WO2000006183A1 - C7F2-A NOVEL POTASSIUM CHANNEL β-SUBUNIT - Google Patents

Abstract

The present invention relates to a newly identified potassium channel β-subunit. The invention also relates to polynucleotides encoding the subunit. The invention further relates to methods using subunit polypeptides and polynucleotides, applicable to diagnosis and treatment in channel-mediated disorders. The invention further relates to drug-screening methods using the polypeptides and polynucleotides to identify agonists and antagonists, applicable to diagnosis and treatment. The invention further encompasses agonists, and antagonists based on the subunit polypeptides and polynucleotides. The invention further relates to procedures for producing the subunit polypeptides and polynucleotides.

Description

C7F2- A NOVEL POTASSIUM CHANNEL β-SUBUNiT

FIELD OF THE INVENTION

The present invention relates to a newly identified potassium channel β- subunit. The invention also relates to polynucleotides encoding the β-subunit. The invention further relates to methods using β-subunit polypeptides and polynucleotides, as a target for diagnosis and treatment in channel-mediated disorders. The invention further relates to drug-screening methods using the polypeptides and polynucleotides to identify agonists and antagonists, applicable to diagnosis and treatment. The invention further encompasses agonists, and antagonists based on the β-subunit polypeptides and polynucleotides. The invention further relates to procedures for producing the β-subunit polypeptides and polynucleotides.

BACKGROUND OF THE INVENTION

The flow of potassium through the plasma membrane affects diverse biological processes including action-potential firing and control of cell volume. Potassium channels are ubiquitous integral membrane proteins serving numerous functions in excitable and nonexcitable cells (McManus, O. B., J. Bioenerg. Biomembr. 23:537-560 (1991)). Many different classes of potassium channels have evolved and have been separated into classes on the basis of their biophysical properties, physiological regulation, and pharmacology (Hille, B., Ionic Channels of Excitable Membranes, Sunderland, M. A. Sinauer (1992); Rudy, B., Neuroscience 25:129-149 (1988)). Major types include voltage-dependent, calcium-activated, and ATP-sensitive channels. Some subtypes exist within the classifications. However, certain functional features are shared among many types of potassium channels (Kukuljan et al., Am. J. Physiol. 268 (Cell Physiol. 37):C535-C556 (1995)).

Potassium channel-forming proteins can be grouped into three families that differ in the number of transmembrane segments. The largest family contains six membrane-spanning segments. Inward rectifiers comprise the second family with subunits having two transmembrane segments. The third family contains only one transmembrane segment. These channels have been studied using recombinant DNA techniques. The information has been reviewed in Kukuljan et al, cited above. High conductance calcium-activated potassium channels are a group of proteins with a number of unique features. The channels are activated by intracellular calcium, as well as membrane depolarization. The channels display a high single-channel conductance and are highly selective for potassium. They are sensitive to specific toxins, such as charybdotoxin that binds to a receptor site located on the external vestibule of the channel and prevents potassium flow by physical occlusion of the pore .

Knaus et al. (J. Biol. Chem. 269:3921-3924 (1994)) reported on the subunit composition of the high conductance calcium-activated potassium channel from smooth muscle. This potassium channel is reported to be composed of two subunits, α and β, of 62 and 31 kilodaltons, respectively. Amino acid sequence analysis showed a high sequence homology with two cloned high conductance potassium channels from Drosophila. An antipeptide antibody directed against the amino acid sequence of one of the -subunit fragments could also immunoprecipitate, under nondenaturing conditions, the β-subunit, demonstrating specific noncovalent association of both subunits. The results indicated that the α-subunit of this specific high conductance potassium channel is a member of a specific family of potassium channels and forms a noncovalent complex with a β-subunit. The reference reported a specific and tight interaction between the two polypeptides. The following model was proposed. The α-subunit is the central ion channel-forming element and contains the receptor for the various blocking toxins. A tetramer α-subunit is noncovalently associated with four β-subunits. The β-subunits are in close proximity (less than 12 A) to the pore-forming and receptor carrying subunit. This high conductance potassium channel β-subunit shares characteristics with the β-subunit of rat brain sodium channels and the γ-subunit of skeletal muscle L-type calcium channels and may be analogous in structure and/or function. It is speculated that this subunit is a conserved constituent of many voltage- and calcium-dependent ' potassium channels. Knaus et al. (J. Biol. Chem. 269:11214- 1218 (1994)) disclosed the primary sequence and immunological characterization of the β-subunit of the high conductance calcium-activated potassium channel from smooth muscle. The amino acid sequence was used to design oligonucleotide probes with which cDNAs encoding the protein were isolated. The protein was reported to contain two hydrophobic (putative transmembrane) domains bearing little sequence homology to subunits of other known ion channels. Reports had suggested that the β-subunit plays a role in modulating the properties of the pore-forming subunit. For example, co-expression of sodium or calcium channel α- and β-subunits had been demonstrated to modulate the currents expressed from the α-subunits alone. The reference also reported small regions of homology with other β-subunits. It is reported, for example, that the β₂- subunit of the rabbit cardiac calcium channel contains a stretch of eight amino acids that are 100% homologous to a region of the β-subunit of the channel under study. McManus et al. (Neuron 14:645-650 (1995)) examined the functional contribution of the β-subunit properties of high conductance potassium channels expressed heterologously in Xenopus oocytes. The reference reported that co- expression of the bovine smooth muscle high conductance potassium channel β- subunit has dramatic effects on the properties of expressed mouse brain α-subunits. The reference noted that expression of an α-subunit alone is sufficient to generate potassium channels that are gated by voltage and intracellular calcium. Nevertheless, channels from oocytes injected with cDNAs encoding both α- and β-subunits were much more sensitive to activation voltage and calcium than channels composed of the α-subunit alone. Expression levels, single channel conductance, and ion selectivity appeared unaffected. Further, channels from oocytes expressing both subunits were sensitive to a potent agonist of native high conductance potassium channels, whereas channels composed of the α-subunit alone were insensitive. Thus, in addition to its effects on channel gating, the β-subunit conferred sensitivity to DHS-I, a potent agonist of native high conductance potassium channels. Accordingly, whereas expression of the β-subunit alone did not result in a functional potassium channel, a coexpression with the α-subunit formed channels with biophysical and pharmacological properties distinct from channels formed by the α- subunit alone. These properties more closely resemble those of native high conductance potassium channels. The report concluded that based on the effect on sensitivity of the channel to voltage and calcium conferred by the β-subunit, that the β-subunit may form part ofthe transduction machinery of the channel. This reference also showed that these properties could be conferred by chimeric multimers in which a β-subunit from one tissue was able to modulate the α-subunit from another tissue. The possibility was raised that regulated expression of β-subunits, as in tissue-specific or developmental-specific regulation, could constitute a mechanism for generating functional diversity among mammalian high conductance potassium channels.

Meera et al. (FEBS. Lett. 552:84-88 (1996)) disclosed the importance of calcium concentration for the functional coupling between α- and β-subunits of high conductance potassium channels. The reference pointed out that these channels are unique in that they are modulated not only by voltage, but also by calcium in the micromolar range. They referred to the β-subunit as "the regulatory subunit for the pore-forming α-subunit." The reference demonstrated that intracellular calcium concentration controls the functional coupling between α- and β-subunits of the complex in a concentration range relevant to cellular excitation. The β-subunit used for the experiments was derived from human smooth muscle. The experiments were performed by injecting cRNA into Xenopus oocytes. Channel currents and number of channels were recorded. The results were reported as demonstrating that a minimum calcium concentration was required to switch α- and β-subunits to a functional activated mode. It was proposed that a rise in local calcium concentration would induce a conformational change in one or both of the subunits, triggering the functional coupling and causing the α-subunit to respond much more efficiently to calcium and voltage. Prior to this work, it was thought that the channels were calcium- and voltage-activated and would never open in the virtual absence of calcium. However, the report demonstrated that the channel α-subunit will open at a low calcium concentration and, in fact, becomes independent of calcium at concentrations lower than 100 nM, operating according to a purely voltage-regulated mode. Similarly, the results provided evidence for a calcium dependent mechanism that switches the α-subunit from a calcium-independent to a calcium-dependent mode and from a β-subunit-null interaction to a β-subunit-activated mode.

The β-subunits of voltage-gated potassium channels have been recently reviewed (Barry et al., Ann. Rev. Physiol. 55:363-394 (1996)).

Oberst et al. (Oncogene 74:1109-1116 (1997)) recently identified a nucleic acid sequence in quail cDNA in which the corresponding gene encodes a 200 amino acid protein with 46-48% amino acid sequence identity to regulatory β-subunits of the bovine, human, and canine high conductance calcium-activated potassium channel. Studies of gene expression in v-w c-transformed quail embryo fibroblasts led to the isolation of a clone hybridizing in the normal, but not in the transformed fibroblasts. Subsequent analysis revealed that the sequence was expressed in all normal avian fibroblasts tested, but was undetectable in a variety of cell lines transformed by a variety of oncogenes or chemical carcinogens. It was suggested that the protein encoded by this sequence is a regulatory subunit of a calcium- activated potassium channel potentially involved in the regulation of cell proliferation.

Rhodes et al. (J. Neurosci. 77:8246-8258 (1997)) examined the association and colocalization of two mammalian β-subunits with several potassium channel α- subunits in adult rat brain. The experiment showed that the two subunits associate with virtually all of the α-subunits examined. It was suggested that the differential expression and association of cytoplasmic β-subunits with pore-forming α-subunits could significantly contribute to the complexity and heterogeneity of voltage-gated potassium channels in excitable cells. The results provided a biochemical and neuroanatomical basis for the differential contribution of α and β subunits to electrophysiologically diverse neuronal potassium currents.

Ion channels are a major target for drug action and development. Accordingly, it is valuable to the field of pharmaceutical development to identify and characterize previously unknown ion channel components. The present invention advances the state of the art by providing a previously unidentified human potassium channel β-subunit. SUMMARY OF THE INVENTION

It is a general object of the invention to modulate ion channels.

Therefore, it is an object of the invention to identify novel ion channel components.

It is a specific object of the invention to provide novel ion channel β-subunit polypeptides, herein referred to as C7F2 polypeptides, that are useful as reagents or targets in assays applicable to treatment and diagnosis of ion-channel-mediated disorders.

It is a further object of the invention to provide polynucleotides corresponding to the novel β-subunit polypeptides that are useful as targets and reagents in assays applicable to treatment and diagnosis of ion-channel-mediated disorders and useful for producing novel ion channel polypeptides by recombinant methods.

A specific object of the invention is to identify compounds that act as agonists or antagonists and modulate the function or expression of the β-subunit. A further specific object of the invention is to provide the compounds that modulate the expression or function of the β-subunit for treatment and diagnosis of ion-channel-related disorders.

The novel β-subunit polypeptides and polynucleotides of the invention are useful for the treatment of β-subunit-associated or related disorders, including, for example, central nervous system (CNS) disorders, cardiovascular system disorders, and musculoskeletal system disorders, β-subunit-associated or related disorders also include disorders of tissues in which the novel β-subunit C7F2 is expressed, e.g., heart, placental, lung, kidney, prostate, testicular, ovarian, spleen, small and large intestine, colon, or thymus tissues, as well as in brain tissues, including cerebellum, cerebral cortex, medulla, spinal cord, occipital lobe, frontal lobe, temporal lobe, putanem, amygdala, caudate, corpus colosum, hippocampus, substantia nigra, sub halamus and thalamus. The invention is thus based on the identification of a novel potassium channel β-subunit.

This β-subunit is useful for modulating ion channels in view of its interaction with the pore-forming α-subunit. Accordingly, by using the β-subunit to modulate α-subunit activity, ion channel modulation is provided.

The β-subunit is also useful per se as a target or reagent for treatment and diagnosis.

The invention thus provides isolated β-subunit polypeptides including a polypeptide having the amino acid sequence shown in SEQ ID NO 1 , or the amino acid sequence encoded by the cDNA deposited as ATCC No. on

("the deposited cDNA").

The invention also provides isolated β-subunit nucleic acid molecules having the sequence shown in SEQ ID NO 2 or in the deposited cDNA.

The invention also provides variant polypeptides having an amino acid sequence that is substantially homologous to the amino acid sequence shown in SEQ ID NO 1 or encoded by the deposited cDNA.

The invention also provides variant nucleic acid sequences that are substantially homologous to the nucleotide sequence shown in SEQ ID NO 2 or in the deposited cDNA. The invention also provides fragments of the polypeptide shown in SEQ ID

NO 1 and nucleotide shown in SEQ ID NO 2, as well as substantially homologous fragments of the polypeptide or nucleic acid.

The invention also provides vectors and host cells for expression of the β- subunit nucleic acid molecules and polypeptides and particularly recombinant vectors and host cells.

The invention also provides methods of making the vectors and host cells and methods for using them to produce the β-subunit nucleic acid molecules and polypeptides.

The invention also provides antibodies that selectively bind the β-subunit polypeptides and fragments. The invention also provides methods of screening for compounds that modulate the expression or activity of the β-subunit polypeptides. Modulation can be at the level of the polypeptide β-subunit or at the level of controlling the expression of nucleic acid expressing the β-subunit polypeptide. The invention also provides a process for modulating β-subunit expression or activity using the screened compounds, including to treat conditions related to expression or activity of the β-subunit polypeptides.

