WO2000053628A2

WO2000053628A2 - Genes encoding human potassium channel proteins

Info

Publication number: WO2000053628A2
Application number: PCT/EP2000/001750
Authority: WO
Inventors: David Malcolm Duckworth; Robert James Godden; Conrad Gerald Chapman; Helen Jane Meadows
Original assignee: Smithkline Beecham Plc
Priority date: 1999-03-05
Filing date: 2000-03-02
Publication date: 2000-09-14
Also published as: EP1165777A2; WO2000053628A3; JP2002542765A

Abstract

DKCN1 polypeptides and polynucleotides and methods for producing such polypeptides by recombinant techniques are disclosed. Also disclosed are methods for utilizing DKCN1 polypeptides and polynucleotides in diagnostic assays.

Description

Novel Compounds

Field of the Invention

This invention relates to newly identified polypeptides and polynucleotides encoding such polypeptides, to their use in diagnosis and in ιdentιf\ ing compounds that may be agonists, antagonists that are potentially useful in therapy, and to production of such polypeptides and polynucleotides

Background of the Invention The drug discovery process is currently undergoing a fundamental revolution as it embraces

"functional genomics", that is, high throughput genome- or gene-based biology This approach as a means to identify genes and gene products as therapeutic targets is rapidly supercedmg earlier approaches based on "positional cloning" A phenotype, that is a biological function or genetic disease, would be identified and this would then be tracked back to the responsible gene, based on its genetic map position

Functional genomics relies heavily on high-throughput DNA sequencing technologies and the various tools of biomformatics to identify gene sequences of potential interest from the many molecular biology databases now available There is a continuing need to identify and charactense further genes and their related polypeptides/proteins, as targets for drug discovery

Summary of the Invention

The present invention relates to DKCNl , in particular DKCNl polypeptides and DKCNl polynucleotides, recombinant materials and methods for their production Such polypeptides and polynucleotides are of interest in relation to methods of treatment of certain diseases, including, but not limited to, cancer, pulmonary disease, cardiovascular diseases, inflammatory diseases, renal disease, pam, psychiatric disorders including depression and schizophrenia, neurodegenerative disease including Alzheimer's, stroke and head trauma, and neurological disorders including migraine, epilepsy, cognitive dysfunction/enhancement attention deficit disorder, addiction, dyskinesias including Parkinson's and Huntington s chorea cerebral palsy, anxiety / social phobia, sleep-related disorders, the induction of sleep, incontinence erectile dysfunction, alopecia, hereinafter referred to as " diseases of the invention In a further aspect, the invention relates to methods for identifying agonists and antagonists (β s; , inhibitors) using the materials provided by the invention, and treating conditions associated with DKCNl imbalance with the identified compounds In a still further aspect, the invention relates to diagnostic assays for detecting diseases associated with inappropπate DKCNl activity or levels Description of the Invention

In a first aspect, the present invention relates to DKCNl polypeptides Such polypeptides include (a) an isolated polvpeptide encoded by a polynucleotide comprising the sequence of SEQ ID NO 1. (b) an isolated poh peptide comprising a polypeptide sequence having at least 95%, 96% 97% 98%, or 99% identity to the polypeptide sequence of SEQ ID NO 2

(c) an isolated poh peptide comprising the poh peptide sequence of SEQ ID NO 2,

(d) an isolated polypeptide having at least 95% 96% 97% 98% or 99% identity to the polypeptide sequence of SEQ ID NO 2 (e) the polypeptide sequence of SEQ ID NO 2 and

(f) an isolated polypeptide having or comprising a polypeptide sequence that has an Identity Index of 0 95, 0 96, 0 97. 0 98, or 0 99 compared to the polypeptide sequence of SEQ ID NO 2,

(g) fragments and \aπants of such polypeptides in (a) to (0

Polypeptides of the present invention are believed to be members of the potassium channel family of polypeptides They are therefore of interest because potassium channels are a group of ion channels that are important in controlling excitability and modulating secretory processes They have a number of roles including neuronal integration volume regulation maintenance of the resting membrane potential and an important role in determining the frequency and duration of action potentials The changes in cell excitability that follow modulation of potassium channels gι\e rise to a broad number of potential therapeutic uses of such modulators

The biological properties of the DKCNl are hereinafter referred to as biological activity of DKCNl " or "DKCNl activity Preferably a polvpeptide of the present invention exhibits at least one biological activity of DKCNl

Polypeptides of the present invention also includes variants of the aforementioned polypeptides, including all allelic forms and splice

Such polypeptides vary from the reference polypeptide by insertions, deletions, and substitutions that may be conservative or non- conservative, or any combination thereof Particularly preferred variants are those in which several for instance from 50 to 30 from 30 to 20 from 20 to 10 from 10 to 5 from 5 to ^'s from 3 to 2, from 2 to 1 or 1 ammo acids are inserted substituted or deleted in anv combination Preferred fragments of polypeptides of the present invention include an isolated polypeptide comprising an amino acid sequence hav ing at least 30 ^0 or 100 contiguous amino acids from the amino acid sequence of SEQ ID NO 2 or an isolated polv peptide comprising an amino acid sequence having at least 30 50 or 100 contiguous ammo acids truncated oi deleted from the amino acid sequence of SEQ ID NO 2 Preferred fragments are biologically active fragments that mediate the biological activity of DKCNl including those with a similar acti itv oi an improved activity or with a decreased undesirable activitv Also preferred are those fragments that are antigenic or lmmunogemc in an animal, especiallv in a human

Fragments of the polypeptides of the inv ention mav be employed for producing the corresponding full-length polypeptide by peptide synthesis, therefore, these variants may be employed as intermediates for producing the full-length polypeptides of the inv ention The polypeptides of the present invention mav be in the form of the mature" protein or may be a part of a larger protein such as a precursor or a fusion protein It is often advantageous to include an additional amino acid sequence that contains secretorv or leader sequences, pro-sequences, sequences that aid in purification, for instance multiple histidine residues, or an additional sequence for stability during recombinant production

Polypeptides of the present invention can be prepared in any suitable manner, for instance by isolation form naturally occuπng sources, from genetically engineered host cells compπsing expression systems ι v ide infra) or by chemical synthesis using for instance automated peptide synthesisers, or a combination of such methods Means for preparing such polypeptides are well understood in the an

In a further aspect, the present invention relates to DKCNl polynucleotides Such polynucleotides include

(a) an isolated pol nucleotide comprising a polynucleotide sequence having at least 95%, 96%, 97%, 98%, or 99% identity to the polynucleotide squence of SEQ ID NO 1 ,

(b) an isolated polynucleotide comprising the polynucleotide of SEQ ID NO 1,

(c) an isolated polynucleotide having at least 95%, 96%. 97%, 98%, or 99% identity to the polynucleotide of SEQ ID NO 1 ,

(d) the isolated poly nucleotide of SEQ ID NO 1 , (e) an isolated poly nucleotide comprising a polynucleotide sequence encoding a polypeptide sequence having at least 95%, 967c 977c. 987c. or 997c identity to the polypeptide sequence of SEQ

ID NO 2,

(0 <m isolated polynucleotide compπsing a polynucleotide sequence encoding the polypeptide of

SEQ ID NO 2, (g) an isolated polv nucleotide having a polynucleotide sequence encoding a polv peptide sequence having at least 957c 96%*. 977c, 987c. or 997c identitv to the polypeptide sequence of SEQ ID

NO.2.

(h) an isolated polv nucleotide encoding the polypeptide of SEQ ID NO 2,

(0 an isolated poly nucleotide having or compπsing a polv nucleotide sequence that has an Identity Index of 0 95, 0 96 0 97, 0 98. or 0 99 compared to the polynucleotide sequence of SEQ ID NO 1 (j) an isolated polynucleotide having or comprising a polynucleotide sequence encoding a polypeptide sequence that has an Identity Index of 0 95, 0 96, 0 97, 0 98, or 0 99 compared to the polypeptide sequence of SEQ ID NO 2 and polynucleotides that are fragments and v ariants of the above mentioned polynucleotides or that are complementary to above mentioned polynucleotides ovei the entire length thereof Preferred fragments of polynucleotides of the present invention include an isolated polynucleotide compπsing an nucleotide sequence hav ing at least 15, 30, 50 or 100 contiguous nucleotides from the sequence of SEQ ID NO 1. or an isolated polynucleotide comprising an sequence having at least 30, 50 or 100 contiguous nucleotides truncated or deleted from the sequence of SEQ ID NO 1

Preferred variants of polynucleotides of the present invention include splice variants, allelic variants, and polymorphisms, including polynucleotides having one or more single nucleotide polymorphisms (SNPs)

Polynucleotides of the present invention also include polynucleotides encoding polypeptide vaπants that compπse the amino acid sequence of SEQ ED NO 2 and in which several, for instance from 50 to 30, from 30 to 20, from 20 to 10, from 10 to 5. from 5 to 3, from 3 to 2. from 2 to 1 or 1 amino acid residues are substituted, deleted or added, in any combination

