GB2372503A - Voltage-gated potassium channel polypeptides - Google Patents

Abstract

The present invention provides an isolated voltage-gated potassium channel polypeptide comprising <SL> <LI>(i) the amino acid sequence of SEQ ID NO: 2 or SEQ ID NO: 4; or <LI>(ii) a variant thereof which capable of forming a channel which can be activated by depolarisation of the cell membrane potential above the reversal potential for K<SP>+</SP> (E<SB>K</SB>); or <LI>(iii) a fragment of (i) or (ii) which capable of forming a channel which can be activated by depolarisation of the cell membrane potential above the reversal potential for K<SP>+</SP> (E<SB>K</SB>). </SL> The polypeptide may be used in a method to identify substances that may be useful in the treatment of pain, alzheimers disease, epilepsy, psychiatric disorders and thyroid disorders.

Description

NEW PROTEIN

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

Background of the Invention Ion channels are involved in a wide variety of neurological and other disorders in man. Voltage-gated potassium channels of the Kv3 family yield delayed rectifier type currents when expressed in heterologous expression systems.

Kv3 channel subtypes have high activation voltage and fast deactivation rates which help repolarise action potentials rapidly without subsequently influencing the action potential generation threshold. The rapid deactivation of Kv3 channel currents leads to a fast recovering afterhyperpolarisation which maximises recovery of sodium channels from inactivation. Thus the fast recovering afterhyperpolarisation is one factor that enables Kv3 expressing neurons to fire at high frequencies and to regulate synaptic transmission. Presynaptic voltage-gated potassium channels affect Ca2+ entry and neurotransmitter release.

Summary of the Invention A novel voltage-gated potassium channel, referred to herein as HIPHUM 59/60/190, is now provided. HIPHUM 59/60/190 is shown to be primarily expressed in brain (whole brain, fetal brain, cerebral cortex, cerebellum, hypothalamus) and thyroid. The novel voltage-gated potassium channel is a screening target for the identification and development of novel pharmaceutical agents, including modulators of voltage-gated potassium 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, sleep disorders such as insomnia, hypersomnia, parasomnia, sleep apnea syndromes and stupor, pain states such as acute postoperative pain, psychogenic pain syndromes, pain from cancer, glossopharyngeal neuralgia, inflammatory pain, neuropathic pain, migraine, trigeminal neuralgia, headache and tension headache, neurodegenerative diseases

such as Alzheimer's disease, Huntington's disease, Parkinson's disease, palsies and paralysis, pyschiatric disorders such as anxiety, depression, bipolar disorder, schizophrenia and paranoid psychoses and thyroid disorders such as euthyroid sick syndrome, hyperthyroidism, hypothyroidism, simple goiter and thyroiditis.

Accordingly, the present invention provides an isolated voltage-gated potassium channel polypeptide comprising: (i) the amino acid sequence of SEQ ID NO: 2 or SEQ ID NO: 4; (ii) a variant thereof which capable of forming a channel which can be activated by depolarisation of the cell membrane potential above the reversal potential for K+ (EK) ; or (iii) a fragment of (i) or (ii) which capable of forming a channel which can be activated by depolarisation of the cell membrane potential above the reversal potential for K+ (EK).

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 or SEQ ID NO: 3 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 98% 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 potassium 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 potassium channel activity and/or expression ; a compound which stimulates or modulates voltage-gated potassium 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 voltagegated potassium 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 potassium 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 potassium channel activity.

Preferably the disorder is selected from epilepsy, juvenile myoclonic epilepsy (JME), temporal lobe epilepsy (TLE), seizure disorders, sleep disorders such as insomnia, hypersomnia, parasomnia, sleep apnea syndromes and stupor, pain states such as acute postoperative pain, psychogenic pain syndromes, pain from cancer, glossopharyngeal neuralgia, inflammatory pain, neuropathic pain, migraine, trigeminal neuralgia, headache and tension headache, neurodegenerative diseases such as Alzheimer's disease, Huntington's disease, Parkinson's disease, palsies and paralysis, pyschiatric disorders such as anxiety, depression, bipolar disorder, schizophrenia and paranoid psychoses and thyroid disorders such as euthyroid sick syndrome, hyperthyroidism, hypothyroidism, simple goiter and thyroiditis.

Brief Description of the Figures Figure 1 shows the relative expression levels ofHIPHUM 59/60/190 in a variety of human tissues.

Brief Description of the Sequences SEQ ID NO : 1 shows the nucleotide and amino acid sequences of the longer splice variant of human protein HIPHUM 59/60/190.

SEQ ID NO: 2 is the amino acid sequence alone of the longer splice variant of HIPHUM 59/60/190.

SEQ ID NO: 3 shows the nucleotide and amino acid sequences of the shorter splice variant of human protein HIPHUM 59/60/190.

SEQ ID NO: 4 is the amino acid sequence alone of the shorter splice variant of HIPHUM 59/60/190.

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 potassium channel, referred to herein as HIPHUM 59/60/190, and variants thereof. Sequence information for HIPHUM 59/60/190 is provided in SEQ ID NO: 1 and SEQ ID NO: 3 (nucleotide and amino acid) and in SEQ ID NO: 2 and SEQ ID NO: 4 (amino acid).

A polypeptide of the invention thus consists essentially of the amino acid sequence of SEQ ID NO: 2 or SEQ ID NO: 4 or of a variant of either sequence, or of a fragment of any 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 ai, Molecular Cloning : a Laboratory Manual, 2"''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 59/60/190. The essential character of HIPHUM 59/60/190 can be defined as follows: HIPHUM 59/60/190 is a voltage-gated potassium channel. Preferably the polypeptide is capable of forming a delayed-rectifier potassium channel which can be activated by depolarisation of the cell membrane potential above the reversal potential for K+ (EK). Preferably a variant polypeptide is one which binds to the same the same Kv3 subfamily members as HIPHUM 59/60/190. Preferably the channel is inhibited by phosphorylation for example, mediated by PKA, PKC or PKG. Preferably, a variant of the longer splice variant of HIPHUM 59/60/190 contains a PKC phosphorylation site. Preferably, a variant of either the splice variant of HIPHUM 59/60/190 contains a PKA phosphorylation site. A polypeptide having the same essential character as HIPHUM 59/60/190 may be identified by monitoring for potassium channel activity of the voltage-gated potassium channel. Potassium channel activity may be monitored electrophysiologically, for example by monitoring the threshold for channel

activation, the rate of inactivation, the rate of recovery from inactivation or the rate of deactivation. Alternatively a conformational change in the potassium channel or changes in intracellular K+ and/or Rb+ ion concentration may be monitored.

