GB2376235A

GB2376235A - Voltage-gated sodium channel beta subunit polypeptide

Info

Publication number: GB2376235A
Application number: GB0108588A
Authority: GB
Inventors: Andrew Jonathan Powell; Daniel Crowther
Original assignee: Glaxo Group Ltd
Current assignee: Glaxo Group Ltd
Priority date: 2001-04-05
Filing date: 2001-04-05
Publication date: 2002-12-11
Also published as: GB0108588D0

Abstract

An isolated voltage-gated sodium channel beta-subunit polypeptide comprising <SL> <LI>(i) the amino acid sequence of SEQ ID NO:2 or <LI>(ii) a variant thereof which is capable of modulating sodium channel alpha-subunit activity or <LI>(iii) a fragment of (i) or (ii) which is capable of modulating sodium channel alpha-subunit activity. </SL> The beta subunit may be a screening target for the identification of modulators of voltage-gated sodium channel activity. These modulators may be used in the treatment of epilepsy, seizure disorders, pain, neuropathies, migraine, headache, anxiety, depression, bipolar disorder and schizophrenia.

Description

NEW PROTEIN Field of the Invention The present invention relates to voltage-gated sodium channel beta-subunit polypeptides.

Background of the Invention Ion channels are involved in a wide variety of neurological and other disorders in man. Voltage-gated sodium channels are formed by a major poreforming a-subunit that may interact with one or more auxiliary ss-subunits. The asubunit alone is sufficient to form a functional voltage-gated channel, however the sodium channel ss-subunits have two functions. Firstly they modulate the properties of channel gating and secondly they mediate extracellular interactions.

Coexpression of the a-subunit with either or both ss-subunits leads to faster activation and inactivation kinetics and a negative shift in the voltage dependence of steady state inactivation. Expression of the ss-subunits is also associated with an increase in peak current suggesting that these subunits play a role in trafficking the pore-forming a-subunits to the membrane. The ss2-subunit also causes an increase in cell membrane surface area possibly by stimulating fusion of intracellular transport vesicles containing sodium channels with the plasma membrane. In neurons, association of a-subunits with ss-subunits may be a prerequisite for the assembly of functional sodium channels on the cell surface.

Cell adhesion molecules of the immunoglobulin (Ig) superfamily interact with other extracellular matrix proteins and the Ig-like domains are believed to be important structural determinants of the interactions. All Ig-superfamily members have either adhesion functions or binding functions that trigger a subsequent event at the cell surface. A key functional feature of the immunoglobulin superfamily of adhesion factors is that homophilic or heterophilic binding occurs between Ig-like domain containing proteins. This often occurs between protein molecules on opposed membrane surfaces and these interactions are involved in cell surface recognition, cell migration, axonal extension in neurons and adhesion with other cells.

The voltage-gated sodium channel auxiliary ss-subunit proteins are members

of the Ig-like superfamily. They contain single extracellular Ig-like domains that are believed to be involved in the localisation of voltage-gated sodium channels to specific cell membrane regions through interactions with extracellular matrix proteins.

Summary of the Invention A novel voltage-gated sodium channel beta-subunit, referred to herein as HIPHUM30, is now provided. HIPHUM30 is shown to be primarily expressed in adrenal gland, brain (including cerebral cortex, cerebellum, hypothalamus), testes, small intestine, rectum, ovary and kidney. The novel voltage-gated sodium channel beta-subunit is a screening target for the identification and development of novel pharmaceutical agents, including modulators of voltage-gated sodium channel activity. These agents may be used in the treatment and/or prophylaxis of disorders such as epilepsy, juvenile myoclonic epilepsy (JME), temporal lobe epilepsy (TLE), seizure disorders, acute postoperative pain, psychogenic pain syndromes, pain from cancer, glossopharyngeal neuralgia, inflammatory pain, neuropathic pain, motor and sensory neuropathies, migraine, trigeminal neuralgia, headache, tension headache, carpal tunnel syndrome, injury and neuroprotection, anxiety, depression, bipolar disorder, schizophrenia and paranoid psychoses.