The invention also provides diagnostic assays for determining the presence, level, or activity of the β-subunit polypeptides or nucleic acid molecules in a biological sample.

The invention also provides diagnostic assays for determining the presence of a mutation in the β-subunit polypeptides or nucleic acid molecules.

DESCRIPTION OF THE DRAWINGS

Figure 1 shows the C7F2 β-subunit nucleotide sequence (SEQ ID NO 2) and the deduced amino acid sequence (SEQ ID NO 1). The amino acid sequence is numbered with respect to nucleotides (below). It is predicted that amino acids 1-19 constitute the amino terminal intracellular domain, 20-40 constitute the first transmembrane domain, 41-167 constitute the extracellular loop, 168- 192 constitute the second transmembrane domain, and 193-210 constitute the carboxyl terminal intracellular domain.

Figures 2 A and B show a sequence comparison of the C7F2 β-subunit amino acid sequence with (A) the sequence of a human calcium-activated potassium channel β-subunit (SEQ ID NO 3) (Meera, P. et al. , FEBS Letters 552:84-88 (1996)) and (B) a quail calcium-activated potassium channel β-subunit (SEQ ID NO 4) (Oberst, C. et al, Oncogene 74:1109-1116 (1997)). Sequences were aligned using the Clustal W (1.74) multiple sequence alignment program using default parameters. Figure 3 shows an analysis of the C7F2 β-subunit amino acid sequence: α βturn and coil regions; hydrophilicity; amphipathic regions; flexible regions; antigenic index; and surface probability. Figure 4 shows a C7F2 β-subunit hydrophobicity plot. Regions of high hydrophobicity corresponding to transmembrane segments occur from amino acids 20-40 and 168-192.

Figure 5 shows an analysis of the C7F2 β-subunit open reading frame for amino acids corresponding to specific functional sites. A glycosylation site is found corresponding to the site at amino acid 56, which would be in the extracellular loop. A second glycosylation site is found at the site of amino acid 93, also in the extracellular loop. A cyclic AMP or cyclic GMP dependent protein kinase phosphorylation site is found at the site of amino acid 210, which is in the carboxy terminal intracellular segment. A protein kinase C phosphorylation site corresponds to the site at amino acid 19, which is just outside the beginning of the first transmembrane domain. This corresponds to the protein kinase A phosphorylation site in one of the β-subunits previously referenced (Knaus, H.G. et al, J. Biol. Chem. 2(59:17274-17278 (1994)). A site corresponding to a casein kinase II phosphorylation site is found at the site of amino acid 14, also in the amino terminal intracellular segment close to the beginning of the first transmembrane segment. A second casein kinase II phosphorylation site is found at the site of amino acid 167, which is in the extracellular loop just adjacent to the beginning of the second transmembrane segment. Figure 6 shows the time constants of activation and deactivation of the mouse maxi-K channel (mSlo) when expressed in HEK293 cells alone or when co- expressed with the C7F2 β-subunit. Note the increase in activation and deactivation time constants when mSlo is co-expressed with C7F2.

Figure 7 shows half-maximal channel activation of the mouse maxi-K channel (mSlo) in the presence of 3 μM Ca⁺⁺when mSlo is expressed alone or co- expressed with the C7F2 β-subunit. Note the consistent and highly significant 20 mV hyperpolarizing (leftward) shift of half-maximal activation of mSlo when co- expressed with C7F2.

Figure 8 shows half-maximal channel activation of the human maxi-K channel (hSlo) in the presence of 3 μM Ca⁺⁺when hSlo is expressed alone or co- expressed with the C7F2 β-subunit. Note the 20-50 mV depolarizing (rightward) shift of half-maximal channel activation of hSlo when co-expressed with C7F2.

DETAILED DESCRIPTION OF THE INVENTION

Polypeptides

The invention is based on the discovery of a novel potassium channel β- subunit. An expressed sequence tag (EST) was identified in a monkey striatum library. This EST had homology to a quail putative potassium channel β-subunit (Oberst et al, cited above) and a human calcium-activated potassium channel β- subunit (Meera et al. , cited above). A human EST was identified with similarity to the 3' end of the monkey EST. This human EST was sequenced and found to be nearly identical to the 3' end of the monkey clone. This EST was used in a Northern blot analysis for expression in various human tissues.

The gene is expressed preferentially in brain with highest expression in the cortical regions but with expression in other regions and in the spinal cord. In the brain the following tissues showed a positive signal upon Northern blotting: cerebellum, cerebral cortex, medulla, spinal cord, occipital lobe, frontal lobe, temporal lobe, putanem, amygdala, caudate, corpus colosum, hippocampus, substantia nigra, subthalamus and thalamus. However, expression is also found in heart, kidney, placenta, lung, prostate, testes, ovary and small and large intestine. Using the sequence as a probe, a full-length human clone from fetal brain was identified and sequenced and designated C7F2.

The invention thus relates to a novel potassium channel β-subunit having the deduced amino acid sequence shown in Figure 1 (SEQ ID NO 1) or having the amino acid sequence encoded by the deposited cDNA, ATCC No. . The deposit will be maintained under the terms of the Budapest Treaty on the International Recognition of the Deposit of Microorganisms. The deposit is provided as a convenience to those of skill in the art and is not an admission that a deposit is required under 35 U.S.C. §112. The deposited sequence, as well as the polypeptide encoded by the sequence, is incorporated herein by reference and controls in the event of any conflict, such as a sequencing error, with description in this application.

The "C7F2 β-subunit polypeptide" or "C7F2 β-subunit protein" refers to the polypeptide in SEQ ID NO 1 or encoded by the deposited cDNA. The term " β- subunit protein" or " β-subunit polypeptide", however, further includes the variants described herein, as well as fragments derived from the full length C7F2 β-subunit polypeptide and variants.

The present invention thus provides an isolated or purified C7F2 potassium channel β-subunit polypeptide and variants and fragments thereof. The C7F2 β-subunit polypeptide is a 210 residue protein exhibiting 5 structural domains. The amino terminal intracellular domain is identified to be within residues 1 to about residue 19 in SEQ ID NO 1. The first transmembrane domain is identified to be within residues from about 20 to about 40 in SEQ ID NO 1. The extracellular loop is identified to be within residues from about 41 to 167 in SEQ ID NO 1. The second transmembrane domain is identified to be within residues from about 168 to about 192 in SEQ ID NO 1. The carboxy terminal intracellular domain is identified to be within residues from about 193 to 210.

As used herein, a polypeptide is said to be "isolated" or "purified" when it is substantially free of cellular material when it is isolated from recombinant and non- recombinant cells, or free of chemical precursors or other chemicals when it is chemically synthesized. A polypeptide, however, can be joined to another polypeptide with which it is not normally associated in a cell and still be considered "isolated" or "purified."

The β-subunit polypeptides can be purified to homogeneity. It is understood, however, that preparations in which the polypeptide is not purified to homogeneity are useful and considered to contain an isolated form of the polypeptide. The critical feature is that the preparation allows for the desired function of the polypeptide, even in the presence of considerable amounts of other components. Thus, the invention encompasses various degrees of purity.

In one embodiment, the language "substantially free of cellular material" includes preparations of the β-subunit polypeptide having less than about 30% (by dry weight) other proteins (i.e., contaminating protein), less than about 20%> other proteins, less than about 10% other proteins, or less than about 5% other proteins. When the β-subunit polypeptide is recombinantly produced, it can also be substantially free of culture medium, i.e., culture medium represents less than about 20%), less than about 10%, or less than about 5% of the volume of the protein preparation.

The language "substantially free of chemical precursors or other chemicals" includes preparations of the β-subunit polypeptide in which it is separated from chemical precursors or other chemicals that are involved in its synthesis. In one embodiment, the language "substantially free of chemical precursors or other chemicals" includes preparations of the polypeptide having less than about 30% (by dry weight) chemical precursors or other chemicals, less than about 20% chemical precursors or other chemicals, less than about 10%o chemical precursors or other chemicals, or less than about 5% chemical precursors or other chemicals. In one embodiment, the β-subunit polypeptide comprises the amino acid sequence shown in SEQ ID NO 1. However, the invention also encompasses sequence variants. Variants include a substantially homologous protein encoded by the same genetic locus in an organism, i.e., an allelic variant. Variants also encompass proteins derived from other genetic loci in an organism, but having substantial homology to the C7F2 β-subunit protein of SEQ ID NO 1. Variants also include proteins substantially homologous to the C7F2 β-subunit protein but derived from another organism, i.e., an ortholog. Variants also include proteins that are substantially homologous to the C7F2 β-subunit protein that are produced by chemical synthesis. Variants also include proteins that are substantially homologous to the C7F2 β-subunit protein that are produced by recombinant methods. It is understood, however, that variants exclude any amino acid sequences disclosed prior to the invention.

As used herein, two proteins (or a region of the proteins) are substantially homologous when the amino acid sequences are at least about 55-60%>, typically at least about 70-75%, more typically at least about 80-85%), and most typically at least about 90-95%) or more homologous. A substantially homologous amino acid sequence, according to the present invention, will be encoded by a nucleic acid sequence hybridizing to the nucleic acid sequence, or portion thereof, of the sequence shown in SEQ ID NO 2 under stringent conditions as more fully described below. To determine the percent homology of two amino acid sequences, or of two nucleic acids, the sequences are aligned for optimal comparison purposes (e.g., gaps can be introduced in the sequence of one protein or nucleic acid for optimal alignment with the other protein or nucleic acid). The amino acid residues or nucleotides at corresponding amino acid positions or nucleotide positions are then compared. When a position in one sequence is occupied by the same amino acid residue or nucleotide as the corresponding position in the other sequence, then the molecules are homologous at that position. As used herein, amino acid or nucleic acid "homology" is equivalent to amino acid or nucleic acid "identity". The percent homology between the two sequences is a function of the number of identical positions shared by the sequences (i.e., per cent homology equals the number of identical positions/total number of positions times 100).

The invention also encompasses polypeptides having a lower degree of identity but having sufficient similarity so as to perform one or more of the same functions performed by the C7F2 β-subunit polypeptide. Similarity is determined by conserved amino acid substitution. Such substitutions are those that substitute a given amino acid in a polypeptide by another amino acid of like characteristics. Conservative substitutions are likely to be phenotypically silent. Typically seen as conservative substitutions are the replacements, one for another, among the aliphatic amino acids Ala, Val, Leu, and He; interchange of the hydroxyl residues Ser and Thr, exchange of the acidic residues Asp and Glu, substitution between the amide residues Asn and Gin, exchange of the basic residues Lys and Arg and replacements among the aromatic residues Phe, Tyr. Guidance concerning which amino acid changes are likely to be phenotypically silent are found in Bowie et al, Science 247:1306-1310 (1990).

TABLE 1. Conservative Amino Acid Substitutions.

Aromatic Phenylalanine

Tryptophan

Tyrosine

Hydrophobic Leucine

Isoleucine

Valine

Polar Glutamine

Asparagine

Basic Arginine

Lysine

Histidine

Acidic Aspartic Acid

Glutamic Acid

Small Alanine

Serine

Threonine

Methionine

Glycine

Both identity and similarity can be readily calculated (Computational Molecular Biology, Lesk, A.M., ed., Oxford University Press, New York, 1988; Biocomputing: Informatics and Genome Projects, Smith, D.W., ed., Academic Press, New York, 1993; Computer Analysis of Sequence Data, Part 1, Griffin, A.M., and Griffin, H.G., eds., Humana Press, New Jersey, 1994; Sequence Analysis in Molecular Biology, von Heinje, G., Academic Press, 1987; and Sequence Analysis Primer, Gribskov, M. and Devereux, J., eds., M Stockton Press, New York, 1991). Preferred computer program methods to determine identify and similarity between two sequences include, but are not limited to, GCG program package (Devereux, J., et al, Nucleic Acids Res. 12(1):381 (1984)), BLASTP, BLASTN, FASTA (Atschul, S.F. et al, J. Molec. Biol. 215:403 (1990)). A variant polypeptide can differ in amino acid sequence by one or more substitutions, deletions, insertions, inversions, fusions, and truncations or a combination of any of these.

Variant polypeptides can be fully functional or can lack function in one or more activities. Thus, in the present case, variations can affect the function, for example, of one or more of the regions corresponding to ligand binding, transmembrane association, phosphorylation, and α-subunit interaction.

Fully functional variants typically contain only conservative variation or variation in non-critical residues or in non-critical regions. Functional variants can also contain substitution of similar amino acids which result in no change or an insignificant change in function. Alternatively, such substitutions may positively or negatively affect function to some degree.

Non-functional variants typically contain one or more non-conservative amino acid substitutions, deletions, insertions, inversions, or truncation or a substitution, insertion, inversion, or deletion in a critical residue or critical region. As indicated, variants can be naturally-occurring or can be made by recombinant means or chemical synthesis to provide useful and novel characteristics for the β-subunit polypeptide. This includes preventing immunogenicity from pharmaceutical formulations by preventing protein aggregation, for example if soluble peptides corresponding to the extracellular loop are used. Useful variations include alteration of ligand binding characteristics. For example, one embodiment involves a variation at the binding site that results in increased or decreased extent or rate of ligand binding. A further useful variation at the same site can result in a higher or lower affinity for ligand. Useful variations also include changes that provide affinity for another ligand. Another useful variation provides for reduced or increased affinity for the α-subunit or for binding by a different α-subunit than the one with which the β-subunit is normally associated. Another useful variation provides for reduced or increased rate or extent of activation of the α-subunit. Another useful variation provides a fusion protein in which one or more segments is operatively fused to one or more segments from another β-subunit. Another useful variation provides for an increase or decrease in phosphorylation or glycosylation.