In another aspect of the invention, a variant is one which does not show the same activity as HIPHUM 59/60/190 but is one which inhibits or enhances a basic function of HIPHUM 59/60/190. For example, a variant polypeptide is one which inhibits formation of potassium channels by binding to HIPHUM 59/60/190 or another Kv3 subfamily member to prevent homomeric or heteromeric channel assembly. A variant polypeptide that enhances channel activity may lack one or more consensus phosphorylation site. A variant of HIPHUM 59/60/190 that lacks a consensus phosphorylation site will show a decreased sensitivity to inactivation by phosphorylation.

Typically, polypeptides with more than about 98% identity preferably at least

99% and particularly preferably at least 99. 5% identity, with the amino acid sequences of SEQ ID NO: 2 or SEQ ID NO: 4, 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 59/60/190.

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 potassium 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 G A P ILV Polar-uncharged CSTM NQ Polar-charged D E 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, 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 59/60/190. 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 Kv3 polypeptide-binding region. Such fragments can be used to construct chimeric receptors preferably with another voltage-gated potassium channel polypeptide, more preferably with another delayed rectifier voltage-gated potassium channel, such as a Kv3 polypeptide. Such fragments of HIPHUM 59/60/190 or a variant thereof can also be used to raise anti-HIPHUM 59/60/190 antibodies. In this embodiment the fragment may comprise an epitope of the HIPHUM 59/60/190 polypeptide and may otherwise not demonstrate the properties of HIPHUM 59/60/190, such as the ability to form functional potassium channels. A preferred fragment comprises the amino acid sequence from positions 42 to 92 of SEQ ID NO : 2 or SEQ ID NO : 4. Further preferred fragments comprise a fragment of SEQ ID NO : 2 or SEQ ID NO : 4 which includes the amino acids at positions 186 to 187, position 502, position 210, position 270 and/or position 529 of SEQ ID NO : 2 or SEQ ID NO : 4.

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 59/60/190 or variants 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 and SEQ ID NO : 3. 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 or SEQ ID NO : 3.

A polynucleotide of the invention can hydridize to the coding sequence or the complement of the coding sequence of SEQ ID NO : 1 or SEQ ID NO : 3 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 or SEQ ID NO : 3 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 or SEQ ID NO : 3. 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 DC 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 or SEQ ID NO : 3 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 : I or SEQ ID NO : 3 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 59/60/190 activity. Alternatively, a polynucleotide encodes a portion of a polypeptide or a polypeptide which inhibits HIPHUM 59/60/190 activity, for example by disrupting the formation of channels containing HIPHUM 59/60/190 by binding HIPHUM 59/60/190 to prevent homomeric channel assembly.

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 or SEQ ID NO: 3 will generally have at least at least 98%, at least 99% or at least 99.5% sequence identity to the coding sequence of SEQ ID NO: 1 or SEQ ID NO: 3 over a region of at least 20, preferably at least 30, for instance at least 40, at least 60, at least 100, at least 200, at least 500, more preferably at least 1000 contiguous nucleotides or most preferably over the full length of SEQ ID NO: 1 or SEQ ID NO: 3.

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. 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 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 98% sequence identity over 25, preferably over 30 nucleotides forms one aspect of the invention, as does a polynucleotide which has at least 99% 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 59/60/190, 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 or SEQ ID NO: 3.

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 ss-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 HIPHUM 59/60/190 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 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 the same Kv3 subfamily members 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 HIPHUM 59/60/190 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 potassium channels and in particular which bind to HIPHUM 59/60/190. Screening methods may also be used to identify agonists or antagonists which may modulate voltage-gated potassium channel activity, inhibitors or activators of HIPHUM 59/60/190 activity, and/or agents which up-regulate or down-regulate HIPHUM 59/60/190 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 potassium 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 other Kv3 subfamily members.

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 other Kv3 subfamily members may also be identified through a yeast 2-hybrid assay or other protein interaction assay such as a co-immunoprecipitation or an ELISA based technique.

Assays may be carried out using cells expressing HIPHUM 59/60/190, and optionally other Kv3 subfamily members, and incubating such cells with the test substance. 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 59/60/190 constitutively may be provided for use in assays for HIPHUM 59/60/190 function. Additional test substances may be introduced in any assay to look for inhibitors or enhancers of HIPHUM 59/60/190-mediated activity, preferably delayed rectifier potassium channel activity.

The ability of a test substance to modulate the HIPHUM 59/60/190 regulated flow of potassium ions through voltage-gated potassium 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 K+/Rb+ 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 calcium ions 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 other Kv3 subfamily members may also be used to assay for modulatory activity of a test

substance.

Preferably, electroplysological assays and/or assays comprising measuring changes in intracellular potassium ion concentration are performed on cells expressing a polypeptide of the invention and other Kv3 subfamily members.

Assays may also be carried out to identify substances which modify HIPHUM 59/60/190 expression, for example substances which up-or down-regulate expression. Such assays may be carried out for example by using antibodies for HIPHUM 59/60/190 to monitor levels of HIPHUM 59/60/190 expression. Other assays which can be used to monitor the effect of a test substance on HIPHUM 59/60/190 expression include using a reporter gene construct driven by the HIPHUM 59/60/190 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 59/60/190 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 lM to lOOuM, more preferably from 1, uM

to 10u. M. Preferably, the potassium channel activity of a polypeptide of the invention in response to a test substance is compared to the activity in response to depolarisation of the cell membrane. 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 by depolarisation.

Another aspect of the present invention is the use of polynucleotides encoding the HIPHUM 59/60/190 polypeptides of the invention to identify mutations in HIPHUM 59/60/190 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 59/60/190 expression. Polynucleotides such as SEQ ID NO: 1 or SEQ ID NO: 3 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 59/60/190 genes. This may comprise determining the level of HIPHUM 59/60/190 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 co-segregates 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 HIPHUM 59/60/190,

and qualitative aspects of HIPHUM 59/60/190 expression and/or composition.