Accordingly, the present invention provides an isolated voltage-gated sodium channel beta-subunit polypeptide comprising: (i) the amino acid sequence of SEQ ID NO: 2; (ii) a variant thereof which is capable of modulating sodium channel a- subunit activity; or (iii) a fragment of (i) or (ii) which is capable of modulating sodium channel a-subunit activity.

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 sodium channel beta-subunit 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 sodium channel betasubunit activity and/or expression; a compound which stimulates or modulates voltage-gated sodium channel beta-subunit activity and which is identifiable by the method referred to above; a method of treating a subject having a disorder that is responsive to voltagegated sodium channel stimulation or modulation, which method comprises administering to said subject an effective amount of substance of the invention; and use of a substance that stimulates or modulates voltage-gated sodium channel beta-subunit 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 sodium channel activity.

Preferably the disorder is selected from epilepsy, juvenile myoclonic epilepsy (JME), temporal lobe epilepsy (TLE), seizure disorders, acute postoperative pain, psychogenic pain syndromes, pain from cancer, glossopharyngeal neuralgia, inflammatory pain, neuropathic pain, motor and sensory neuropathies, migraine, trigeminal neuralgia, headache, tension headache, carpal tunnel syndrome, injury & neuroprotection, anxiety, depression, bipolar disorder, schizophrenia and paranoid psychoses.

Brief Description of the Sequences SEQ ID NO: I shows the nucleotide and amino acid sequences of human protein HIPHUM30.

SEQ ID NO: 2 shows the amino acid sequence alone of HIPHUM30.

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 sodium channel betasubunit, referred to herein as HIPHUM30, and variants thereof. Sequence information for HIPHUM30 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 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 purity 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 HIPHUM30. The essential character of HIPHUM30 can be defined as follows: HIPHUM30 is a voltage-gated sodium channel beta-subunit. Preferably the polypeptide is capable of modulating sodium channel activity. Preferably a variant polypeptide is one which interacts with the same sodium channel a-subunit as HIPHUM30. Preferably the polypeptide is capable of binding to a protein expressed on the surface of a cell other than the cell in which the polypeptide is expressed. Preferably the polypeptide is capable of determining the intracellular location of voltage-gated sodium channels. The polypeptide may mediate voltage-gated sodium channel clustering. Preferably, the polypeptide is capable of modulating sodium channel a-subunit activity.

A polypeptide having the same essential character as HIPHUM30 may be identified by monitoring for a function of the voltage-gated sodium channel betasubunit selected from modulation of voltage-gated sodium channel activity, interaction with a voltage-gated sodium channel a-subunit, interaction with an extracellular immunoglobulin domain containing protein such as tenascin-C or tenascin-R, voltage-gated sodium channel clustering or the location of the polypeptide within a cell.

The extracellular Ig-like domain of the ss2-subunit is formed by amino acids 6-123. HIPHUM30 has two extracellular Ig-like domains (residues 37 to 122 and

158 to 217). Preferably the extracellular Ig domain mediates extracellular interactions of HIPHUM30 with extracellular proteins. Like the 2-subunit, HIPHUM30 has a single transmembrane domain (residues 241-265) and an amino- terminal signal sequence (residues 1-21).

HIPHUM30 has 3 potential N-linked glycosylation sites in its predicted extracellular domain, at residues 102-105,108-111 and 204-207. HIPHUM30 has

consensus sites for PKC phosphorylation at residues 295-297 and 393-395 and has a consensus site for CK-II phosphorylation at 304-307 so may be regulated by phosphorylation by protein kinase C or by casein kinase II.

In another aspect of the invention, a variant is one which does not show the same activity as HIPHUM30 but is one which inhibits a basic function of HIPHUM30. For example, a variant polypeptide is one which inhibits the voltagegated sodium channel modulatory activity of HIPHUM30, for example by binding to a sodium channel a-subunit to prevent activity mediated by binding of HIPHUM30 to the sodium channel a-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 HIPHUM30.

