequence and may represent a HIPHUM 39 or other cation channel subunit-binding region. Such fragments can be used to construct chimeric receptors preferably with another voltage-gated ion channel. Such fragments of HIPHUM 39 or a variant thereof can also be used to raise anti-HIPHUM 39 antibodies. In this embodiment the fragment may comprise an epitope of the HIPHUM 39 polypeptide and may otherwise not demonstrate the cation channel subunits binding or other properties of HIPHUM 39.

Polypeptides of the invention may be chemically modified, e. g. posttranslationally modified. For example, they may be glycosylated or comprise modified amino acid residues. They may also be modified by the addition of histidine residues to assist their purification or by the addition of a signal sequence to promote insertion into the cell membrane. Such modified polypeptides fall within the scope of the term"polypeptide"of the invention.

The invention also includes nucleotide sequences that encode for HIPHUM 39 or a variant thereof as well as nucleotide sequences which are complementary thereto. The nucleotide sequence may be RNA or DNA including genomic DNA, synthetic DNA or cDNA. Preferably the nucleotide sequence is a DNA sequence and most preferably, a cDNA sequence. Nucleotide sequence information is provided in SEQ ID NO: 1. Such nucleotides can be isolated from human cells or synthesised according to methods well known in the art, as described by way of example in Sambrook et al, 1989.

Typically a polynucleotide of the invention comprises a contiguous sequence of nucleotides which is capable of hybridizing under selective conditions to the coding sequence or the complement of the coding sequence of SEQ ID NO: 1.

A polynucleotide of the invention can hydridize to the coding sequence or the complement of the coding sequence of SEQ ID NO: 1 at a level significantly above background. Background hybridization may occur, for example, because of other cDNAs present in a cDNA library. The signal level generated by the interaction between a polynucleotide of the invention and the coding sequence or complement of

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the coding sequence of SEQ ID NO: 1 is typically at least 10 fold, preferably at least 100 fold, as intense as interactions between other polynucleotides and the coding sequence of SEQ ID NO: 1. The intensity of interaction may be measured, for example, by radiolabelling the probe, e. g. with 32p. Selective hybridisation may typically be achieved using conditions of medium to high stringency. However, such hybridisation may be carried out under any suitable conditions known in the art (see Sambrook et al, 1989). For example, if high stringency is required suitable

conditions include from 0. 1 to 0. 2 x SSC at 60 C up to 65 C. If lower stringency is required suitable conditions include 2 x SSC at 60oC.

The coding sequence of SEQ ID NO: 1 may be modified by nucleotide substitutions, for example from 1,2 or 3 to 10,25, 50 or 100 substitutions. The polynucleotide of SEQ ID NO: 1 may alternatively or additionally be modified by one or more insertions and/or deletions and/or by an extension at either or both ends.

A polynucleotide may include one or more introns, for example may comprise genomic DNA. Additional sequences such as signal sequences which may assist in insertion of the polypeptide in a cell membrane may also be included. The modified polynucleotide generally encodes a polypeptide which has HIPHUM 39 activity.

Alternatively, a polynucleotide encodes a cation channel subunits-binding portion of a polypeptide or a polypeptide which inhibits HIPHUM 39 activity. Degenerate substitutions may be made and/or substitutions may be made which would result in a conservative amino acid substitution when the modified sequence is translated, for example as shown in the Table above.

A nucleotide sequence which is capable of selectively hybridizing to the complement of the DNA coding sequence of SEQ ID NO: 1 will generally have at least 60%, at least 70%, at least 80%, at least 90%, at least 95%, at least 98% or at least 99% sequence identity to the coding sequence of SEQ ID NO: 1 over a region of at least 20, preferably at least 30, for instance at least 40, at least 60, more preferably at least 100, at least 500, at least 1000, at least 1500 contiguous nucleotides or most preferably over the full length of SEQ ID NO: 1 or the fulll length of the coding sequence of SEQ ID NO: 1.

For example the UWGCG Package provides the BESTFIT program which can be used to calculate homology (for example used on its default settings)

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(Devereux et al (1984) Nucleic Acids Research 12, p387-395). The PILEUP and BLAST algorithms can be used to calculate homology or line up sequences (typically on their default settings), for example as described in Altschul (1993) J. Mol. Evol.

36: 290-300; Altschul et al (1990) J. Mol. Biol. 215: 403-10.

Software for performing BLAST analyses is publicly available through the National Centre for Biotechnology Information (http ://www. ncbi. nlm. nih. gov/).

This algorithm involves first identifying high scoring sequence pair (HSPs) by identifying short words of length W in the query sequence that either match or satisfy some positive-valued threshold score T when aligned with a word of the same length in a database sequence. T is referred to as the neighbourhood word score threshold (Altschul et al, 1990). These initial neighbourhood word hits act as seeds for initiating searches to find HSPs containing them. The word hits are extended in both directions along each sequence for as far as the cumulative alignment score can be increased. Extensions for the word hits in each direction are halted when: the cumulative alignment score falls off by the quantity X from its maximum achieved value; the cumulative score goes to zero or below, due to the accumulation of one or more negative-scoring residue alignments; or the end of either sequence is reached.

The BLAST algorithm parameters W, T and X determine the sensitivity and speed of the alignment. The BLAST program uses as defaults a word length (W) of 11, the BLOSUM62 scoring matrix (see Henikoffand Henikoff (1992) Proc. Natl. Acad.

Sci. USA 89: 10915-10919) alignments (B) of 50, expectation (E) of 10, M=5, N=4, and a comparison of both strands.

The BLAST algorithm performs a statistical analysis of the similarity between two sequences; see e. g. , Karlin and Altschul (1993) Proc. Natl. Acad. Sci.

USA 90: 5873-5787. One measure of similarity provided by the BLAST algorithm is the smallest sum probability (P (N) ), which provides an indication of the probability by which a match between two nucleotide or amino acid sequences would occur by chance. For example, a sequence is considered similar to another sequence if the smallest sum probability in comparison of the first sequence to the second sequence is less than about 1, preferably less than about 0.1, more preferably less than about 0. 01, and most preferably less than about 0. 001.

Any combination of the above mentioned degrees of sequence identity and

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minimum sizes may be used to define polynucleotides of the invention, with the more stringent combinations (i. e. higher sequence identity over longer lengths) being preferred. Thus, for example a polynucleotide which has at least 90% sequence identity over 25, preferably over 30 nucleotides forms one aspect of the invention, as does a polynucleotide which has at least 95% sequence identity over 40 nucleotides.

The nucleotides according to the invention have utility in production of the proteins according to the invention, which may take place in vitro, in vivo or ex vivo.

The nucleotides may be involved in recombinant protein synthesis or indeed as therapeutic agents in their own right, utilised in gene therapy techniques. Nucleotides complementary to those encoding HIPHUM 39, or antisense sequences, may also be used in gene therapy.

Polynucleotides of the invention may be used as a primer, e. g. a PCR primer, a primer for an alternative amplification reaction, a probe e. g. labelled with a revealing label by conventional means using radioactive or non-radioactive labels, or the polynucleotides may be cloned into vectors.

Such primers, probes and other fragments will preferably be at least 10, preferably at least 15 or at least 20, for example at least 25, at least 30 or at least 40 nucleotides in length. They will typically be up to 40,50, 60,70, 100 or 150 nucleotides in length. Probes and fragments can be longer than 150 nucleotides in length, for example up to 200,300, 400,500, 600,700, 1000,1500 or 2000 nucleotides in length, or even up to a few nucleotides, such as five or ten nucleotides, short of the coding sequence of SEQ ID NO: 1.