Alternative diagnostic methods for the detection of HIPHUM 59/60/190 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: 18741878), 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 59/60/190 related pathologies. In this way, it is possible to correlate the amount or kind of HIPHUM 59/60/190 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 potassium 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, sleep disorders such as insomnia, hypersomnia, parasomnia, sleep apnea syndromes

and stupor, pain states such as acute postoperative pain, psychogenic pain syndromes, pain from cancer, glossopharyngeal neuralgia, inflammatory pain, neuropathic pain, migraine, trigeminal neuralgia, headache and tension headache, neurodegenerative diseases such as Alzheimer's disease, Huntington's disease, Parkinson's disease, palsies and paralysis, pyschiatric disorders such as anxiety, depression, bipolar disorder, schizophrenia, paranoid psychoses and thyroid disorders such as euthyroid sick syndrome, hyperthyroidism, hypothyroidism, simple goiter and thyroiditis.

Additional disease states that may be treated include agnosia, akathisia, amnesias, anxiety disorders, bipolar disease, coma, delirium, dyskinesia, Friedreich ataxia, idiopathic orthostatic hypotension, Shy-Drager syndrome, post traumatic stress disorder, tardive dyskinesia and tremor.

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, 17th 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 HIPHUM 59/60/190 or a variant thereof which inhibits or enhances HIPHUM 59/60/190 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 59/60/190 or a variant thereof with an impaired function such as a dominant negative mutant that disrupts the function of the whole voltage-gated potassium 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 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 Ipg to Img, preferably to Ipg to lOug nucleic acid for particle mediated gene delivery and 10jj g to 1 mg for other routes.

The following Examples illustrate the invention.

Example 1: Characterisation of the sequence A voltage-gated potassium channel, designated as HIPHUM 59/60/190 has been identified. The nucleotide and amino acid sequences of HIPHUM 59/60/190 have been determined and two splice variants have been identified. The nucleotide and amino acid sequences of the two variants are set out below in SEQ ID NOs: 1 to

4. Suitable primers and probes were designed and used to analyse tissue expression. HIPHUM 59/60/190 was found to be primarily expressed in brain (whole brain, fetal brain, cerebral cortex, cerebellum, hypothalamus) and thyroid.

The chromosomal localization was also mapped. Human HIPHUM 59/60/190 has been mapped to 12ql4-ql5.

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 potassium channel subunit are generated for use in the assay. 96 and 384 well plate, high throughput screens (HTS) are employed using fluorescence based K+/Rb + 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 K+/Rb+ binding fluorescent dye is as follows. Mammalian cells stably over-expressing the polypeptide of the invention together with appropriate voltage-gated potassium channel subunit proteins for making a potassium 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 MgSO4, 5mM KCI, pH 7.4) is added. The cells are then loaded with the K+/Rb+ 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 the cell membrane. 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 K+/Rb+ can be measured directly in a Fluorescence Imaging Plate Reader (FLIPR; Molecular Devices).

A typical electropysiology protocol using electrophysiology in oocytes expressing HIPHUM 59/60/190 is as follows. HIPHUM 59/60/190 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 KCI. Oocytes are voltage-clamped at a set holding membrane potential (for example, between-lOOmV to-80mV) in ND96 solution (superfused at 2ml per min.) and depolarising voltage pulses are applied to activate the channels.

Potassium currents elicited by these voltage pulses are recorded. Voltageprotocols 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 (0.5ml volume) and superfused with an extracellular recording solution at 2 ml minot. 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 au., 1981). 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 of2-4MC'. 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 KC1, 1.2mM Mg12, ImM CaC12, 1 lmM glucose, 5mM HEPES (titrated with NaOH to pH 7.4 at 25OC) using microelectrode pipettes containing 130mM KCI, 3mM NaCl, ImM MgCI2, 5mM K-EGTA (ethylene glycol-bis (p-aminoethyl ester) N, N, N', N-tetra acetic acid, K salt), lOmM HEPES, 5mM Glucose, 3mM Mg ATP (pH7.3 at 25OC). 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-80mV) 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 (8085%) 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 unpaired 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 agonist or antagonist of the voltage-gated potassium channel.

SEQUENCE LISTING < 110 > GLAXO GROUP LIMITED < 120 > NEW PROTEIN < 130 > P80203 GCW/SER < 140 > < 141 > < 160 > 4 < 170 > Patientin Ver. 2. 1 < 210 > 1 < 211 > 2106 < 212 > DNA < 213 > Homo sapiens < 220 > < 221 > CDS < 222 > (193).. (2106) < 400 > 1 caccccagcg cccagggaag cggctcaacc acttgaatcc ggaaaacgcc aacaagtagt 60 ttctcgtcgg agaagggcgg ctcacctggg cgccaagact cagtcccgct gcccagagaa 120 cctcgtccac tcggaaacca aagcagaacc acttttctct cggtctcgtt aagtcatgtc 180 tgagtcacag ag latg gge aag ate gag aac aac'gag agg gtg ate etc aat 231 Met Gly Lys Ile Glu Asn Asn Glu Arg Val Ile Leu Asn 1 5 10 gtc ggg ggc ace egg cac gaa ace tac cgc age ace etc aag ace ctg 279 Val Gly Gly Thr Arg His Glu Thr Tyr Arg Ser Thr Leu Lys Thr Leu 15 20 25 cct gga aca cgc ctg gcc ctt ctt gcc tee tee gag ccc cca ggc gac 327 Pro Gly Thr Arg Leu Ala Leu Leu Ala Ser Ser Glu Pro Pro Gly Asp 30 35 40 45 tgc ttg ace acg gcg ggc gac aag ctg cag ccg tcg ccg cct cca etc, 375 Cys Leu Thr Thr Ala Gly Asp Lys Leu Gln Pro Ser Pro Pro Pro Leu 50 55 60 teg ccg ccg ccg aga gcg ccc ccg ctg tee ccc ggg cca ggc ggc tgc 423 Ser Pro Pro Pro Arg Ala Pro Pro Leu Ser Pro Gly Pro Gly Gly Cys 65 70 75 ttc gag ggc ggc gcg ggc aac tgc agt tee cgc ggc ggc agg gcc age 471 Phe Glu Gly Gly Ala Gly Asn Cys Ser Ser Arg Gly Gly Arg Ala Ser 80 85 90 gac cat ccc ggt ggc ggc cgc gag ttc ttc ttc gac egg cac ccg ggc 519 Asp His Pro Gly Gly Gly Arg Glu Phe Phe Phe Asp Arg His Pro Gly