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 sodium channel beta-subunit. 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 ILV 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, 300 or 400 amino acids in length is considered to fall within the scope of the invention as long as it demonstrates a basic biological functionality ofHIPHUM30.

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 sodium channel a-subunit-binding region or the immunoglobulin domain.

Such fragments can be used to construct chimeric subunits preferably with another immunoglobulin domain containing polypeptide, more preferably with another voltage-gated sodium channel beta-subunit. Such fragments ofHIPHUM30 or a variant thereof can also be used to raise anti-HIPHUM30 antibodies. In this embodiment the fragment may comprise an epitope of the HIPHUM30 polypeptide and may otherwise not demonstrate the sodium channel a-subunit binding or other properties of HIPHUM30.

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 tor HIPHUM30 or 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 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 60 oc.

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 HIPHUM30 activity.

Alternatively, a polynucleotide encodes a sodium channel a-subunit-binding portion of a polypeptide or a polypeptide which inhibits HIPHUM30 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 contiguous nucleotides or most preferably over the full length 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) (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 Henikoff and 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. Nl. . 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 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 HIPHUM30, 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. iney win typically De up to 4U, U, bU,/U, 1 UU or iU nucleotides in length. Probes and fragments can be longer than 150 nucleotides in length, for example up to 200,300, 400,500, 600,700 or 1000 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 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 p-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 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 HIPHUM30 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/BacMam 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, for example, following injection of plasmid DNA or in vitro transcribed complementary RNA, in particular for use in an assay of the invention. A polypeptide of the invention may be purified from any suitable cell type from any species for reconstitution into lipid bilayers 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 sodium channel asubunit 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.

Antibodies may be linked to a revealing label and thus may be suitable for use in methods of in vivo HIPHUM30 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 used to identify substances that bind to voltage-gated sodium channel beta-subunits and in particular which bind to HIPHUM30. Screening methods may also be used to identify modulators of voltage-gated sodium channel activity, inhibitors or activators of HIPHUM30 activity, and/or agents which up-regulate or down-regulate HIPHUM30 expression. Activators and inhibitors of voltage-gated sodium channels may be state-dependent modulators, for example, they may be substances that have voltage-dependent or use-dependent interactions with a polypeptide of the invention.

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 sodium channel beta-subunit activity or voltage-gated sodium 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 a sodium channel a-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 a sodium channel a-subunit or extracellular protein 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 HIPHUM30, and incubating such cells with the test substance optionally in the presence of a sodium channel asubunit. The results of the assay are compared to the results obtained using the same assay in the absence of the test substance. Cells expressing HIPHUM30 constitutively may be provided for use in assays for HIPHUM30 function.

Additional test substances may be introduced in any assay to look for inhibitors or enhancers of binding of HIPHUM30 to a sodium channel a-subunit or inhibitors or enhancers ofHIPHUM30-mediated activity, preferably modulation of voltage-gated sodium channel activity.

In preferred aspects, a host cell is provided expressing the polypeptide and an

appropriate voltage-gated sodium channel a-subunit and containing a sodium sensitive photoprotein. Such photoproteins increase or decrease light emission on the influx or efflux of sodium ions and can be detected using an imaging system. A reporter gene assay using such photoproteins can be used to assay for modulation of sodium channel activity. In such reporter gene assays the intracellular sodium ion concentration is linked to a signalling cascade that causes up-or down-regulation of a readily detectable reporter protein such as luciferase. The assay enables determination of whether a test substance modulates the HIPHUM30 regulated flow of sodium ions through sodium channels in target cells.

The ability of a test substance to modulate the HIPHUM30 regulated flow of sodium ions through voltage-gated sodium channels may also be determined using fluorescence based assays using a Fluorometric Imaging Plate Reader (FLIPR) and membrane voltage sensitive dyes, such as DiBac, or sodium sensitive dyes.