The present invention also includes expression vectors that comprise nucleotide sequences encoding the proteins or variants thereof of the invention. Such expression vectors are routinely constructed in the art of molecular biology and may for example involve the use of plasmid DNA and appropriate initiators, promoters, enhancers and other elements, such as for example polyadenylation signals which may be necessary, and which are positioned in the correct orientation, in order to allow for protein expression. Other suitable vectors would be apparent to persons skilled in the art. By way of further example in this regard we refer to Sambrook et al. 1989.

Polynucleotides according to the invention may also be inserted into the

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vectors described above in an antisense orientation in order to provide for the production of antisense RNA. Antisense RNA or other antisense polynucleotides may also be produced by synthetic means. Such antisense polynucleotides may be used as test compounds in the assays of the invention or may be useful in a method of treatment of the human or animal body by therapy.

Preferably, a polynucleotide of the invention or for use in the invention in a vector is operably linked to a control sequence which is capable of providing for the expression of the coding sequence by the host cell, i. e. the vector is an expression vector. The term"operably linked"refers to a juxtaposition wherein the components described are in a relationship permitting them to function in their intended manner.

A regulatory sequence, such as a promoter,"operably linked"to a coding sequence is positioned in such a way that expression of the coding sequence is achieved under conditions compatible with the regulatory sequence.

The vectors may be for example, plasmid, virus or phage vectors provided with a origin of replication, optionally a promoter for the expression of the said polynucleotide and optionally a regulator of the promoter. The vectors may contain one or more selectable marker genes, for example an ampicillin resistence gene in the case of a bacterial plasmid or a resistance gene for a fungal vector. Vectors may be used in vitro, for example for the production of DNA or RNA or used to transfect or transform a host cell, for example, a mammalian host cell. The vectors may also be adapted to be used in vivo, for example in a method of gene therapy.

Promoters and other expression regulation signals may be selected to be compatible with the host cell for which expression is designed. For example, yeast

promoters include S. cerevisiae GAL4 and ADH promoters, S. pombe nmtl and adh promoter. Mammalian promoters include the metallothionein promoter which can be induced in response to heavy metals such as cadmium.. Viral promoters such as the SV40 large T antigen promoter or adenovirus promoters may also be used. All these promoters are readily available in the art.

Mammalian promoters, such as -actin promoters, may be used. Tissuespecific promoters are especially preferred. Viral promoters may also be used, for example the Moloney murine leukaemia virus long terminal repeat (MMLV LTR), the rous sarcoma virus (RSV) LTR promoter, the SV40 promoter, the human

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cytomegalovirus (CMV) IE promoter, adenovirus, HSV promoters (such as the HSV IE promoters), or HPV promoters, particularly the HPV upstream regulatory region (URR). Viral promoters are readily available in the art.

The vector may further include sequences flanking the polynucleotide giving rise to polynucleotides which comprise sequences homologous to eukaryotic genomic sequences, preferably mammalian genomic sequences, or viral genomic sequences. This will allow the introduction of the polynucleotides of the invention into the genome of eukaryotic cells or viruses by homologous recombination. In particular, a plasmid vector comprising the expression cassette flanked by viral sequences can be used to prepare a viral vector suitable for delivering the polynucleotides of the invention to a mammalian cell. Other examples of suitable viral vectors include herpes simplex viral vectors and retroviruses, including lentiviruses, adenoviruses, adeno-associated viruses and HPV viruses. Gene transfer techniques using these viruses are known to those skilled in the art. Retrovirus vectors for example may be used to stably integrate the polynucleotide giving rise to the polynucleotide into the host genome. Replication-defective adenovirus vectors by contrast remain episomal and therefore allow transient expression.

The invention also includes cells that have been modified to express the HIPHUM 39 polypeptide or a variant thereof. Such cells include transient, or preferably stable higher eukaryotic cell lines, such as mammalian cells or insect cells, using for example a baculovirus expression system, lower eukaryotic cells, such as yeast or prokaryotic cells such as bacterial cells. Particular examples of cells which may be modified by insertion of vectors encoding for a polypeptide according to the invention include mammalian HEK293T, CHO, HeLa, BHK, 3T3 and COS cells.

Preferably the cell line selected will be one which is not only stable, but also allows for mature glycosylation and cell surface expression of a polypeptide. Expression may be achieved in transformed oocytes. A polypeptide of the invention may be expressed in cells of a transgenic non-human animal, preferably a mouse. A transgenic non-human animal expressing a polypeptide of the invention is included within the scope of the invention. A polypeptide of the invention may also be expressed in Xenopus laevis oocytes, in particular for use in an assay of the invention. A polypeptide of the invention may be purified from any suitable cell

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type from any species for reconstitution into lipid bilayer or vesicles.

According to another aspect, the present invention also relates to antibodies, specific for a polypeptide of the invention. Such antibodies are for example useful in purification, isolation or screening methods involving immunoprecipitation techniques or, indeed, as therapeutic agents in their own right.

Antibodies may be raised against specific epitopes of the polypeptides according to the invention. Such antibodies may be used to block cation channel subunits binding to the polypeptide. An antibody, or other compound,"specifically binds"to a protein when it binds with preferential or high affinity to the protein for which it is specific but does substantially bind not bind or binds with only low affinity to other proteins. A variety of protocols for competitive binding or immunoradiometric assays to determine the specific binding capability of an antibody are well known in the art (see for example Maddox et al, J. Exp. Med. 158, 1211-1226,1993). Such immunoassays typically involve the formation of complexes between the specific protein and its antibody and the measurement of complex formation.

Antibodies of the invention may be antibodies to human polypeptides or fragments thereof. For the purposes of this invention, the term"antibody", unless specified to the contrary, includes fragments which bind a polypeptide of the invention. Such fragments include Fv, F (ab') and F (ab') 2 fragments, as well as single chain antibodies. Furthermore, the antibodies and fragment thereof may be chimeric antibodies, CDR-grafted antibodies or humanised antibodies.

Antibodies may be used in a method for detecting polypeptides of the invention in a biological sample, which method comprises: I providing an antibody of the invention; II incubating a biological sample with said antibody under conditions which allow for the formation of an antibody-antigen complex; and III determining whether antibody-antigen complex comprising said antibody is formed.

A sample may be for example a tissue extract, blood, serum and saliva.

Antibodies of the invention may be bound to a solid support and/or packaged into kits in a suitable container along with suitable reagents, controls, instructions, etc.

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Antibodies may be linked to a revealing label and thus may be suitable for use in methods of in vivo HIPHUM 39 imaging.

Antibodies of the invention can be produced by any suitable method. Means for preparing and characterising antibodies are well known in the art, see for example Harlow and Lane (1988)"Antibodies : A Laboratory Manual", Cold Spring Harbor Laboratory Press, Cold Spring Harbor, NY. For example, an antibody may be produced by raising antibody in a host animal against the whole polypeptide or a fragment thereof, for example an antigenic epitope thereof, herein after the "immunogen".

A method for producing a polyclonal antibody comprises immunising a suitable host animal, for example an experimental animal, with the immunogen and isolating immunoglobulins from the animal's serum. The animal may therefore be inoculated with the immunogen, blood subsequently removed from the animal and the IgG fraction purified.