95 100 105 gtc ttc gcc tat gtg etc aat tac tac cgc ace ggc aag ctg cac tgc 567 Val Phe Ala Tyr Val Leu Asn Tyr Tyr Arg Thr Gly Lys Leu His Cys 110 115 120 125 ccc gca gac gtg tgc ggg ccg etc ttc gag gag gag ctg gcc ttc tgg 615 Pro Ala Asp Val Cys Gly Pro Leu Phe Glu Glu Glu Leu Ala Phe Trp 130 135 140 ggc ate gac gag ace gac gtg gag ccc tgc tgc tgg atg ace tac egg 663 Gly Ile Asp Glu Thr Asp Val Glu Pro Cys Cys Trp Met Thr Tyr Arg 145 150 155 cag cac cgc gac gcc gag gag gcg ctg gac ate ttc gag ace ccc gac 711 Gln His Arg Asp Ala Glu Glu Ala Leu Asp Ile Phe Glu Thr Pro Asp 160 165 170 etc att ggc ggc gac ccc ggc gac gac gag gac ctg gcg gcc aag agg 759 Leu Ile Gly Gly Asp Pro Gly Asp Asp Glu Asp Leu Ala Ala Lys Arg 175 180 185 ctg ggc ate gag gac gcg gcg ggg etc ggg ggc ccg gac ggc aaa tct 807 Leu Gly Ile Glu Asp Ala Ala Gly Leu Gly Gly Pro Asp Gly Lys Ser 190 195 200 205 ggc cgc tgg agg agg ctg cag ccc cgc atg tgg gcc etc ttc gaa gac 855 Gly Arg Trp Arg Arg Leu Gln Pro Arg Met Trp Ala Leu Phe Glu Asp 210 215 220 ccc tac teg tee aga gcc gcc agg ttt att get ttt get tct tta ttc 903 Pro Tyr Ser Ser Arg Ala Ala Arg Phe Ile Ala Phe Ala Ser Leu Phe 225 230 235 ttc ate ctg gtt tea att aca act ttt tgc ctg gaa aca cat gaa get 951 Phe Ile Leu Val Ser Ile Thr Thr Phe Cys Leu Glu Thr His Glu Ala 240 245 250 ttc aat att gtt aaa aac aag aca gaa cca gtc ate aat ggc aca agt 999 Phe Asn Ile Val Lys Asn Lys Thr Glu Pro Val Ile Asn Gly Thr Ser 255 260 265 gtt gtt cta cag tat gaa att gaa acg gat cct gcc ttg acg tat gta 1047 Val Val Leu Gln Tyr Glu Ile Glu Thr Asp Pro Ala Leu Thr Tyr Val 270 275 280 285 gaa gga gtg tgt gtg gtg tgg ttt act ttt gaa ttt tta gtc cgt att 1095 Glu Gly Val Cys Val Val Trp Phe Thr Phe Glu Phe Leu Val Arg Ile 290 295 300 gtt ttt tea ccc aac aaa ctt gaa ttc ate aaa aat etc ttg aat ate 1143 Val Phe Ser Pro Asn Lys Leu Glu Phe Ile Lys Asn Leu Leu Asn Ile 305 310 315 att gac ttt gtg gcc ate cta cct ttc tac tta gag gtg gga etc agt 1191 Ile Asp Phe Val Ala Ile Leu Pro Phe Tyr Leu Glu Val Gly Leu Ser 320 325 330

ggg ctg tea tee aaa get get aaa gat gtg ctt ggc ttc etc agg gtg 1239 Gly Leu Ser Ser Lys Ala Ala Lys Asp Val Leu Gly Phe Leu Arg Val 335 340 345 gta agg ttt gtg agg ate ctg aga att ttc aag etc ace cgc cat ttt 1287 Val Arg Phe Val Arg Ile Leu Arg Ile Phe Lys Leu Thr Arg His Phe 350 355 360 365 gta ggt ctg agg gtg ctt gga cat act ctt cga get agt act aat gaa 1335 Val Gly Leu Arg Val Leu Gly His Thr Leu Arg Ala Ser Thr Asn Glu 370 375 380 ttt ttg ctg ctg ata att ttc ctg get cta gga gtt ttg ata ttt get 1383 Phe Leu Leu Leu Ile Ile Phe Leu Ala Leu Gly Val Leu Ile Phe Ala 385 390 395 ace atg ate tac tat gcc gag aga gtg gga get caa cct aac gac cct 1431 Thr Met Ile Tyr Tyr Ala Glu Arg Val Gly Ala Gln Pro Asn Asp Pro 400 405 410 tea get agt gag cac aca cag ttc aaa aac att ccc att ggg ttc tgg 1479 Ser Ala Ser Glu His Thr Gln Phe Lys Asn Ile Pro Ile Gly Phe Trp 415 420 425 tgg get gta gtg ace atg act ace ctg ggt tat ggg gat atg tac ccc 1527 Trp Ala Val Val Thr Met Thr Thr Leu Gly Tyr Gly Asp Met Tyr Pro 430 435 440 445 caa aca tgg tea ggc atg ctg gtg gga gcc ctg tgt get ctg get gga 1575 Gin Thr Trp Ser Gly Met Leu Val Gly Ala Leu Cys Ala Leu Ala Gly 450 455 460 gtg ctg aca ata gcc atg cca gtg cct gtc att gtc aat aat ttt gga 1623 Val Leu Thr Ile Ala Met Pro Val Pro Val Ile Val Asn Asn Phe Gly 465 470 475 atg tac tac tee ttg gca atg gca aag cag aaa ctt cca agg aaa aga 1671 Met Tyr Tyr Ser Leu Ala Met Ala Lys Gln Lys Leu Pro Arg Lys Arg 480 485 490 aag aag cac ate cct cct get cct cag gca age tea cct act ttt tgc 1719 Lys Lys His Ile Pro Pro Ala Pro Gln Ala Ser Ser Pro Thr Phe Cys 495 500 505 aag aca gaa tta aat atg gcc tgc aat agt aca cag agt gac aca tgt 1767 Lys Thr Glu Leu Asn Met Ala Cys Asn Ser Thr Gln Ser Asp Thr Cys 510 515 520 525 ctg ggc aaa gac aat cga ctt ctg gaa cat aac aga tea gtg tta tea 1815 Leu Gly Lys Asp Asn Arg Leu Leu Glu His Asn Arg Ser Val Leu Ser 530 535 540 ggt gac gac agt aca gga agt gag ccg cca cta tea ccc cca gaa agg 1863 Gly Asp Asp Ser Thr Gly Ser Glu Pro Pro Leu Ser Pro Pro Glu Arg 545 550 555