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 sodium ions or guanidine in cells expressing a polypeptide of the invention and a voltage-gated sodium channel a-subunit. The structural conformation or intracellular location of a voltage-gated sodium channel comprising a polypeptide of the invention may also be monitored.

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

Preferably, electrophysiological assays and/or assays comprising measuring changes in intracellular sodium ion concentration are performed on cells expressing a polypeptide of the invention and sodium channel a-subunit.

Assays may also be carried out to identify substances which modify HIPHUM30 expression, for example substances which up-or down-regulate expression. Such assays may be carried out for example by using antibodies for HIPHUM30 to monitor levels of HIPHUM30 expression. Other assays which can be used to monitor the effect of a test substance on HIPHUM30 expression include using a reporter gene construct driven by the HIPHUM30 regulatory sequences as the

promoter sequence and monitoring for expression of the reporter polypeptide.

Further possible assays could utilise membrane fractions from overexpression of HIPHUM30 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 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 InM to lOOOM, preferably from luM to lOOuM, more preferably from luM to 1 Oj. LM. 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 HIPHUM30 polypeptides of the invention to identify mutations in HIPHUM30 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 HIPHUM30 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 HIPHUM30 genes. This may comprise determining the level of HIPHUM30 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 of intron/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, "PCR In Situ Hybridization: Protocols And Applications", Raven Press, NY). Such analysis techniques include, DNA or RNA blotting analyses, single stranded conformational polymorphism analyses, in situ hybridization assays, and polymerase chain reaction analyses. Such analyses may

reveal both quantitative aspects of the expression pattern of a HIPHUM30, and qualitative aspects of HIPHUM30 expression and/or composition.

Alternative diagnostic methods for the detection of HIPHUM30 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 targets rap I 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 HIPHUM30 related pathologies. In this way, it is possible to correlate the amount or kind of HIPHUM30 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 sodium 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 epilepsy, juvenile myoclonic epilepsy (JME), temporal lobe epilepsy (TLE), seizure disorders, acute postoperative pain, psychogenic pain syndromes, pain from cancer, glossopharyngeal neuralgia, inflammatory pain, neuropathic pain, motor and sensory neuropathies, migraine, trigeminal neuralgia, headache, tension headache, carpal tunnel syndrome, injury & neuroprotection, anxiety, depression, bipolar disorder, schizophrenia and paranoid psychoses.

Additional disease states that may be treated include Alzheimer's disease, Huntington's disease, Parkinson's disease, amyloidosis, palsies, paralysis, Krabbe's disease, Lou Gehrig's disease, meningitis, Pelizaeus-Merezbacher disease, polycythemia vera, Schilder's disease, Scholz's disease, tuberous sclerosis, Creutzfeldt-Jackob disease (and other prion diseases), encephalitis including acute viral encephalititis, multiple sclerosis, amyotrophic lateral sclerosis (ALS), rheumatoid arthritis, psioriasis, dermatitus, astrocytoma, CNS paraneoplastic syndromes, ependymoma, glioblasoma multiforme, medulloblastoma, meningioma,

neuroblastoma, oligodendrogiioma, schwannoma, soft tissue sarcoma, small bowel tumors, colorectal tumors, benign and malignant tumors, basal cell carcinomas, nonsmall cell lung carcinomas, obesity, metabolic X syndrome, amenorrhea, diverticular disease, ileus, intestinal obstruction and pseudomembranous enterocolitis.

In disease conditions HIPHUM30 may be involved in up-or down-regulation of voltage-gated sodium channel activity, or membrane relocalisation of the channel, thereby causing increased or decreased membrane excitability.

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, 17"'Ed. 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 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 HIPHUM30 or a variant thereof which inhibits or enhances HIPHUM30 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 HIPHUM30 or a variant thereof with an impaired function such as a dominant negative mutant that prevents endogenous HIPHUM30 from modulating voltage-gated sodium channel activity.