A method for producing a monoclonal antibody comprises immortalising cells which produce the desired antibody. Hybridoma cells may be produced by fusing spleen cells from an inoculated experimental animal with tumour cells (Kohler and Milstein (1975) Nature 256,495-497).

An immortalized cell producing the desired antibody may be selected by a conventional procedure. The hybridomas may be grown in culture or injected intraperitoneally for formation of ascites fluid or into the blood stream of an allogenic host or immunocompromised host. Human antibody may be prepared by in vitro immunisation of human lymphocytes, followed by transformation of the lymphocytes with Epstein-Barr virus.

For the production of both monoclonal and polyclonal antibodies, the experimental animal is suitably a goat, rabbit, rat or mouse. If desired, the immunogen may be administered as a conjugate in which the immunogen is coupled, for example via a side chain of one of the amino acid residues, to a suitable carrier.

The carrier molecule is typically a physiologically acceptable carrier. The antibody obtained may be isolated and, if desired, purified.

An important aspect of the present invention is the use of polypeptides according to the invention in screening methods. The screening methods may be

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used to identify substances that bind to voltage-gated ion channels and in particular which bind to HIPHUM 39. Screening methods may also be used to identify agonists or antagonists which may modulate voltage-gated ion channel activity, inhibitors or activators of HIPHUM 39 activity, and/or agents which up-regulate or down-regulate HIPHUM 39 expression.

Any suitable format may be used for the assay. In general terms such screening methods may involve contacting a polypeptide of the invention with a test substance and monitoring for binding of the test substance to the polypeptide or measuring receptor activity. A polypeptide of the invention may be incubated with a test substance. Modulation of voltage-gated ion channel activity may be determined.

In a preferred aspect, the assay is a cell-based assay. Preferably the assay may be carried out in a single well of a microtitre plate. Assay formats which allow high throughput screening are preferred.

Modulator activity can be determined by contacting cells expressing a polypeptide of the invention with a substance under investigation and by monitoring an effect mediated by the polypeptide. The cells expressing the polypeptide may be in vitro or in vivo. The polypeptide of the invention may be naturally or recombinantly expressed. Preferably, the assay is carried out in vitro using cells expressing recombinant polypeptide. Preferably, control experiments are carried out on cells which do not express the polypeptide of the invention to establish whether the observed responses are the result of activation of the polypeptide. Typically the cells will express one or more additional voltage-gated cation channel subunit.

The binding of a test substance to a polypeptide of the invention can be determined directly. For example, a radiolabelled test substance can be incubated with the polypeptide of the invention and binding of the test substance to the polypeptide can be monitored. Typically, the radiolabelled test substance can be incubated with cell membranes containing the polypeptide until equilibrium is reached. The membranes can then be separated from a non-bound test substance and dissolved in scintillation fluid to allow the radioactive content to be determined by scintillation counting. Non-specific binding of the test substance may also be determined by carrying out a competitive binding assay.

Substances that inhibit the interaction of a polypeptide of the invention with

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one or more additional voltage-gated cation channel subunit may also be identified through a yeast 2-hybrid assay or other protein interaction assay such as a coimmunoprecipitation or an ELISA based technique.

Assays may be carried out using cells expressing HIPHUM 39, and incubating such cells with the test substance optionally in the presence of one or more additional voltage-gated cation channel subunit. The results of the assay are compared to the results obtained using the same assay in the absence of the test substance. Cells expressing HIPHUM 39 constitutively may be provided for use in assays for HIPHUM 39 function. Additional test substances may be introduced in any assay to look for inhibitors or enhancers of binding of HIPHUM 39 to one or more additional voltage-gated cation channel subunit or inhibitors or enhancers of HIPHUM 39-mediated activity, preferably cation channel activity.

In preferred aspects, a host cell is provided expressing the polypeptide and containing a calcium sensitive photoprotein such as NFAT-luciferase or aequorin.

Such photoproteins increase or decrease light emission on the influx or efflux of calcium ions and can be detected using an imaging system. A reporter gene assay using such photoproteins can be used to assay for modulation of calcium channel activity. The assay enables determination of whether a test substance modulates the HIPHUM 39 regulated flow of calcium ions through calcium channels in target cells.

The ability of a test substance to modulate the HIPHUM 39 regulated flow of calcium or sodium ions may also be determined using fluorescence based assays using a Fluorometric Imaging Plate Reader (FLIPR) and membrane voltage sensitive dyes, such as DiBac, or calcium or sodium sensitive dyes Fura2, Fura red, Fluo3 and Fluo 4 (Molecular Probes). FRET/BRET based membrane voltage sensitive dyes with VIPR may also be used.

Assays may also be carried out by measuring the influx or efflux of radioactive calcium, barium or sodium ions or guanidine in cells expressing a polypeptide of the invention.

Electrophysiological recordings of cell membrane currents or membrane potentials from cells expressing a polypeptide of the invention and one or more additional voltage-gated cation channel subunit may also be used to assay for modulatory activity of a test substance.

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Reporter gene assays that link the intracellular calcium or sodium ion concentration to a signaling cascade that causes up-regulation or down-regulation of a readily detectable reporter protein such as luciferase may be used to monitor HIPHUM 39 activity.

Preferably, electrophysiological assays, reporter gene assays and/or assays comprising measuring changes in intracellular calcium or sodium ion concentration are performed on cells expressing a polypeptide of the invention and cation channel subunits.

Assays may also be carried out to identify substances which modify HIPHUM 39 expression, for example substances which up-or down-regulate expression. Such assays may be carried out for example by using antibodies for HIPHUM 39 to monitor levels of HIPHUM 39 expression. Other assays which can be used to monitor the effect of a test substance on HIPHUM 39 expression include using a reporter gene construct driven by the HIPHUM 39 regulatory sequences as the promoter sequence and monitoring for expression of the reporter polypeptide.

Further possible assays could utilise membrane fractions from overexpression of HIPHUM 39 polypeptide either in X laevis oocytes or cell lines such as HEK293, CHO, COS7, BHK, 3T3 and HeLa cells.

Additional control experiments may be carried out.

Suitable test substances which can be tested in the above assays include combinatorial libraries, defined chemical entities and compounds, peptide and peptide mimetics, oligonucleotides and natural product libraries, such as display (e. g. phage display libraries) and antibody products.

Typically, organic molecules will be screened, preferably small organic molecules which have a molecular weight of from 50 to 2500 daltons. Candidate products can be biomolecules including, saccharides, fatty acids, steroids, purines, pyrimidines, derivatives, structural analogs or combinations thereof. Candidate agents are obtained from a wide variety of sources including libraries of synthetic or natural compounds. Known pharmacological agents may be subjected to directed or random chemical modifications, such as acylation, alkylation, esterification, amidification, etc. to produce structural analogs.

Test substances may be used in an initial screen of, for example, 10

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substances per reaction, and the substances of these batches which show inhibition or activation tested individually. Test substances may be used at a concentration of from lnM to lOOOjjJvt, preferably from l, uM to lOOuM, more preferably from luM to lOp. M. Preferably, the activity of a test substance is compared to the activity shown by a known activator or inhibitor. A test substance which acts as an inhibitor may produce a 50% inhibition of activity of the channel. Alternatively a test substance which acts as an activator may produce 50% of the maximal activity produced using a known activator.

