GB2369362A - Modulators of inwardly rectifying potassium channels - Google Patents

Abstract

The present invention provides the use of a substance which modulates inwardly rectifying potassium channel (Kir) activity of a polypeptide, which polypeptide comprises: <SL> <LI>(a) the amino acid sequence of SEQ ID NO: 2; or <LI>(b) a variant thereof which is a potassium channel activated by hyperpolarisation of cell membrane potential below the reversal potential for potassium; or <LI>(c) a fragment of (a) or (b) which is a potassium channel activated by hyperpolarisation of cell membrane potential below the reversal potential for potassium; </SL> in the manufacture of a medicament for the treatment or prophylaxis of a cerebellar or spinocerebellar disorder, a thyroid disorder or a kidney disorder.

Description

POLYPEPTIDE Field of the Invention The present invention relates to inwardly rectifying potassium channel polypeptides.

Background of the Invention Ion channels are involved in a wide variety of neurological and other disorders in man. Inwardly rectifying potassium channels (Kir) underlie voltage dependent conductance of an inward potassium current. At low membrene potentials, below the reversal potential for potassium, these channels conduct very little current. Members of the Kir channel family have a broad range of functions.

Kir 1 subtypes are involved in maintenance of potassium homoeostasis and renal potassium secretion. Kir2 subtypes are involved in the maintenance of resting membrane potential and modulation of action potential waveforms in neurons and myocardium. Kir3 subtypes are all G-protein-activated strongly rectifying potassium channels and are believed to underlie G-protein-coupled receptor activated currents involved in modulation of heart rate and neuronal excitability. Kir 4 subtypes are involved in the maintenance of potassium homeostasis in kidney and glia. The two Kir6 subtypes associate with the sulphonylurea receptor yielding the molecular correlate of the ATP-sensitive potassium channel. These channels link the metabolic state of different cell types to membrane excitability, insulin secretion, cytoprotection and maintenance of vascular tone. The function of KirS is unknown.

Summary of the Invention A novel expression pattern of an inwardly rectifying potassium channel, referred to herein as HIPHUM61, is now provided. HIPHUM61 is shown to be primarily expressed in thyroid, cerebellum and kidney. HIPHUM is a screening target for the identification and development of novel pharmaceutical agents, including modulators of inwardly rectifying potassium channel activity for use in the treatment or prophylaxis of cerebellar, spinocerebellar, thyroid and kidney disorders.

These disorders include cerebellar and spinocerebellar disorders such as acute viral

encephalitis ! astrocytoma, amyloidosis, ateriovenous malformations, brain abscess, brain infarction, CNS paraneoplastic syndromes, Creutzfeldt-Jackob disease (and other prion diseases), glioblastoma multiforme, neuro-opthalmologic disorders, neuropathic pain, oligodendroglioma, transient ischaemic attacks (TIA) and spinocerebellar ataxia, thyroid disorders such as euthyroid sick syndrome, follicular carcinoma, giant cell thyroid cancer, hyperthyroidism, medullary carcinoma, papillary carcinoma, simple goiter and thyroiditis and kidney disorders such as acute poststreptococcal glomerulonephritis, acute pyelonephritis, acute renal failure, acute tubular necrosis, Alport's syndrome, chronic glomerulonephritis, chronic renal failure, cystinuria, diabetic nephropathy, Fanconi's syndrome, glomerulonephritis, Grawitz's tumor, hydronephrosis, hypemephroma, medullary cystic disease, medullary sponge kidney, nephrocarcinoma, nephrotic syndrome, polycystic kidney disease, renal calculi, renal cell carcinoma, renal infarction, renal tubular acidosis, renal vein thrombosis, renovascular hypertension and Wilm's Tumor.

Accordingly, the present invention provides the use of a substance which modulates inwardly rectifying potassium channel activity of a polypeptide, which polypeptide comprises: (a) the amino acid sequence of SEQ ID NO: 2 or (b) a variant thereof which is a potassium channel activated by hyperpolarisation of cell membrane potential below the reversal potential for potassium or (c) a fragment of (a) or (b) which is a potassium channel activated by hyperpolarisation of cell membrane potential below the reversal potential for potassium in the manufacture of a medicament for the treatment or prophylaxis of a cerebellar or spinocerebellar disorder, a thyroid disorder or a kidney disorder.

The invention also provides: a method of treating a subject having a cerebellar or spinocerebellar disorder, a thyroid disorder or a kidney disorder, which method comprises: (i) contacting a test substance and a polypeptide comprising: (a) the amino acid sequence of SEQ ID NO: 2 or (b) a variant thereof which is a potassium channel activated by