In a further aspect, the present invention provides polynucleotides that are RNA transcripts of the DNA sequences of the present invention Accordingly, there is provided an RNA polynucleotide that

(a) comprises an RNA transcript of the DNA sequence encoding the polypeptide of SEQ ID NO.2;

(b) is the RNA transcript of the DNA sequence encoding the polypeptide of SEQ ID NO.2, (c) comprises an RNA transcπpt of the DNA sequence of SEQ ID NO 1 , or

(d) is the RNA transcript of the DNA sequence of SEQ ID NO 1 , and RNA polynucleotides that are complementary thereto

The polynucleotide sequence of SEQ ID NO 1 shows homology with rat TWIK-related acid- sensitive potassium channel (D Leonoudakis et al . J Neuroscience 18 868-877 1998) The polynucleotide sequence of SEQ ID NO 1 is a cDNA sequence that encodes the polypeptide of SEQ ID NO 2 The polynucleotide sequence encoding the poly peptide of SEQ ID NO 2 may be identical to the polypeptide encoding sequence of SEQ ID NO 1 oi it may be a sequence other than SEQ ID NO 1. which, as a result of the redundancy (degeneracy) of the genetic code, also encodes the polypeptide of SEQ ID NO 2 The polypeptide of the SEQ ID NO 2 is related to other proteins of the potassium channel family, having homology and/or structural similarity with rat

TWIK-related acid-sensitive potassium channel (D. Leonoudakis et al., J. Neuroscience 18: 868-877,

1998).

Preferred polypeptides and polynucleotides of the present invention are expected to have, inter alia, similar biological functions/properties to their homologous polypeptides and polynucleotides. Furthermore, preferred polypeptides and polynucleotides of the present invention have at least one DKCNl activity. he present invention also relates to partial or other polynucleotide and polypeptide sequences which were first identified prior to the determination of the corresponding full length sequences of SEQ ID NO: 1 and SEQ ID NO:2.

Accordingly, in a further aspect, the present invention provides for an isolated polynucleotide which:

(a) comprises a nucleotide sequence which has at least 95% identity, preferably at least 97-99% identity to SEQ ID NO:3 over the entire length of SEQ ED NO:3; (b) has a nucleotide sequence which has at least 95% identity, preferably at least 97-99% identity, to SEQ ID NO:3 over the entire length of SEQ ID NO:3:

(c) comprises the polynucleotide of SEQ ID NO:3; or

(d) has a nucleotide sequence encoding a polypeptide which has at least 95% identity, even more preferably at least 97-99% identity, to the amino acid sequence of SEQ ID NO:4, over the entire length of SEQ ID NO:4; as well as the polynucleotide of SEQ ID NO:3.

The present invention further provides for a polypeptide which:

(a) comprises an amino acid sequence which has at least 957c identity, preferably at least 97-99% identity, to that of SEQ ID NO:4 over the entire length of SEQ ID NO:4; (b) has an amino acid sequence which is at least 957c identity, preferably at least 97-99% identity, to the amino acid sequence of SEQ ID NO:4 over the entire length of SEQ ID NO:4;

(c) comprises the amino acid of SEQ ID NO:4: and

(d) is the polypeptide of SEQ ID NO:4; as well as polypeptides encoded by a polynucleotide comprising the sequence contained in SEQ ID NO:3.

The nucleotide sequence of SEQ ID NO:3 and the peptide sequence encoded thereby are derived from EST (Expressed Sequence Tag) sequences. It is recognised by those skilled in the art that there will inevitably be some nucleotide sequence reading errors in EST sequences (see Adams, M.D. et al. Nature 377 (supp) 3, 1995). Accordingly, the nucleotide sequence of SEQ ID NO:3 and the peptide sequence encoded therefrom are therefore subject to the same inherent limitations in sequence accuracy Furthermore, the peptide sequence encoded by SEQ ID NO 3 comprises a region of identity or close homology and/or close structural similarity (for example a conservative amino acid difference) with the closest homologous or structurally similar protein Polynucleotides of the present in ention may be obtained using standard cloning and screening techniques from a cDNA librarv derived from mRNA in cells of human infant brain, (see for instance, Sambrook et al , Molecular Cloning A Laboratory Manual, 2nd Ed , Cold Spring Harbor Laboratory Press, Cold Spring Harbor, N Y ( 1989)) Polynucleotides of the invention can also be obtained from natural sources such as genomic DNA libraries or can be synthesized using well known and commercially available techniques When polynucleotides of the present invention are used for the recombinant production of polypeptides of the present invention, the polynucleotide may include the coding sequence for the mature polypeptide, by itself, or the coding sequence for the mature polypeptide in reading frame with other coding sequences, such as those encoding a leader or secretory sequence, a pre-, or pro- or prepro- protein sequence, or other fusion peptide poπions For example, a marker sequence that facilitates punfication of the fused polypeptide can be encoded In certain preferred embodiments of this aspect of the invention, the marker sequence is a hexa-histidine peptide, as provided in the pQE vector (Qiagen, Inc ) and described in Gentz et al , Proc Natl Acad Sci USA ( 1989) 86 821-824, or is an HA tag The polynucleotide may also contain non coding 5' and 3' sequences, such as transcribed, non-translated sequences, splicing and polyadenylation signals, πbosome binding sites and sequences that stabilize mRNA

Polynucleotides that are identical, or have sufficient identity to a polynucleotide sequence of SEQ ID NO 1, may be used as hybridization probes for cDNA and genomic DNA or as primers for a nucleic acid amplification reaction (for instance, PCR) Such probes and primers may be used to isolate full-length cDNAs and genomic clones encoding polypeptides of the present invention and to isolate cDNA and genomic clones of other genes (including genes encoding paralogs from human sources and orthologs and paralogs from species other than human) that have a high sequence similaπty to SEQ ID NO 1, typically at least 957c identity Preferred probes and primers will generally comprise at least 15 nucleotides preferablv at least 30 nucleotides and may have at least 50, if not at least 100 nucleotides Particularly preferred probes will have between 30 and 50 nucleotides Particularly preferred primers will have between 20 and 25 nucleotides

A polynucleotide encoding a polypeptide of the present invention, including homologs from species other than human, may be obtained by a process compπsing the steps of screening a library under stringent hybridization conditions vv ith a labeled probe having the sequence of SEQ ID NO 1 or a fragment thereof, preferably of at least 15 nucleotides and isolating full-length cDNA and genomic clones containing said polynucleotide sequence Such hybridization techniques are well known to the skilled artisan Preferred stringent hybridization conditions include overnight incubation at 42°C in a solution comprising 50% formamide 5xSSC (150mM NaCI, 15mM tπsodium citrate), 50 mM sodium phosphate (pH7 6) 5\ Denhardt's solution, 10 % dextran sulfate, and 20 microgram/ml denatured sheared salmon sperm DNA followed by washing the filters in 0 lx SSC at about 65°C Thus the present invention also includes isolated polynucleotides preferably with a nucleotide sequence of at least 100 obtained by screening a library under stπngent hybridization conditions with a labeled probe hav ing the sequence of SEQ ID NO 1 or a fragment thereof, preferably of at least 15 nucleotides

The skilled artisan will appreciate that in manv cases an isolated cDNA sequence will be incomplete, in that the region coding for the polypeptide does not extend all the way through to the 5' terminus This is a consequence of reverse transcriptase, an enzyme with inherently low "processivity" (a measure of the ability of the enzyme to remain attached to the template duπng the polymerisation reaction), failing to complete a DNA copy of the mRNA template duπng first strand cDNA synthesis There are several methods available and well known to those skilled in the art to obtain full-length cDNAs, or extend short cDNAs, for example those based on the method of Rapid Amplification of cDNA ends (RACE) (see, for example Frohman et al , Proc Nat Acad Sci USA 85, 8998-9002, 1988) Recent modifications of the technique exemplified by the Marathon (trade mark) technology (Clontech Laboratories Inc ) for example have significantly simplified the search for longer cDNAs In the Marathon (trade mark) technology, cDNAs have been prepared from mRNA extracted from a chosen tissue and an 'adaptor' sequence ligated onto each end Nucleic acid amplification (PCR) is then earned out to amplify the ' missing" 5 ' end of the cDNA using a combination of gene specific and adaptor specific ohgonucleotide primers The PCR reaction is then repeated using 'nested primers that is, primers designed to anneal within the amplified product (typically an adaptor specific primer that anneals further 3 in the adaptor sequence and a gene specific primer that anneals further 5 in the known gene sequence) The products of this reaction can then be analysed bv DNA sequencing and a full-length cDNA constructed either bv joining the product directly to the existing cDNA to give a complete sequence, or carrying out a separate full-length PCR using the new sequence information for the design of the 5' primer