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 of transfection 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 I pg to I mg, preferably to I pg to lOug nucleic acid for particle mediated gene delivery and I g to Img for other routes.

The following Examples illustrate the invention.

Example 1: Characterisation of the sequence A human voltage-gated sodium channel beta-subunit, designated as HIPHUM30 has been identified. 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.

HIPHUM30 was found to be primarily expressed in adrenal gland, brain (including cerebral cortex, cerebellum, hypothalamus), testes, small intestine, rectum, ovary and kidney. HIPHUM30 was also found to be expressed in skeletal muscle, colon, ileum, omentum, urinary bladder, trachea, thyroid, uterus and oesophagus.

The chromosomal localization was also mapped. Human HIPHUM30 has

been mapped to jqn-qzi.

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 an appropriate voltage-gated sodium channel a-subunit are generated for use in the assay. 96 and 384 well plate, high throughput screens (HTS) are employed using fluorescence based 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 sodium binding fluorescent dye is as follows. Mammalian cells stably over-expressing the polypeptide of the invention together with appropriate voltage-gated sodium channel a-subunit proteins for

making a 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 MgSOmM KCI, pH 7.4) is added. The cells are then loaded with the sodium indicator dye of choice for 30 minutes. The test compounds are added to the wells and pre-incubated for a period of 10 minutes. The channel is activated by depolarising voltage pulses. 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 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 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 sodium channel beta-subunit.

A typical electropysiology protocol using two electrode voltage-clamp on oocytes expressing HIPHUM30 is as follows. HIPHUM30 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 KCl. Oocytes are voltage-clamped at a set holding membrane potential (for example, between-ICOmV to-80mV) in ND96 solution (superfused at 2ml per min.) and depolarising voltage pulses are applied to activate the channels. 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 non-specific'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 (0. 5ml volume) and superfused with an extracellular recording solution at 2 ml min-1. 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. Wholecell currents are recorded using an Axopatch 200B amplifier (Axon Instruments) or other voltage-clamp amplifier (e. g. HEKA), using standard electrophysiological methods (Hamill et aL, (1981) Pflugers 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 (1-1 OGQ) 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 NaCI, 4.7mM KCI, 1.2mM MgC12, ImM CaCI2, I1mM glucose, 5mM HEPES (titrated with NaOH to pH 7.4 at 25oC) using microelectrode pipettes containing 120mM CsF, 15mM NaCl, I OmM Cs-EGTA (ethylene glycol-bis (p-aminoethyl ester) N, N, N', N-tetra acetic acid, Cs salt), 10mM 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-lOOmV to-80m V) 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 with 95% confidence limits. Statistical comparisons are made using paired or unaired Student's t-test and considered of significance when P < 0. 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 sodium channel beta-subunit.

SEQUENCE LISTING < 110 > GLAXO GROUP LIMITED < 120 > NEW PROTEIN < 130 > QG 1046 < 160 > 2 < 170 > Patentin version 3. 0 < 210 > 1 < 211 > 1494 < 212 > DNA < 213 > Homo sapiens < 220 > < 221 > CDS < 222 > (154) (1449) < 400 > 1

gtctgacagt cccagctctg cgcggggaac agcggcccgg cgctgggtgt gggaggacca 60 ggctgcccca agagcgcgga gactcacgcc cgctcctctc ctgttgcgac cgggagccgg 120 gtaggaggca ggcgcgctcc ctgcggcccc ggg atg act tct cag cgt tec cct 174 Met Thr Ser Gln Arg Ser Pro 1 5

ctg gcg cct ttg ctg etc ctc tct ctg cac ggt gtt gca gca tec ctg 222 Leu Ala Pro Leu Leu Leu Leu Ser Leu His Gly Val Ala Ala Ser Leu 10 15 20 gaa gtg tea gag age cct ggg agt ate cag gtg gcc egg ggt cag aca 270 Glu Val Ser Glu Ser Pro Gly Ser Ile Gln Val Ala Arg Gly Gln Thr