hyperpolarisation of cell membrane potential below the reversal potential for potassium or (c) a fragment of (a) or (b) which is a potassium channel activated by hyperpolarisation of cell membrane potential below the reversal potential for potassium; (ii) determining the effect of the test substance on the activity and/or expression of the said polypeptide, thereby determining whether the test substance modulates inwardly rectifying potassium channel activity and/or expression; and (iii) administering to said subject an effective amount of a substance which modulates inwardly rectifying potassium channel activity; and a method of treating a subject having a cerebellar or spinocerebellar disorder, a thyroid disorder or a kidney disorder, which method comprises: (i) contacting a test substance and a polynucleotide encoding a inwardly rectifying potassium channel polypeptide which is a potassium channel activated by hyperpolarisation of cell membrane potential below the reversal potential for potassium, which polynucleotide comprises : (a) the nucleic acid sequence of SEQ ID NO: 1 and/or a sequence complementary thereto; (b) a sequence which hybridises under stringent conditions to a sequence as defined in (a); (c) a sequence that is degenerate as a result of the genetic code to a sequence as defined in (a) or (b); or (d) a sequence having at least 60% identity to a sequence as defined in (a), (b) or (c); (ii) determining the effect of the test substance on the activity and/or expression of the polypeptide encoded by the said polynucleotide, thereby determining whether the test substance modulates inwardly rectifying potassium channel activity and/or expression; and (iii) administering to said subject an effective amount of a substance which modulates inwardly rectifying potassium channel activity.

Preferably the disorder is selected from cerebellar and spinocerebellar disorders such as acute viral encephalitis, astrocytoma, amyloidosis, ateriovenous malformations, brain abscess, brain infarction, CNS paraneoplastic syndromes, Creutzfeldt-Jackob disease (and other prion diseases), glioblastoma multiforme, neuro-opthalmologic disorders, neuropathic pain, oligodendroglioma, transient ischaemic attacks (TIA) and spinocerebellar ataxia, thyroid disorders such as euthyroid sick syndrome, follicular carcinoma, giant cell thyroid cancer, hyperthyroidism, medullary carcinoma, papillary carcinoma, simple goiter and thyroiditis and kidney disorders such as acute poststreptococcal glomerulonephritis. acute pyelonephritis, acute renal failure, acute tubular necrosis, Alport's syndrome, chronic glomerulonephritis, chronic renal failure, cystinuria, diabetic nephropathy, Fanconi's syndrome, glomerulonephritis, Grawitz's tumor, hydronephrosis, hypernephroma, medullary cystic disease, medullary sponge kidney, nephrocarcinoma, nephrotic syndrome, polycystic kidney disease, renal calculi, renal cell carcinoma, renal infarction, renal tubular acidosis, renal vein thrombosis, renovascular hypertension and Wilm's Tumor.

Brief Description of the Sequences SEQ ID NO : 1 shows the nucleotide and amino acid sequences of human protein HIPHUM61.

SEQ ID NO : 2 is the amino acid sequence alone ofHIPHUM61.

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 inwardly rectifying potassium channel, referred to herein as HIPHUM61, and variants thereof. Sequence information for HIPHUM61 is provided in SEQ ID NO : 1 (nucleotide and amino acid) and in SEQ ID NO : 2. A polypeptide of the invention thus consists essentially

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

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

Routine methods, can be employed to 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'd 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 HIPHUM61. The essential character of HIPHUM61 can be defined as follows : HIPHUM61 is a inwardly rectifying potassium channel. Preferably the polypeptide is a potassium channel activated by hyperpolarisation of cell membrane potential below the reversal potential for potassium. Preferably a variant polypeptide is one which binds to the same Kir family polypeptide as HIPHUM61. A polypeptide having the same essential character as HIPHUM61 may be identified by monitoring for a function of the inwardly rectifying potassium channel selected from interacting with other Kir family members to form a heteromultimeric inwardly rectifying potassium channel and forming a homomeric or heteromeric inwardly rectifying potassium channel that may be regulated by interaction with a kinase, a phosphatase, a G-protein and/or a PDZ-domain containing protein..

In another aspect of the invention, a variant is one which does not show the same activity as HIPHUM61 but is one which inhibits a basic function of HIPHUM61. For example, a variant polypeptide is one which inhibits homomeric or heteromeric channel formation of HIPHUM61, for example by binding to a Kir family polypeptide to prevent activity mediated by binding of HIPHUM61 to a Kir

family polypeptide.

Typically, polypeptides with more than about 65% identity preferably at least 80% or at least 90% and particularly preferably at least 95% at least 97% or at least 99% identity, with the amino acid sequences of SEQ ID NO : 2, are considered as variants of the proteins. Such variants may include allelic variants and the deletion, modification or addition of single amino acids or groups of amino acids within the protein sequence, as long as the peptide maintains a basic biological functionality of HIPHUM61.

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 an inwardly rectifying 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 Non-polar GAP ILV Polar-uncharged CSTM NQ Polar-charged DE KR AROMATIC H F W Y 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 or 200 amino acids in length is considered to fall within the scope of the invention as long as it demonstrates a basic biological functionality ofHIPHUM61. 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 a Kir family polypeptide-binding region. Such fragments can be used to construct chimeric receptors preferably with another homomeric or heteromeric Kir channel polypeptide, more preferably with another inwardly rectifying potassium channel.

Such fragments ofHIPHUM61 or a variant thereof can also be used to raise anti HIPHUM61 antibodies. In this embodiment the fragment may comprise an epitope of the HIPHUM61 polypeptide and may otherwise not demonstrate the a Kir family polypeptide binding or other properties of HIPHUM61.