Recombinant polypeptides of the present invention may be prepared bv processes well known in the art from genetically engineered host cells comprising expression sv stems Accordingly in a further aspect, the present invention relates to expression systems comprising a polvnucleoude or polynucleotides of the present invention to host cells which are genetically engineered with such

- 7 -

SUBSTITLTE SHEET (RULE 26) expression svtems and to the production of polypeptides of the invention bv recombinant techniques Cell-free translation systems can also be employed to produce such proteins using RNAs deπved from the DNA constructs of the present invention

For recombinant production host cells can be genetically engineered to incorporate expression systems or portions thereof for polynucleotides of the present invention Polynucleotides may be introduced into host cells by methods described in many standard laboiatorv manuals, such as Davis et al Basic Methods in Molecular Biology ( 1986) and Sambrook et al ( ibid) Preferred methods of introducing polynucleotides into host cells include, for instance, calcium phosphate transfection, DEAE-dextran mediated transfection. transvection, microinjection cationic pid- mediated transfection electroporation, transduction scrape loading, ballistic introduction or infection

Representativ e examples of appropriate hosts include bacterial cells, such as Sti eptococci, Staphylococci, E coll Sti eptomvces and Bacillus subtilis cells, fungal cells, such as yeast cells and Aspergillus cells, insect cells such as Dtosophύa S2 and Spodoptera Sf9 cells, animal cells such as CHO, COS, HeLa, C127, 3T3, BHK, HEK 293 and Bowes melanoma cells and plant cells

A great vaπety of expression systems can be used, for instance, chromosomal, episomal and virus-derived systems, e g , vectors derived from bacterial plasmids, from bacteπophage, from transposons, from yeast episomes, from insertion elements, from yeast chromosomal elements, from viruses such as baculoviruses, papova viruses, such as SV40, vaccinia viruses, adenoviruses, fowl pox viruses, pseudorabies viruses and retroviruses, and vectors derived from combinations thereof, such as those derived from plasmid and bacteπophage genetic elements, such as cosmids and phagemids The expression systems may contain control regions that regulate as well as engender expression Generally any system or vector that is able to maintain, propagate or express a polynucleotide to produce a polypeptide in a host may be used The appropriate polynucleotide sequence may be inserted into an expression system by any of a variety of well-known and routine techniques, such as. for example, those set forth in Sambrook et al , (ibid) Appropriate secretion signals may be incorporated into the desired polypeptide to allow secretion of the translated protein into the lumen of the endoplasmic reticulum the peπplasmic space or the extiacellular environment These signals may be endogenous to the polypeptide or thev mav be heteiologous signals If a polypeptide of the present inv ention is to be expressed for use in screening assays, it is generally preferred that the polypeptide be produced at the surface of the cell In this e ent, the cells may be harvested prior to use in the screening assav If the polypeptide is secreted into the medium, the medium can be recoveied in order to recover and purify the polv peptide If produced intracellularly. the cells must fu st be lysed before the polypeptide is iecovered Polypeptides of the present invention can be recovered and purified from recombinant cell cultures by well-known methods including ammonium sulfate or ethanol precipitation, acid extraction, anion or cation exchange chromatography phosphocellulose chromatography, hydrophobic interaction chromatography, affinity chromatography, hydroxylapatite chromatography and lectm chromatography Most preferablv high performance liquid chromatography is employed for purification Well known techniques for refolding proteins may be employed to regenerate active conformation when the polypeptide is denatured duπng intracellular synthesis, isolation and/or puπfication

Polynucleotides of the present invention may be used as diagnostic reagents, through detecting mutations in the associated gene Detection of a mutated form of the gene characteπsed by the polynucleotide of SEQ ED NO 1 in the cDNA or genomic sequence and which is associated with a dysfunction will provide a diagnostic tool that can add to. or define, a diagnosis of a disease, or susceptibility to a disease, which results from under-expression, over-expression or altered spatial or temporal expression of the gene Individuals carrying mutations in the gene may be detected at the DNA level by a vaπety of techniques well known in the art

Nucleic acids for diagnosis may be obtained from a subject's cells, such as from blood, urine, saliva, tissue biopsy or autopsy material The genomic DNA may be used directly for detection or it may be amplified enzymatically by using PCR. preferably RT-PCR, or other amplification techniques pπor to analysis RNA or cDNA may also be used in similar fashion Deletions and insertions can be detected by a change in size of the amplified product in comparison to the normal genotype Point mutations can be identified by hybridizing amplified DNA to labeled DKCNl nucleotide sequences Perfectly matched sequences can be distinguished from mismatched duplexes by RNase digestion or by differences in melting temperatures DNA sequence difference may also be detected by alterations in the electrophoretic mobility of DNA fragments in gels, with or without denaturing agents, or by direct DNA sequencing (see for instance, Myers et al . Science ( 1985) 230.1242) Sequence changes at specific locations may also be revealed by nuclease protection assays, such as RNase and S 1 protection or the chemical cleavage method (see Cotton et al , Proc Natl Acad Sci USA ( 1985) 85 4397-4401 )

An array of o gonucleotides probes compπsing DKCNl polynucleotide sequence or fragments thereof can be constructed to conduct efficient screening of e t> genetic mutations Such arrays are preferably high density arrays or grids Arrav technology methods are well known and have general applicability and can be used to address a aπety of questions in molecular genetics including gene expression, genetic linkage, and genetic variability, see, for example, M Chee et al , Science, 274, 610-613 (1996) and other references cited therein Detection of abnormally decreased or increased levels of polypeptide or mRNA expression may also be used for diagnosing or determining susceptibility of a subject to a disease of the invention Decreased or increased expiession can be measured at the RNA level using any of the methods well known in the art for the quantitation of polynucleotides, such as for example, nucleic acid amplification, for instance PCR, RT-PCR, RNase protection. Northern blotting and other hybridization methods Assay techniques that can be used to determine le els of a protein, such as a polypeptide of the present invention in a sample derived from a host are well-known to those of skill in the art Such assay methods include radioimmunoassays, competitive-binding assays, Western Blot analysis and ELISA assays Thus in another aspect, the present invention relates to a diagonostic kit compπsing

(a) a polynucleotide of the present invention, preferably the nucleotide sequence of SEQ ID NO 1, or a fragment or an RNA transcript thereof.

(b) a nucleotide sequence complementary to that of (a)

(c) a polypeptide of the present invention, preferably the polypeptide of SEQ ID NO 2 or a fragment thereof, or

(d) an antibody to a polypeptide of the present inv ention, preferably to the polypeptide of SEQ ID NO 2

It will be appreciated that in any such kit (a), (b). (c) or (d) may comprise a substantial component Such a kit will be of use in diagnosing a disease or susceptibility to a disease, particularly diseases of the invention, amongst others

The polynucleotide sequences of the present invention are valuable for chromosome localisation studies The sequence is specifically targeted to and can hybridize with, a particular location on an individual human chromosome The mapping of relevant sequences to chromosomes according to the present invention is an important first step in correlating those sequences with gene associated disease Once a sequence has been mapped to a precise chromosomal location, the physical position of the sequence on the chromosome can be correlated with genetic map data Such data are found in, for example. V McKusick. Mende an Inheritance in Man (available on-line through Johns Hopkins University Welch Medical Library) The relationship between genes and diseases that have been mapped to the same chromosomal region are then identified through linkage analysis (co-inheritance of physically adjacent genes) Pi ease human chromosomal localisations for a genomic sequence (gene fragment etc ) can be determined using Radiation Hybrid (RH) Mapping (Walter, M Spillett, D , Thomas, P , Weissenbach. J . and Goodfello P . ( 1994) A method for constructing radiation hybrid maps of whole genomes, Nature Genetics 7. 22-28) A numbei of RH panels are available from Research Genetics (Huntsville. AL USA) e g the GeneBπdge4 RH panel (Hum Mol Genet 1996 Mar;5(^'3):339-46 A radiation hybrid map of the human genome. Gyapay G, Schmitt K. Fizames C, Jones H, Vega-Czarny N. Spillett D. Muselet D, Prud^'Homme JF, Dib C, Auffray C, Morissette J. Weissenbach J. Goodfellow PN). To determine the chromosomal location of a gene using this panel. 93 PCRs are performed using primers designed from the gene of interest on RH DNAs. Each of these DNAs contains random human genomic fragments maintained in a hamster background (human / hamster hybrid cell lines). These PCRs result in 93 scores indicating the presence or absence of the PCR product of the gene of interest. These scores are compared with scores created using PCR products from genomic sequences of known location. This comparison is conducted at http://www.genome.wi.mit.edu/. The gene of the present invention maps to human chromosome 8q24.2-qtel.