Another aspect of the present invention is the use of polynucleotides encoding the HIPHUM 39 polypeptides of the invention to identify mutations in HIPHUM 39 genes which may be implicated in human disorders. Identification of such mutations may be used to assist in diagnosis or susceptibility to such disorders and in assessing the physiology of such disorders. Polynucleotides may also be used in hybridisation studies to monitor for up-or down-regulation of HIPHUM 39 expression. Polynucleotides such as SEQ ID NO: 1 or fragments thereof may be used to identify allelic variants, genomic DNA and species variants.

The present invention provides a method for detecting variation in the expressed products encoded by HIPHUM 39 genes. This may comprise determining the level of HIPHUM 39 expressed in cells or determining specific alterations in the expressed product. Sequences of interest for diagnostic purposes include, but are not limited to, the conserved portions as identified by sequence similarity and conservation ofintron/exon structure. The diagnosis may be performed in conjunction with kindred studies to determine whether a mutation of interest cosegregates with disease phenotype in a family.

Diagnostic procedures may be performed on polynucleotides isolated from an individual or alternatively, may be performed in situ directly upon tissue sections (fixed and/or frozen) of patient tissue obtained from biopsies or resections, such that no nucleic acid purification is necessary. Appropriate procedures are described in, for example, Nuovo, G. J., 1992, "PCRIn Situ Hybridization : Protocols And Applications", Raven Press, NY). Such analysis techniques include, DNA or RNA blotting analyses, single stranded conformational polymorphism analyses, in situ

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hybridization assays, and polymerase chain reaction analyses. Such analyses may reveal both quantitative aspects of the expression pattern of a HIPHUM 39, and qualitative aspects of HIPHUM 39 expression and/or composition.

Alternative diagnostic methods for the detection of HIPHUM 39 nucleic acid molecules may involve their amplification, e. g. by PCR (the experimental embodiment set forth in U. S. Patent No. 4,683, 202), ligase chain reaction (Barany, 1991, Proc. Natl. Acad. Sci. USA 88: 189-193), self sustained sequence replication (Guatelli et al., 1990, Proc. Natl. Acad. Sci. USA 87: 1874-1878), transcriptional amplification system (Kwoh et al., 1989, Proc. Natl. Acad. Sci. 15 USA 86: 1173- 1177), Q-Beta Replicase (Lizardi et al., 1988, Bio/Technology 6: 1197) or any other nucleic acid amplification method, followed by the detection of the amplified molecules using techniques well known to those of skill in the art. These detection schemes are especially useful for the detection of nucleic acid molecules if such molecules are present in very low numbers.

Particularly suitable diagnostic methods are chip-based DNA technologies such as those described by Hacia et al., 1996, Nature Genetics 14: 441-447 and Shoemaker et al., 1996, Nature Genetics 14: 450-456. Briefly, these techniques involve quantitative methods for analyzing large numbers of nucleic acid sequence targets rapidly and accurately. By tagging with oligonucleotides or using fixed probe arrays, one can employ chip technology to segregate target molecules as high density arrays and screen these molecules on the basis of hybridization.

Following detection, the results seen in a given patient may be compared with a statistically significant reference group of normal patients and patients that have HIPHUM 39 related pathologies. In this way, it is possible to correlate the amount or kind of HIPHUM 39 encoded product detected with various clinical states or predisposition to clinical states.

Another aspect of the present invention is the use of the substances that have been identified by screening techniques referred to above in the treatment of disease states, which are responsive to regulation of voltage-gated ion channel activity. The treatment may be therapeutic or prophylactic. The condition of a patient suffering from such a disease state can thus be improved.

In particular, such substances may be used in the treatment of from irritable

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bowel syndrome (IBS), inflammatory pain, neuropathic pain, acute postoperative pain, headache, cluster headache, tension headache, migraine, glossopharyngeal neuralgia, tooth ache, ear ache, trigeminal neuralgia, cancer associated pain, psychogenic pain syndromes, intestinal pain, ileus, intestinal obstruction pain, abdominal pain, premenstrual syndrome, haemorrhoid pain, peptic ulcer pain, ulcerative colitis pain, medulloblastoma, hypertension, cardiac arrhythmias, congestive heart failure, Huchard's disease, acute bronchitis, acute respiratory failure in chronic obstructive pulmonary disease (COPD), adult respiratory distress syndrome, asthma, chronic obstructive pulmonary disease (COPD), emphysema, pulmonary hypertension, respiratory bronchiolitis-associated interstitial lung disease, respiratory distress syndrome, infant sudden death syndrome and allergic asthma.

Additional disease states that may be treated include aphasia, apraxia, coma, dyskinesias, dystonia, facial nerve disorders, hearing disorders, taste disorders, athletic heart syndrome, cardiac tamponade, dilated cardiomyopathy, congenital acyanotic defects, Raynaud's syndrome, allergic bronchopulmonary aspergillosis, berylliosis, respiratory acidosis, respiratory alkalosis, croup and tinnitus.

Substances identified according to the screening methods outlined above may be formulated with standard pharmaceutically acceptable carriers and/or excipients as is routine in the pharmaceutical art. For example, a suitable substance may be dissolved in physiological saline or water for injections. The exact nature of a formulation will depend upon several factors including the particular substance to be administered and the desired route of administration. Suitable types of formulation are fully described in Remington's Pharmaceutical Sciences, Mack Publishing Company, Eastern Pennsylvania, 176 lid. 1985, the disclosure of which is included herein of its entirety by way of reference.

The substances may be administered by enteral or parenteral routes such as via oral, buccal, anal, pulmonary, intravenous, intra-arterial, intramuscular, intraperitoneal, topical or other appropriate administration routes.

A therapeutically effective amount of a modulator is administered to a patient. The dose of a modulator may be determined according to various parameters, especially according to the substance used; the age, weight and condition of the patient to be treated; the route of administration; and the required regimen. A

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physician will be able to determine the required route of administration and dosage for any particular patient. A typical daily dose is from about 0.1 to 50 mg per kg of body weight, according to the activity of the specific modulator, the age, weight and conditions of the subject to be treated, the type and severity of the degeneration and the frequency and route of administration. Preferably, daily dosage levels are from 5 mg to 2 g.

Nucleic acid encoding HIPHUM 39 or a variant thereof which inhibits or enhances HIPHUM 39 activity or antisense nucleic acid may be administered to the mammal. Nucleic acid, such as RNA or DNA, and preferably, DNA, is provided in the form of a vector, such as the polynucleotides described above, which may be expressed in the cells of the mammal.

Nucleic acid administered to the mammal for gene therapy may encode functional HIPHUM 39 or a variant thereof with an impaired function such as a dominant negative mutant that disrupts the function of the whole cation channel.

Nucleic acid encoding the polypeptide may be administered by any available technique. For example, the nucleic acid may be introduced by needle injection, preferably intradermally, subcutaneously or intramuscularly. Alternatively, the nucleic acid may be delivered directly across the skin using a nucleic acid delivery device such as particle-mediated gene delivery. The nucleic acid may be administered topically to the skin, or to mucosal surfaces for example by intranasal, oral, intravaginal or intrarectal administration.

Uptake of nucleic acid constructs may be enhanced by several known transfection techniques, for example those including the use oftransfection agents.

Examples of these agents includes cationic agents, for example, calcium phosphate and DEAE-Dextran and lipofectants, for example, lipofectam and transfectam. The dosage of the nucleic acid to be administered can be altered. Typically the nucleic acid is administered in the range of Ipg to Img, preferably to Ipg to zug nucleic acid for particle mediated gene delivery and lOug to Img for other routes.

The following Examples illustrate the invention.