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 HIPHUM61 or variant thereof as well as nucleotide sequences which are complementary thereto.

The nucleotide sequence may be RNA or DNA including genomic DNA, synthetic DNA or cDNA. Preferably the nucleotide sequence is a DNA sequence and most preferably, a cDNA sequence. Nucleotide sequence information is provided in SEQ ID NO: 1. Such nucleotides can be isolated from human cells or synthesised according to methods well known in the art, as described by way of example in Sambrook et al, 1989.

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

A polynucleotide of the invention can hydridize to the coding sequence or the complement of the coding sequence of SEQ ID NO: 1 at a level significantly above background. Background hybridization may occur, for example, because of other cDNAs present in a cDNA library. The signal level generated by the interaction between a polynucleotide of the invention and the coding sequence or complement of the coding sequence of SEQ ID NO: 1 is typically at least 10 fold, preferably at least 100 fold, as intense as interactions between other polynucleotides and the coding

sequence of SEQ ID NO : 1. The intensity of interaction may be measured, for example, by radiolabelling the probe, e. g. with 32p Selective hybridisation may typically be achieved using conditions of medium to high stringency. However, such hybridisation may be carried out under any suitable conditions known in the art (see Sambrook et al, 1989. For example, if high stringency is required suitable conditions include from 0.1 to 0.2 x SSC at 60 C up to 65 C. If lower stringency is required suitable conditions include 2 x SSC at 60 DC.

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

A polynucleotide may include one or more introns, for example may comprise genomic DNA. Additional sequences such as signal sequences which may assist in insertion of the polypeptide in a cell membrane may also be included. The modified polynucleotide generally encodes a polypeptide which has HIPHUM61 activity.

Alternatively, a polynucleotide encodes Kir polypeptide-binding portion of a polypeptide or a polypeptide which inhibits HIPHUM61 activity. Degenerate substitutions may be made and/or substitutions may be made which would result in a conservative amino acid substitution when the modified sequence is translated, for example as shown in the Table above.

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

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

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

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

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

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

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

The BLAST algorithm performs a statistical analysis of the similarity between two sequences; see e. g. , Karlin and Altschul (1993) Proto 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 90% sequence

identity over 25, preferably over 30 nucleotides forms one aspect of the invention, as does a polynucleotide which has at least 95% sequence identity over 40 nucleotides.

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

The nucleotides may be involved in recombinant protein synthesis or indeed as therapeutic agents in their own right, utilised in gene therapy techniques. Nucleotides complementary to those encoding HIPHUM61, 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 nucleotides in length, or even up to a few nucleotides, such as five or ten nucleotides, short of the coding sequence of SEQ ID NO : 1.

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

Polynucleotides according to the invention may also be inserted into the vectors described above in an antisense orientation in order to provide for the production of antisense RNA. Antisense RNA or other antisense polynucleotides may also be produced by synthetic means. Such antisense polynucleotides may be

used as test compounds in the assays of the invention or may be useful in a method of treatment of the human or animal body by therapy.

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

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

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

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

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

Mammalian promoters, such as 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 lentivimses, 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 HIPHUM61 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 binding of a polypeptide of the invention to other Kir polypeptides to form a channel. 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 e 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: 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 HIPHUM61 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 zn 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 inwardly rectifying potassium channels and in particular which bind to HIPHUM61. Screening methods may also be used to identify agonists or antagonists which may modulate inwardly rectifying potassium

channel activity, inhibitors or activators ofHIPHUM61 activity, and/or agents which up-regulate or down-regulate HIPHUM61 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 inwardly rectifying 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 Kir family members such as Kir4.1, Kir3.2, or Kir7.1.

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 Kir family members such as Kir4.1, Kir3.2, or Kir7. 1 may also be identified through a yeast 2-hybrid assay or other protein interaction assay such as a coimmunoprecipitation or an ELISA based technique.

Assays may be carried out using cells expressing HIPHUM61, and incubating such cells with the test substance optionally in the presence of other Kir family members such as Kir4.1, Kir3. 2, or Kir7.1. The results of the assay are compared to the results obtained using the same assay in the absence of the test substance. Cells expressing HIPHUM61 constitutively may be provided for use in assays for HIPHUM61 function. Additional test substances may be introduced in any assay to look for inhibitors or enhancers of binding of HIPHUM61 to other Kir family members such as Kir4.1, Kir3.2, or Kir7. 1 or inhibitors or enhances of HIPHUM61- mediated activity, preferably homomeric or heteromeric channel formation.

In preferred aspects, a host cell is provided expressing the polypeptide and containing a K+/Rb+ sensitive photoprotein. Such photoproteins increase or decrease light emission on the influx or efflux ofK/Rb* ions and can be detected using an imaging system. A reporter gene assay using such photoproteins can be used to assay for modulation of potassium channel activity. The assay enables determination of whether a test substance modulates the HIPHUM61 regulated flow ofK/Rb ions through potassium channels in target cells.

The ability of a test substance to modulate the HIPHUM61 regulated flow of calcium ions through homomeric or heteromeric Kir 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.