The polynucleotide sequences of the present invention are also valuable tools for tissue expression studies. Such studies allow the determination of expression patterns of polynucleotides of the present invention which may give an indication as to the expression patterns of the encoded polypeptides in tissues, by detecting the mRNAs that encode them. The techniques used are well known in the art and include in situ hydridisation techniques to clones arrayed on a grid, such as cDNA microarray hybridisation (Schena et al. Science. 270. 467-470, 1995 and Shalon et al. Genome Res, 6, 639-645, 1996) and nucleotide amplification techniques such as PCR. A preferred method uses the TAQMAN (Trade mark) technology available from Perkin Elmer. Results from these studies can provide an indication of the normal function of the polypeptide in the organism. In addition, comparative studies of the normal expression pattern of mRNAs with that of mRNAs encoded by an alternative form of the same gene (for example, one having an alteration in polypeptide coding potential or a regulatory mutation) can provide valuable insights into the role of the polypeptides of the present invention, or that of inappropriate expression thereof in disease. Such inappropriate expression may be of a temporal, spatial or simply quantitative nature.

The polypeptides of the present invention are expressed in brain and pituitary as well as other tissues.

A further aspect of the present invention relates to antibodies. The polypeptides of the invention or their fragments, or cells expressing them, can be used as immunogens to produce antibodies that are immunospecific for polypeptides of the present invention. The term

"immunospecific" means that the antibodies have substantially greater affinity for the polypeptides of the invention than their affinity for other related polypeptides in the prior art.

Antibodies generated against polypeptides of the present invention may be obtained by administering the polypeptides or epitope-bearing fragments, or cells to an animal, preferably a non- human animal, using routine protocols. For preparation of monoclonal antibodies, any technique which provides antibodies produced by continuous cell line cultures can be used Examples include the hybπdoma technique (Kohlei G and Milstein C Nature ( 1975) 256 495-497), the tπoma technique, the human B-cell hybπdoma technique (Kozbov et al Immunology Today (1983) 4 72) and the EBV-hybπdoma technique (Cole et al Monoclonal Antibodies and Cancer Therapy, 77-96, Alan R Liss, Inc , 1985)

Techniques for the production of single chain antibodies, such as those described in U S Patent No 4 946,778 can also be adapted to produce single chain antibodies to polypeptides of this invention Also, transgenic mice or other organisms including other mammals, may be used to express humanized antibodies The above-described antibodies may be employed to isolate or to identify clones expressing the polypeptide or to purify the polypeptides by affinity chromatography Antibodies against polypeptides of the present invention may also be employed to treat diseases of the invention, amongst otheis

Polypeptides and polynucleotides of the present invention may also be used as vaccines

Accordingly, in a further aspect, the present invention relates to a method for inducing an immunological response in a mammal that comprises inoculating the mammal with a polypeptide of the present invention, adequate to produce antibody and/or T cell immune response, including, for example, cytokine-producing T cells or cytotoxic T cells, to protect said animal from disease, whether that disease is already established within the individual or not An immunological response in a mammal may also be induced by a method comprises delivering a polypeptide of the present invention via a vector directing expression of the polynucleotide and coding for the polypeptide in vivo in order to induce such an immunological response to produce antibody to protect said animal from diseases of the invention One way of administering the vector is by accelerating it into the desired cells as a coating on particles or otherwise Such nucleic acid vector may comprise DNA, RNA, a modified nucleic acid or a DNA/RNA hybrid For use a vaccine, a polypeptide or a nucleic acid vector will be normally provided as a v accine formulation (composition) The formulation may further comprise a suitable carrier Since a polypeptide may be broken down in the stomach, it is preferablv administered parenterally (for instance, subcutaneous, intramuscular intravenous or intradermal injection) Formulations suitable for parenteral administration include aqueous and non aqueous sterile injection solutions that may contain anti-oxidants. buffers, bacteπostats and solutes that render the formulation mstonic with the blood of the recipient, and aqueous and non-aqueous sterile suspensions that may include suspending agents or thickening agents The formulations may be presented in unit-dose or multi-dose containers, for example, sealed ampoules and vials and mav be stored in a fieeze-dπed condition requiring only the addition of the sterile liquid carrier immediately prior to use The vaccine formulation may also include adjuv ant sv stems for enhancing the immunogenicity of the formulation, such as oil-in water systems and other systems known in the art The dosage will depend on the specific activity of the vaccine and can be readilv determined bv routine experimentation

Polypeptides of the present invention ha e one or more biological functions that are of relevance in one or more disease states, in particular the diseases of the invention hereinbefore mentioned It is therefore useful to to identify compounds that stimulate or inhibit the function or level of the polypeptide Accordingly, in a further aspect, the present invention provides for a method of screening compounds to identify those that stimulate or inhibit the function or level of the polypeptide Such methods identify agonists or antagonists that may be employed for therapeutic and prophylactic purposes for such diseases of the invention as hereinbefore mentioned Compounds may be identified from a variety of sources for example, cells, cell-free preparations, chemical hbraπes, collections of chemical compounds, and natural product mixtures Such agonists or antagonists so-identified may be natural or modified substrates, ligands, receptors, enzymes, etc , as the case may be, of the polypeptide, a structural or functional mimetic thereof (see Cohgan et al , Current Protocols in Immunology 1 (2) Chapter 5 ( 1991 )) or a small molecule Such small molecules preferably have a molecular weight below 2,000 daltons. more preferably between 300 and 1,000 daltons, and most preferably between 400 and 700 daltons It is preferred that these small molecules are organic molecules

The screening method may simplv measure the binding of a candidate compound to the polypeptide, or to cells or membranes bearing the polypeptide, or a fusion protein thereof, by means of a label directly or indirectly associated with the candidate compound Alternatively, the screening method may involve measuring or detecting (qualitatively or quantitatively) the competitive binding of a candidate compound to the polypeptide against a labeled competitor (e g. agonist or antagonist) Further, these screening methods may test whether the candidate compound results in a signal generated bv activation or inhibition of the polypeptide, using detection systems appropriate to the cells bearing the polypeptide Inhibitors of activation are generally assayed in the presence of a known agonist and the effect on activation by the agonist by the presence of the candidate compound is observed Further the screening methods may simply comprise the steps of mixing a candidate compound with a solution containing a polypeptide of the present invention to foim a mixture measuring a DKCN l activity in the mixture, and comparing the DKCNl activ ity of the mixture to a control mixture which contains no candidate compound

- n - Polypeptides of the present invention may be employed in conventional low capacity screening methods and also in high-throughput screening (HTS) formats Such HTS formats include not only the well-established use of 96- and, more recently, 384-well micotiter plates but also emerging methods such as the nanowell method described by Schullek et al. Anal Biochem., 246, 20-29, (1997)

Fusion proteins, such as those made from Fc portion and DKCNl polypeptide, as hereinbefore described, can also be used for high-throughput screening assays to identify antagonists for the polypeptide of the present inv ention ( see D Bennett et al , J Mol Recognition, 8:52-58 (1995), and K Johanson et al , J Biol Chem, 270(16) 9459-9471 ( 1995)) In a preferred embodiment cells transiently or stably expressing DKCNl potassium channel are incubated in cell culture medium in the presence of labelled rubidium (86Rb) for 3 hours. The cells are washed with a tyrode solution to remove the 86Rb not taken up by the cells. Tyrode with or without potential channel blockers or openers is then applied and samples collected at 5 minutes intervals Total 86Rb remaining in the cells is measured after solubihsing the cells at the end of the assay and all samples are measured by Cherenkov counting. Openers or blockers can be identified by a reduction or potentiation in the fractional rate of efflux of 86Rb over cells incubated only in Tyrode

The polynucleotides, polypeptides and antibodies to the polypeptide of the present invention may also be used to configure screening methods for detecting the effect of added compounds on the production of mRNA and polypeptide in cells For example, an ELISA assay may be constructed for measuring secreted or cell associated levels of polypeptide using monoclonal and polyclonal antibodies by standard methods known in the art This can be used to discover agents that may inhibit or enhance the production of polypeptide (also called antagonist or agonist, respectively) from suitably manipulated cells or tissues A polypeptide of the present invention may be used to identify membrane bound or soluble receptors, if any, through standard receptor binding techniques known in the art These include, but are not limited to, ligand binding and crosslmking assays in which the polypeptide is labeled with a radioactive isotope (for instance. ' -^D, chemically modified (for instance, biotinylated), or fused to a peptide sequence suitable for detection or purification, and incubated with a source of the putative receptor (cells, cell membranes, cell supematants. tissue extracts, bodily fluids) Other methods include biophysical techniques such as surface plasmon resonance and spectroscopy These screening methods may also be used to identify agonists and antagonists of the polypeptide that compete with the binding of the polypeptide to its leceptois, if any Standard methods for conducting such assays are well understood in the art Examples of antagonists of polypeptides of the present invention include antibodies or. in some cases, o gonucleotides or proteins that are closely related to the gands, substrates, receptors, enzymes, etc , as the case may be. of the polypeptide. e <ξ . tx fragment of the hgands. substrates, receptors, enzymes, etc , or a small molecule that bind to the polypeptide of the present invention but do not elicit a response, so that the activity of the polypeptide is prevented