25 30 35 gca gtc ctg ccc tgc act ttc act ace age get gcc etc att aac etc 318 Ala Val Leu Pro Cys Thr Phe Thr Thr Ser Ala Ala Leu Ile Asn Leu 40 45 50 55 aat gtc att tqq atg gtc act cct etc tec aat gcc aac caa cct gaa 366 Asn Val Ile Trp Met Val Thr Pro Leu Ser Asn Ala Asn Gln Pro Glu 60 65 70 cag gtc ate ctg tat cag ggt gga cag atg ttt gat ggt gcc ccc egg 414 Gln Val Ile Leu Tyr Gln Gly Gly Gln Met Phe Asp Gly Ala Pro Arg 75 80 85 ttc cac ggt agg gta gga ttt aca ggc ace atg cca get ace aat gtc 462 Phe His Gly Arg Val Gly Phe Thr Gly Thr Met Pro Ala Thr Asn Val 90 95 100 tct ate ttc att aat aac act cag tta tea gac act ggc ace tac cag 510 Ser Ile Phe Ile Asn Asn Thr Gln Leu Ser Asp Thr Gly Thr Tyr Gln 105 110 115 tgc ctg gtc aac aac ctt cca gac ata ggg ggc agg aac att ggg gtc 558 Cys Leu Val Asn Asn Leu Pro Asp Ile Gly Gly Arg Asn Ile Gly Val 120 125 130 135 ace ggt etc aca gtg tta gtt ccc cct tct gcc cca cac tgc caa ate 606 Thr Gly Leu Thr Val Leu Val Pro Pro Ser Ala Pro His Cys Gln Ile 140 145 150 caa gga tec cag gat att ggc age gat gtc ate ctg etc tgt age tea 654 Gln Gly Ser Gln Asp Ile Gly Ser Asp Val Ile Leu Leu Cys Ser Ser 155 160 165 gag gaa ggc att cct cga cca act tac ctt tgg gag aag tta gac aat 702 Glu Glu Gly Ile Pro Arg Pro Thr Tyr Leu Trp Glu Lys Leu Asp Asn 170 175 180 ace etc aaa cta cct cca aca get act cag gac cag gtc cag gga aca 750 Thr Leu Lys Leu Pro Pro Thr Ala Thr Gln Asp Gln Val Gln Gly Thr 185 190 195 gtc ace ate egg aac ate agt gcc ctg tct tea ggt ttg tac cag tgc 798 Val Thr Ile Arg Asn Ile Ser Ala Leu Ser Ser Gly Leu Tyr Gln Cys 200 205 210 215 gtg get tct aat get att gga ace age ace tgt ctt ctg gat etc cag 846 Val Ala Ser Asn Ala Ile Gly Thr Ser Thr Cys Leu Leu Asp Leu Gln 220 225 230 gtt att tea ccc cag ccc agg aac att gga cta ata get gga gcc att 894 Val Ile Ser Pro Gln Pro Arg Asn Ile Gly Leu Ile Ala Gly Ala Ile 235 240 245 ggc act ggt gca gtt att ate att ttt tgc att gca cta att tta ggg 942 Gly Thr Gly Ala Val Ile Ile Ile Phe Cys Ile Ala Leu Ile Leu Gly 250 255 260