etc ccc ate aga cgc tct agt ace aga gac aaa aac aga aga ggg gaa 1911 Leu Pro Ile Arg Arg Ser Ser Thr Arg Asp Lys Asn Arg Arg Gly Glu 560 565 570 aca tgt ttc cta ctg acg aca ggt gat tac acg tgt get tct gat gga 1959 Thr Cys Phe Leu Leu Thr Thr Gly Asp Tyr Thr Cys Ala Ser Asp Gly 575 580 585 ggg ate agg aaa ggt tat gaa aaa tee cga age tta aac aac ata gcg 2007 Gly Ile Arg Lys Gly Tyr Glu Lys Ser Arg Ser Leu Asn Asn Ile Ala 590 595 600 605 ggc ttg gca ggc aat get ctg agg etc tct cca gta aca tea ccc tac 2055 Gly Leu Ala Gly Asn Ala Leu Arg Leu Ser Pro Val Thr Ser Pro Tyr 610 615 620 aac tct cct tgt cct ctg agg cgc tct lcga tct ccc atc cca tct atcl 2103 Asn Ser Pro Cys Pro Leu Arg Arg Ser Arg Ser Pro Ile Pro Ser Ile 625 630 635 ttg 2106 Leu < 210 > 2 < 211 > 638 < 212 > PRT < 213 > Homo sapiens < 400 > 2

Met Gly Lys Ile Glu Asn Asn Glu Arg Val Ile Leu Asn Val Gly Gly 1 5 10 15 Thr Arg His Glu Thr Tyr Arg Ser Thr Leu Lys Thr Leu Pro Gly Thr 20 25 30 Arg Leu Ala Leu Leu Ala Ser Ser Glu Pro Pro Gly Asp Cys Leu Thr 35 40 45 Thr Ala Gly Asp Lys Leu Gln Pro Ser Pro Pro Pro Leu Ser Pro Pro 50 55 60

Pro Arg Ala Pro Pro Leu Ser Pro Gly Pro Gly Gly Cys Phe Glu Gly 65 70 75 80 Gly Ala Gly Asn Cys Ser Ser Arg Gly Gly Arg Ala Ser Asp His Pro 85 90 95 Gly Gly Gly Arg Glu Phe Phe Phe Asp Arg His Pro Gly Val Phe Ala 100 105 110 Tyr Val Leu Asn Tyr Tyr Arg Thr Gly Lys Leu His Cys Pro Ala Asp 115 120 125 Val Cys Gly Pro Leu Phe Glu Glu Glu Leu Ala Phe Trp Gly Ile Asp 130 135 140

Glu Thr Asp Val Glu Pro Cys Cys Trp Met Thr Tyr Arg Gln His Arg 145 150 155 160 Asp Ala Glu Glu Ala Leu Asp Ile Phe Glu Thr Pro Asp Leu Ile Gly 165 170 175 Gly Asp Pro Gly Asp Asp Glu Asp Leu Ala Ala Lys Arg Leu Gly Ile 180 185 190 Glu Asp Ala Ala Gly Leu Gly Gly Pro Asp Gly Lys Ser Gly Arg Trp 195 200 205 " Arg Arg Leu Gln Pro Arg Met Trp Ala Leu Phe Glu Asp Pro Tyr Ser 210 215 220 Ser Arg Ala Ala Arg Phe Ile Ala Phe Ala Ser Leu Phe Phe Ile Leu 225 230 235 240 Val Ser Ile Thr Thr Phe Cys Leu Glu Thr His Glu Ala Phe Asn Ile 245 250 255 Val Lys Asn Lys Thr Glu Pro Val Ile Asn Gly Thr Ser Val Val Leu 260 265 270 Gln Tyr Glu Ile Glu Thr Asp Pro Ala Leu Thr Tyr Val Glu Gly Val 275 280 285 Cys Val Val Trp Phe Thr Phe Glu Phe Leu Val Arg Ile Val Phe Ser 290 295 300 Pro Asn Lys Leu Glu Phe Ile Lys Asn Leu Leu Asn Ile Ile Asp Phe 305 310 315 320 Val Ala Ile Leu Pro Phe Tyr Leu Glu Val Gly Leu Ser Gly Leu Ser 325 330 335 Ser Lys Ala Ala Lys Asp Val Leu Gly Phe Leu Arg Val Val Arg Phe 340 345 350 Val Arg Ile Leu Arg Ile Phe Lys Leu Thr Arg His Phe Val Gly Leu 355 360 365 Arg Val Leu Gly His Thr Leu Arg Ala Ser Thr Asn Glu Phe Leu Leu 370 375 380 Leu Ile Ile Phe Leu Ala Leu Gly Val Leu Ile Phe Ala Thr Met Ile 385 390 395 400 Tyr Tyr Ala Glu Arg Val Gly Ala Gln Pro Asn Asp Pro Ser Ala Ser 405 410 415 Glu His Thr Gln Phe Lys Asn Ile Pro Ile Gly Phe Trp Trp Ala Val 420 425 430 Val Thr Met Thr Thr Leu Gly Tyr Gly Asp Met Tyr Pro Gln Thr Trp 435 440 445

Ser Gly Met Leu Val Gly Ala Leu Cys Ala Leu Ala Gly Val Leu Thr 450 455 460 Ile Ala Met Pro Val Pro Val Ile Val Asn Asn Phe Gly Met Tyr Tyr 465 470 475 480 Ser Leu Ala Met Ala Lys Gln Lys Leu Pro Arg Lys Arg Lys Lys His 485 490 495 Ile Pro Pro Ala Pro Gln Ala Ser Ser Pro Thr Phe Cys Lys Thr Glu 500 505 510 Leu Asn Met Ala Cys Asn Ser Thr Gln Ser Asp Thr Cys Leu Gly Lys 515 520 525 Asp Asn Arg Leu Leu Glu His Asn Arg Ser Val Leu Ser Gly Asp Asp 530 535 540 Ser Thr Gly Ser Glu Pro Pro Leu Ser Pro Pro Glu Arg Leu Pro Ile 545 550 555 560 Arg Arg Ser Ser Thr Arg Asp Lys Asn Arg Arg Gly Glu Thr Cys Phe 565 570 575

Leu Leu Thr Thr Gly Asp Tyr Thr Cys Ala Ser Asp Gly Gly Ile Arg 580 585 590 Lys Gly Tyr Glu Lys Ser Arg Ser Leu Asn Asn Ile Ala Gly Leu Ala 595 600 605 Gly Asn Ala Leu Arg Leu Ser Pro Val Thr Ser Pro Tyr Asn Ser Pro 610 615 620