Screening methods may also involve the use of transgenic technology and DKCNl gene The art of constructing transgenic animals is w ell established For example, the DKCNl gene may be introduced through microinjection into the male pronucleus of fertilized oocvtes, retroviral transfer into pre- or post-implantation embryos, or injection of genetically modified, such as by electroporation. embryonic stem cells into host blastocysts Particularly useful transgenic animals are so-called "knock- ' animals in which an animal gene is replaced by the human equivalent within the genome of that animal Knock-in transgenic animals are useful in the drug discovery process, for target validation where the compound is specific for the human target Other useful transgenic animals are so-called "knock-out animals in which the expression of the animal ortholog of a polypeptide of the present invention and encoded by an endogenous DNA sequence in a cell is partially or completely annulled The gene knock-out may be targeted to specific cells or tissues, may occur only in certain cells or tissues as a consequence of the limitations of the technology, or may occur in all or substantially all, cells in the animal Transgenic animal technology also offers a whole animal expression-cloning system in which introduced genes are expressed to give large amounts of polypeptides of the present invention

Screening kits for use in the above described methods form a further aspect of the present invention Such screening kits comprise

(a) a polypeptide of the present invention

(b) a recombinant cell expressing a polypeptide of the present invention, (c) a cell membrane expressing a polypeptide of the present invention, or

(d) an antibody to a polypeptide of the present inv ention which polypeptide is preferably that of SEQ ID NO 2

It will be appreciated that in any such kit (a), (b) (c) or (d) may compπse a substantial component

Glossary

The following definitions are prov ided to facilitate undei standing of certain terms used frequently hereinbefore

"Antibodies^' as used herein includes polvclonal and monoclonal antibodies, chimeπc, single chain, and humanized antibodies as well as Fab fragments including the products of an Fab or other immunoglobu n expression librarv

"Isolated" means altered "by the hand of man from its natural state. ; e , if it occurs in nature, it has been changed or removed fiom its original environment or both For example, a polynucleotide or a polypeptide naturally present in a li ing organism is not isolated," but the same polynucleotide or polypeptide separated from the coexisting materials of its natural state is "isolated' , as the term is employed herein Moreover a polynucleotide or polypeptide that is introduced into an organism by transformation genetic manipulation oi by any other recombinant method is "isolated even if it is still present in said organism which organism may be living or non-living "Polynucleotide" generally refers to any polyπbonucleotide (RNA) or polydeoxnbonucleotide (DNA), which may be unmodified or modified RNA or DNA "Polynucleotides" include, without limitation, single- and double-stranded DNA, DNA that is a mixture of single- and double-stranded regions single- and double-stranded RNA, and RNA that is mixture of single- and double-stranded regions hybrid molecules comprising DNA and RNA that may be single-stranded or, more typically, double-stranded or a mixture of single- and double-stranded regions In addition, "polynucleotide" refers to triple-stranded regions comprising RNA or DNA or both RNA and DNA The term "polynucleotide" also includes DNAs or RNAs containing one or more modified bases and DNAs or RNAs with backbones modified for stability or for other reasons "Modified" bases include, for example, tπtylated bases and unusual bases such as inosine A variety of modifications may be made to DNA and RNA; thus, "polynucleotide" embraces chemically, enzymatically or metabolically modified forms of polynucleotides as typically found in nature, as well as the chemical forms of DNA and RNA characteristic of viruses and cells "Polynucleotide^" also embraces relatively short polynucleotides, often referred to as ohgonucleotides "Polypeptide ' refers to any polypeptide comprising two or more amino acids joined to each other by peptide bonds or modified peptide bonds, l e , peptide isosteres ' Polypeptide" refers to both short chains, commonly referred to as peptides, ohgopeptides or ohgomers, and to longer chains, generally referred to as proteins Polypeptides may contain amino acids other than the 20 gene-encoded amino acids "Polypeptides^' include amino acid sequences modified either by natural processes such as post-tianslational processing, or bv chemical modification techniques that are ell known in the art Such modifications are well descπbed in basic texts and in more detailed monographs, as well as in a oluminous research literature Modifications may occur anywhere in a polypeptide, including the peptide backbone, the amino acid side-chains and the amino or carboxyl termini It will be appreciated that the same type of modification may be present to the same or vaiying degrees at sevei al sites in a given polypeptide Also, a given polypeptide may contain many types of modifications. Polypeptides may be branched as a result of ubiquitination, and they may be cyclic, with or without branching. Cyclic, branched and branched cyclic polypeptides may result from post-translation natural processes or may be made by synthetic methods. Modifications include acetylation. acylation, ADP-ribosylation, amidation, biotinylation, covalent attachment of flavin, covalent attachment of a heme moiety, covalent attachment of a nucleotide or nucleotide derivative, covalent attachment of a lipid or lipid derivative, covalent attachment of phosphotidylinositol. cross-linking, cyclization, disulfide bond formation, demethylation, formation of covalent cross-links, formation of cystine, formation of pyroglutamate, formylation, gamma-carboxylation. glycosylation. GPI anchor formation, hydroxylation, iodination, methylation. myristoylation. oxidation, proteolytic processing, phosphorylation, prenylation, racemization, selenoylation, sulfation, transfer-RNA mediated addition of amino acids to proteins such as arginylation. and ubiquitination (see, for instance, Proteins - Structure and Molecular Properties, 2nd Ed., T. E. Creighton, W. H. Freeman and Company, New York, 1993; Wold, F., Post-translational Protein Modifications: Perspectives and Prospects, 1-12, in Post-translational Covalent Modification of Proteins, B. C. Johnson, Ed., Academic Press, New York, 1983; Seifter et al, "Analysis for protein modifications and nonprotein cofactors", Meth Enzymol, 182, 626-646. 1990, and Rattan et al., "Protein Synthesis: Post-translational Modifications and Aging", Ann NY Acad Sci, 663, 48-62, 1992).

"Fragment" of a polypeptide sequence refers to a polypeptide sequence that is shorter than the reference sequence but that retains essentially the same biological function or activity as the reference polypeptide. "Fragment" of a polynucleotide sequence refers to a polynucloetide sequence that is shorter than the reference sequence of SEQ ID NO: 1.

"Variant" refers to a polynucleotide or polypeptide that differs from a reference polynucleotide or polypeptide, but retains the essential properties thereof. A typical variant of a polynucleotide differs in nucleotide sequence from the reference polynucleotide. Changes in the nucleotide sequence of the variant may or may not alter the amino acid sequence of a polypeptide encoded by the reference polynucleotide. Nucleotide changes may result in amino acid substitutions, additions, deletions, fusions and truncations in the polypeptide encoded by the reference sequence, as discussed below. A typical variant of a polypeptide differs in amino acid sequence from the reference polypeptide. Generally, alterations are limited so that the sequences of the reference polypeptide and the variant are closely similar overall and, in many regions. identical. A variant and reference polypeptide may differ in amino acid sequence by one or more substitutions, insertions, deletions in any combination. A substituted or inserted amino acid residue may or may not be one encoded by the genetic code. Typical conservative substitutions include Gly, Ala; Val. He. Leu; Asp, Glu: Asn. Gin: Ser, Thr; Lys. Arg; and Phe and Tyr. A variant of a polynucleotide or polypeptide may be naturally occurring such as an allele, or it may be a variant that is not known to occur naturally Non-naturally occurring variants of polynucleotides and polypeptides may be made by mutagenesis techniques or by direct synthesis. Also included as variants are polypeptides having one or more post-translational modifications, for instance glycosylation, phosphorylation, methylation. ADP πbosylation and the like Embodiments include methylation of the N-termmal amino acid, phosphorylations of seπnes and threonines and modification of C-terminal glycines

"Allele" refers to one of two or more alternative forms of a gene occuπng at a given locus in the genome "Polymorphism" refers to a variation in nucleotide sequence (and encoded polypeptide sequence, if relevant) at a given position in the genome within a population.

"Single Nucleotide Polymorphism" (SNP) refers to the occurence of nucleotide variability at a single nucleotide position m the genome, within a population An SNP may occur within a gene or within intergenic regions of the genome SNPs can be assayed using Allele Specific Amplification (ASA). For the process at least 3 primers are required. A common primer is used in reverse complement to the polymorphism being assayed This common primer can be between 50 and 1500 bps from the polymorphic base The other two (or more) primers are identical to each other except that the final 3' base wobbles to match one of the two (or more) alleles that make up the polymorphism Two (or more) PCR reactions are then conducted on sample DNA, each using the common primer and one of the Allele Specific Primers.