gca ttc ttt tac tgg aga age aaa aat aaa gag gag gaa gaa gaa gaa 990 Ala Phe Phe Tyr Trp Arg Ser Lys Asn Lys Glu Glu Glu Glu Glu Glu 265 270 275 att cct aat gaa ata aga gag gat gat ctt cca ccc aag tgt tct tct 1038 Ile Pro Asn Glu Ile Arg Glu Asp Asp Leu Pro Pro Lys Cys Ser Ser 280 285 290 295 gcc aaa gca ttt cac act gag att tec tec teg gac aac aac aca cta 1086 Ala Lys Ala Phe His Thr Glu Ile Ser Ser Ser Asp Asn Asn Thr Leu 300 305 310 ace tct tec aat gcc tac aac agt cga tac tgg age aac aat cca aaa 1134 Thr Ser Ser Asn Ala Tyr Asn Ser Arg Tyr Trp Ser Asn Asn Pro Lys 315 320 325 gtt cat aga aac aca gag tea gtc age cac ttc agt gac ttg ggc caa 1182 Val His Arg Asn Thr Glu Ser Val Ser His Phe Ser Asp Leu Gly Gln 330 335 340 tct ttc tct ttc cac tea ggc aat gcc aac ata cca tec att tat get 1230 Ser Phe Ser Phe His Ser Gly Asn Ala Asn Ile Pro Ser Ile Tyr Ala 345 350 355 aat ggg ace cat ctg gtc ccg ggt caa cat aag act ctg gta gtg aca 1278 Asn Gly Thr His Leu Val Pro Gly Gln His Lys Thr Leu Val Val Thr 360 365 370 375 gcc aac aga ggg tea tea cca cag gtg atg tec agg age aat ggc tea 1326 Ala Asn Arg Gly Ser Ser Pro Gln Val Met Ser Arg Ser Asn Gly Ser 380 385 390 gtc agt agg aag cct egg cct cca cac act cat tec tac ace ate age 1374 Val Ser Arg Lys Pro Arg Pro Pro His Thr His Ser Tyr Thr Ile Ser 395 400 405 cac gca aca ctg gaa cga att ggt gca gta cct gtc atg gta cca gcc 1422 His Ala Thr Leu Glu Arg Ile Gly Ala Val Pro Val Met Val Pro Ala 410 415 420 cag agt egg gcc ggg tec ttg gta tag gacatgagga aatgttgtgt 1469 Gln Ser Arg Ala Gly Ser Leu Val 425 430 tcagaaatga ataatggaat gccct 1494 < 210 > 2 < 211 > 431 < 212 > PRT < 213 > Homo sapiens

< 400 > 2 Met Thr Ser Gln Arg Ser Pro Leu Ala Pro Leu Leu Leu Leu Ser Leu 1 5 10 15

His Gly Val Ala Ala Ser Leu Glu Val Ser Glu Ser Pro Gly Ser Ile 20 25 30 Gln Val Ala Arg Gly Gln Thr Ala Val Leu Pro Cys Thr Phe Thr Thr 35 40 45 Ser Ala Ala Leu Ile Asn Leu Asn Val Ile Trp Met Val Thr Pro Leu 50 55 60 Ser Asn Ala Asn Gln Pro Glu Gln Val Ile Leu Tyr Gln Gly Gly Gln 65 70 75 80

Met Phe Asp Gly Ala Pro Arg Phe His Gly Arg Val Gly Phe Thr Gly 85 90 95 Thr Met Pro Ala Thr Asn Val Ser Ile Phe Ile Asn Asn Thr Gln Leu 100 105 110 Ser Asp Thr Gly Thr Tyr Gln Cys Leu Val Asn Asn Leu Pro Asp Ile 115 120 125

Gly Gly Arg Asn Ile Gly Val Thr Gly Leu Thr Val Leu Val Pro Pro 130 135 140 Ser Ala Pro His Cys Gln Ile Gln Gly Ser Gln Asp Ile Gly Ser Asp 145 150 155 160

Val Ile Leu Leu Cys Ser Ser Glu Glu Gly Ile Pro Arg Pro Thr Tyr 165 170 175 Leu Trp Glu Lys Leu Asp Asn Thr Leu Lys Leu Pro Pro Thr Ala Thr 180 185 190 Gln Asp Gln Val Gln Gly Thr Val Thr Ile Arg Asn Ile Ser Ala Leu 195 200 205