Assays may also be carried out by measuring the influx or efflux of radioactive K+/Rb+ 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 Kir family members such as Kir4.1, Kir3.2, or Kir7.1 may also be used to assay for modulatory activity of a test substance.

Preferably, electroplysological assays and/or assays comprising measuring changes in intracellular K+/Rb+ ion concentration are performed on cells expressing a

polypeptide of the invention and a a Kir family polypeptide.

P Assays monitoring conformational changes of the inwardly rectifying potassium channels of the invention may also be used to identify activation,

modulation or inhibition of potassium channel activity.

Assays may also be carried out to identify substances which modify HIPHUM61 expression, for example substances which up-or down-regulate expression. Such assays may be carried out for example by using antibodies for HIPHUM61 to monitor levels of HIPHUM61 expression. Other assays which can be used to monitor the effect of a test substance on HIPHUM61 expression include using a reporter gene construct driven by the HIPHUM61 regulatory sequences as the promoter sequence and monitoring for expression of the reporter polypeptide.

Further possible assays could utilise membrane fractions from overexpression of HIPHUM61 polypeptide either in lavis 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.

ZD 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 lnM to 1OOOu0 M, preferably from luM to lOOu. M, more preferably from IjM to 10pu. Preferably, the activity of a test substance is compared to the activity shown by a known activator or inhibitor. A test substance which acts as an inhibitor may produce a 50% inhibition of activity of the receptor. Alternatively a test

substance which acts as an activator may produce 50% of the maximal activity produced using a known activator.

Another aspect of the present invention is the use of polynucleotides encoding the HIPHUM61 polypeptides of the invention to identify mutations in HIPHUM61 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 HIPHUM61 expression. Polynucleotides such as SEQ ID NO: 1 or fragments thereof may be used to identify allelic variants, genomic DNA and species variants.

The present invention provides a method for detecting variation in the expressed products encoded by HIPHUM61 genes. This may comprise determining the level of HIPHUM61 expressed in cells or determining specific alterations in the expressed product. Sequences of interest for diagnostic purposes include, but are not limited to, the conserved portions as identified by sequence similarity and conservation of intron/exon structure. The diagnosis may be performed in conjunction with kindred studies to determine whether a mutation of interest cosegregates with disease phenotype in a family.

Diagnostic procedures may be performed on polynucleotides isolated from an individual or alternatively, may be performed in situ directly upon tissue sections (fixed and/or frozen) of patient tissue obtained from biopsies or resections, such that no nucleic acid purification is necessary. Appropriate procedures are described in, for example, Nuovo, G. J., 1992, "PCR In Situ Hybridization: Protocols And Applications", Raven Press, NY). Such analysis techniques include, DNA or RNA blotting analyses, single stranded conformational polymorphism analyses, in situ hybridization assays, and polymerase chain reaction analyses. Such analyses may reveal both quantitative aspects of the expression pattern of a HIPHUM61, and

qualitative aspects of HIPHUM61 expression and/or composition.

Alternative diagnostic methods for the detection of HIPHUM61 nucleic acid molecules may involve their amplification, e. g. by PCR (the experimental embodiment set forth in U. S. Patent No. 4, 683, 202), ligase chain reaction (Barany, 1991, Proc. Natl. Acad. Sci. USA 88 : 189-193), self sustained sequence replication

(Guatelli et al.. 1990, Proc. Natl. Acad. Sci. USA 87 : 1874-1878), transcriptional amplification system (Kwoh et al., 1989, Proc. Natl. Acad. Sci. 15 USA 86 : 1173 1177), Q-Beta Replicase (Lizardi et al., 1988, Bio/Technology 6: 1197) or any other nucleic acid amplification method, followed by the detection of the amplified molecules using techniques well known to those of skill in the art. These detection schemes are especially useful for the detection of nucleic acid molecules if such molecules are present in very low numbers.

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

Following detection, the results seen in a given patient may be compared with a statistically significant reference group of normal patients and patients that have HIPHUM61 related pathologies. In this way, it is possible to correlate the amount or kind of HIPHUM61 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 inwardly rectifying 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 cerebellar and spinocerebellar disorders such as acute viral encephalitis, astrocytoma, amyloidosis, ateriovenous malformations, brain abscess, brain infarction, CNS paraneoplastic syndromes, Creutzfeldt-Jackob disease (and other prion diseases), glioblastoma multiforme, neuro-opthalmologic disorders, neuropathic pain, oligodendroglioma, transient ischaemic attacks (TIA) and spinocerebellar ataxia, thyroid disorders such as euthyroid sick syndrome, follicular carcinoma, giant cell thyroid cancer, hyperthyroidism, medullary carcinoma, papillary carcinoma, simple goiter and

thyroiditis and kidney disorders such as acute poststreptococcal glomerulonephritis, acute pyelonephritis, acute renal failure, acute tubular necrosis, Alport's syndrome, chronic glomerulonephritis, chronic renal failure, cystinuria, diabetic nephropathy, Fanconi's syndrome, glomerulonephritis, Grawitz's tumor, hydronephrosis, hypemephroma, medullary cystic disease, medullary sponge kidney, nephrocarcinoma, nephrotic syndrome, polycystic kidney disease, renal calculi, renal cell carcinoma, renal infarction, renal tubular acidosis, renal vein thrombosis, renovascular hypertension and Wilm's Tumor.