Example 1: Characterisation of the sequence A voltage-gated ion channel, designated as HIPHUM 39 has been identified.

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The nucleotide and amino acid sequences of the receptor have been determined.

These are set out below in SEQ ID NOs: I and 2. Suitable primers and probes were designed and used to analyse tissue expression. HIPHUM 39 was found to be primarily expressed in medulla oblongata.

The chromosomal localization was also mapped. Human HIPHUM 39 has been mapped to 11q13.

Example 2: Screening for substances which exhibit protein modulating activity Mammalian cells, such as HEK293, CHO, COS, BHK, 3T3 or HeLa cells, or Xenopus oocytes over-expressing a polypeptide of the invention together with one or more appropriate voltage-gated cation channel subunit are generated for use in the assay. 96 and 384 well plate, high throughput screens (HTS) are employed using fluorescence based calcium or sodium indicator molecules or voltage sensitive indicator molecules. Secondary screening involves electrophysiological assays utilising two electrodes, voltage clamp or patch clamp technology. Tertiary screens involve the study of modulators in rat and mouse models of disease relevant to the target.

A brief screening assay protocol based on a calcium or sodium binding fluorescent dye is as follows. Mammalian cells stably over-expressing the polypeptide of the invention together with appropriate voltage-gated cation channel subunit proteins for making a calcium or sodium channel are cultured in 96 or 384 well plates. One T225cm 3 flask is sufficient for setting up ten 96 well plates with a volume of 100ml cell culture medium in each well. These plates are set up the night before each assay run. The culture media is removed and 100ml of assay buffer (125mM Choline chloride, 50mM HEPES, 5. 5mM Glucose, 0. 8mM MgSOSmM KCI, pH 7.4) is added. The cells are then loaded with the calcium or sodium indicator dye of choice for 30 minutes. The test compounds are added to the wells and preincubated for a period of 10 minutes. The channel is activated by Changes in the membrane potential. Modulation of the activity of a polypeptide of the invention results in either an increase or a decrease in the activity of the channel and the change in intracellular calcium or sodium can be measured directly in a Fluorescence Imaging Plate Reader, (FLIPR; Molecular Devices). Channel activity is monitored in the presence and absence of a test substance and modulation of channel activity by

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the test substance is compared in the presence and absence of the test substance to determine whether the test substance is an agonist or antagonist of the voltage-gated ion channel.

A typical electropysiology protocol using electrophysiology in oocytes expressing HIPHUM 39 is as follows. HIPHUM 39 is expressed in Xenopus laevis oocytes either : i) by injection of plasmid DNA that allows the expression of the ion channel cDNA or gene by virtue of an upstream promoter (for example the CMV promoter), or preferably ii) by injection of in vitro transcribed, m'G (5') pp (5') GTP-capped, complementary RNA synthesised from the ion channel cDNA by virtue of an upstream Sp6, T3 or T7 promoter and Sp6, T3 or T7 RNA polymerase.

Typically, 20-50ng of plasmid DNA or cRNA is injected per oocyte and whole-cell currents are recorded using two-microelectrode voltage-clamp (Geneclamp amplifier, Axon instruments Inc.) 1 to 7 days post-injection. Typical microelectrodes have a resistance of 0.5 to 2MQ and are filed with

3M KC1. Oocytes are voltage-clamped at a set holding membrane potential (for example, between-lOOmV to-80m V) in ND96 solution (superfused at 2ml per min.) and depolarising voltage pulses are applied to activate the channels. Calcium or sodium currents elicited by these voltage pulses are recorded. Voltage-protocols can be generated using pCLAMP8 software (Axon Instruments) and a P/N leak subtraction protocol is used throughout (to remove artefacts generated by nonspecific'leak'current across the membrane). In these experiments the effects of a test compound on current mediated by the channel is studied by inclusion of the compound in the extracellular buffer which is superfused across the oocyte.

A typical electrophysiology assay using mammalian cells expressing a polypeptide of the invention is as follows.

Cells are grown on a glass coverslip, placed into a recording chamber (O. Sml volume) and superfused with an extracellular recording solution at 2 ml min-'. Drugs are applied either via addition to the bath perfusate, or alternatively using a rapid perfusion system which consists of a series of reservoirs connected to a small microfil tube that is placed in close proximity to the voltage-clamped cell. Whole-

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cell currents are recorded using an Axopatch 200B amplifier (Axon Instruments) or other voltage-clamp amplifier (e. g. HEKA), using standard electrophysiological methods (Hamill et at., (1981) Pfluges Arch. , 391: 85-100). Patch pipettes are fabricated from 1.5mm outside diameter borosilicate capillary glass (Clark Electromedical) using a micropipette puller (Sutter model P97), and fire polished (Narishige Microforge) to give final tip resistances of2-4MQ. A silver/silver chloride pellet is used as the bath reference electrode and the potential difference between this and the recording electrode will be adjusted for zero current flow before seal formation. Cells are visualised using a Diaphot200 inverted microscope (Nikon) with modulation contrast optics at a final magnification ofx400. High resistance seals (1-10GQ) between pipette and neuronal cell membranes are achieved by gentle suction, and the'whole cell'configuration attained by applying further suction.

Cells are patch-clamped in an extracellular buffer containing 140mM NaCl, 4.7mM KCI, 1.2mM MgC12, ImM CaC12, HmM glucose, 5mM HEPES (titrated with NaOH to pH 7.4 at 25oC) using microelectrode pipettes containing 120mM CsF, 15mM NaCl, IOmM Cs-EGTA (ethylene glycol-bis (ss-aminoethyl ester) N, N, N', N-tetra acetic acid, Cs salt), lOmM HEPES (titrated with CsOH to pH7.25 at 25 C). Patch electrodes should have resistances of 2 to 6MQ when filled with the pipette-filling solution. Cells are voltage-clamped at a set holding membrane potential (for example, between -IOOmV to -80mV) in and depolarising voltage pulses are applied to activate the channels.

Voltage command protocols are generated, and current records stored, via a digidata 1200 analog/digital interface (Axon Instruments) controlled by microcomputer (Hewlett Packard Kayak XA) using pCLAMP8 Clampex software (Axon Instruments). Signals are prefiltered at 5kHz bandwidth and sampled at 20kHz. Capacitance transients and series resistance errors are compensated for (80- 85%) using the amplifier circuitry, and linear leakage currents subtracted using an on-line'P-4'procedure provided by the commercial software package.

Data are analysed using pCLAMP8/Clampfit (Axon Instruments), ORIGIN (MicroCal) and DAISI data handling and graphical presentation software packages.

Results can be presented as either arithmetic mean s. e mean or geometric mean

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with 95% confidence limits. Statistical comparisons are made using paired or unpaired Student's t-test and considered of significance when P < O. 05.

Channel activity is monitored in the presence and absence of a test substance and modulation of channel activity by the test substance is compared in the presence and absence of the test substance to determine whether the test substance is an activator or inhibitor of the voltage-gated ion channel.