Amino acids that are essential for function can be identified by methods known in the art, such as site-directed mutagenesis or alanine-scanning mutagenesis (Cunningham et al, Science 244: 1081-1085 (1989)). The latter procedure introduces single alanine mutations at every residue in the molecule. The resulting mutant molecules are then tested for biological activity such as ligand binding, α-subunit association or activation, or channel currents. Sites that are critical for ligand binding and α-subunit modulation can also be determined by structural analysis such as crystallization, nuclear magnetic resonance or photoaffmity labeling (Smith et al, J. Mol. Biol. 224:899-904 (1992); de Vos et al. Science 255:306-312 (1992)). The invention also includes polypeptide fragments of the C7F2 β-subunit protein. Fragments can be derived from the amino acid sequence shown in SEQ ID NO 1. However, the invention also encompasses fragments of the variants of the β- subunit protein as described herein.

The fragments to which the invention pertains, however, are not to be construed as encompassing fragments that may be disclosed prior to the present invention.

Fragments can retain one or more of the biological activities of the protein, for example the ability to bind to an α-subunit or ligand. Biologically active fragments can comprise a domain or motif, e.g., an extracellular domain, one or more transmembrane domains, α-subunit binding domain, or intracellular domains or functional parts thereof. Such peptides can be, for example, 7, 10, 15, 20, 30, 35, 36, 37, 38, 39, 40, 50, 100 or more amino acids in length.

Possible fragments include, but are not limited to: 1) peptides comprising from about amino acid 1 to about amino acid 19 of SEQ ID NO 1; 2) peptides comprising from about amino acid 20 to about amino acid 40 of SEQ ID NO 1 ; 3) peptides comprising from about amino acid 41 to about amino acid 167 of SEQ ID NO 1; 4) peptides comprising from about amino acid 168 to about amino acid 192; and 5) peptides comprising from about amino acid 193 to amino acid 210, or combinations of these fragments such as two, three, or four domains. Other fragments include fragments containing the various functional sites described herein such as phosphorylation sites such as around amino acids 210, 19, 14, and 167, and glycosylation sites around amino acids 56 and 93. Fragments, for example, can extend in one or both directions from the functional site to encompass 5, 10, 15, 20, 30, 40, 50, or up to 100 amino acids. Further, fragments can include subfragments of the specific domains mentioned above, which subfragments retain the function of the domain from which they are derived. Fragments also include amino acid sequences greater than 71 amino acids. Fragments also include antigenic fragments and specifically those shown to have a high antigenic index in Figure 3. Further specific fragments include amino acids 1 to 29, 306 to 326, and fragments including but larger than amino acids 1-29, 30-65, 67-252, 254-305, 306-326, 330-338, 342-347, 353-361, and 366-382.

Accordingly, possible fragments include fragments defining the site of association between the β and α subunits, fragments defining a ligand binding site, fragments defining a glycosylation site, fragments defining membrane association, and fragments defining phosphorylation sites. By this is intended a discrete fragment that provides the relevant function or allows the relevant function to be identified. In a preferred embodiment, the fragment contains the site(s) of α and β subunit association.

The invention also provides fragments with immunogenic properties. These contain an epitope-bearing portion of the C7F2 β-subunit protein and variants. These epitope-bearing peptides are useful to raise antibodies that bind specifically to a β- subunit polypeptide or region or fragment. These peptides can contain at least 7, at least 14, or between at least about 15 to about 30 amino acids. Peptides having a high antigenic index are shown in Figure 3.

Non-limiting examples of antigenic polypeptides that can be used to generate antibodies include peptides derived from the extracellular domain. The epitope-bearing β-subunit and polypeptides may be produced by any conventional means (Houghten, R.A., Proc. Natl. Acad. Sci. USA 52:5131-5135 (1985)). Simultaneous multiple peptide synthesis is described in U.S. Patent No. 4,631,211. Fragments can be discrete (not fused to other amino acids or polypeptides) or can be within a larger polypeptide. Further, several fragments can be comprised within a single larger polypeptide. In one embodiment a fragment designed for expression in a host can have heterologous pre- and pro-polypeptide regions fused to the amino terminus of the β-subunit fragment and an additional region fused to the carboxyl terminus of the fragment.

The invention thus provides chimeric or fusion proteins. These comprise a β- subunit protein operatively linked to a heterologous protein having an amino acid sequence not substantially homologous to the β-subunit protein. "Operatively linked" indicates that the β-subunit protein and the heterologous protein are fused in- frame. The heterologous protein can be fused to the N-terminus or C-terminus of the β-subunit protein.

In one embodiment the fusion protein does not affect β-subunit function er se. For example, the fusion protein can be a GST-fusion protein in which the β- subunit sequences are fused to the C-terminus of the GST sequences. Other types of fusion proteins include, but are not limited to, enzymatic fusion proteins, for example beta-galactosidase fusions, yeast two-hybrid GAL fusions, poly-His fusions and Ig fusions. Such fusion proteins, particularly poly-His fusions, can facilitate the purification of recombinant β-subunit protein. In certain host cells (e.g., mammalian host cells), expression and/or secretion of a protein can be increased by using a heterologous signal sequence. Therefore, in another embodiment, the fusion protein contains a heterologous signal sequence at its N-terminus.

EP-A-O 464 533 discloses fusion proteins comprising various portions of immunoglobin constant regions. The Fc is useful in therapy and diagnosis and thus results, for example, in improved pharmacokinetic properties (EP-A 0232 262). In drug discovery, for example, human proteins have been fused with Fc portions for the purpose of high-throughput screening assays to identify antagonists. Bennett et al., Journal of Molecular Recognition 5:52-58 (1995) and Johanson et al., The Journal of Biological Chemistry 270,16:9459-9411 (1995). Thus, this invention also encompasses soluble fusion proteins containing a β-subunit polypeptide and various portions of the constant regions of heavy or light chains of immunoglobulins of various subclass (IgG, IgM, IgA, IgE). Preferred as immunoglobulin is the constant part of the heavy chain of human IgG, particularly IgGl, where fusion takes place at the hinge region. For some uses it is desirable to remove the Fc after the fusion protein has been used for its intended purpose, for example when the fusion protein is to be used as antigen for immunizations. In a particular embodiment, the Fc part can be removed in a simple way by a cleavage sequence which is also incoφorated and can be cleaved with factor Xa.

A chimeric or fusion protein can be produced by standard recombinant DNA techniques. For example, DNA fragments coding for the different protein sequences are ligated together in-frame in accordance with conventional techniques. In another embodiment, the fusion gene can be synthesized by conventional techniques including automated DNA synthesizers. Alternatively, PCR amplification of gene fragments can be carried out using anchor primers which give rise to complementary overhangs between two consecutive gene fragments which can subsequently be annealed and re-amplified to generate a chimeric gene sequence (see Ausubel et al., Current Protocols in Molecular Biology, 1992). Moreover, many expression vectors are commercially available that already encode a fusion moiety (e.g., a GST protein). A β-subunit protein-encoding nucleic acid can be cloned into such an expression vector such that the fusion moiety is linked in-frame to the β-subunit protein. Another form of fusion protein is one that directly affects β-subunit functions. Accordingly, a β-subunit polypeptide encompassed by the present invention in which one or more of the β-subunit segments has been replaced by homologous segments from another β-subunit. Various permutations are possible. The various segments include the intracellular amino and carboxy terminal domains, the two transmembrane domains, and the extracellular loop domain. More specifically, the functional domains include the domain containing the ligand binding site, the domains containing the phosphorylation sites, and the domain containing the site that functions to bind α-subunit or modulate α-subunit activation. Any of these domains or subregions thereof containing a specific site can be replaced with the corresponding domain or subregion from another β-subunit protein, or other subunit protein that modulates α-subunit activation. Accordingly, one or more of the specific domains or functional subregions can be combined with those from another subunit that modulates an α-subunit. Thus, chimeric β-subunits can be formed in which one or more of the native domains or subregions has been replaced.

The invention also encompasses chimeric channels in which an α-subunit other than the one with which the β-subunit is naturally found is substituted. The β- subunit can therefore be tested for the ability to modulate other α-subunits. Using assays directed towards these α-subunits as end points, allows the assessment of the β-subunit function. With this type of construct, an α-subunit can be made responsive to a ligand by which it is not normally activated. Thus, by substitution of the β- subunit, a ligand binding to that β-subunit can be used to modulate the activity of the α-subunit.

The isolated β-subunit protein can be purified from cells that naturally express it, such as from brain, heart, kidney, prostate, placenta, lung, testes, ovary and intestine, purified from cells that have been altered to express it (recombinant), or synthesized using known protein synthesis methods. In one embodiment, the protein is produced by recombinant DNA techniques.

For example, a nucleic acid molecule encoding the β-subunit polypeptide is cloned into an expression vector, the expression vector introduced into a host cell and the protein expressed in the host cell. The protein can then be isolated from the cell by an appropriate purification scheme using standard protein purification techniques. Polypeptides often contain amino acids other than the 20 amino acids commonly referred to as the 20 naturally-occurring amino acids. Further, many amino acids, including the terminal amino acids, may be modified by natural processes, such as processing and other post-translational modifications, or by chemical modification techniques well known in the art. Common modifications that occur naturally in polypeptides are described in basic texts, detailed monographs, and the research literature, and they are well known to those of skill in the art. Accordingly, the polypeptides also encompass derivatives or analogs in which a substituted amino acid residue is not one encoded by the genetic code, in which a substituent group is included, in which the mature polypeptide is fused with another compound, such as a compound to increase the half-life of the polypeptide (for example, polyethylene glycol), or in which the additional amino acids are fused to the mature polypeptide, such as a leader or secretory sequence or a sequence for purification of the mature polypeptide or a pro-protein sequence.

Known modifications include, but are not limited to, acetylation, acylation, ADP-ribosylation, amidation, 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 crosslinks, 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.

Such modifications are well known to those of skill in the art and have been described in great detail in the scientific literature. Several particularly common modifications, glycosylation, lipid attachment, sulfation, gamma-carboxylation of glutamic acid residues, hydroxylation and ADP-ribosylation, for instance, are described in most basic texts, such as Proteins - Structure and Molecular Properties, 2nd Ed., T.E. Creighton, W. H. Freeman and Company, New York (1993). Many detailed reviews are available on this subject, such as by Wold, F., Posttranslational Covalent Modification of Proteins, B.C. Johnson, Ed., Academic Press, New York 1- 12 (1983); Seifter et al, Meth. Enzymol. 182: 626-646 (1990) and Rattan et al, Ann. N Y. Acad. Sci. 663:48-62 (1992).

As is also well known, polypeptides are not always entirely linear. For instance, polypeptides may be branched as a result of ubiquitination, and they may be circular, with or without branching, generally as a result of post-translation events, including natural processing event and events brought about by human manipulation which do not occur naturally. Circular, branched and branched circular polypeptides may be synthesized by non-translational natural processes and by synthetic methods. Modifications can occur anywhere in a polypeptide, including the peptide backbone, the amino acid side-chains and the amino or carboxyl termini. Blockage of the amino or carboxyl group in a polypeptide, or both, by a covalent modification, is common in naturally-occurring and synthetic polypeptides. For instance, the amino terminal residue of polypeptides made in E. coli, prior to proteolytic processing, almost invariably will be N-formylmethionine.

The modifications can be a function of how the protein is made. For recombinant polypeptides, for example, the modifications will be determined by the host cell posttranslational modification capacity and the modification signals in the polypeptide amino acid sequence. Accordingly, when glycosylation is desired, a polypeptide should be expressed in a glycosylating host, generally a eukaryotic cell.

Insect cells often carry out the same posttranslational glycosylations as mammalian cells and, for this reason, insect cell expression systems have been developed to efficiently express mammalian proteins having native patterns of glycosylation.

Similar considerations apply to other modifications.

The same type of modification may be present in the same or varying degree at several sites in a given polypeptide. Also, a given polypeptide may contain more than one type of modification.

Polypeptide Uses

The β-subunit polypeptides, as well as the β-subunit nucleic acid molecules, modulators of these polypeptides, and antibodies (also referred to herein as "active compounds") of the invention are useful in the modulation, diagnosis, and treatment of β-subunit-associated or related disorders, also referred to as C7F2-associated or related disorders. Such disorders include, for example, central nervous system (CNS) disorders, cardiovascular system disorders, and musculoskeletal system disorders.