Additional disease states that may be treated include cerebellar outflow degeneration (cod).

Substances identified according to the screening methods outlined above may be formulated with standard pharmaceutically acceptable carriers and/or excipients as is routine in the pharmaceutical art. For example, a suitable substance may be dissolved in physiological saline or water for injections. The exact nature of a formulation will depend upon several factors including the particular substance to be administered and the desired route of administration. Suitable types of formulation are fully described in Remington's Pharmaceutical Sciences, Mack Publishing Company, Eastern Pennsylvania, 17"'Ed. 1985. the disclosure of which is included herein of its entirety by way of reference.

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

A therapeutically effective amount of a modulator is administered to a patient. The dose of a modulator may be determined according to various parameters, especially according to the substance used; the age, weight and condition of the patient to be treated; the route of administration ; and the required regimen. A physician will be able to determine the required route of administration and dosage for any particular patient. A typical daily dose is from about 0. 1 to 50 mg per kg of body weight, according to the activity of the specific modulator, the age, weight and conditions of the subject to be treated, the type and severity of the degeneration and the frequency and route of administration. Preferably, daily dosage levels are from 5 mg to 2 g.

Nucleic acid encoding HIPHUM61 or a variant thereof which inhibits or enhances HIPHUM61 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 HIPHUM61 or a variant thereof with an impaired function such as a dominant negative mutant that disrupts the function of the whole homomeric or heteromeric Kir 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 10u, g nucleic acid for particle mediated gene delivery and 10u. g to Img for other routes.

The following Examples illustrate the invention.

Example 1: Characterisation of the sequence An inwardly rectifying potassium channel, designated as HIPHUM61 has been identified. The nucleotide and amino acid sequences of the receptor have been determined. These are set out below in SEQ ID NOs: 1 and 2. Suitable primers and probes were designed and used to analyse tissue expression. HIPHUM61 was found to be primarily expressed in thyroid, cerebellum and kidney.

The chromosomal localization was also mapped. Human HIPHUM61 has

been mapped to chromosome 17q24-q25. 1.

Example 2 : Screening for substances which exhibit protein modulating activity Mammalian cells, such as HEK293, CHO, COS, BHK, 3T3 or HeLa cells over-expressing a polypeptide of the invention together with one or more appropriate Kir channel polypeptide are generated for use in the assay. 96 and 384 well plate, high throughput screens (HTS) are employed using fluorescence based potassium or rubidium 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 alone or together with appropriate Kir polypeptides for making a heteromeric 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 hyperpolaristaion of 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).

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 inwardly rectifying potassium channel.

SEQUENCE LISTING

< 110 > GLAXO GROUP LTD < 120 > POLYPEPTIDE < 130 > QG 1019 < 140 > < 141 > < 160 > 2 < 170 > Patent In Ver. 2. 1 < 210 > 1 < 211 > 1473 < 212 > DNA < 213 > Homo sapins < 220 > < 221 > CDS < 222 > (1).. (1473)

< 400 > 1 atg gcg tea gca cca aat gag tat gcg gca ggg gtt tat aaa gtc tee 48 Met Ala Ser Ala Pro Asn Glu Tyr Ala Ala Gly Val Tyr Lys Val Ser 1 5 10 15 tgt aaa cag gaa gtg tct cag tct gat gta act get acg cag tac gcg 96 Cys Lys Gln Glu Val Ser Gln Ser Asp Val Thr Ala Thr Gln Tyr Ala 20 25 30 gac ggc etc tct ttc cgt ctt cag egg gta gtt cta act gaa aac cca 144 Asp Gly Leu Ser Phe Arg Leu Gln Arg Val Val Leu Thr Glu Asn Pro 35 40 45 aac caa gaa ata gca aca agt cta gaa ttc tta cta cta caa aac tea 192 Asn Gln Glu Ile Ala Thr Ser Leu Glu Phe Leu Leu Leu Gln Asn Ser 50 55 60 cct gga tee cta agg gca cag caa aga atg age tat tac ggc age age 240 Pro Gly Ser Leu Arg Ala Gln Gln Arg Met Ser Tyr Tyr Gly Ser Ser 65 70 75 80 tat cat att ate aat gcg gac gca aaa tac cca ggc tac ccg cca gag 288 Tyr His Ile Ile Asn Ala Asp Ala Lys Tyr Pro Gly Tyr Pro Pro Glu 85 90 95 cac att ata get gag aag aga aga gca aga aga cga tta ctt cac aaa 336 His Ile Ile Ala Glu Lys Arg Arg Ala Arg Arg Arg Leu Leu His Lys 100 105 110 gat ggc age tgt aat gtc tac ttc aag cac att ttt gga gaa tgg gga 384 Asp Gly Ser Cys Asn Val Tyr Phe Lys His Ile Phe Gly Glu Trp Gly 115 120 125