"Splice Variant" as used herein refers to cDNA molecules produced from RNA molecules initially transcπbed from the same genomic DNA sequence but which have undergone alternative RNA splicing. Alternative RNA splicing occurs when a primary RNA transcript undergoes splicing, generally for the removal of introns, which results in the production of more than one mRNA molecule each of that may encode different ammo acid sequences. The term splice variant also refers to the proteins encoded by the above cDNA molecules

"Identity" reflects a relationship between two or more polypeptide sequences or two or more polynucleotide sequences, determined by comparing the sequences. In general, identity refers to an exact nucleotide to nucleotide or amino acid to amino acid correspondence of the two polynucleotide or two polypeptide sequences, respectively, ovei the length of the sequences being compared

"% Identity" - For sequences where there is not an exact correspondence, a "% identity" may be determined. In geneial. the two sequences to be compared are aligned to give a maximum correlation between the sequences This may include inserting "gaps" in either one or both sequences, to enhance the degree of alignment. A 7c identity may be determined over the whole length of each of the sequences being compared (so-called global alignment) that is particularly suitable for sequences of the same or very similar length, or over shorter, defined lengths (so- called local alignment), that is more suitable for sequences of unequal length

"Similarity is a further, more sophisticated measure of the relationship between two polypeptide sequences In general, "similarity means a comparison between the ammo acids of two polypeptide chains, on a residue by residue basis, taking into account not only exact correspondences between a between pairs of residues, one from each of the sequences being compared (as for identity) but also, where there is not an exact correspondence, whether, on an evolutionary basis, one residue is a likely substitute for the other This likelihood has an associated "score" from which the "% similarity ' of the two sequences can then be determined Methods for comparing the identity and similarity of two or more sequences are well known in the art Thus for instance, programs available in the Wisconsin Sequence Analysis Package, version 9 1 (Devereux J et al, Nucleic Acids Res. 12, 387-395, 1984. available from Genetics Computer Group, Madison, Wisconsin, USA), for example the programs BESTFIT and GAP, may be used to determine the % identity between two polynucleotides and the % identity and the % similarity between two polypeptide sequences BESTFIT uses the local homology" algorithm of Smith and Waterman (J Mol Biol, 147,195-197, 1981 , Advances in Applied Mathematics, 2, 482-489, 1981) and finds the best single region of similarity between two sequences BESTFIT is more suited to comparing two polynucleotide or two polypeptide sequences that are dissimilar in length, the program assuming that the shortei sequence represents a portion of the longer In comparison, GAP aligns two sequences, finding a maximum similarity", according to the algorithm of Neddleman and Wunsch (J Mol Biol. 48, 443-453. 1970) GAP is more suited to comparing sequences that are approximately the same length and an alignment is expected over the entire length Preferably, the parameters "Gap Weight" and "Length Weight" used in each program are 50 and 3, for polynucleotide sequences and 12 and 4 for polypeptide sequences, respectively Preferably, % identities and similarities are determined when the two sequences being compared are optimally aligned

Other programs for determining identity and/or similarity between sequences are also known in the art. for instance the BLAST family of piograms (Altschul S F et al, J Mol Biol, 215. 403-410 1990. Altschul S F et al, Nucleic Acids Res 25 389-3402 1997, a ailable from the

National Center for Biotechnology Information (NCBI) Bethesda, Maryland. USA and accessible through the home page of the NCBI at www ncbi nlm nih gov) and FASTA (Pearson W R, Methods in Enzy ology, 183, 63-99, 1990, Peaison W R and Lipman D J, Proc Nat Acad Sci USA, 85, 2444-2448.1988. available as part of the Wisconsin Sequence Analv sis Package) Preferably, the BLOSUM62 amino acid substitution matrix (Henikoff S and Henikoff J G, Proc Nat Acad Sci USA, 89, 10915-10919 1992) is used in polypeptide sequence comparisons including where nucleotide sequences are first translated into amino acid sequences before comparison

Preferably the program BESTFIT is used to determine the % identity of a query polynucleotide or a polypeptide sequence with respect to a reference polynucleotide or a polypeptide sequence, the query and the ieference sequence being optimally aligned and the parameters of the program set at the default value, as hereinbefore described

"Identity Index" is a measure of sequence relatedness which may be used to compare a candidate sequence (polynucleotide or polypeptide) and a reference sequence Thus, for instance, a candidate polynucleotide sequence having, for example, an Identity Index of 0 95 compared to a reference polynucleotide sequence is identical to the reference sequence except that the candidate polynucleotide sequence may include on average up to five differences per each 100 nucleotides of the reference sequence Such differences are selected from the group consisting of at least one nucleotide deletion, substitution, including transition and transversion, or insertion These differences may occur at the 5' or 3' terminal positions of the reference polynucleotide sequence or anywhere between these terminal positions, interspersed either individually among the nucleotides in the reference sequence or in one or more contiguous groups within the reference sequence In other words, to obtain a polynucleotide sequence having an Identity Index of 0 95 compared to a reference polynucleotide sequence, an average of up to 5 in every 100 of the nucleotides of the in the reference sequence may be deleted, substituted or inserted, or any combination thereof, as hereinbefore described The same applies mutatis mutandis for other values of the Identity Index, for instance 0 96, 0 97, 0 98 and 0 99

Similarly, for a polypeptide, a candidate polypeptide sequence having, for example, an Identity Index of 0 95 compared to a reference polypeptide sequence is identical to the reference sequence except that the polypeptide sequence may include an average of up to five differences per each 100 amino acids of the reference sequence Such differences are selected from the group consisting of at least one amino acid deletion, substitution, including conservative and non- conservative substitution, or insertion These differences may occur at the amino- or carboxy- termmal positions of the reference polypeptide sequence or anywhere between these terminal positions, interspersed either individually among the ammo acids in the reference sequence or in one or more contiguous gioups within the lefeience sequence In other words to obtain a polypeptide sequence having an Identity Index of 0 95 compared to a reference polypeptide sequence, an average of up to 5 in every 100 of the amino acids in the reference sequence may be deleted, substituted oi inserted, or any combination thereof, as hereinbefore described The same applies mutatis mutandis for other values of the Identity Index, for instance 0 96 0 97, 0 98 and

0 99

The relationship between the number of nucleotide or amino acid differences and the

Identity Index may be expressed in the following equation n_a < \_a - (\_a • I), in which n_a is the number of nucleotide or amino acid differences x_a is the total number of nucleotides or amino acids in SEQ ID NO 1 or SEQ ID NO 2, respectively, I is the Identity Index ,

• is the symbol for the multiplication operator, and in which any non-integer product of x_a and I is rounded down to the nearest integer pπor to subtracting it from x_a

"Homolog" is a generic term used in the art to indicate a polynucleotide or polypeptide sequence possessing a high degree of sequence relatedness to a reference sequence Such relatedness may be quantified by determining the degree of identity and/or similarity between the two sequences as hereinbefore defined Falling within this generic term are the terms "ortholog", and "paralog" "Ortholog" refers to a polynucleotide or polypeptide that is the functional equivalent of the polynucleotide or polypeptide in another species "Paralog ' refers to a polynucleotideor polypeptide that within the same species which is functionally similar

"Fusion protein" refers to a protein encoded by two, often unrelated, fused genes or fragments thereof In one example, EP-A-0 464 533-A discloses fusion proteins comprising various portions of constant region of immunoglobulin molecules together with another human protein or part thereof In many cases, employing an immunoglobulin Fc region as a part of a fusion protein is advantageous for use in therapy and diagnosis resulting in, for example, improved pharmacokinetic properties [see, e g , EP-A 0232 262] On the other hand, for some uses it would be desirable to be able to delete the Fc part after the fusion protein has been expressed, detected and purified

All publications and references including but not limited to patents and patent applications, cited in this specification are herein incorporated bv reference in their entiretv as if each individual publication or reference were specificallv and individually indicated to be incorpoiated by reference herein as being fully set forth Any patent application to which this application claims priority is also incorporated by reference herein in its entirety in the manner descπbed above foi publications and references Examples

Example 1: TaqMan Analysis of mRNA distribution

The expression pattern of DKCNl was investigated using TaqMan fluorescent PCR (Perkin Elmer) and human cDNAs prepared from various brain areas and peripheral tissues. All TaqMan analysis was earned out according to the manufacturers instructions using the following ohgonucleotides:

Forward pπmer: 5 '-CCC ATC GCC TAT TAG CTC CA Reverse primer: 5 '-CGG CGT TTC ATC AGC CTC

TaqMan probe: 5'-CTC CTG GGT TAC ACA GCT TTA CCG ACC AC

20 tissues examined in the TaqMan experiments were- Brain*, pituitary, heart, lung, liver, foetal liver, kidney, skeletal muscle, stomach, small/large intestine, spleen, lymphocytes (PBMC), macrophages, adipose, pancreas, prostate (4 males), placenta, cartilage, bone (l male, 3 females) and bone marrow

(* Brain = an equal-part mix of the 18 most distinct brain regions representing 75% of sample and 25% of sample is spinal cord. This approach was designed to maximise the chance of detecting genes expressed spefically in small brain sub-regions.)