CNS disorders include, but are not limited to, cognitive and neurodegenerative disorders such as Alzheimer's disease and dementias related to Alzheimer's disease

(such as Pick's disease), senile dementia, Huntington's disease, amyotrophic lateral sclerosis, Parkinson's disease and other Lewy diffuse body diseases, Gilles de la Tourette's syndrome, multiple sclerosis, amyotrophic lateral sclerosis, progressive supranuclear palsy, epilepsy, and Jakob-Creutzfieldt disease autonomic function disorders such as hypertension and sleep disorders, and neuropsychiatric disorders, such as depression, schizophrenia, schizoaffective disorder, korsakoff s psychosis, learning or memory disorders, e.g., amnesia or age-related memory loss, attention deficit disorder, dysthymic disorder, major depressive disorder, mania, obsessive- compulsive disorder, psychoactive substance use disorders, anxiety, phobias, panic disorder, as well as bipolar affective disorder, e.g.. severe bipolar affective (mood) disorder (BP-I), bipolar affective (mood) disorder with hypomania and major depression (BP-II), neurological disorders, e.g., migraine, and obesity. Further CNS- related disorders include, for example, those listed in the American Psychiatric Association's Diagnostic and Statistical manual of Mental Disorders (DSM), the most current version of which is incoφorated herein by reference in its entirety. β-subunit-associated or related disorders can detrimentally affect conveyance of sensory impulses from the periphery to the brain (e.g., pain disorders) and/or conductance of motor impulses from the brain to the periphery; integration of reflexes; inteφretation of sensory impulses (e.g., pain); or emotional, intellectual (e.g., learning and memory), or motor processes. Cardiovascular system disorders include, but are not limited to, arteriosclerosis, ischemia reperfusion injury, restenosis, arterial inflammation, vascular wall remodeling, ventricular remodeling, rapid ventricular pacing, coronary microembolism, tachycardia, bradycardia, pressure overload, aortic bending, coronary artery ligation, vascular heart disease, atrial fibrilation, long-QT syndrome, congestive heart failure, sinus node disfunction, angina, heart failure, hypertension, atrial fibrillation, atrial flutter, dilated cardiomyopathy, idiopathic cardiomyopathy, myocardial infarction, coronary artery disease, coronary artery spasm, or arrhythmia. C7F2-mediated or related disorders also include disorders of the musculoskeletal system such as paralysis and muscle weakness, e.g., ataxia, myotonia, and myokymia. β-subunit-associated or related disorders also include disorders of tissues in which C7F2 is expressed, e.g., heart, placental, lung, kidney, prostate, testicular, ovarian, spleen, small and large intestine, colon, or thymus tissues, as well as in brain tissues, including cerebellum, cerebral cortex, medulla, spinal cord, occipital lobe, frontal lobe, temporal lobe, putanem, amygdala, caudate, coφus colosum, hippocampus, substantia nigra, subthalamus and thalamus.

The β-subunit polypeptides and nucleotide sequences encoding the polypeptides find use in modulating a β-subunit function or activity. By "modulating" is intended the upregulating or downregulating of a response. That is, the β-subunit polypeptide and nucleic acid compositions of the invention affect the targeted activity in either a positive or negative fashion.

The β-subunit-associated or related activities include, but are not limited to, an activity that involves a potassium channel, e.g., a potassium channel in a neuronal cell or a muscle cell, associated with receiving, conducting, and transmitting signals in, for example, the nervous system. Potassium-channel mediated activities include release of neurotransmitters, e.g., dopamine or norepinephrine, from cells, e.g., neuronal cells; modulation of resting potential of membranes, wave forms and frequencies of action potentials, and thresholds of excitation; and modulation of processes such as integration of sub-threshold synaptic responses and the conductance of back-propagating action potentials in, for example, neuronal cells or muscle cells, β-subunit-associated or related activities also include activities which involve a potassium channel in nonneuronal cells, e.g., placental, lung, kidney, prostate, testicular, ovarian, spleen, small intestine, colon, or thymus cells, such as membrane potential, cell volume, and pH regulation, β-subunit-associated or related activities include activities involved in muscle function such as maintenance of muscle membrane potential, regulation of muscle contraction andrelaxation, and coordination. A preferred β-subunit activity is modulation or regulation of the pore- forming α-subunit of a potassium channel, particularly activation of the α-subunit.

Accordingly, in one aspect, this invention provides a method for identifying a compound suitable for treating a β-subunit-associated or related disorder by contacting a C7F2 β-subunit polypeptide, or a cell expressing a C7F2 β-subunit polypeptide, with a test compound and determining whether the C7F2 β-subunit polypeptide binds to the test compound, thereby identifying a compound suitable for treating a β-subunit-associated or related disorder.

The β-subunit polypeptides are useful for producing antibodies specific for the C7F2 β-subunit protein, regions, or fragments.

The β-subunit polypeptides are also useful in drug screening assays, in cell- based or cell-free systems. Cell-based systems can be native i.e., cells that normally express the β-subunit protein, as a biopsy or expanded in cell culture. In one embodiment, however, cell-based assays involve recombinant host cells expressing the β-subunit protein.

The polypeptides can be used to identify compounds that modulate β-subunit activity. Both C7F2 β-subunit protein and appropriate variants and fragments can be used in high throughput screens to assay candidate compounds for the ability to bind to the β-subunit. These compounds can be further screened against a functional β- subunit to determine the effect of the compound on the β-subunit activity.

Compounds can be identified that activate (agonist) or inactivate (antagonist) the β- subunit to a desired degree.

The β-subunit polypeptides can be used to screen a compound for the ability to stimulate or inhibit interaction between the β-subunit protein and a target molecule that normally interacts with the β-subunit protein. The target can be ligand or another channel subunit with which the β-subunit protein normally interacts (for example, the α-subunit in the potassium channel). The target can be a molecule that modifies the β-subunit such as by phosphorylation, for example, casein kinase II. The assay includes the steps of combining the β-subunit protein with a candidate compound under conditions that allow the β-subunit protein or fragment to interact with the target molecule, and to detect the formation of a complex between the protein and the target or to detect the biochemical consequence of the interaction with the β-subunit protein and the target, such as ion currents or any of the associated effects of the currents, phosphorylation, change in cell volume, mutagenesis, or transformation. The invention also encompasses chimeric channels in which a β-subunit is associated with a heterologous α-subunit. Thus, the β-subunit can be used to modulate heterologous α-subunits, as a target for drug screening and in diagnosis and treatment. Candidate compounds include, for example, 1) small organic and inorganic molecules (e.g., molecules obtained from combinatorial and natural product libraries); 2) phosphopeptides (e.g., members of random and partially degenerate, directed phosphopeptide libraries, see, e.g., Songyang et al, Cell 72:161-11 (1993)); 3) antibodies (e.g., polyclonal, monoclonal, humanized, anti-idiotypic, chimeric, and single chain antibodies as well as Fab, F(ab')2_> Fab expression library fragments, and epitope-binding fragments of antibodies); and 4) peptides such as soluble peptides, including Ig-tailed fusion peptides and members of random peptide libraries (see, e.g., Lam et al, Nature 354:82-84 (1991); Houghten et al, Nature 554:84-86 (1991)) and combinatorial chemistry-derived molecular libraries made of D- and/or L- configuration amino acids.

The invention provides other end points to identify compounds that modulate (stimulate or inhibit) β-subunit activity. The assays typically involve an assay of events in channeling that indicate β-subunit activity. A preferred assay involves the activation of the α-subunit. Assays allowing the assessment of β-subunit activity are known to those of skill in the art and can be found, for example, in McManus et al. (1995), Knaus et al. (1996), Knaus et al. (1994), Meera et al., and Oberst et al, cited above.

Binding and/or modulating (activating or inhibiting) compounds can also be screened by using chimeric subunit proteins in which the ligand binding or α-subunit binding region is replaced by a heterologous region. For example, an α-subunit binding region can be used that interacts with a different α-subunit than that which is recognized by the native β-subunit. Accordingly, a different end-point assay is available. Alternatively, one or two transmembrane regions can be replaced with transmembrane portions specific to a host cell that is different from the native host cell from which the native β-subunit is derived. This allows for assays to be performed in other than the original host cell. Alternatively, the ligand binding region can be replaced by a region binding a different ligand, thus, enabling an assay for test compounds that interact with the heterologous ligand binding region but still cause channeling function, including α-subunit activation.

The β-subunit polypeptides are also useful in competition binding assays in methods designed to discover compounds that interact with the β-subunit. Thus, a compound is exposed to a β-subunit polypeptide under conditions that allow the compound to bind or to otherwise interact with the polypeptide. Competing β- subunit polypeptide is also added to the mixture. If the test compound interacts with the competing β-subunit polypeptide, it decreases the amount of complex formed or activity from the β-subunit target. This type of assay is particularly useful in cases in which compounds are sought that interact with specific regions of the β-subunit. Thus, the polypeptide that competes with the target β-subunit region is designed to contain peptide sequences corresponding to the region of interest.

A β-subunit is also useful for assessing function of a given α-subunit. Thus, alteration in channel currents, number of receptors, cell transformation, or any other biological end point can be assessed using the β-subunit of the present invention in cell-based or cell-free assays with a given α-subunit. Mutation in the α-subunit can be detected by any of the various end points. Moreover, mutations in the β-subunit that complement (i.e., correct) mutations in the α-subunit can be identified through cell-based or cell-free assays. Such assays could even be performed at the level of the organism, as with a transgenic animal (see below).

To perform cell free drug screening assays, it is desirable to immobilize either the β-subunit protein, or fragment, or its target molecule to facilitate separation of complexes from uncomplexed forms of one or both of the proteins, as well as to accommodate automation of the assay.

Techniques for immobilizing proteins on matrices can be used in the drug screening assays. In one embodiment, a fusion protein can be provided which adds a domain that allows the protein to be bound to a matrix. For example, glutathione-S- transferase/flh385 fusion proteins can be adsorbed onto glutathione sepharose beads (Sigma Chemical, St. Louis, MO) or glutathione derivatized microtitre plates, which

35 are then combined with the cell lysates (e.g., S-labeled) and the candidate compound, and the mixture incubated under conditions conducive to complex formation (e.g., at physiological conditions for salt and pH). Following incubation, the beads are washed to remove any unbound label, and the matrix immobilized and radiolabel determined directly, or in the supernatant after the complexes are dissociated. Alternatively, the complexes can be dissociated from the matrix, separated by SDS-PAGE, and the level of β-subunit-binding protein found in the bead fraction quantitated from the gel using standard electrophoretic techniques. For example, either the polypeptide or its target molecule can be immobilized utilizing conjugation of biotin and streptavidin using techniques well known in the art. Alternatively, antibodies reactive with the protein but which do not interfere with binding of the protein to its target molecule can be derivatized to the wells of the plate, and the protein trapped in the wells by antibody conjugation. Preparations of a β-subunit-binding protein and a candidate compound are incubated in the β-subunit protein-presenting wells and the amount of complex trapped in the well can be quantitated. Methods for detecting such complexes, in addition to those described above for the GST-immobilized complexes, include immunodetection of complexes using antibodies reactive with the β-subunit protein target molecule, or which are reactive with β-subunit protein and compete with the target molecule; as well as enzyme-linked assays which rely on detecting an enzymatic activity associated with the target molecule.

Modulators of β-subunit protein activity identified according to these drug screening assays can be used to treat a subject with a disorder mediated by the β- subunit. These methods of treatment include the steps of administering the modulators of protein activity in a pharmaceutical composition as described herein, to a subj ect in need of such treatment.

The β-subunit polypeptides also are useful to provide a target for diagnosing a disease or predisposition to disease mediated by the β-subunit protein. Accordingly, methods are provided for detecting the presence, or levels of, the β- subunit protein in a cell, tissue, or organism. The method involves contacting a biological sample with a compound capable of interacting with the β-subunit protein such that the interaction can be detected. One agent for detecting β-subunit protein is an antibody capable of selectively binding to β-subunit protein. A biological sample includes tissues, cells and biological fluids isolated from a subject, as well as tissues, cells and fluids present within a subject. The β-subunit protein also provides a target for diagnosing active disease, or predisposition to disease, in a patient having a variant β-subunit protein. Thus, β- subunit protein can be isolated from a biological sample, assayed for the presence of a genetic mutation that results in aberrant β-subunit protein. This includes amino acid substitution, deletion, insertion, rearrangement, (as the result of aberrant splicing events), and inappropriate post-translational modification. Analytic methods include altered electrophoretic mobility, altered tryptic peptide digest, altered β-subunit activity in cell-based or cell-free assay, alteration in ligand, α-subunit, or antibody- binding pattern, altered isoelectric point, direct amino acid sequencing, and any other of the known assay techniques useful for detecting mutations in a protein. In vitro techniques for detection of β-subunit protein include enzyme linked immunosorbent assays (ELISAs), Western blots, immunoprecipitations and immunofluorescence. Alternatively, the protein can be detected in vivo in a subject by introducing into the subject a labeled anti- β-subunit antibody. For example, the antibody can be labeled with a radioactive marker whose presence and location in a subject can be detected by standard imaging techniques. Particularly useful are methods which detect the allelic variant of a β-subunit protein expressed in a subject and methods which detect fragments of a β-subunit protein in a sample.

The β-subunit polypeptides are also useful in pharmacogenomic analysis. Pharmacogenomics deal with clinically significant hereditary variations in the response to drugs due to altered drug disposition and abnormal action in affected persons. See, e.g., Eichelbaum, M. (1996) Clin. Exp. Pharmacol. Physiol. 23(10- l l):983-985 and Linder, M.W. (1997) Clin. Chem. 43(2):254-266. The clinical outcomes of these variations result in severe toxicity of therapeutic drugs in certain individuals or therapeutic failure of drugs in certain individuals as a result of individual variation in metabolism. Thus, the genotype of the individual can determine the way a therapeutic compound acts on the body or the way the body metabolizes the compound. Further, the activity of drug metabolizing enzymes effects both the intensity and duration of drug action. Thus, the pharmacogenomics of the individual permit the selection of effective compounds and effective dosages of such compounds for prophylactic or therapeutic treatment based on the individual's genotype. The discovery of genetic polymoφhisms in some drug metabolizing enzymes has explained why some patients do not obtain the expected drug effects, show an exaggerated drug effect, or experience serious toxicity from standard drug dosages. Polymoφhisms can be expressed in the phenotype of the extensive metabolizer and the phenotype of the poor metabolizer. Accordingly, genetic polymoφhism may lead to allelic protein variants of the β-subunit protein in which one or more of the β-subunit functions in one population is different from those in another population. The polypeptides thus allow a target to ascertain a genetic predisposition that can affect treatment modality. Thus, in a ligand-based treatment, for example, polymoφhism may give rise to sites that are more or less active in ligand binding, and channel activation. Accordingly, ligand choice or dosage could be modified to maximize the therapeutic effect within a given population containing a polymoφhism. As an alternative to genotyping, specific polymoφhic polypeptides could be identified.