age tat gtg gtt gac ate ttc ace act ctt gtg gac ace aag tgg cgc 432 Ser Tyr Val Val Asp Ile Phe Thr Thr Leu Val Asp Thr Lys Trp Arg 130 135 140 cat atg ttt gtg ata ttt tct tta tct tat att etc teg tgg ttg ata 480 His Met Phe Val Ile Phe Ser Leu Ser Tyr Ile Leu Ser Trp Leu Ile 145 150 155 160 ttt ggc tct gtc ttt tgg etc ata gee ttt cat cat ggc gat cta tta 528 Phe Gly Ser Val Phe Trp Leu Ile Ala Phe His His Gly Asp Leu Leu 165 170 175 aat gat cca gac ate aca cct tgt gtt gac aac gtc cat tct ttc aca 576 Asn Asp Pro Asp Ile Thr Pro Cys Val Asp Asn Val His Ser Phe Thr 180 185 190 ggg gcc ttt ttg ttc tee cta gag ace caa ace ace ata gga tat ggt 624 Gly Ala Phe Leu Phe Ser Leu Glu Thr Gln Thr Thr Ile Gly Tyr Gly 195 200 205 tat cgc tgt gtt act gaa gaa tgt tct gtg gcc gtg etc atg gtg ate 672 Tyr Arg Cys Val Thr Glu Glu Cys Ser Val Ala Val Leu Met Val Ile 210 215 220 etc cag tee ate tta agt tgc ate ata aat ace ttt ate att gga get 720 Leu Gln Ser Ile Leu Ser Cys Ile Ile Asn Thr Phe Ile Ile Gly Ala 225 230 235 240 gcc ttg gcc aaa atg gca act get cga aag aga gcc caa ace att cgt 768 Ala Leu Ala Lys Met Ala Thr Ala Arg Lys Arg Ala Gln Thr Ile Arg 245 250 255 ttc age tac ttt gca ctt ata ggt atg aga gat ggg aag ctt tgc etc 816 Phe Ser Tyr Phe Ala Leu Ile Gly Met Arg Asp Gly Lys Leu Cys Leu 260 265 270 atg tgg cgc att ggt gat ttt egg cca aac cac gtg gta gaa gga aca 864 Met Trp Arg Ile Gly Asp Phe Arg Pro Asn His Val Val Glu Gly Thr 275 280 285 gtt aga gcc caa ctt etc cgc tat aca gaa gac agt gaa ggg agg atg 912 Val Arg Ala Gln Leu Leu Arg Tyr Thr Glu Asp Ser Glu Gly Arg Met 290 295 300 acg atg gca ttt aaa gac etc aaa tta gtc aac gac caa ate ate ctg 960 Thr Met Ala Phe Lys Asp Leu Lys Leu Val Asn Asp Gln Ile Ile Leu 305 310 315 320 gtc ace ccg gta act att gtc cat gaa att gac cat gag age cct ctg 1008 Val Thr'Pro Val Thr Ile Val His Glu Ile Asp His Glu Ser Pro Leu 325 330 335 tat gcc ctt gac cgc aaa gca gta gcc aaa gat aac ttt gag att ttg 1056 Tyr Ala Leu Asp Arg Lys Ala Val Ala Lys Asp Asn Phe Glu Ile Leu 340 345 350 gtg aca ttt ate tat act ggt gat tee act gga aca tct cac caa tct 1104

Val Thr Phe Ile Tyr Thr Gly Asp Ser Thr Gly Thr Ser His Gln Ser 355 360 365 aga age tee tat gtt ccc cga gaa att etc tgg ggc cat agg ttt aat 1152 Arg Ser Ser Tyr Val Pro Arg Glu Ile Leu Trp Gly His Arg Phe Asn 370 375 380 gat gtc ttg gaa gtt aag agg aag tat tac aaa gtg aac tgc tta cag 1200 Asp Val Leu Glu Val Lys Arg Lys Tyr Tyr Lys Val Asn Cys Leu Gln 385 390 395 400 ttt gaa gga agt gtg gaa gta tat gcc ccc ttt tgc agt gcc aag caa 1248 Phe Glu Gly Ser Val Glu Val Tyr Ala Pro Phe Cys Ser Ala Lys Gln 405 410 415 ttg gac tgg aaa gac cag cag etc cac ata gaa aaa gca cca cca gtt 1296 Leu Asp Trp Lys Asp Gln Gln Leu His Ile Glu Lys Ala Pro Pro Val 420 425 430 cga gaa tee tgc acg teg gac ace aag gcg aga cga agg tea ttt agt 1344 Arg Glu Ser Cys Thr Ser Asp Thr Lys Ala Arg Arg Arg Ser Phe Ser 435 440 445 gca gtt gcc att gtc age age tgt gaa aac cct gag gag ace ace act 1392 Ala Val Ala Ile Val Ser Ser Cys Glu Asn Pro Glu Glu Thr Thr Thr 450 455 460 tee gcc aca cat gaa tat agg gaa aca cct tat cag aaa get etc ctg 1440 Ser Ala Thr His Glu Tyr Arg Glu Thr Pro Tyr Gln Lys Ala Leu Leu 465 470 475 480 act tta aac aga ate tct gta gaa tee caa atg 1473 Thr Leu Asn Arg Ile Ser Val Glu Ser Gln Met 485 490 < 210 > 2 < 211 > 491 < 212 > PRT < 213 > Homo sapins < 400 > 2 Met Ala Ser Ala Pro Asn Glu Tyr Ala Ala Gly Val Tyr Lys Val Ser 1 5 10 15 Cys Lys Gln Glu Val Ser Gln Ser Asp Val Thr Ala Thr Gln Tyr Ala 20 25 30 Asp Gly Leu Ser Phe Arg Leu Gln Arg Val Val Leu Thr Glu Asn Pro 35 40 45 Asn Gln Glu Ile Ala Thr Ser Leu Glu Phe Leu Leu Leu Gln Asn Ser 50 55 60 Pro Gly Ser Leu Arg Ala Gln Gln Arg Met Ser Tyr Tyr Gly Ser Ser 65 70 75 80