19 brain regions were examined in the TaqMan experiments (2 males, 2 females or as indicated). The regions were: amygdala, caudate nucleus, cerebellum, cingulate gyrus, globus pallidus, hippocampus, hypothalamus, locus coeruleus. medial frontal gyrus, medulla oblongata, nucleus accumbens, parahippocampal gyrus, putamen, stπatum. substantia nigra, superior frontal gyrus, thalamus, spinal cord and dorsal root ganglion (1 control female, 1 Multiple sclerosis male, 2 Multiple sclerosis females)

The results indicate that expression of DKCNl is seen in the pituitary and brain, predominantly the cerebellum, and at very low levels in other tissues

Example 2: Electrophysiological recordings in oocytes

Using standard methods, Xenopus laevis oocytes were removed, dissociated and defolliculated. Individual defolliculated oocytes were subsequently injected into their nuclei with 0 5-1 5 ng of appropπate cDNA constructs tor DKCNl The cDNA constructs employed were pcDNA3 1 plasmids which either contained or lacked h-DKCNl , the latter being used for control recording purposes Following injection the oocvtes were incubated at 22°C in modified Barth s solution (MBS) supplemented with gentamvcin (0 lmg/mi) and used for electrophysiological recordings within 1-4 days

For electrophvsiological recordings oocytes were placed in a recording chamber and continuously superfused with a solution containing in mM NaCl 93, KC1 5, HEPES 5, MgCl₂ I and CaCl₂ 1 8 Electrodes were low resistance (0 5-3MΩ) and were filled with 3M KC1 Electrophysiology on HEK293 cells utilised the whole cell patch clamp method For this cells were placed on the stage of an inverted microscope and continuously superfused with a solution containing, in mM NaCl 130, KC1 5, Glucose 30, HEPES 15, CaCh 2 and MgCl 1 , pH 7 3 with NaOH The recording pipette solution contained m mM KC1 140, HEPES 10 MgCh 4 and EGTA 10, pH7 3 w ith KOH

The resting membrane potential of oocytes expressing h-DKCNl was -78.9 ± 4 7 mV (n=9), approximately 44 mV more hyperpolaπsed than that of control pcDNA3 1 injected oocytes, -34 2 ± 2 3 mV (n=l 1) A similar negative shift in resting membrane potential was also observed in HEK293 cells expressing h-DKCNl Thus expression of human DKCNl channels dπves resting membrane potential towards more negative values This indicates a role for this channel generating resting membrane potential and therefore controlling the electπcal excitability of cells

The properties of the DKCNl conductance was further investigated in voltage-clamp expeπments performed on oocytes expressing this channels From a holding potential of -80 mV depolaπsmg 500 ms voltage pulses resulted in the activation of outward currents These currents were non-inactivating at all but very positive potentials d e +50 mV) where a slight degree of inactivation was apparent Similar currents were not observed in control pcDN A3 1 -injected oocytes

The current present in DKCNl injected oocytes as shown to be predominantly K⁺ selective by varying [K⁺]₀ between 5 mM and 98 mM (to do this direct replacement of Na⁺ with K⁺ was earned out) Increasing the [K⁺]₀ from 5 mM to 98 mM shifted the current reversal potential towards 0 mV A 51 1 ± 1 8 mV change in reversal potential as produced bv a 10 fold change in [K⁺]₀ - a v alue close to that (58 2 mV) predicted bv the Nernst equation for a purely potassium selective channel

h-DKCNl -mediated currents recorded from oocvtes were inhibited by reductions in extracellular pH below 7 4 Close to maximal inhibition was observed around pH 5 0 Increasing the pH above 7 4 produced only a very small potentiation of DKCN- 1 -mediated current This effect was not voltage-dependent between membrane potentials of -50mV and +50mV

Example 3: Electrophysiological recordings in HEK293 cells

HEK293 cells were cultured under standard conditions and were transiently transfected with pcDNA3 1-h-DKCNl using the hpofectamine plus reagent As a marker of successful transfection a plasmid containing a green fluorescent protein (GFP) gene was co-transfected with pcDNA3 1 -h-DKC l Control recordings were made from both untransfected and GFP-only transfected HEK293 cells

A similar negative shift in resting membrane potential to that for oocytes transfected with h-DKCNl (Example 1) was observed in HEK293 cells expressing h-DKCNl Thus expression of human DKCNl channels drives resting membrane potential towards more negative values. This indicates a role for this channel in generating resting membrane potential and therefore controlling the electπcal excitability of cells

Voltage clamp recordings from DKCN-1 -transfected HEK293 cells were used to additionally characterise the kinetics of this channel In these recordings 100 ms αuration depolarising test pulses to a potential of +60 mV from a holding potential of -80 V elicited a current with a very rapidly activation phase followed bv more slowly activating component of time constant 10 9 1 3 ms This current did not noticeably inactivate over the duration of the test pulse λt a test potential of +60mV these currents were considerably greater in amplitude, 1918 6pA 344 3 (n=l 1) than those recorded from GFP-transfected wild type HEK 293 cells, 179 6pA 26 3 (n=8) SEQUENCE INFORMATION SEQ ID NO: 1

ATGAAGAGGCAGAACGTGCGGACTCTGTCCCTCATCGTCTGCACCTTCACCTACCTGCTG GTGGGCGCCGCCGTGTTCGACGCCCTCGAGTCGGACCACGAGATGCGCGAGGAGGAGAAA CTCAAAGCCGAGGAGATCCGGATCAAGGGGAAGTACAACATCAGCAGCGAGGACTACCGG CAGCTGGAGCTGGTGATCCTGCAGTCGGAACCGCACCGCGCCGGCGTCCAGTGGAAATTC GCCGGCTCCTTCTACTTTGCGATCACGGTCATCACCACCATAGGTTATGGGCACGCTGCA CCTGGCACCGATGCGGGCAAGGCCTTCTGCATGTTCTACGCCGTGCTGGGCATCCCGCTG ACACTGGTCATGTTCCAGAGCCTGGGCGAGCGCATGAACACCTTCGTGCGCTACCTGCTG AAGCGCATTAAGAAGTGCTGTGGCATGCGCAACACTGACGTGTCTATGGAGAACATGGTG ACTGTGGGCTTCTTCTCCTGCATGGGGACGCTGTGCATCGGGGCGGCCGCCTTCTCCCAG TGTGAGGAGTGGAGCTTCTTCCACGCCTACTACTACTGCTTCATCACGTTGACTACCATT GGGTTCGGGGACTACGTGGCCCTGCAGACCAAGGGCGCCCTGCAGAAGAAGCCGCTCTAC GTGGCCTTTAGCTTTATGTATATCCTGGTGGGGCTGACGGTCATCGGGGCCTTCCTCAAC CTGGTCGTCCTCAGGTTCTTGACCATGAACAGTGAGGATGAGCGGCGGGATGCTGAAGAG AGGGCATCCCTCGCCGGAAACCGCAACAGCATGGTCATTCACATCCCTGAGGAGCCGCGG CCCAGCCGGCCCAGGTACAAGGCGGACGTCCCGGACCTGCAGTCTGTGTGCTCCTGCACC TGCTACCGCTCGCAGGACTATGGCGGCCGCTCGGTGGCACCGCAGAACTCCTTCAGCGCC AAGCTTGCCCCCCACTACTTCCACTCCATCTCTTACAAGATCGAGGAGATCTCACCAAGC ACATTAAAAAACAGCCTCTTCCCATCGCCTATTAGCTCCATCTCTCCTGGGTTACACAGC TTTACCGACCACCAGAGGCTGATGAAACGCCGGAAGTCCGTTTAG

SEQ ID NO:2 KRQNVRTLS IVCTFTYLLVGAAVFDA ESDHEMREEΞKL AEEIRIKGKYNISSEDYR QLE VI QSEPHRAGVQ KFAGSFYFAITVITTIGYGHAAPGTDAGKAFCMFYAV GIPL TLVMFQSLGERMNTFVRYLLKRIKKCCGMR TDVSMENMVTVGFFSCMGTLCIGAAAFSQ CEE SFFHAYYYCFIT TTIGFGDYVA Q KGALQKKP YVAFSFMYILVG TVIGAF N LW RF TMNSEDΞRRDAEERASLAGNRMSI-IVIHIPEΞPRPSRPRYKADVPD QSVCSCT CYRSQDYGGRSVAPQNSFSAKLAPHYFKSrSYKIEΞISPSTLKNSLFPSPISSISPGLHS FTDHQRLMKRRKSV