Cys Pro Leu Arg Arg Ser Arg Ser Pro Ile Pro Ser Ile Leu 625 630 635 < 210 > 3 < 211 > 2031 < 212 > DNA < 213 > Homo sapiens < 220 > < 221 > CDS < 222 > (193).. (2031) < 400 > 3 caccccagcg cccagggaag cggctcaacc acttgaatcc ggaaaacgcc aacaagtagt 60 ttctcgtcgg agaagggcgg ctcacctggg cgccaagact cagtcccgct gcccagagaa 120 cctcgtccac tcggaaacca aagcagaacc acttttctct cggtctcgtt aagtcatgtc 180 tgagtcacag ag atg ggc aag ate gag aac aac gag agg gtg ate ctc aat 231

Met Gly Lys Ile Glu Asn Asn Glu Arg Val Ile Leu Asn 1 5 10

gtc ggg ggc ace egg cac gaa ace tac cgc age ace etc aag ace ctg 279 Val Gly Gly Thr Arg His Glu Thr Tyr Arg Ser Thr Leu Lys Thr Leu 15 20 25 cct gga aca cgc ctg gcc ctt ctt gcc tee tee gag ccc cca ggc gac 327 Pro Gly Thr Arg Leu Ala Leu Leu Ala Ser Ser Glu Pro Pro Gly Asp 30 35 40 45 tgc ttg ace acg gcg ggc gac aag ctg cag ccg teg ccg cct cca ctg 375 Cys Leu Thr Thr Ala Gly Asp Lys Leu Gln Pro Ser Pro Pro Pro Leu 50 55 60 teg ccg ccg ccg aga gcg ccc ccg ctg tee ccc ggg cca ggc ggc tgc 423 Ser Pro Pro Pro Arg Ala Pro Pro Leu Ser Pro Gly Pro Gly Gly Cys 65 70 75 ttc gag ggc ggc gcg ggc aac tgc agt tee cgc ggc ggc agg gcc age 471 Phe Glu Gly Gly Ala Gly Asn Cys Ser Ser Arg Gly Gly Arg Ala Ser 80 85 90 gac cat ccc ggt ggc ggc cgc gag ttc ttc ttc gac egg cac ccg ggc 519 Asp His Pro Gly Gly Gly Arg Glu Phe Phe Phe Asp Arg His Pro Gly 95 100 105 gtc ttc gcc tat gtg etc aat tac tac cgc ace ggc aag ctg cac tgc 567 Val Phe Ala Tyr Val Leu Asn Tyr Tyr Arg Thr Gly Lys Leu His Cys 110 115 120 125 ccc gca gac gtg tgc ggg ccg etc ttc gag gag gag ctg gcc ttc tgg 615 Pro Ala Asp Val Cys Gly Pro Leu Phe Glu Glu Glu Leu Ala Phe Trp 130 135 140 ggc ate gac gag ace gac gtg gag ccc tgc tgc tgg atg ace tac egg 663 Gly Ile Asp Glu Thr Asp Val Glu Pro Cys Cys Trp Met Thr Tyr Arg 145 150 155 cag cac cgc gac gcc gag gag gcg ctg gac ate ttc gag ace ccc gac 711 Gln His Arg Asp Ala Glu Glu Ala Leu Asp Ile Phe Glu Thr Pro Asp 160 165 170 etc att ggc ggc gac ccc ggc gac gac gag gac ctg gcg gcc aag agg 759 Leu Ile Gly Gly Asp Pro Gly Asp Asp Glu Asp Leu Ala Ala Lys Arg 175 180 185 ctg ggc ate gag gac gcg gcg ggg etc ggg ggc ccg gac ggc aaa tct 807 Leu Gly Ile Glu Asp Ala Ala Gly Leu Gly Gly Pro Asp Gly Lys Ser 190 195 200 205 ggc cgc tgg agg agg ctg cag ccc cgc atg tgg gcc etc ttc gaa gac 855 Gly Arg Trp Arg Arg Leu Gln Pro Arg Met Trp Ala Leu Phe Glu Asp 210 215 220 ccc tac teg tee aga gcc gcc agg ttt att get ttt get tct tta ttc 903 Pro Tyr Ser Ser Arg Ala Ala Arg Phe Ile Ala Phe Ala Ser Leu Phe 225 230 235

ttc ate ctg gtt tea att aca act ttt tgc ctg gaa aca cat gaa get 951 Phe Ile Leu Val Ser Ile Thr Thr Phe Cys Leu Glu Thr His Glu Ala 240 245 250 ttc aat att gtt aaa aac aag aca gaa cca gtc ate aat ggc aca agt 999 Phe Asn Ile Val Lys Asn Lys Thr Glu Pro Val Ile Asn Gly Thr Ser 255 260 265 gtt gtt cta cag tat gaa att gaa acg gat cct gcc ttg acg tat gta 1047 Val Val Leu Gln Tyr Glu Ile Glu Thr Asp Pro Ala Leu Thr Tyr Val 270 275 280 285 gaa gga gtg tgt gtg gtg tgg ttt act ttt gaa ttt tta gtc cgt att 1095 Glu Gly Val Cys Val Val Trp Phe Thr Phe Glu Phe Leu Val Arg Ile 290 295 300 gtt ttt tea ccc aac aaa ctt gaa ttc ate aaa aat etc ttg aat ate 1143 Val Phe Ser Pro Asn Lys Leu Glu Phe Ile Lys Asn Leu Leu Asn Ile 305 310 315 att gac ttt gtg gcc ate cta cct ttc tac tta gag gtg gga etc agt 1191 Ile Asp Phe Val Ala Ile Leu Pro Phe Tyr Leu Glu Val Gly Leu Ser 320 325 330 ggg ctg tea tee aaa get get aaa gat gtg ctt ggc ttc etc agg gtg 1239 Gly Leu Ser Ser Lys Ala Ala Lys Asp Val Leu Gly Phe Leu Arg Val 335 340 345 gta agg ttt gtg agg ate ctg aga att ttc aag etc ace cgc cat ttt 1287 Val Arg Phe Val Arg Ile Leu Arg Ile Phe Lys Leu Thr Arg His Phe 350 355 360 365 gta ggt ctg agg gtg ctt gga cat act ctt cga get agt act aat gaa 1335 Val Gly Leu Arg Val Leu Gly His Thr Leu Arg Ala Ser Thr Asn Glu 370 375 380 ttt ttg ctg ctg ata att ttc ctg get cta gga gtt ttg ata ttt get 1383 Phe Leu Leu Leu Ile Ile Phe Leu Ala Leu Gly Val Leu Ile Phe Ala 385 390 395 ace atg ate tac tat gcc gag aga gtg gga get caa cct aac gac cct 1431 Thr Met Ile Tyr Tyr Ala Glu Arg Val Gly Ala Gln Pro Asn Asp Pro 400 405 410 tea get agt gag cac aca cag ttc aaa aac att ccc att ggg ttc tgg 1479 Ser Ala Ser Glu His Thr Gln Phe Lys Asn Ile Pro Ile Gly Phe Trp 415 420 425 tgg get gta gtg ace atg act ace ctg ggt tat ggg gat atg tac ccc 1527 Trp Ala Val Val Thr Met Thr Thr Leu Gly Tyr Gly Asp Met Tyr Pro 430 435 440 445 caa aca tgg tea ggc atg ctg gtg gga gcc ctg tgt get ctg get gga 1575 Gln Thr Trp Ser Gly Met Leu Val Gly Ala Leu Cys Ala Leu Ala Gly 450 455 460 gtg ctg aca ata gcc atg cca gtg cct gtc att gtc aat aat ttt gga 1623