The β-subunit polypeptides are also useful for monitoring therapeutic effects during clinical trials and other treatment. Thus, the therapeutic effectiveness of an agent that is designed to increase or decrease gene expression, protein levels or β- subunit activity can be monitored over the course of treatment using the β-subunit polypeptides as an end-point target.

The β-subunit polypeptides are also useful for treating a β-subunit-associated disorders. Accordingly, methods for treatment include the use of soluble subunit or fragments of the β-subunit protein that compete for molecules interacting with the extracellular portions of the subunit. These β-subunits or fragments can have a higher affinity for the molecule so as to provide effective competition. Antibodies

The invention also provides antibodies that selectively bind to the subunit protein and its variants and fragments. An antibody is considered to selectively bind, even if it also binds to other proteins that are not substantially homologous with the β-subunit protein. These other proteins share homology with a fragment or domain of the β-subunit protein. This conservation in specific regions gives rise to antibodies that bind to both proteins by virtue of the homologous sequence. In this case, it would be understood that antibody binding to the subunit protein is still selective. Regions showing a high antigenic index are shown in Figure 3. Antibodies are preferably prepared from these regions or from discrete fragments in these regions. However, antibodies can be prepared from any region of the peptide as described herein.

Antibodies can be polyclonal or monoclonal. An intact antibody, or a fragment thereof (e.g. Fab or F(ab ')₂) can be used.

Detection can be facilitated by coupling (i.e., physically linking) the antibody to a detectable substance. Examples of detectable substances include various enzymes, prosthetic groups, fluorescent materials, luminescent materials, bioluminescent materials, and radioactive materials. Examples of suitable enzymes include horseradish peroxidase, alkaline phosphatase, β-galactosidase, or acetylcholinesterase; examples of suitable prosthetic group complexes include streptavidin/biotin and avidin biotin; examples of suitable fluorescent materials include umbelliferone, fluorescein, fluorescein isothiocyanate, rhodamine, dichlorotriazinylamine fluorescein, dansyl chloride or phycoerythrin; an example of a luminescent material includes luminol; examples of bioluminescent materials include luciferase, luciferin, and aequorin, and examples of suitable radioactive material i •ncl ,ud ,e 125 I_τ, 131 I_τ, 35 S_c or 3 H_U.

To generate antibodies, an isolated β-subunit polypeptide is used as an immunogen to generate antibodies using standard techniques for polyclonal and monoclonal antibody preparation. Either the full-length protein or antigenic peptide fragment can be used. Fragments having a high antigenic index are shown in Figure 3. A preferred fragment produces an antibody that diminishes or completely prevents association between the α and β subunits. Accordingly, a preferred antibody is one that diminishes or completely inhibits association between the two subunits. An antigenic fragment will typically comprise at least 7 contiguous amino acid residues. The antigenic peptide can comprise, however, at least 12, at least 14 amino acid residues, at least 15 amino acid residues, at least 20 amino acid residues, or at least 30 amino acid residues. In one embodiment, fragments correspond to regions that are located on the surface of the protein, e.g., hydrophilic regions.

An appropriate immunogenic preparation can be derived from native, recombinantly expressed, protein or chemically synthesized peptides.

Antibody Uses

The antibodies can be used to isolate a β-subunit protein by standard techniques, such as affinity chromatography or immunoprecipitation. The antibodies can facilitate the purification of the natural β-subunit protein from cells and recombinantly produced β-subunit protein expressed in host cells.

The antibodies are useful to detect the presence of β-subunit protein in cells or tissues to determine the pattern of expression of the β-subunit among various tissues in an organism and over the course of normal development. The antibodies can be used to detect β-subunit protein in situ, in vitro, or in a cell lysate or supernatant in order to evaluate the abundance and pattern of expression.

The antibodies can be used to assess abnormal tissue distribution or abnormal expression during development. Antibody detection of fragments of the full length β-subunit protein can be used to identify β-subunit turnover.

Further, the antibodies can be used to assess β-subunit expression in disease states such as in active stages of the disease or in an individual with a predisposition toward disease related to β-subunit function. When a disorder is caused by an inappropriate tissue distribution, developmental expression, or level of expression of the β-subunit protein, the antibody can be prepared against the normal β-subunit protein. If a disorder is characterized by a specific mutation in the β-subunit protein, antibodies specific for this mutant protein can be used to assay for the presence of the specific mutant β-subunit protein.

The antibodies can also be used to assess normal and aberrant subcellular localization of cells in the various tissues in an organism.

Antibodies can be developed against the whole β-subunit or portions of the β-subunit, for example, the intracellular regions, the extracellular region, the transmembrane regions, and specific functional sites such as the site of ligand binding, the site of interaction with the α-subunit, or sites that are phosphorylated, for example by casein kinase II.

The diagnostic uses can be applied, not only in genetic testing, but also in monitoring a treatment modality. Accordingly, where treatment is ultimately aimed at correcting β-subunit expression level or the presence of aberrant β-subunits and aberrant tissue distribution or developmental expression, antibodies directed against the β-subunit or relevant fragments can be used to monitor therapeutic efficacy.

Additionally, antibodies are useful in pharmocogenomic analysis. Thus, antibodies prepared against polymoφhic β-subunit proteins can be used to identify individuals that require modified treatment modalities.

The antibodies are also useful as diagnostic tools as an immunological marker for aberrant β-subunit protein analyzed by electrophoretic mobility, isoelectric point, tryptic peptide digest, and other physical assays known to those in the art.

The antibodies are also useful for tissue typing. Thus, where a specific β- subunit protein has been correlated with expression in a specific tissue, antibodies that are specific for this β-subunit protein can be used to identify a tissue type.

The antibodies are also useful in forensic identification. Accordingly, where an individual has been correlated with a specific genetic polymoφhism resulting in a specific polymoφhic protein, an antibody specific for the polymoφhic protein can be used as an aid in identification. The antibodies are also useful for inhibiting subunit function, for example, blocking ligand binding or α-subunit binding and/or activation. Subunit function involving the extracellular loop is particularly amenable to antibody inhibition.

These uses can also be applied in a therapeutic context in which treatment involves inhibiting subunit function. Antibodies can be prepared against specific fragments containing sites required for function or against intact β-subunit associated with a cell.

The invention also encompasses kits for using antibodies to detect the presence of a β-subunit protein in a biological sample. The kit can comprise antibodies such as a labeled or labelable antibody and a compound or agent for detecting β-subunit protein in a biological sample; means for determining the amount of β-subunit protein in the sample; and means for comparing the amount of β- subunit protein in the sample with a standard. The compound or agent can be packaged in a suitable container. The kit can further comprise instructions for using the kit to detect β-subunit protein.

Polynucleotides

The nucleotide sequence in SEQ ID NO 2 was obtained by sequencing the deposited human full length cDNA. Accordingly, the sequence of the deposited clone is controlling as to any discrepancies between the two and any reference to the sequence of SEQ ID NO 2 includes reference to the sequence of the deposited cDNA.

The specifically disclosed cDNA comprises the coding region, 5' and 3' untranslated sequences (SEQ ID NO 2). In one embodiment, the subunit nucleic acid comprises only the coding region.

The human C7F2 β-subunit cDNA is approximately 1737 nucleotides in length and encodes a full length protein that is approximately 210 amino acid residues in length. The nucleic acid is expressed in brain, heart, kidney, placenta, lung, prostate, testes, ovary, and small and large intestine. Structural analysis of the amino acid sequence of SEQ ID NO 1 is provided in Figure 4, a hydropathy plot.

The figure shows the putative structure of the two transmembrane domains, the extracellular loop and the two intracellular domains. As used herein, the term "transmembrane domain" (or "region" or "segment") refers to a structural amino acid motif which includes a hydrophobic helix that spans the plasma membrane.

The invention provides isolated polynucleotides encoding a C7F2 β-subunit protein. The term "C7F2 β-subunit polynucleotide" or "C7F2 β-subunit nucleic acid" refers to the sequence shown in SEQ ID NO 2 or in the deposited cDNA. The term "β-subunit polynucleotide" or "β-subunit nucleic acid" further includes variants and fragments of the C7F2 polynucleotide.

An "isolated" β-subunit nucleic acid is one that is separated from other nucleic acid present in the natural source of the β-subunit nucleic acid. Preferably, an "isolated" nucleic acid is free of sequences which naturally flank the nucleic acid (i.e., sequences located at the 5' and 3' ends of the nucleic acid) in the genomic DNA of the organism from which the nucleic acid is derived. However, there can be some flanking nucleotide sequences, for example up to about 5KB. The important point is that the nucleic acid is isolated from flanking sequences such that it can be subjected to the specific manipulations described herein such as recombinant expression, preparation of probes and primers, and other uses specific to the β-subunit nucleic acid sequences.

Moreover, an "isolated" nucleic acid molecule, such as a cDNA molecule, can be substantially free of other cellular material, or culture medium when produced by recombinant techniques, or chemical precursors or other chemicals when chemically synthesized. However, the nucleic acid molecule can be fused to other coding or regulatory sequences and still be considered isolated.

For example, recombinant DNA molecules contained in a vector are considered isolated. Further examples of isolated DNA molecules include recombinant DNA molecules maintained in heterologous host cells or purified (partially or substantially) DNA molecules in solution. Isolated RNA molecules include in vivo or in vitro RNA transcripts of the isolated DNA molecules of the present invention. Isolated nucleic acid molecules according to the present invention further include such molecules produced synthetically. The β-subunit polynucleotides can encode the mature protein plus additional amino or carboxy terminal amino acids, or amino acids interior to the mature polypeptide (when the mature form has more than one polypeptide chain, for instance). Such sequences may play a role in processing of a protein from precursor to a mature form, facilitate protein trafficking, prolong or shorten protein half-life or facilitate manipulation of a protein for assay or production, among other things. As generally is the case in situ, the additional amino acids may be processed away from the mature protein by cellular enzymes.

The β-subunit polynucleotides include, but are not limited to, the sequence encoding the mature polypeptide alone, the sequence encoding the mature polypeptide and additional coding sequences, such as a leader or secretory sequence (e.g., a pre-pro or pro-protein sequence), the sequence encoding the mature polypeptide, with or without the additional coding sequences, plus additional non- coding sequences, for example introns and non-coding 5' and 3' sequences such as transcribed but non-translated sequences that play a role in transcription, mRNA processing (including splicing and polyadenylation signals), ribosome binding and stability of mRNA. In addition, the polynucleotide may be fused to a marker sequence encoding, for example, a peptide that facilitates purification. β-subunit polynucleotides can be in the form of RNA, such as mRNA, or in the form DNA, including cDNA and genomic DNA obtained by cloning or produced by chemical synthetic techniques or by a combination thereof. The nucleic acid, especially DNA, can be double-stranded or single-stranded. Single-stranded nucleic acid can be the coding strand (sense strand) or the non-coding strand (anti-sense strand). One β-subunit nucleic acid comprises the nucleotide sequence shown in SEQ

ID NO 2, corresponding to human fetal brain cDNA.

The invention further provides variant subunit polynucleotides, and fragments thereof, that differ from the nucleotide sequence shown in SEQ ID NO 2 due to degeneracy of the genetic code and thus encode the same protein as that encoded by the nucleotide sequence shown in SEQ ID NO 2. The invention also provides β-subunit nucleic acid molecules encoding the variant polypeptides described herein. Such polynucleotides may be naturally occurring, such as allelic variants (same locus), homologs (different locus), and orthologs (different organism), or may be constructed by recombinant DNA methods or by chemical synthesis. Such non-naturally occurring variants may be made by mutagenesis techniques, including those applied to polynucleotides, cells, or organisms. Accordingly, as discussed above, the variants can contain nucleotide substitutions, deletions, inversions and insertions.

Variation can occur in either or both the coding and non-coding regions. The variations can produce both conservative and non-conservative amino acid substitutions.