Tyr His Ile Ile Asn Ala Asp Ala Lys Tyr Pro Gly Tyr Pro Pro Glu 85 90 95 His Ile Ile Ala Glu Lys Arg Arg Ala Arg Arg Arg Leu Leu His Lys 100 105 110 Asp Gly Ser Cys Asn Val Tyr Phe Lys His Ile Phe Gly Glu Trp Gly 115 120 125 Ser Tyr Val Val Asp Ile Phe Thr Thr Leu Val Asp Thr Lys Trp Arg 130 135 140 His Met Phe Va1 lie Phe Ser Leu Ser Tyr He Leu Ser Trp Leu He 145 150 155 160 Phe Gly Ser Val Phe Trp Leu Ile Ala Phe His His Gly Asp Leu Leu 165 170 175 Asn Asp Pro Asp Ile Thr Pro Cys Val Asp Asn Val His Ser Phe Thr 180 185 190 Gly Ala Phe Leu Phe Ser Leu Glu Thr Gln Thr Thr Ile Gly Tyr Gly 195 200 205 Tyr Arg Cys Val Thr Glu Glu Cys Ser Val Ala Val Leu Met Val Ile 210 215 220 Leu Gln Ser Ile Leu Ser Cys Ile Ile Asn Thr Phe He Ile Gly Ala 225 fi30 235 240 Ala Leu Ala Lys Met Ala Thr Ala Arg Lys Arg Ala Gln Thr Ile Arg 245 250 255 Phe Ser Tyr Phe Ala Leu Ile Gly Met Arg Asp Gly Lys Leu Cys Leu 260 265 270 Met Trp Arg Ile Gly Asp Phe Arg Pro Asn His Val Val Glu Gly Thr 275 280 285 Val Arg Ala Gln Leu Leu Arg Tyr Thr Glu Asp Ser Glu Gly Arg Met 290 295 300 Thr Met Ala Phe Lys Asp Leu Lys Leu Val Asn Asp Gin He He Leu 305 310 315 320 Val Thr Pro Val Thr Ile Val His Glu Ile Asp His Glu Ser Pro Leu 325 330 335 Tyr Ala Leu Asp Arg Lys Ala Val Ala Lys Asp Asn Phe Glu Ile Leu 340 345 350 Val Thr Phe Ile Tyr Thr Gly Asp Ser Thr Gly Thr Ser His Gln Ser 355 360 365 Arg Ser Ser Tyr Val Pro Arg Glu Ile Leu Trp Gly His Arg Phe Asn 370 375 380

Asp Val Leu Glu Val Lys Arg Lys Tyr Tyr Lys Val Asn Cys Leu Gln 385 390 395 400 Phe Glu Gly Ser Val Glu Val Tyr Ala Pro Phe Cys Ser Ala Lys Gln 405 410 415 Leu Asp Trp Lys Asp Gln Gln Leu His Ile Glu Lys Ala Pro Pro Val 420 425 430 Arg Glu Ser Cys Thr Ser Asp Thr Lys Ala Arg Arg Arg Ser Phe Ser 435 440 445

Ala Val Ala Ile Val Ser Ser Cys Glu Asn Pro Glu Glu Thr Thr Thr 450 455 460 Ser Ala Thr His Glu Tyr Arg Glu Thr Pro Tyr Gln Lys Ala Leu Leu 465 470 475 480 Thr Leu Asn Arg Ile Ser Val Glu Ser Gln Met 485 490

Claims (14)

1. Use of a substance which modulates inwardly rectifying potassium channel activity of a polypeptide, which polypeptide comprises: (a) the amino acid sequence of SEQ ID NO: 2; or (b) a variant thereof which is a potassium channel activated by hyperpolarisation of cell membrane potential below the reversal potential for potassium; or (c) a fragment of (a) or (b) which is a potassium channel activated by hyperpolarisation of cell membrane potential below the reversal potential for potassium; in the manufacture of a medicament for the treatment or prophylaxis of a cerebellar or spinocerebellar disorder, a thyroid disorder or a kidney disorder.