Val Leu Thr Ile Ala Met Pro Val Pro Val Ile Val Asn Asn Phe Gly 465 470 475 atg tac tac tee ttg gca atg gca aag cag aaa ctt cca agg aaa aga 1671 Met Tyr Tyr Ser Leu Ala Met Ala Lys Gln Lys Leu Pro Arg Lys Arg 480 485 490 aag aag cac ate cct cct get cct cag gca age tea cct act ttt tgc 1719 Lys Lys His Ile Pro Pro Ala Pro Gln Ala Ser Ser Pro Thr Phe Cys 495 500 505 aag aca gaa tta aat atg gcc tgc aat agt aca cag agt gac aca tgt 1767 Lys Thr Glu Leu Asn Met Ala Cys Asn Ser Thr Gln Ser Asp Thr Cys 510 515 520 525 ctg ggc aaa gac aat cga ctt ctg gaa cat aac aga tea gtg tta tea 1815 Leu Gly Lys Asp Asn Arg Leu Leu Glu His Asn Arg Ser Val Leu Ser 530 535 540 ggt gac gac agt aca gga agt gag ccg cca cta tea ccc cca gaa agg 1863 Gly Asp Asp Ser Thr Gly Ser Glu Pro Pro Leu Ser Pro Pro Glu Arg 545 550 555 etc ccc ate aga cgc tct agt ace aga gac aaa aac aga aga ggg gaa 1911 Leu Pro Ile Arg Arg Ser Ser Thr Arg Asp Lys Asn Arg Arg Gly Glu 560 565 570 aca tgt ttc cta ctg acg aca ggt gat tac acg tgt get tct gat gga 1959 Thr Cys Phe Leu Leu Thr Thr Gly Asp Tyr Thr Cys Ala Ser Asp Gly 575 580 585 ggg ate agg aaa gat aac tgc aaa gag, gtt gtc att act ggt tac acg 2007 Gly Ile Arg Lys Asp Asn Cys Lys Glu Val Val Ile Thr Gly Tyr Thr 590 595 600 605 caa gcc gag gcc aga tct ctt act 2031 Gln Ala Glu Ala Arg Ser Leu Thr 610 < 210 > 4 < 211 > 613 < 212 > PRT < 213 > Homo sapiens < 400 > 4 Met Gly Lys Ile Glu Asn Asn Glu Arg Val Ile Leu Asn Val Gly Gly 1 5 10 15 Thr Arg His Glu Thr Tyr Arg Ser Thr Leu Lys Thr Leu Pro Gly Thr 20 25 30 Arg Leu Ala Leu Leu Ala Ser Ser Glu Pro Pro Gly Asp Cys Leu Thr 35 40 45 Thr Ala Gly Asp Lys Leu Gln Pro Ser Pro Pro Pro Leu Ser Pro Pro 50 55 60

Pro Arg Ala Pro Pro Leu Ser Pro Gly Pro Gly Gly Cys Phe Glu Gly 65 70 75 80 Gly Ala Gly Asn Cys Ser Ser Arg Gly Gly Arg Ala Ser Asp His Pro 85 90 95 Gly Gly Gly Arg Glu Phe Phe Phe Asp Arg His Pro Gly Val Phe Ala 100 105 110 Tyr Val Leu Asn Tyr Tyr Arg Thr Gly Lys Leu His Cys Pro Ala Asp 115 120 125 Val Cys Gly Pro Leu Phe Glu Glu Glu Leu Ala Phe Trp Gly Ile Asp 130 135 140 Glu Thr Asp Val Glu Pro Cys Cys Trp Met Thr Tyr Arg Gln His Arg 145 150 155 160 Asp Ala Glu Glu Ala Leu Asp Ile Phe Glu Thr Pro Asp Leu Ile Gly 165 170 175 Gly Asp Pro Gly Asp Asp Glu Asp Leu Ala Ala Lys Arg Leu Gly Ile 180 185 190 Glu Asp Ala Ala Gly Leu Gly Gly Pro Asp Gly Lys Ser Gly Arg Trp 195 200 205 Arg Arg Leu Gln Pro Arg Met Trp Ala Leu Phe Glu Asp Pro Tyr Ser 210 215 220 Ser Arg Ala Ala Arg Phe Ile Ala Phe Ala Ser Leu Phe Phe Ile Leu 225 230 235 240 Val Ser Ile Thr Thr Phe Cys Leu Glu Thr His Glu Ala Phe Asn Ile 245 250 255 Val Lys Asn Lys Thr Glu Pro Val Ile Asn Gly Thr Ser Val Val Leu 260 265 270 Gln Tyr Glu Ile Glu Thr Asp Pro Ala Leu Thr Tyr Val Glu Gly Val 275 280 285 Cys Val Val Trp Phe Thr Phe Glu Phe Leu Val Arg Ile Val Phe Ser 290 295 300 Pro Asn Lys Leu Glu Phe Ile Lys Asn Leu Leu Asn Ile Ile Asp Phe 305 310 315 320 Val Ala Ile Leu Pro Phe Tyr Leu Glu Val Gly Leu Ser Gly Leu Ser 325 330 335 Ser Lys Ala Ala Lys Asp Val Leu Gly Phe Leu Arg Val Val Arg Phe 340 345 350 Val Arg Ile Leu Arg Ile Phe Lys Leu Thr Arg His Phe Val Gly Leu 355 360 365