Orthologs, homologs, and allelic variants can be identified using methods well known in the art. These variants comprise a nucleotide sequence encoding a β- subunit that is at least about 55-60%), typically at least about 70-75%>, more typically at least about 80-85%o, and most typically at least about 90-95%) or more homologous to the nucleotide sequence shown in SEQ ID NO: 2 or a fragment of this sequence. Such nucleic acid molecules can readily be identified as being able to hybridize under stringent conditions, to the nucleotide sequence shown in SEQ ID NO 2 or a fragment of the sequence. It is understood that stringent hybridization does not indicate substantial homology where it is due to general homology, such as poly A sequences, or sequences common to all or most proteins, all K^* channel β-subunits, or all channel β-subunits. Moreover, it is understood that variants do not include any of the nucleic acid sequences that may have been disclosed prior to the invention. As used herein, the term "hybridizes under stringent conditions" is intended to describe conditions for hybridization and washing under which nucleotide sequences encoding a β-subunit at least 55-60%> homologous to each other typically remain hybridized to each other. The conditions can be such that sequences at least about 65%o, at least about 70%>, or at least about 75% or more homologous to each other typically remain hybridized to each other. Such stringent conditions are known to those skilled in the art and can be found in Current Protocols in Molecular

Biology, John Wiley & Sons, N.Y. (1989), 6.3.1-6.3.6. One example of stringent hybridization conditions are hybridization in 6X sodium chloride/sodium citrate (SSC) at about 45°C, followed by one or more washes in 0.2 X SSC, 0.1% SDS at 50-65°C. In one embodiment, an isolated β-subunit nucleic acid molecule that hybridizes under stringent conditions to the sequence of SEQ ID NO 2 corresponds to a naturally-occurring nucleic acid molecule. As used herein, a "naturally- occurring" nucleic acid molecule refers to an RNA or DNA molecule having a nucleotide sequence that occurs in nature (e.g., encodes a natural protein).

The invention also provides polynucleotides that comprise a fragment of the full length β-subunit polynucleotides. The fragment can be single or double stranded and can comprise DNA or RNA. The fragment can be derived from either the coding or the non-coding sequence, e.g., transcriptional regulatory sequence.

In one embodiment, β-subunit coding region nucleic acid is at least 216 nucleotides in length and hybridizes under stringent conditions to the nucleic acid molecule comprising the nucleotide sequence of SEQ ID NO 2. Fragments also include those nucleic acid sequences encoding the specific domains described herein. Fragments also include nucleic acids encoding the entire coding sequence. Fragments also include nucleic acids encoding the mature protein. Fragments also include nucleic acid sequences encoding two or more domains. Fragments also include nucleic acid sequences corresponding to the amino acids at the specific functional sites described herein. Fragments further include nucleic acid sequences encoding a portion of the amino acid sequence described herein but further including flanking nucleotide sequences at the 5' and/or 3' regions. Other fragments can include subfragments of the specific domains or sites described herein. Fragments also include nucleic acid sequences corresponding to specific amino acid sequences described above or fragments thereof. In these embodiments, the nucleic acid is at least 20, 30, 40, 50, 100, 250 or 500 nucleotides in length. Nucleic acid fragments, according to the present invention, are not to be construed as encompassing those fragments that may have been disclosed prior to the invention.

However, it is understood that a β-subunit fragment includes any nucleic acid sequence that does not include the entire gene. β-subunit nucleic acid fragments include nucleic acid molecules encoding a polypeptide comprising an amino terminal intracellular domain including amino acid residues from 1 to about 19, a polypeptide comprising the first transmembrane domain (amino acid residues from about 20 to about 40), a polypeptide comprising the extracellular loop domain (amino acid residues from about 41 to about 167), a polypeptide comprising the second transmembrane domain (amino acid residues from about 168 to about 192) and a polypeptide comprising the carboxy terminal intracellular domain (amino acid residues from about 193 to 210). Where the location of the domains have been predicted by computer analysis, one of ordinary skill would appreciate that the amino acid residues constituting these domains can vary depending on the criteria used to define the domains.

The invention also provides β-subunit nucleic acid fragments that encode epitope bearing regions of the β-subunit proteins described herein.

The isolated β-subunit polynucleotide sequences, and especially fragments, are useful as DNA probes and primers.

For example, the coding region of a β-subunit gene can be isolated using the known nucleotide sequence to synthesize an oligonucleotide probe. A labeled probe can then be used to screen a cDNA library, genomic DNA library, or mRNA to isolate nucleic acid corresponding to the coding region. Further, primers can be used in PCR reactions to clone specific regions of β-subunit genes.

A probe/primer typically comprises substantially purified oligonucleotide. The oligonucleotide typically comprises a region of nucleotide sequence, as described above, that hybridizes under stringent conditions to at least about 20, typically about 25, more typically about 40, 50 or 75 consecutive nucleotides of SEQ ID NO 2 sense or anti-sense strand or other β-subunit polynucleotides. A probe further comprises a label, e.g., radioisotope, fluorescent compound, enzyme, or enzyme co-factor.

Polynucleotide Uses The β-subunit polynucleotides are useful as a hybridization probe for cDNA and genomic DNA to isolate a full-length cDNA and genomic clones encoding the polypeptide described in SEQ ID NO 1 and to isolate cDNA and genomic clones that correspond to variants producing the same polypeptide shown in SEQ ID NO 1 or the other variants described herein. Variants can be isolated from the same tissue and organism from which the polypeptide shown in SEQ ID NO 1 was isolated, different tissues from the same organism, or from different organisms. This method is useful for isolating genes and cDNA that are developmentally controlled and therefore may be expressed in the same tissue at different points in the development of an organism.

The probe can correspond to any sequence along the entire length of the gene encoding the β-subunit. Accordingly, it could be derived from 5' noncoding regions, the coding region, as specified above, and 3' noncoding regions.

The nucleic acid probe can be, for example, the full-length cDNA of SEQ ID NO 1 , or a fragment thereof, as described above. The probe can be an oligonucleotide of at least 20, 30, 50, 100, 250 or 500 nucleotides in length and sufficient to specifically hybridize under stringent conditions to mRNA or DNA. Fragments of the polynucleotides described herein are also useful to synthesize larger fragments or full-length polynucleotides described herein. For example, a fragment can be hybridized to any portion of an mRNA and a larger or full-length cDNA can be produced. The fragments are also useful to synthesize antisense molecules of desired length and sequence.

The β-subunit polynucleotides are also useful as primers for PCR to amplify any given region of a β-subunit polynucleotide.

The β-subunit polynucleotides are also useful for constructing recombinant vectors. Such vectors include expression vectors that express a portion of, or all of, the β-subunit polypeptides. Vectors also include insertion vectors, used to integrate into another polynucleotide sequence, such as into the cellular genome, to alter in situ expression of β-subunit genes and gene products. For example, an endogenous β- subunit coding sequence can be replaced via homologous recombination with all or part of the coding region containing one or more specifically introduced mutations. The β-subunit polynucleotides are also useful as probes for determining the chromosomal positions of the β-subunit polynucleotides by means of in situ hybridization methods.

The β-subunit polynucleotide probes are also useful to determine patterns of the presence of the gene encoding the β-subunits and their variants with respect to tissue distribution, for example whether gene duplication has occurred and whether the duplication occurs in all or only a subset of tissues. The genes can be naturally occurring or can have been introduced into a cell, tissue, or organism exogenously. The β-subunit polynucleotides are also useful for designing ribozymes corresponding to all, or a part, of the mRNA produced from genes encoding the polynucleotides described herein.

The β-subunit polynucleotides are also useful for constructing host cells expressing a part, or all, of the β-subunit polynucleotides and polypeptides.

The β-subunit polynucleotides are also useful for constructing transgenic animals expressing all, or a part, of the β-subunit polynucleotides and polypeptides.

The β-subunit polynucleotides are also useful for making vectors that express part, or all, of the β-subunit polypeptides.

The β-subunit polynucleotides are also useful as hybridization probes for determining the level of β-subunit nucleic acid expression. Accordingly, the probes can be used to detect the presence of, or to determine levels of, β-subunit nucleic acid in cells, tissues, and in organisms. The nucleic acid whose level is determined can be DNA or RNA. Accordingly, probes corresponding to the polypeptides described herein can be used to assess gene copy number in a given cell, tissue, or organism. This is particularly relevant in cases in which there has been an amplification of the β-subunit genes.

Alternatively, the probe can be used in an in situ hybridization context to assess the position of extra copies of the β-subunit genes, as on extrachromosomal elements or as integrated into chromosomes in which the β-subunit gene is not normally found, for example as a homogeneously staining region. These uses are relevant for diagnosis of disorders involving an increase or decrease in β-subunit expression relative to normal results. In vitro techniques for detection of mRNA include Northern hybridizations and in situ hybridizations. In vitro techniques for detecting DNA includes Southern hybridizations and in situ hybridization.

Probes can be used as a part of a diagnostic test kit for identifying cells or tissues that express a β-subunit protein, such as by measuring a level of a subunit- encoding nucleic acid in a sample of cells from a subject e.g., mRNA or genomic DNA, or determining if a β-subunit gene has been mutated.

Nucleic acid expression assays are useful for drug screening to identify compounds that modulate β-subunit nucleic acid expression or activity. The invention thus provides a method for identifying a compound that can be used to treat a disorder associated with nucleic acid expression of the β-subunit gene. The method typically includes assaying the ability of the compound to modulate the expression of the β-subunit nucleic acid and thus identifying a compound that can be used to treat a disorder characterized by undesired β-subunit nucleic acid expression.

The assays can be performed in cell-based and cell-free systems. Cell-based assays include cells naturally expressing the β-subunit nucleic acid or recombinant cells genetically engineered to express specific nucleic acid sequences.

Alternatively, candidate compounds can be assayed in vivo in patients or in transgenic animals.

The assay for β-subunit nucleic acid expression can involve direct assay of nucleic acid levels, such as mRNA levels, or on collateral compounds involved in the signal pathway (such as cyclic AMP or phosphatidylinositol turnover). Further, the expression of genes that are up- or down-regulated in response to the β-subunit protein signal pathway can also be assayed. In this embodiment the regulatory regions of these genes can be operably linked to a reporter gene such as luciferase.

Thus, modulators of β-subunit gene expression can be identified in a method wherein a cell is contacted with a candidate compound and the expression of mRNA determined. The level of expression of β-subunit mRNA in the presence of the candidate compound is compared to the level of expression of β-subunit mRNA in the absence of the candidate compound. The candidate compound can then be identified as a modulator of nucleic acid expression based on this comparison and be used, for example to treat a disorder characterized by aberrant nucleic acid expression. When expression of mRNA is statistically significantly greater in the presence of the candidate compound than in its absence, the candidate compound is identified as a stimulator of nucleic acid expression. When nucleic acid expression is statistically significantly less in the presence of the candidate compound than in its absence, the candidate compound is identified as an inhibitor of nucleic acid expression.

Accordingly, the invention provides methods of treatment, with the nucleic acid as a target, using a compound identified through drug screening as a gene modulator to modulate β-subunit nucleic acid expression. Modulation includes both up-regulation (i.e. activation or agonization) or down-regulation (suppression or antagonization) of nucleic acid expression.

Alternatively, a modulator for β-subunit nucleic acid expression can be a small molecule or drug identified using the screening assays described herein as long as the drug or small molecule inhibits the β-subunit nucleic acid expression.

The β-subunit polynucleotides are also useful for monitoring the effectiveness of modulating compounds on the expression or activity of the β-subunit gene in clinical trials or in a treatment regimen. Thus, the gene expression pattern can serve as a barometer for the continuing effectiveness of treatment with the compound, particularly with compounds to which a patient can develop resistance. The gene expression pattern can also serve as a marker indicative of a physiological response of the affected cells to the compound. Accordingly, such monitoring would allow either increased administration of the compound or the administration of alternative compounds to which the patient has not become resistant. Similarly, if the level of nucleic acid expression falls below a desirable level, administration of the compound could be commensurately decreased.

The β-subunit polynucleotides are also useful in diagnostic assays for qualitative changes in β-subunit nucleic acid, and particularly in qualitative changes that lead to pathology. The polynucleotides can be used to detect mutations in β- subunit genes and gene expression products such as mRNA. The polynucleotides can be used as hybridization probes to detect naturally occurring genetic mutations in the β-subunit gene and thereby determining whether a subject with the mutation is at risk for a disorder caused by the mutation. Mutations include deletion, addition, or substitution of one or more nucleotides in the gene, chromosomal rearrangement such as inversion or transposition, modification of genomic DNA such as aberrant methylation patterns or changes in gene copy number such as amplification. Detection of a mutated form of the β-subunit gene associated with a dysfunction provides a diagnostic tool for an active disease or susceptibility to disease when the disease results from overexpression, underexpression, or altered expression of a β- subunit protein.

Individuals carrying mutations in the β-subunit gene can be detected at the nucleic acid level by a variety of techniques. Genomic DNA can be analyzed directly or can be amplified by using PCR prior to analysis. RNA or cDNA can be used in the same way. In certain embodiments, detection of the mutation involves the use of a probe/primer in a polymerase chain reaction (PCR) (see, e.g. U.S. Patent Nos. 4,683,195 and 4,683,202), such as anchor PCR or RACE PCR, or, alternatively, in a ligation chain reaction (LCR) (see, e.g., Landegran et al, Science 247:1077-1080 (1988); and Nakazawa et al, PNAS 97:360-364 (1994)), the latter of which can be particularly useful for detecting point mutations in the gene (see Abravaya et al, Nucleic Acids Res. 25:675-682 (1995)). This method can include the steps of collecting a sample of cells from a patient, isolating nucleic acid (e.g., genomic, mRNA or both) from the cells of the sample, contacting the nucleic acid sample with one or more primers which specifically hybridize to a gene under conditions such that hybridization and amplification of the gene (if present) occurs, and detecting the presence or absence of an amplification product, or detecting the size of the amplification product and comparing the length to a control sample. Deletions and insertions can be detected by a change in size of the amplified product compared to the normal genotype. Point mutations can be identified by hybridizing amplified DNA to normal RNA or antisense DNA sequences. Alternatively, mutations in a β-subunit gene can be directly identified, for example, by alterations in restriction enzyme digestion patterns determined by gel electrophoresis.