2. A use according to claim 1 wherein the cerebellar or spinocerebellar disorder is selected from acute viral encephalititis, astrocytoma, amyloidosis, ateriovenous malformations, brain abscess, brain infarction, CNS paraneoplastic syndromes, Creutzfeldt-Jackob disease (and other prion diseases), glioblasoma multiforme, neuro-ophthalmologic disorders, neuropathic pain, oligodendroglioma, transient ischemic attacks (TIA) and spinocerebellar ataxia.

3. A use according to claim 1 wherein the thyroid disorder is selected from euthyroid sick syndrome, follicular carcinoma, giant cell thyroid cancer, hyperthyroidism, hypothyroidism, medullary carcinoma, papillary carcinoma, simple goiter and thyroiditis.

4. A use according to claim 1 wherein the kidney disorder is selected from acute poststreptococcal glomerulonephritis, acute pyelonephritis, acute renal failure, acute tubular necrosis, Alport's syndrome, chronic glomerulonephritis, chronic renal failure, cystinuria, diabetic nephropathy, Fanconi's syndrome, glomerulonephritis, Grawitz's tumor, hydronephrosis, hypernephroma, medullary cystic disease, medullary sponge kidney, nephrocarcinoma, nephrotic syndrome, polycystic kidney disease, renal calculi, renal cell carcinoma, renal infarction, renal tubular acidosis, renal vein thrombosis, renovascular hypertension and Wilm's tumor.

5. A method of treating a subject having a cerebellar or spinocerebellar disorder, a thyroid disorder or a kidney disorder, which method comprises:

(i) contacting a test substance and a polypeptide comprising : (a) the amino acid sequence of SEQ ID NO : 2 ; or (b) a variant thereof which is a potassium channel activated by hyperpolarisation of cell membrane potential below the reversal potential for potassium ; or (c) a fragment of (a) or (b) which is a potassium channel activated by hyperpolarisation of cell membrane potential below the reversal potential for potassium; (ii) determining the effect of the test substance on the activity and/or expression of the said polypeptide, thereby determining whether the test substance modulates inwardly rectifying potassium channel activity and/or expression; and (iii) administering to said subject an effective amount of a substance which modulates inwardly rectifying potassium channel activity.

6. A method according to claim 5 wherein the variant (b) has at least 80% identity to the amino acid sequence of SEQ ID NO: 2.

7. A method of treating a subject having a cerebellar or spinocerebellar disorder, a thyroid disorder or a kidney disorder, which method comprises: (i) contacting a test substance and a polynucleotide encoding a inwardly rectifying potassium channel polypeptide which is a potassium channel activated by hyperpolarisation of cell membrane potential below the reversal potential for potassium which polynucleotide comprises: (a) the nucleic acid sequence of SEQ ID NO: 1 and/or a sequence complementary thereto; (b) a sequence which hybridises under stringent conditions to a sequence as defined in (a); (c) a sequence that is degenerate as a result of the genetic code to a sequence as defined in (a) or (b); or (d) a sequence having at least 60% identity to a sequence as defined in (a), (b) or (c); (ii) determining the effect of the test substance on the activity and/or expression of the polypeptide encoded by the said polynucleotide,

thereby determining whether the test substance modulates inwardly rectifying potassium channel activity and/or expression : and (iii) administering to said subject an effective amount of a substance which modulates inwardly rectifying potassium channel activity.

8. A method according to any one of claims 5 to 7 wherein the polypeptide is expressed in a cell.

9. A method according to claim 8 wherein the cell expresses another polypeptide of the inwardly rectifying potassium channel family.

10. A method according to claim 9 wherein step (ii) comprises monitoring inwardly rectifying potassium channel activity.

11. A method according to claim 9 wherein step (ii) comprises monitoring any interaction between the said polypeptide with the said polypeptide of the inwardly rectifying potassium channel family.

12. A method according to any one of claims 5 to 11 wherein the cerebellar or spinocerebellar disorder is selected from acute viral encephalititis, astrocytoma, amyloidosis, ateriovenous malformations, brain abscess, brain infarction, CNS paraneoplastic syndromes, Creutzfeldt-Jackob disease (and other prion diseases), glioblasoma multiforme, neuro-ophthalmologic disorders, neuropathic pain, oligodendroglioma, transient ischemic attacks (TIA) and spinocerebellar ataxia.

13. A use according to any one of claims 5 to 11 wherein the thyroid disorder is selected from euthyroid sick syndrome, follicular carcinoma, giant cell thyroid cancer, hyperthyroidism, hypothyroidism, medullary carcinoma, papillary carcinoma, simple goiter and thyroiditis.

14. A use according to any one of claims 5 to 11 wherein the kidney disorder is selected from acute poststreptococcal glomerulonephritis, acute pyelonephritis, acute renal failure, acute tubular necrosis, Alport's syndrome, chronic glomerulonephritis, chronic renal failure, cystinuria, diabetic nephropathy, Fanconi's syndrome, glomerulonephritis, Grawitz's tumor, hydronephrosis, hypemephroma, medullary cystic disease, medullary sponge kidney, nephrocarcinoma, nephrotic syndrome, polycystic kidney disease, renal calculi, renal cell carcinoma, renal infarction, renal tubular acidosis, renal vein thrombosis, renovascular hypertension and Wilm's tumor.