Arg Val Leu Gly His Thr Leu Arg Ala Ser Thr Asn Glu Phe Leu Leu 370 375 380 Leu Ile Ile Phe Leu Ala Leu Gly Val Leu Ile Phe Ala Thr Met Ile 385 390 395 400 Tyr Tyr Ala Glu Arg Val Gly Ala Gln Pro Asn Asp Pro Ser Ala Ser 405 410 415 Glu His Thr Gln Phe Lys Asn Ile Pro Ile Gly Phe Trp Trp Ala Val 420 425 430 Val Thr Met Thr Thr Leu Gly Tyr Gly Asp Met Tyr Pro Gln Thr Trp 435 440 445 Ser Gly Met Leu Val Gly Ala Leu Cys Ala Leu Ala Gly Val Leu Thr 450 455 460 Ile Ala Met Pro Val Pro Val Ile Val Asn Asn Phe Gly Met Tyr Tyr 465 470 475 480 Ser Leu Ala Met Ala Lys Gln Lys Leu Pro Arg Lys Arg Lys Lys His 485 490 495 Ile Pro Pro Ala Pro Gln Ala Ser Ser Pro Thr Phe Cys Lys Thr Glu 500 505 510 Leu Asn Met Ala Cys Asn Ser Thr Gln Ser Asp Thr Cys Leu Gly Lys 515 520 525 Asp Asn Arg Leu Leu Glu His Asn Arg Ser Val Leu Ser Gly Asp Asp 530 535 540 Ser Thr Gly Ser Glu Pro Pro Leu Ser Pro Pro Glu Arg Leu Pro Ile 545 550 555 560 Arg Arg Ser Ser Thr Arg Asp Lys Asn Arg Arg Gly Glu Thr Cys Phe 565 570 575 Leu Leu Thr Thr Gly Asp Tyr Thr Cys Ala Ser Asp Gly Gly Ile Arg 580 585 590

Lys Asp Asn Cys Lys Glu Val Val Ile Thr Gly Tyr Thr Gln Ala Glu 595 600 605 Ala Arg Ser Leu Thr 610

Claims (18)

1. CLAIMS 1. An isolated voltage-gated potassium channel polypeptide comprising (i) the amino acid sequence of SEQ ID NO: 2 or SEQ ID NO: 4; or (ii) a variant thereof which capable of forming a channel which can be activated by depolarisation of the cell membrane potential above the reversal potential for K+ (EK) ; or (iii) a fragment of (i) or (ii) which capable of forming a channel which can be activated by depolarisation of the cell membrane potential above the reversal potential for K+ (es).
2. 2. A polypeptide according to claim 1 wherein the variant (ii) has at least 98% identity to the amino acid sequence of SEQ ID NO: 2 or SEQ ID NO: 4.
3. 3. A polynucleotide encoding a polypeptide according to claim 1 or 2.
4. 4. A polynucleotide according to claim 3 which is a cDNA sequence.
5. 5. A polynucleotide encoding a voltage-gated potassium channel polypeptide which is capable of forming a channel which can be activated by depolarisation of the cell membrane potential above the reversal potential K+ (EK), which polynucleotide comprises: (a) the nucleic acid sequence of SEQ ID NO: 1 or SEQ ID NO : 3 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 98% identity to a sequence as defined in (a), (b) or (c).
6. 6. An expression vector comprising a polynucleotide according to any one of claims 3 to 5.
7. 7. A host cell comprising an expression vector according to claim 6.
8. 8. An antibody specific for a polypeptide according to claim 1 or 2.
9. 9. A method for the identification of a substance that modulates voltagegated potassium 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 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 potassium channel activity and/or expression.
10. 10. A method according to claim 9 wherein the polypeptide is expressed in a cell.
11. 11. A method according to claim 10 wherein the cell expresses other Kv3 subfamily members.
12. 12. A method according to any one of claims 9 to 11 wherein step (ii) comprises monitoring any voltage-gated potassium channel activity.
13. 13. A substance which modulates voltage-gated potassium channel activity and which is identifiable by a method according to any one of claims 9 to 12.
14. 14. A method of treating a subject having a disorder that is responsive to voltage-gated potassium channel modulation, which method comprises administering to said subject an effective amount of a substance according to claim 13.
15. 15. A method according to claim 14 wherein the disorder is selected from epilepsy, juvenile myoclonic epilepsy (JME), temporal lobe epilepsy (TLE), seizure disorders, sleep disorders such as insomnia, hypersomnia, parasomnia, sleep apnea syndromes and stupor, pain states such as acute postoperative pain, psychogenic pain syndromes, pain from cancer, glossopharyngeal neuralgia, inflammatory pain, neuropathic pain, migraine, trigeminal neuralgia, headache and tension headache, neurodegenerative diseases such as Alzheimer's disease, Huntington's disease, Parkinson's disease, palsies and paralysis, pyschiatric disorders such as anxiety, depression, bipolar disorder, schizophrenia, paranoid psychoses and thyroid disorders such as euthyroid sick syndrome, hyperthyroidism, hypothyroidism, simple goiter and thyroiditis.
16. 16. Use of a substance as defined in claim 13 in the manufacture of a medicament for treatment or prophylaxis of a disorder that is responsive to

    stimulation or modulation of voltage-gated potassium channel activity.
17. 17. A use according to claim 16 wherein the disorder is selected from epilepsy, juvenile myoclonic epilepsy (JME), temporal lobe epilepsy (TLE), seizure disorders, sleep disorders such as insomnia, hypersomnia, parasomnia, sleep apnea syndromes and stupor, pain states such as acute postoperative pain, psychogenic pain syndromes, pain from cancer, glossopharyngeal neuralgia, inflammatory pain, neuropathic pain, migraine, trigeminal neuralgia, headache and tension headache, neurodegenerative diseases such as Alzheimer's disease, Huntington's disease, Parkinson's disease, palsies and paralysis, pyschiatric disorders such as anxiety, depression, bipolar disorder, schizophrenia, paranoid psychoses and thyroid disorders such as euthyroid sick syndrome, hyperthyroidism, hypothyroidism, simple goiter and thyroiditis.
18. 18. 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.