Further, sequence-specific ribozymes (U.S.Patent No. 5,498,531) can be used to score for the presence of specific mutations by development or loss of a ribozyme cleavage site.

Perfectly matched sequences can be distinguished from mismatched sequences by nuclease cleavage digestion assays or by differences in melting temperature. Sequence changes at specific locations can also be assessed by nuclease protection assays such as RNase and SI protection or the chemical cleavage method.

Furthermore, sequence differences between a mutant subunit gene and a wild-type gene can be determined by direct DNA sequencing. A variety of automated sequencing procedures can be utilized when performing the diagnostic assays (Biotechniques 79:448 (1995)), including sequencing by mass spectrometry (see, e.g., PCT International Publication No. WO 94/16101 ; Cohen et al, Adv. Chromatogr. 5(5:127-162 (1996); and Griffin et al., Appl. Biochem. Biotechnol. 35:147-159 (1993)).

Other methods for detecting mutations in the gene include methods in which protection from cleavage agents is used to detect mismatched bases in RNA/RNA or RNA/DNA duplexes (Myers et al, Science 250:1242 (1985); Cotton et al, PNAS 55:4397 (1988); Saleeba et al, Meth. Enzymol. 277:286-295 (1992)), electrophoretic mobility of mutant and wild type nucleic acid is compared (Orita et al, PNAS 86:2166 (1989); Cotton et al., Mutat. Res. 255:125-144 (1993); and Hayashi et al, Genet. Anal. Tech. Appl 9:73-79 (1992)), and movement of mutant or wild-type fragments in polyacrylamide gels containing a gradient of denaturant is assayed using denaturing gradient gel electrophoresis (Myers et al, Nature 575:495 (1985)). Examples of other techniques for detecting point mutations include, selective oligonucleotide hybridization, selective amplification, and selective primer extension. The β-subunit polynucleotides are also useful for testing an individual for a genotype that while not necessarily causing the disease, nevertheless affects the treatment modality. Thus, the polynucleotides can be used to study the relationship between an individual's genotype and the individual's response to a compound used for treatment (pharmacogenomic relationship). In the present case, for example, a mutation in the β-subunit gene that results in altered affinity for ligand, for example, could result in an excessive or decreased drug effect with standard concentrations of ligand. Alternatively, for example, a mutation in the subunit gene that results in an altered interaction with the α-subunit could result in an increased or decreased drug effect with standard concentrations of a drug that affects this functional interaction. Accordingly, the β-subunit polynucleotides described herein can be used to assess the mutation content of the β-subunit gene in an individual in order to select an appropriate compound or dosage regimen for treatment.

Thus polynucleotides displaying genetic variations that affect treatment provide a diagnostic target that can be used to tailor treatment in an individual. Accordingly, the production of recombinant cells and animals containing these polymoφhisms allow effective clinical design of treatment compounds and dosage regimens.

The β-subunit polynucleotides are also useful for chromosome identification when the sequence is identified with an individual chromosome and to a particular location on the chromosome. First, the DNA sequence is matched to the chromosome by in situ or other chromosome-specific hybridization. Sequences can also be correlated to specific chromosomes by preparing PCR primers that can be used for PCR screening of somatic cell hybrids containing individual chromosomes from the desired species. Only hybrids containing the chromosome containing the gene homologous to the primer will yield an amplified fragment. Sublocalization can be achieved using chromosomal fragments. Other strategies include prescreening with labeled flow-sorted chromosomes and preselection by hybridization to chromosome-specific libraries. Further mapping strategies include fluorescence in situ hybridization which allows hybridization with probes shorter than those traditionally used. Reagents for chromosome mapping can be used individually to mark a single chromosome or a single site on the chromosome, or panels of reagents can be used for marking multiple sites and/or multiple chromosomes. Reagents corresponding to noncoding regions of the genes actually are preferred for mapping puφoses. Coding sequences are more likely to be conserved within gene families, thus increasing the chance of cross hybridizations during chromosomal mapping.

The β-subunit polynucleotides can also be used to identify individuals from small biological samples. This can be done for example using restriction fragment- length polymoφhism (RFLP) to identify an individual. Thus, the polynucleotides described herein are useful as DNA markers for RFLP (See U.S. Patent No. 5,272,057). Furthermore, the β-subunit sequence can be used to provide an alternative technique which determines the actual DNA sequence of selected fragments in the genome of an individual. Thus, the β-subunit sequences described herein can be used to prepare two PCR primers from the 5' and 3' ends of the sequences. These primers can then be used to amplify DNA from an individual for subsequent sequencing.

Panels of corresponding DNA sequences from individuals prepared in this manner can provide unique individual identifications, as each individual will have a unique set of such DNA sequences. It is estimated that allelic variation in humans occurs with a frequency of about once per each 500 bases. Allelic variation occurs to some degree in the coding regions of these sequences, and to a greater degree in the noncoding regions. The β-subunit sequences can be used to obtain such identification sequences from individuals and from tissue. The sequences represent unique fragments of the human genome. Each of the sequences described herein can, to some degree, be used as a standard against which DNA from an individual can be compared for identification puφoses.

If a panel of reagents from the sequences is used to generate a unique identification database for an individual, those same reagents can later be used to identify tissue from that individual. Using the unique identification database, positive identification of the individual, living or dead, can be made from extremely small tissue samples. The β-subunit polynucleotides can also be used in forensic identification procedures. PCR technology can be used to amplify DNA sequences taken from very small biological samples, such as a single hair follicle, body fluids (eg. blood, saliva, or semen). The amplified sequence can then be compared to a standard allowing identification of the origin of the sample.

The β-subunit polynucleotides can thus be used to provide polynucleotide reagents, e.g., PCR primers, targeted to specific loci in the human genome, which can enhance the reliability of DNA-based forensic identifications by, for example, providing another "identification marker" (i.e. another DNA sequence that is unique to a particular individual). As described above, actual base sequence information can be used for identification as an accurate alternative to patterns formed by restriction enzyme generated fragments. Sequences targeted to the noncoding region are particularly useful since greater polymoφhism occurs in the noncoding regions, making it easier to differentiate individuals using this technique. Fragments are at least 12 bases.

The β-subunit polynucleotides can further be used to provide polynucleotide reagents, e.g., labeled or labelable probes which can be used in, for example, an in situ hybridization technique, to identify a specific tissue. This is useful in cases in which a forensic pathologist is presented with a tissue of unknown origin. Panels of β-subunit probes can be used to identify tissue by species and/or by organ type.

In a similar fashion, these primers and probes can be used to screen tissue culture for contamination (i.e. screen for the presence of a mixture of different types of cells in a culture).

Alternatively, the β-subunit polynucleotides can be used directly to block transcription or translation of β-subunit gene expression by means of antisense or ribozyme constructs. Thus, in a disorder characterized by abnormally high or undesirable β-subunit gene expression, nucleic acids can be directly used for treatment.

The β-subunit polynucleotides are thus useful as antisense constructs to control β-subunit gene expression in cells, tissues, and organisms. A DNA antisense polynucleotide is designed to be complementary to a region of the gene involved in transcription, preventing transcription and hence production of β-subunit protein. An antisense RNA or DNA polynucleotide would hybridize to the mRNA and thus block translation of mRNA into β-subunit protein.

Examples of antisense molecules useful to inhibit nucleic acid expression include antisense molecules complementary to a fragment of the 5' untranslated region of SEQ ID NO 2 which also includes the start codon and antisense molecules which are complementary to a fragment of the 3' untranslated region of SEQ ID NO 2.

Alternatively, a class of antisense molecules can be used to inactivate mRNA in order to decrease expression of β-subunit nucleic acid. Accordingly, these molecules can treat a disorder characterized by abnormal or undesired subunit nucleic acid expression. This technique involves cleavage by means of ribozymes containing nucleotide sequences complementary to one or more regions in the mRNA that attenuate the ability of the mRNA to be translated. Possible regions include coding regions and particularly coding regions corresponding to the functional activities of the β-subunit protein.

The β-subunit polynucleotides also provide vectors for gene therapy in patients containing cells that are aberrant in β-subunit gene expression. Thus, recombinant cells, which include the patient's cells that have been engineered ex vivo and returned to the patient, are introduced into an individual where the cells produce the desired β-subunit protein to treat the individual.

The invention also encompasses kits for detecting the presence of a β-subunit nucleic acid in a biological sample. For example, the kit can comprise reagents such as a labeled or labelable nucleic acid or agent capable of detecting β-subunit nucleic acid in a biological sample; means for determining the amount of β-subunit nucleic acid in the sample; and means for comparing the amount of β-subunit nucleic acid in the sample with a standard. The compound or agent can be packaged in a suitable container. The kit can further comprise instructions for using the kit to detect β- subunit mRNA or DNA.

Vectors/Host Cells The invention also provides vectors containing the β-subunit polynucleotides and to host cells containing the β-subunit polynucleotides. As described more fully below, vectors can be used for cloning or expression but are preferably used for expression of the β-subunit. Preferably expression systems include host cells in which both the α and β subunits are expressed. The term "vector" refers to a vehicle, preferably a nucleic acid molecule, that can transport the β-subunit polynucleotides. When the vector is a nucleic acid molecule, the β-subunit polynucleotides are covalently linked to the vector nucleic acid. With this aspect of the invention, the vector includes a plasmid, single or double stranded phage, a single or double stranded RNA or DNA viral vector, or artificial chromosome, such as a BAC, PAC, YAC, OR MAC.

A vector can be maintained in the host cell as an extrachromosomal element where it replicates and produces additional copies of the β-subunit polynucleotides. Alternatively, the vector may integrate into the host cell genome and produce additional copies of the β-subunit polynucleotides when the host cell replicates.

The invention provides vectors for the maintenance (cloning vectors) or vectors for expression (expression vectors) of the β-subunit polynucleotides. The vectors can function in procaryotic or eukaryotic cells or in both (shuttle vectors).

Expression vectors contain cis-acting regulatory regions that are operably linked in the vector to the β-subunit polynucleotides such that transcription of the polynucleotides is allowed in a host cell. The polynucleotides can be introduced into the host cell with a separate polynucleotide capable of affecting transcription. Thus, the second polynucleotide may provide a trans-acting factor interacting with the cis- regulatory control region to allow transcription of the β-subunit polynucleotides from the vector. Alternatively, a trans-acting factor may be supplied by the host cell. Finally, a trans-acting factor can be produced from the vector itself.

It is understood, however, that in some embodiments, transcription and or translation of the β-subunit polynucleotides can occur in a cell-free system.

The regulatory sequence to which the polynucleotides described herein can be operably linked include promoters for directing mRNA transcription. These include, but are not limited to, the left promoter from bacteriophage λ, the lac, TRP, and TAC promoters from E. coli, the early and late promoters from SV40, the CMV immediate early promoter, the adenovirus early and late promoters, and retrovirus long-terminal repeats.

In addition to control regions that promote transcription, expression vectors may also include regions that modulate transcription, such as repressor binding sites and enhancers. Examples include the SV40 enhancer, the cytomegalovirus immediate early enhancer, polyoma enhancer, adenovirus enhancers, and retrovirus LTR enhancers.

In addition to containing sites for transcription initiation and control, expression vectors can also contain sequences necessary for transcription termination and, in the transcribed region a ribosome binding site for translation. Other regulatory control elements for expression include initiation and termination codons as well as polyadenylation signals. The person of ordinary skill in the art would be aware of the numerous regulatory sequences that are useful in expression vectors. Such regulatory sequences are described, for example, in Sambrook et al, Molecular Cloning: A Laboratory Manual. 2nd. ed., Cold Spring Harbor Laboratory Press, Cold Spring Harbor, NY (1989).

A variety of expression vectors can be used to express a β-subunit polynucleotide. Such vectors include chromosomal, episomal, and virus-derived vectors, for example vectors derived from bacterial plasmids, from bacteriophage, from yeast episomes, from yeast chromosomal elements, including yeast artificial chromosomes, from viruses such as baculoviruses, papovaviruses such as SV40, Vaccinia viruses, adenoviruses, poxviruses, pseudorabies viruses, and retroviruses. Vectors may also be derived from combinations of these sources such as those derived from plasmid and bacteriophage genetic elements, eg. cosmids and phagemids. Appropriate cloning and expression vectors for prokaryotic and eukaryotic hosts are described in Sambrook et al, Molecular Cloning: A Laboratory Manual. 2nd. ed., Cold Spring Harbor Laboratory Press, Cold Spring Harbor, NY (1989). The regulatory sequence may provide constitutive expression in one or more host cells (i.e. tissue specific) or may provide for inducible expression in one or more cell types such as by temperature, nutrient additive, or exogenous factor such as a hormone or other ligand. A variety of vectors providing for constitutive and inducible expression in prokaryotic and eukaryotic hosts are well known to those of ordinary skill in the art. The β-subunit polynucleotides can be inserted into the vector nucleic acid by well-known methodology. Generally, the DNA sequence that will ultimately be expressed is joined to an expression vector by cleaving the DNA sequence and the expression vector with one or more restriction enzymes and then ligating the fragments together. Procedures for restriction enzyme digestion and ligation are well known to those of ordinary skill in the art.