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SEQUENCE LISTING < 110 > GLAXO GROUP LIMITED < 120 > PROTEIN < 130 > QG1038 (P80909) < 160 > 2 < 170 > Patent In version 3.0 < 210 > 1 < 211 > 2557 < 212 > DNA < 213 > Homo sapiens < 220 > < 221 > CDS < 222 > (92).. (2380) < 400 > 1 tgagcttggc tcaggaaaga agggaaatcc aggcggggcc tgttgggccc aggtcttgag 60

ctcttttggc tccagagttc ccagcacagt c atg gat caa aac tea gtg cct 112 Met Asp Gin Asn Ser Val Pro 1 5

gaa aag get cag aat gag gca gac ace aat aac gca gat, agg ttc ttt 160 Glu Lys Ala Gin Asn Glu Ala Asp Thr Asn Asn Ala Asp Arg Phe Phe 10 15 20 cgc tct cac tea tea ccc cca cac cac agg cca ggc cac age aga get 208 Arg Ser His Ser Ser Pro Pro His His Arg Pro Gly His. Ser Arg Ala 25 30 35..

<Desc/Clms Page number 27>

etc cac cat tac gag ttg cac cat cac ggc gtg ccc cac caa cgt ggt 256 Leu His His Tyr Glu Leu His His His Gly Val Pro His Gln Arg Gly 40 45 50 55 gaa tct cac cac cct ccg gag ttc caa gac ttc cac gac caa gcc ttg 304 Glu Ser His His Pro Pro Glu Phe Gin Asp Phe His Asp Gln Ala Leu 60 65 70 tec tec cat gtc cac caa tct cac cac cac age gag gca egg aat cac 352 Ser Ser His Val His Gln Ser His His His Ser Glu Ala Arg Asn His 75 80 85

ggc aga gcc cat ggc ccc aca ggc ttt ggt ctg get ccc tct caa ggc 400 Gly Arg Ala His Gly Pro Thr Gly Phe Gly Leu Ala Pro Ser Gln Gly 90 95 100

gcc gtc ccc tec cac cgt tec tac ggt gag gac tac cat gat gag etc 448 Ala Val Pro Ser His Arg Ser Tyr Gly Glu Asp Tyr His Asp Glu Leu 105 110 115

caa cgt gat ggc agg agg cat cat gat ggg tec caa tac ggt ggg ttc 496 Gin Arg Asp Gly Arg Arg His His Asp Gly Ser Gln Tyr Gly Gly Phe 120 125 130 135

cat cag cag agt gac tec cat tac cat agg ggg tct cac cat ggc aga 544 His Gln Gln Ser Asp Ser His Tyr His Arg Gly Ser His His Gly Arg 140 145 150

ccc caa tat etc ggt gag aat tta tec cac tat tec tct ggc gtg ccc 592 Pro Gln Tyr Leu Gly Glu Asn Leu Ser His Tyr Ser Ser Gly Val Pro 155 160 165

cac cac ggt gag get tec cac cat ggt ggg tec tac etc ccc cat gga 640 His His Gly Glu Ala Ser His His Gly Gly Ser Tyr Leu Pro His Gly 170 175 180

ccc aat ccc tac agt gag tec ttc cac cac age gag get tec cac ctt 688 Pro Asn Pro Tyr Ser Glu Ser Phe His His Ser Glu Ala Ser His Leu 185 190 195

age ggg etc caa cac gat gag tec cag cat cac caa gtc ccc cac cgt 736 Ser Gly Leu Gln His Asp Glu Ser Gin His His Gln Val Pro His Arg 200 205 210 215

ggc tgg ccc cac cat cac caa gtc cac cac cat ggc agg tec cgt cat 784 Gly Trp Pro His His His Gln Val His His His Gly Arg Ser Arg His 220 225 230

cat gaa gcc cac cag cat gga aag tct cct cat cac gga gag ace att 832 His Glu Ala His Gln His Gly Lys Ser Pro His His Gly Glu Thr lIe

?' < ?/in ?

<Desc/Clms Page number 28>

tec cct cat tec tct gtg ggg tec tac cag cgt ggg ata tct gac tat 880 Ser Pro His Ser Ser Val Gly Ser Tyr Gln Arg Gly lIe Ser Asp Tyr 250 255 260

cac age gag tac cac caa ggt gat cac cac ccc agt gag tac cac cat 928 His Ser Glu Tyr His Gln Gly Asp His His Pro Ser Glu Tyr His His 265 270 275

ggc gac cat ccc cac cac aca cag cac cac tac cac cag ace cac egg 976 Gly Asp His Pro His His Thr Gln His His Tyr His Gin Thr His Arg 280 285 290 295

cac cga gac tac cat cag cac caa gac cac cac ggc gcg tat cat tec 1024 His Arg Asp Tyr His Gln His Gln Asp His His Gly Ala Tyr His Ser 300 305 310

agt tac etc cat ggc gac tac gtc cag age act tec caa etc tct ate 1072 Ser Tyr Leu His Gly Asp Tyr Val Gin Ser Thr Ser Gln Leu Ser lie 315 320 325

cca cac aca tec egg age ctg att cac gat gee ccc ggc cct get get 1120 Pro His Thr Ser Arg Ser Leu He His Asp Ala Pro Gly Pro Ala Ala 330 335 340

tct cgt aca gga gtc ttc ccc tat cac gta gca cac cca egg ggc teg 1168 Ser Arg Thr Gly Val Phe Pro Tyr His Val Ala His Pro Arg Gly Ser 345 350 355

get cac age atg act egg tec tec age aca ate cgc tea cgt gtc ace 1216 Ala His Ser Met Thr Arg Ser Ser Ser Thr lie Arg Ser Arg Val Thr 360 365 370 375

cag atg tec aaa aaa gtc cat ace cag gat ate tec ace aaa cat tea 1264 Gln Met Ser Lys Lys Val His Thr Gln Asp lie Ser Thr Lys His Ser 380 385 390

gaa gac tgg ggc aaa gaa gaa ggg caa ttt cag aaa cgc aaa ace ggc 1312 Glu Asp Trp Gly Lys Glu Glu Gly Gin Phe Gin Lys Arg Lys Thr Gly 395 400 405

agg etc cag egg ace cgc aag aag gga cac tct ace aat etc ttc cag 1360 Arg Leu Gin Arg Thr Arg Lys Lys Gly His Ser Thr Asn Leu Phe Gln 410 415 420

tgg ctg tgg gaa aag cta ace ttc etc att cag ggc ttc egg gaa atg 1408 Trp Leu Trp Glu Lys Leu Thr Phe Leu He Gin Gly Phe Arg Glu Met 425 430 435

ate egg aac ctg ace caa tec ttg gee ttt gaa act ttc ate ttc ttc 1456 He Arg Asn Leu Thr Gln Ser Leu Ala Phe Glu Thr Phe He Phe Phe