SEQ ID NO:3

GTGGGACGCGCGCGGCTGTGAGCCTGCGGGACATGCCCCCCGCGCCGGCTCCTTGCTGGC GGCCATGAAGAGGCAGAACGTGCGGACTCTGTCCCTCATCGTCTGCACCTTCACCTACCT GCTGGTGGGCGCCGCCGTGTTCGACGCCCTCGAGTCGGACCACGAGATGCGCGAGGAGGA GAAACTCAAAGCCGAGGAGATCCGGATCAAGGGGAAGTACAACATCAGCAGCGAGGACTA CCGGCAGCTGGAGCTGGTGATCCTGCAGTCGGAACCGCACCGCGCCGGCGTCCAGTGGAA ATTCGCCGGCTCCTTCTACTTTGCGATCACGGTCATCACCACCATAGGTTATGGGCACGC TGCACCTGGCACCGATGCGGGCAAGGCCTTCTGCATGTTCTACGCCGTGCTGGGCATCCC GCTGACACTGGTCA GTTCCAGAGCCTGGGCGAGCGCATGAA.CACCTTCGTGCGCTACCT GCTGAAGCGCATTAAGAAGTGCTGTGGCATGCGCAACACTGACGTGTCTATGGAGAACAT GGTGACTGTGGGCTTCTTCTCCTGCATGGGGACGCTGTGCATCGGGGCGGCCGCCTTCTC CCAGTGTGAGGAGTGGAGCTTCTTCCACGCCTACTACTACTGCTTCATCACGTTGACTAC CATTGGGTTCGGGGACTACGTGGCCCTGCAGACCAAGGGCGCCCTGCAGAAGAAGCCGCT CTACGTGGCCTTTAGCTTTATGTATATCCTGGTGGGGCTGACGGTCATCGGGGCCTTCCT CAACCTGGTCGTCCTCAGGTTCTTGACCATGAACAGTGAGGATGAGCGGCGGGATGCTGA AGAGAGGGCATCCCTCGCCGGAAACCGCAACAGCATGGTCATTCACATCCCTGAGGAGCC GCGGCCCAGCCGGCCCAGGTACAAGGCGGACGTCCCGGACC GCAGTCTGTGTGCTCCTG CACCTGCTACCGCTCGCAGGACTATGGCGGCCGCTCGGTGGCACCGCAGAACTCCTTCAG CGCCAAGCTTGCCCCCCACTACTTCCACTCCATCTCTTACAAGATCGAGGAGATCTCACC AAGCACATTAAAAAACAGCCTCTTCCCATCGCCTATTAGCTCCATCTCTCCTGGGTTACA CAGCTTTACCGACCACCAGAGGCTGATGAAACGCCGGAAGTCCGTTTAGGTGTGGGGAGG GAAATGGGACAGAAAAGTCATTTGTCATAGTTTGTGTTAATTTCCATTGGTCCAACTCGT CTTTTCTTATTTATTTATTATTATTATTGKCATCATTATTACTTTCTCTCCTTCCTCCTT TCTTGGTCTCTTGGTCTCATTTTCCCCCACCTTTCCAGCCAGACAGAGCAGGCCAAAGGG AAATACAGGCCCATCCTCCTCTGAAACTCACATCTGAGCATGAAGCATGGATCTCCTCCT T

SEQ ID NO:4

MKRQNVRTLS IVCTFTYL VGAAVFDALESDHEMREEEK AEEIRIKGKYNISSEDYR

Q ELVI QSEPHRAGVQ KFAGSFYFAITVITTIGYGHAAPGTDAGKAFCMFYAV GIPL TLVMFQS GERMNTFVRYLLKRIKKCCGMRNTDVSMENMVTVGFFSCMGTLCIGAAAFSQ

CEEWSFFHAYYYCFITLTTIGFGDYVALQTKGALQKKPLYVAFSFMYI VG TVIGAFLN

LWLRF TMNSEDERRDAEERASLAGNRNSMVIHIPEEPRPSRPRYKADVPD QSVCSCT

CYRSQDYGGRSVAPQNSFSAK APHYFHSISYKIEEISPSTLKNS FPSPISSISPG HS FTDHQRLMKRRKSV

Claims

1 An isolated polypeptide selected from the group consisting of

(a) an isolated polypeptide encoded by a polynucleotide comprising the seqlience of SEQ ID NO: l;

(b) an isolated pol peptide comprising a polypeptide sequence having at least 957c identity to the polypeptide sequence of SEQ ED NO.2;

(c) an isolated polypeptide having at least 95% identity to the polypeptide sequence of SEQ ED NO:2; and (d) fragments and vaπants of such polypeptides in (a) to (e).

2. The isolated polypeptide as claimed in claim 1 comprising the polypeptide sequence of SEQ ID NO:2.

3. The isolated polypeptide as claimed in claim 1 which is the polypeptide sequence of SEQ ID NO:2.

4. An isolated polynucleotide selected from the group consisting of.

(a) an isolated polynucleotide comprising a polynucleotide sequence having at least 957o identity to the polynucleotide sequence of SEQ ED NO: 1 ,

(b) an isolated polynucleotide having at least 95% identity to the polynucleotide of SEQ ED NO: 1 ,

(c) an isolated polynucleotide comprising a polynucleotide sequence encoding a polypeptide sequence having at least 95% identity to the polypeptide sequence of SEQ ED NO:2,

(d) an isolated polynucleotide having a polynucleotide sequence encoding a polypeptide sequence having at least 95% identity to the polypeptide sequence of SEQ ED NO:2;

(e) an isolated polynucleotide with a nucleotide sequence of at least 100 nucleotides obtained by screening a library under stπngent hybπdization conditions with a labeled probe having the sequence of SEQ ED NO' 1 or a fragment thereof having at least 15 nucleotides,

(0 a polynucleotide which is the RNA equivalent of a polynucleotide of (a) to (e). or a polynucleotide sequence complementary to said isolated polynucleotide and polynucleotides that are vaπants and fragments of the above mentioned polynucleotides or that are complementary to above mentioned polynucleotides, over the entire length thereof.

5. An isolated polynucleotide as claimed in claim 4 selected from the group consisting of (a) an isolated polynucleotide compπsing the polynucleotide of SEQ ED NO 1 ,

(b) the isolated polynucleotide of SEQ ED NO- 1 ,

(c) an isolated polynucleotide compπsing a polynucleotide sequence encoding the polypeptide of SEQ ED NO.2; and

(d) an isolated polynucleotide encoding the polypeptide of SEQ ED NO 2

6. An expression system compπsing a polynucleotide capable of producing a polypeptide of claim 1 when said expression vector is present in a compatible host cell

7 A recombinant host cell comprising the expression vector of claim 6 or a membrane thereof expressing the polypeptide of claim 1

8 A process for producing a polypeptide of claim 1 comprising the step of cultuπng a host cell as defined in claim 7 under conditions sufficient for the production of said polypeptide and recoveπng the polypeptide from the culture medium

9 An antibody immunospecific for the polypeptide of any one of claims 1 to 3

10 A method for screening to identify compounds that stimulate or inhibit the function or level of the polypeptide of claim 1 compnsing a method selected from the group consisting of

(a) measunng or, detecting, quantitatively or qualitatively, the binding of a candidate compound to the polypeptide (or to the cells or membranes expressing the polypeptide) or a fusion protein thereof by means of a label directly or indirectly associated with the candidate compound,

(b) measunng the competition of binding of a candidate compound to the polypeptide (or to the cells or membranes expressing the polypeptide) or a fusion protein thereof m the presence of a labeled competitior,

(c) testing whether the candidate compound results in a signal generated by activation or inhibition of the polypeptide, using detection systems appropriate to the cells or cell membranes expressing the polypeptide,

(d) mixing a candidate compound with a solution containing a polypeptide of claim 1, to form a mixture, measunng activity of the polypeptide in the mixture, and comparing the activity of the mixture to a control mixture which contains no candidate compound, or (e) detecting the effect of a candidate compound on the production of mRNA encoding said polypeptide or said polypeptide in cells, using for instance, an ELISA assay

1 1 An isolated polynucleotide selected form the group consisting of

(a) an isolated polynucleotide comprising a nucleotide sequence which has at least 95% identity to SEQ ID NO 3 over the entire length of SEQ ID NO 3

(b) an isolated polynucleotide comprising the polynucleotide of SEQ ID NO 3, or

(c) the polynucleotide of SEQ ID NO 3

(d) an isolated polynucleotide comprising a nucleotide sequence encoding a polypeptide which has at least 95% identity to the amino acid sequence of SEQ ID NO 4 over the entire length of SEQ ID NO 4

12. A polypeptide selected from the group consisting of

(a) a polypeptide which comprises an amino acid sequence which has at least 95% identity to that of SEQ ID NO:4 over the entire length of SEQ ID NO.4;

(b) a polypeptide in which the amino acid sequence has at least 95% identity to the amino acid sequence of SEQ ID NO:4 over the entire length of SEQ ID NO:4;

(c) a polypeptide which comprises the amino acid of SEQ ID NO:4;

(d) a polypeptide which is the polypeptide of SEQ ID NO:4; or

(e) a polypeptide which is encoded by a polynucleotide comprising the sequence contained in SEQ ID NO;3.