Ser Ser Gly Leu Tyr Gln Cys Val Ala Ser Asn Ala Ile Gly Thr Ser

210 215 220 Thr Cys Leu Leu Asp Leu Gln Val Ile Ser Pro Gln Pro Arg Asn Ile 225 230 235 240

Gly Leu Ile Ala Gly Ala Ile Gly Thr Gly Ala Val Ile Ile Ile Phe 245 250 255 Cys Ile Ala Leu Ile Leu Gly Ala Phe Phe Tyr Trp Arg Ser Lys Asn 260 265 270 Lys Glu Glu Glu Glu Glu Glu Ile Pro Asn Glu Ile Arg Glu Asp Asp 275 280 285 Leu Pro Pro Lys Eys Ser Ser Ala Lys Ala Phe His Thr Glu Ile Ser 290 295 300 Ser Ser Asp Asn Asn Thr Leu Thr Ser Ser Asn Ala Tyr Asn Ser Arg 305 310 315 320 Tyr Trp Ser Asn Asn Pro Lys Val His Arg Asn Thr Glu Ser Val Ser 325 330 335 His Phe Ser Asp Leu Gly Gln Ser Phe Ser Phe His Ser Gly Asn Ala 340 345 350

Asn Ile Pro Ser Ile Tyr Ala Asn Gly Thr His Leu Val Pro Gly Gln 355 360 365 His Lys Thr Leu Val Val Thr Ala Asn Arg Gly Ser Ser Pro Gln Val 370 375 380 Met Ser Arg Ser Asn Gly Ser Val Ser Arg Lys Pro Arg Pro Pro His 385 390 395 400

Thr His Ser Tyr Thr Ile Ser His Ala Thr Leu Glu Arg Ile Gly Ala 405 410 415 Val Pro Val Met Val Pro Ala Gln Ser Arg Ala Gly Ser Leu Val 420 425 430

Claims

CLAIMS 1. An isolated voltage-gated sodium channel beta-subunit polypeptide comprising (i) the amino acid sequence of SEQ ID NO: 2 or (ii) a variant thereof which is capable of modulating sodium channel a- subunit activity or (iii) a fragment of (i) or (ii) which is capable of modulating sodium channel a-subunit activity.
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 sodium channel betasubunit polypeptide which is capable of modulating sodium channel a-subunit activity 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 1 or 2.
9. A method for the identification of a substance that modulates voltagegated sodium channel beta-subunit 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

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expression vector according to claim 6 or a host cell according to 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 sodium channel beta-subunit 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 sodium channel a-subunit.
12. A method according to claim 11 wherein step (ii) comprises monitoring any voltage-gated sodium channel activity.
13. A method according to claim 11 wherein step (ii) comprises monitoring any interaction between the said polypeptide with the said sodium channel a-subunit.
14. A substance which modulates voltage-gated sodium channel betasubunit 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 sodium 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 epilepsy, juvenile myoclonic epilepsy (JME), temporal lobe epilepsy (TLE), seizure disorders, acute postoperative pain, psychogenic pain syndromes, pain from cancer, glossopharyngeal neuralgia, inflammatory pain, neuropathic pain, motor and sensory neuropathies, migraine, trigeminal neuralgia, headache, tension headache, carpal tunnel syndrome, injury & neuroprotection, anxiety, depression, bipolar disorder, schizophrenia and paranoid psychoses.
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 sodium channel activity.

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18. A use according to claim 17 wherein the disorder is selected from epilepsy, juvenile myoclonic epilepsy (JME), temporal lobe epilepsy (TLE), seizure disorders, acute postoperative pain, psychogenic pain syndromes, pain from cancer, glossopharyngeal neuralgia, inflammatory pain, neuropathic pain, motor and sensory neuropathies, migraine, trigeminal neuralgia, headache, tension headache, carpal tunnel syndrome, injury & neuroprotection, anxiety, depression, bipolar disorder, schizophrenia and paranoid psychoses.
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.