<Desc/Clms Page number 29>

440 445 450 455

gtt gtc tgc etc aac ace gtc atg ctg gtg gcc cag ace ttc get gaa 1504 Val Val Cys Leu Asn Thr Val Met Leu Val Ala Gln Thr Phe Ala Glu 460 465 470

gtc gag ate egg ggc gag tgg tac ttc atg gcc ttg gac tec ata ttc 1552 Val Glu lie Arg Gly Glu Trp Tyr Phe Met Ala Leu Asp Ser lIe Phe 475 480 485

ttc tgc ate tac gtg gtg gaa gcc ctg etc aag ate ate gee ctg ggc 1600 Phe Cys lie Tyr Val Val Glu Ala Leu Leu Lys lie lie Ala Leu Gly 490 495 500

etc teg tac ttc ttt gac ttc tgg aac aat ttg gac ttc ttc att atg 1648 Leu Ser Tyr Phe Phe Asp Phe Trp Asn Asn Leu Asp Phe Phe Ile Met 505 510 515 gcc atg gcc gtg ctg gac ttc ttg ctg atg cag ace cac tec ttc gee 1696 Ala Met Ala Val Leu Asp Phe Leu Leu Met Gln Thr His Ser Phe Ala 520 525 530 535

ate tac cac caa age etc ttc egg ate etc aag gtc ttc aag age ctg 1744 lie Tyr His Gln Ser Leu Phe Arg lie Leu Lys Val Phe Lys Ser Leu 540 545 550

egg gcc ctg agg gca ate egg gtc ctg egg agg etc age ttc ctg ace 1792 Arg Ala Leu Arg Ala lie Arg Val Leu Arg Arg Leu Ser Phe Leu Thr 555 560 565

age gtc cag gaa gtg aca ggg ace ctg ggc cag tec ttg ccg tec ate 1840 Ser Val Gln Glu Val Thr Gly Thr Leu Gly Gln Ser Leu Pro Ser lie 570 575 580

gca gee ate etc ate etc atg ttt ace tgc etc ttc etc ttc tec gcg 1888 Ala Ala He Leu l1e Leu Met Phe Thr Cys Leu Phe Leu Phe Ser Ala 585 590 595

gtc etc egg gca ctg ttc cgc aaa tct gac ccc aag cgc ttc cag aac 1936 Val Leu Arg Ala Leu Phe Arg Lys Ser Asp Pro Lys Arg Phe Gln Asn 600 605 610 615

ate ttc ace ace ate ttc ace etc ttc ace ttg etc acg ctg gat gac 1984 lie Phe Thr Thr lie Phe Thr Leu Phe Thr Leu Leu Thr Leu Asp Asp 620 625 630

tgg tec etc ate tac atg gac age cgt gcc cag ggc gcc tgg tac ate 2032 Trp Ser Leu lie Tyr Met Asp Ser Arg Ala Gin Gly Ala Trp Tyr He 635 640 645

att ccc ate etc ata att tac ate ate ate cag tac ttc ate ttc etc 2080

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He Pro He Leu He He Tyr He He He Gln Tyr Phe He Phe Leu 650 655 660 aac ctg gtg att act gtc ctg gtg gat age ttc cag acg gcg ctg ttc 2128 Asn Leu Val lie Thr Val Leu Val Asp Ser Phe Gln Thr Ala Leu Phe 665 670 675

aaa ggc ctt gag aaa gcg aag cag gag agg gcc gcc egg ate caa gag 2176 Lys Gly Leu Glu Lys Ala Lys Gln Glu Arg Ala Ala Arg He Gln Glu 680 685 690 695

aag ctg ctg gaa gac tea ctg acg gag etc aga get gca gag ccc aaa 2224 Lys Leu Leu Glu Asp Ser Leu Thr Glu Leu Arg Ala Ala Glu Pro Lys 700 705 710

gag gtg gcg agt gaa ggc ace atg ctg aag egg etc ate gag aaa aag 2272 Glu Val Ala Ser Glu Gly Thr Met Leu Lys Arg Leu He Glu Lys Lys 715 720 725

ttt ggg ace atg act gag aag cag cag aag ttc cgc tec cag gca gcc 2320 Phe Gly Thr Met Thr Glu Lys Gln Gln Lys Phe Arg Ser Gln Ala Ala 730 735 740

gtc ate gat gag att gtg gac ace aca ttt gag get gga gaa gag gac 2368 Val He Asp Glu He Val Asp Thr Thr Phe Glu Ala Gly Glu Glu Asp 745 750 755 ttc agg aat tga ccccaggagg acaccagata cagacttcag cccctggcag 2420 Phe Arg Asn 760 tctgcccacc tgggtgcact gggacgggtc cccagatctg ctggaatgat tgtccgggcc 2480 ctgcagagca ggggccccaa cagagttttt aaaccccaaa aaaaaaaaaa aaaaaaaaaa 2540 aaaaaaaaaa aaaaaaa 2557 < 210 > 2 < 211 > 762 < 212 > PRT < 213 > Homo sapiens < 400 > 2 Met Asp Gln Asn Ser Val Pro Glu Lys Ala Gln Asn Glu Ala Asp Thr 1 5 10 15

<Desc/Clms Page number 31>

Asn Asn Ala Asp Arg Phe Phe Arg Ser His Ser Ser Pro Pro His His 20 25 30 Arg Pro Gly His Ser Arg Ala Leu His His Tyr Glu Leu His His His 35 40 45 Gly Val Pro His Gln Arg Gly Glu Ser His His Pro Pro Glu Phe Gln 50 55 60 Asp Phe His Asp Gln Ala Leu Ser Ser His Val His Gin Ser His His 65 70 75 80 His Ser Glu Ala Arg Asn His Gly Arg Ala His Gly Pro Thr Gly Phe 85 90 95 Gly Leu Ala Pro Ser Gln Gly Ala Val Pro Ser His Arg Ser Tyr Gly 100 105 110 Glu Asp Tyr His Asp Glu Leu Gin Arg Asp Gly Arg Arg His His Asp 115 120 125 Gly Ser Gin Tyr Gly Gly Phe His Gln Gln Ser Asp Ser His Tyr His 130 135 140 Arg Gly Ser His His Gly Arg Pro Gln Tyr Leu Gly Glu Asn Leu Ser 145 150 155 160 His Tyr Ser Ser Gly Val Pro His His Gly Glu Ala Ser His His Gly 165 170 175 Gly Ser Tyr Leu Pro His Gly Pro Asn Pro Tyr Ser Glu Ser Phe His 180 185 190 His Ser Glu Ala Ser His Leu Ser Gly Leu Gln His Asp Glu Ser Gln 195 200 205 His His Gln Val Pro His Arg Gly Trp Pro His His His Gln Val His 210 215 220 His His Gly Arg Ser Arg His His Glu Ala His Gln His Gly Lys Ser 225 230 235 240 Pro His His Gly Glu Thr lie Ser Pro His Ser Ser Val Gly Ser Tyr 245 250 255 Gin Arg Gly Ile Ser Asp Tyr His Ser Glu Tyr His Gln Gly Asp His 260 265 270

<Desc/Clms Page number 32>

His Pro Ser Glu Tyr His His Gly Asp His Pro His His Thr Gln His 275 280 285 His Tyr His Gln Thr His Arg His Arg Asp Tyr His Gln His Gln Asp 290 295 300 His His Gly Ala Tyr His Ser Ser Tyr Leu His Gly Asp Tyr Val Gln 305 310 315 320 Ser Thr Ser Gln Leu Ser He Pro His Thr Ser Arg Ser Leu lIe His 325 330 335 Asp Ala Pro Gly Pro Ala Ala Ser Arg Thr Gly Val Phe Pro Tyr His 340 345 350 Val Ala His Pro Arg Gly Ser Ala His Ser Met Thr Arg Ser Ser Ser 355 360 365 Thr He Arg Ser Arg Val Thr Gln Met Ser Lys Lys Val His Thr Gln 370 375 380 Asp He Ser Thr Lys His Ser Glu Asp Trp Gly Lys Glu Glu Gly Gln 385 390 395 400 Phe Gln Lys Arg Lys Thr Gly Arg Leu Gln Arg Thr Arg Lys Lys Gly 405 410 415 His Ser Thr Asn Leu Phe Gln Trp Leu Trp Glu Lys Leu Thr Phe Leu 420 425 430 He Gln Gly Phe Arg Glu Met He Arg Asn Leu Thr Gln Ser Leu Ala 435 440 445 Phe Glu Thr Phe He Phe Phe Val Val Cys Leu Asn Thr Val Met Leu 450 455 460 Val Ala Gln Thr Phe Ala Glu Val Glu He Arg Gly Glu Trp Tyr Phe 465 470 475 480 Met Ala Leu Asp Ser He Phe Phe Cys He Tyr Val Val Glu Ala Leu 485 490 495 Leu Lys He He Ala Leu Gly Leu Ser Tyr Phe Phe Asp Phe Trp Asn 500 505 510 Asn Leu Asp Phe Phe He Met Ala Met Ala Val Leu Asp Phe Leu Leu 515 520 525

<Desc/Clms Page number 33>

Met Gln Thr His Ser Phe Ala lIe Tyr His Gln Ser Leu Phe Arg Ile 530 535 540 Leu Lys Val Phe Lys Ser Leu Arg Ala Leu Arg Ala lIe Arg Val Leu 545 550 555 560 Arg Arg Leu Ser Phe Leu Thr Ser Val Gln Glu Val Thr Gly Thr Leu 565 570 575 Gly Gln Ser Leu Pro Ser lIe Ala Ala lie Leu Ile Leu Met Phe Thr 580 585 590 Cys Leu Phe Leu Phe Ser Ala Val Leu Arg Ala Leu Phe Arg Lys Ser 595 600 605 Asp Pro Lys Arg Phe Gln Asn lie Phe Thr Thr lIe Phe Thr Leu Phe 610 615 620 Thr Leu Leu Thr Leu Asp Asp Trp Ser Leu lIe Tyr Met Asp Ser Arg 625 630 635 640

Ala Gln Gly Ala Trp Tyr 11e Ile Pro lie Leu Ile Ile Tyr Ile Ile 645 650 655 lIe Gln Tyr Phe lIe Phe Leu Asn Leu Val lIe Thr Val Leu Val Asp 660 665 670 Ser Phe Gln Thr Ala Leu Phe Lys Gly Leu Glu Lys Ala Lys Gln Glu 675 680 685 Arg Ala Ala Arg Ile Gln Glu Lys Leu Leu Glu Asp Ser Leu Thr Glu 690 695 700 Leu Arg Ala Ala Glu Pro Lys Glu Val Ala Ser Glu Gly Thr Met Leu 705 710 715 720 Lys Arg Leu lIe Glu Lys Lys Phe Gly Thr Met Thr Glu Lys Gln Gln 725 730 735 Lys Phe Arg Ser Gln Ala Ala Val He Asp Glu He Val Asp Thr Thr 740 745 750 Phe Glu Ala Gly Glu Glu Asp Phe Arg Asn 755 760

Claims (19)

1. An isolated voltage-gated ion channel polypeptide comprising (i) the amino acid sequence of SEQ ID NO: 2 or (ii) a variant thereof which is capable of forming part of a cation channel that can be activated by a change in membrane potential or (iii) a fragment of (i) or (ii) which is capable of forming part of a cation channel that can be activated by a change in membrane potential.

2. A polypeptide according to claim 1 wherein the variant (ii) has at least 80% identity to the amino acid sequence of SEQ ID NO: 2.

3. A polynucleotide encoding a polypeptide according to claim 1 or 2.

4. A polynucleotide according to claim 3 which is a cDNA sequence.

5. A polynucleotide encoding a voltage-gated ion channel polypeptide which is capable of forming part of a cation channel that can be activated by a change in membrane potential which polynucleotide comprises: (a) the nucleic acid sequence of SEQ ID NO: 1 and/or a sequence complementary thereto; (b) a sequence which hybridises under stringent conditions to a sequence as defined in (a); (c) a sequence that is degenerate as a result of the genetic code to a sequence as defined in (a) or (b); or (d) a sequence having at least 60% identity to a sequence as defined in (a), (b) or (c).

6. An expression vector comprising a polynucleotide according to any one of claims 3 to 5.

7. A host cell comprising an expression vector according to claim 6.

8. An antibody specific for a polypeptide according to claim I or 2.

9. A method for the identification of a substance that modulates voltagegated ion channel activity and/or expression, which method comprises: (i) contacting a test substance and a polypeptide according to claim 1 or 2, a polynucleotide according to any one of claims 3 to 5, an expression vector according to claim 6 or a host cell according to

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claim 7, and (ii) determining the effect of the test substance on the activity and/or expression of the said polypeptide or the polypeptide encoded by said polynucleotide, thereby to determine whether the test substance modulates voltage-gated ion channel activity and/or expression.

10. A method according to claim 9 wherein the polypeptide is expressed in a cell.

11. A method according to claim 10 wherein the cell expresses a cation channel subunits.

12. A method according to claim 11 wherein step (ii) comprises monitoring any cation channel activity.

13. A method according to claim 11 wherein step (ii) comprises monitoring any interaction between the said polypeptide with the said cation channel subunits.

14. A substance which modulates voltage-gated ion channel activity and which is identifiable by a method according to any one of claims 9 to 13.

15. A method of treating a subject having a disorder that is responsive to voltage-gated ion channel modulation, which method comprises administering to said subject an effective amount of a substance according to claim 14.

16. A method according to claim 15 wherein the disorder is selected from from irritable bowel syndrome (IBS), inflammatory pain, neuropathic pain, acute postoperative pain, headache, cluster headache, tension headache, migraine, glossopharyngeal neuralgia, tooth ache, ear ache, trigeminal neuralgia, cancer associated pain, psychogenic pain syndromes, intestinal pain, ileus, intestinal obstruction pain, abdominal pain, premenstrual syndrome, haemorrhoid pain, peptic ulcer pain, ulcerative colitis pain, medulloblastoma, hypertension, cardiac arrhythmias, congestive heart failure, Huchard's disease, acute bronchitis, acute respiratory failure in chronic obstructive pulmonary disease (COPD), adult respiratory distress syndrome, asthma, chronic obstructive pulmonary disease (COPD), emphysema, pulmonary hypertension, respiratory bronchiolitis-associated interstitial lung disease, respiratory distress syndrome, infant sudden death syndrome and allergic asthma.

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17. Use of a substance as defined in claim 14 in the manufacture of a medicament for treatment or prophylaxis of a disorder that is responsive to stimulation or modulation of voltage-gated ion channel activity.

18. A use according to claim 17 wherein the disorder is selected from from irritable bowel syndrome (IBS), inflammatory pain, neuropathic pain, acute postoperative pain, headache, cluster headache, tension headache, migraine, glossopharyngeal neuralgia, tooth ache, ear ache, trigeminal neuralgia, cancer associated pain, psychogenic pain syndromes, intestinal pain, ileus, intestinal obstruction pain, abdominal pain, premenstrual syndrome, haemorrhoid pain, peptic ulcer pain, ulcertative colitis pain, medulloblastoma, hypertension, cardiac arrhythmias, congestive heart failure, Huchard's disease, acute bronchitis, acute respiratory failure in chronic obstructive pulmonary disease (COPD), adult respiratory distress syndrome, asthma, chronic obstructive pulmonary disease (COPD), emphysema, pulmonary hypertension, respiratory bronchiolitis-associated interstitial lung disease, respiratory distress syndrome, infant sudden death syndrome and allergic asthma.

19. A method of producing a polypeptide according to claim 1 or 2, which method comprises maintaining a host cell as defined in claim 7 under conditions suitable for obtaining expression of the polypeptide and isolating the said polypeptide.