The vector containing the appropriate polynucleotide can be introduced into an appropriate host cell for propagation or expression using well-known techniques. Bacterial cells include, but are not limited to, E. coli, Streptomyces, and Salmonella typhimurium. Eukaryotic cells include, but are not limited to, yeast, insect cells such as Drosophila, animal cells such as COS and CHO cells, and plant cells.

As described herein, it may be desirable to express the polypeptide as a fusion protein. Accordingly, the invention provides fusion vectors that allow for the production of the β-subunit polypeptides. Fusion vectors can increase the expression of a recombinant protein, increase the solubility of the recombinant protein, and aid in the purification of the protein by acting for example as a ligand for affinity purification. A proteolytic cleavage site may be introduced at the junction of the fusion moiety so that the desired polypeptide can ultimately be separated from the fusion moiety. Proteolytic enzymes include, but are not limited to, factor Xa, thrombin, and enterokinase. Typical fusion expression vectors include pGEX (Smith et al. (1988) Gene 67:31 -40), pMAL (New England Biolabs, Beverly, MA) and pRIT5 (Pharmacia, Piscataway, NJ) which fuse glutathione S-transferase (GST), maltose E binding protein, or protein A, respectively, to the target recombinant protein. Examples of suitable inducible non-fusion E. coli expression vectors include pTrc (Amann et al, Gene (59:301-315 (1988)) and pET l id (Studier et al, Gene Expression Technology: Methods in Enzymology 755:60-89 (1990)). Recombinant protein expression can be maximized in a host bacteria by providing a genetic background wherein the host cell has an impaired capacity to proteolytically cleave the recombinant protein. (Gottesman, S., Gene Expression Technology: Methods in Enzymology 185, Academic Press, San Diego, California (1990) 119-128). Alternatively, the sequence of the polynucleotide of interest can be altered to provide preferential codon usage for a specific host cell, for example E. coli. (Wada et al, Nucleic Acids Res. 20:2111-2118 (1992)).

The β-subunit polynucleotides can also be expressed by expression vectors that are operative in yeast. Examples of vectors for expression in yeast e.g., S. cerevisiae include pYepSecl (Baldari, et al, EMBOJ. 6:229-234 (1987)), pMFa (Kurjan et al, Cell 50:933-943 (1982)), pJRY88 (Schultz et al, Gene 54:113-123 (1987)), and pYES2 (Invitrogen Coφoration, San Diego, CA).

The β-subunit polynucleotides can also be expressed in insect cells using, for example, baculovirus expression vectors. Baculovirus vectors available for expression of proteins in cultured insect cells (e.g., Sf 9 cells) include the pAc series (Smith et al, Mol. Cell Biol. 5:2156-2165 (1983)) and the pVL series (Lucklow et al, Virology 770:31-39 (1989)).

In certain embodiments of the invention, the polynucleotides described herein are expressed in mammalian cells using mammalian expression vectors. Examples of mammalian expression vectors include pCDM8 (Seed, B. Nature 529:840 (1987)) and pMT2PC (Kaufman et al, EMBOJ. (5:187-195 (1987)).

The expression vectors listed herein are provided by way of example only of the well-known vectors available to those of ordinary skill in the art that would be useful to express the β-subunit polynucleotides. The person of ordinary skill in the art would be aware of other vectors suitable for maintenance propagation or expression of the polynucleotides described herein. These are found for example in Sambrook, J., Fritsh, E. F., and Maniatis, T. Molecular Cloning: A Laboratory Manual. 2nd, ed., Cold Spring Harbor Laboratory, Cold Spring Harbor Laboratory Press, Cold Spring Harbor, NY, 1989. The invention also encompasses vectors in which the nucleic acid sequences described herein are cloned into the vector in reverse orientation, but operably linked to a regulatory sequence that permits transcription of antisense RNA. Thus, an antisense transcript can be produced to all, or to a portion, of the polynucleotide sequences described herein, including both coding and non-coding regions. Expression of this antisense RNA is subject to each of the parameters described above in relation to expression of the sense RNA (regulatory sequences, constitutive or inducible expression, tissue-specific expression).

The invention also relates to recombinant host cells containing the vectors described herein. Host cells therefore include prokaryotic cells, lower eukaryotic cells such as yeast, other eukaryotic cells such as insect cells, and higher eukaryotic cells such as mammalian cells.

The recombinant host cells are prepared by introducing the vector constructs described herein into the cells by techniques readily available to the person of ordinary skill in the art. These include, but are not limited to, calcium phosphate transfection, DEAE-dextran-mediated transfection, cationic lipid-mediated transfection, electroporation, transduction, infection, lipofection, and other techniques such as those found in Sambrook, et al. (Molecular Cloning: A Laboratory Manual. 2nd, ed., Cold Spring Harbor Laboratory, Cold Spring Harbor Laboratory Press, Cold Spring Harbor, NY, 1989).

Host cells can contain more than one vector. Thus, different nucleotide sequences can be introduced on different vectors of the same cell. Similarly, the β- subunit polynucleotides can be introduced either alone or with other polynucleotides that are not related to the β-subunit polynucleotides such as those providing transacting factors for expression vectors. When more than one vector is introduced into a cell, the vectors can be introduced independently, co-introduced or joined to the β- subunit polynucleotide vector.

In the case of bacteriophage and viral vectors, these can be introduced into cells as packaged or encapsulated virus by standard procedures for infection and transduction. Viral vectors can be replication-competent or replication-defective. In the case in which viral replication is defective, replication will occur in host cells providing functions that complement the defects. Vectors generally include selectable markers that enable the selection of the subpopulation of cells that contain the recombinant vector constructs. The marker can be contained in the same vector that contains the polynucleotides described herein or may be on a separate vector. Markers include tetracycline or ampicillin- resistance genes for prokaryotic host cells and dihydrofolate reductase or neomycin resistance for eukaryotic host cells. However, any marker that provides selection for a phenotypic trait will be effective.

While the mature proteins can be produced in bacteria, yeast, mammalian cells, and other cells under the control of the appropriate regulatory sequences, cell- free transcription and translation systems can also be used to produce these proteins using RNA derived from the DNA constructs described herein.

Where secretion of the polypeptide is desired, appropriate secretion signals are incoφorated into the vector. The signal sequence can be endogenous to the β- subunit polypeptides or heterologous to these polypeptides. Where the polypeptide is not secreted into the medium, the protein can be isolated from the host cell by standard disruption procedures, including freeze thaw, sonication, mechanical disruption, use of ly sing agents and the like. The polypeptide can then be recovered and purified by well-known purification methods including ammonium sulfate precipitation, acid extraction, anion or cationic exchange chromatography, phosphocellulose chromatography, hydrophobic-interaction chromatography, affinity chromatography, hydroxylapatite chromatography, lectin chromatography, or high performance liquid chromatography.

It is also understood that depending upon the host cell in recombinant production of the polypeptides described herein, the polypeptides can have various glycosylation patterns, depending upon the cell, or maybe non-glycosylated as when produced in bacteria. In addition, the polypeptides may include an initial modified methionine in some cases as a result of a host-mediated process.

Uses of Vectors and Host Cells The host cells expressing the polypeptides described herein, and particularly recombinant host cells, have a variety of uses. First, the cells are useful for producing β-subunit proteins or polypeptides that can be further purified to produce desired amounts of β-subunit protein or fragments. Thus, host cells containing expression vectors are useful for polypeptide production.

Host cells are also useful for conducting cell-based assays involving the β- subunit or β-subunit fragments. Thus, a recombinant host cell expressing a native β- subunit is useful to assay for compounds that stimulate or inhibit β-subunit function. This includes ligand binding, gene expression at the level of transcription or translation, α-subunit interaction, and ability to be phosphorylated.

Accordingly, in preferred embodiments the host cells express both the α and β subunits or relevant portions thereof. Therefore, cell-based and cell-free assays are provided in which both α and β subunits (or relevant portions thereof) provide assays useful for detection of β-subunit function. In a preferred embodiment, the invention provides a cell-based assay in which the cell expresses both the α and β subunits.

Assay end points include ligand binding, α-subunit association or activation, channel currents, phosphorylation, and conformational changes in either the α or β subunit. Interaction of the α and β subunit can be measured in assays based on dual label energy transfer, methods in which reactants are separately labeled with an energy transfer donor and acceptor, such that energy transfer results when the donor and acceptor are brought into close proximity to each other, producing a detectable lifetime change. Assay methods for detection of a complex formed between the α and β subunits include determining fluorescence emission or fluorescence quenching or other energy transfer between labels on the two subunits. One example is a fluorescein homoquenching method in which a subunit is labeled with fluorescein positioned such that when the other subunit is bound the fluorescein molecules quench one another and the fluorescence of the solution decreases. This analytical technique is well-known and within the skill of those in the art. See, for example, U.S. Patent No. 5,631,169; U.S. Patent No. 5,506,107; U.S. Patent No. 5,716,784; and U.S. Patent No. 5,763,189.

Host cells are also useful for identifying subunit mutants in which these functions are affected. If the mutants naturally occur and give rise to a pathology, host cells containing the mutations are useful to assay compounds that have a desired effect on the mutant β-subunit (for example, stimulating or inhibiting function) which may not be indicated by their effect on the native β-subunit.

Recombinant host cells are also useful for expressing the chimeric polypeptides described herein to assess compounds that activate or suppress activation by means of a heterologous intracellular or extracellular domain.

Alternatively, one or more heterologous transmembrane domains can be used to assess the effect of a desired extracellular domain on any given host cell. In this embodiment, a transmembrane domain compatible with the specific host cell is used to make the chimeric polypeptide. Further, mutant β-subunits can be designed in which one or more of the various functions is engineered to be increased or decreased (i.e., ligand binding or α-subunit activation) and used to augment or replace β-subunit proteins in an individual. Thus, host cells can provide a therapeutic benefit by replacing an aberrant β-subunit or providing an aberrant β-subunit that provides a therapeutic result. In one embodiment, the cells provide β-subunits that are abnormally active.

In another embodiment, the cells provide β-subunits that are abnormally inactive. These β-subunits can compete with endogenous β-subunits in the individual.

In another embodiment, cells expressing β-subunits that cannot be activated, are introduced into an individual in order to compete with endogenous β-subunits for ligand or α-subunit. For example, in the case in which excessive ligand is part of a treatment modality, it may be necessary to inactivate this ligand at a specific point in treatment. Providing cells that compete for the ligand, but which cannot be affected by β-subunit activation would be beneficial. Homologously recombinant host cells can also be produced that allow the in situ alteration of endogenous β-subunit polynucleotide sequences in a host cell genome. This technology is more fully described in WO 93/09222, WO 91/12650 and U.S. 5,641,670. Briefly, specific polynucleotide sequences corresponding to the β-subunit polynucleotides or sequences proximal or distal to a β-subunit gene are allowed to integrate into a host cell genome by homologous recombination where expression of the gene can be affected. In one embodiment, regulatory sequences are introduced that either increase or decrease expression of an endogenous sequence. Accordingly, a β-subunit protein can be produced in a cell not normally producing it, or increased expression of β-subunit protein can result in a cell normally producing the protein at a specific level. Alternatively, the entire gene can be deleted. Still further, specific mutations can be introduced into any desired region of the gene to produce mutant β-subunit proteins. Such mutations could be introduced, for example, into the specific functional regions such as the ligand-binding site or the α- subunit interaction site.

In one embodiment, the host cell can be a fertilized oocyte or embryonic stem cell that can be used to produce a transgenic animal containing the altered β-subunit gene. Alternatively, the host cell can be a stem cell or other early tissue precursor that gives rise to a specific subset of cells and can be used to produce transgenic tissues in an animal. See also Thomas et al, Cell 57:503 (1987) for a description of homologous recombination vectors. The vector is introduced into an embryonic stem cell line (e.g., by electroporation) and cells in which the introduced gene has homologously recombined with the endogenous β-subunit gene is selected (see e.g., Li, E. et al, Cell (59:915 (1992)). The selected cells are then injected into a blastocyst of an animal (e.g., a mouse) to form aggregation chimeras (see e.g., Bradley, A. in Teratocarcinomas and Embryonic Stem Cells: A Practical Approach, E.J. Robertson, ed. (IRL, Oxford, 1987) pp. 113-152). A chimeric embryo can then be implanted into a suitable pseudopregnant female foster animal and the embryo brought to term. Progeny harboring the homologously recombined DNA in their germ cells can be used to breed animals in which all cells of the animal contain the homologously recombined DNA by germline transmission of the transgene. Methods for constructing homologous recombination vectors and homologous recombinant animals are described further in Bradley, A. (1991) Current Opinion in Biotechnology 2:823-829 and in PCT International Publication Nos. WO 90/11354; WO 91/01140; and WO 93/04169.

The genetically engineered host cells can be used to produce non-human transgenic animals. A transgenic animal is preferably a mammal, for example a rodent, such as a rat or mouse, in which one or more of the cells of the animal include a transgene. A transgene is exogenous DNA which is integrated into the genome of a cell from which a transgenic animal develops and which remains in the genome of the mature animal in one or more cell types or tissues of the transgenic animal. These animals are useful for studying the function of a β-subunit protein and identifying and evaluating modulators of β-subunit protein activity.

Other examples of transgenic animals include non-human primates, sheep, dogs, cows, goats, chickens, and amphibians.

In one embodiment, a host cell is a fertilized oocyte or an embryonic stem cell into which β-subunit polynucleotide sequences have been introduced. A transgenic animal can be produced by introducing nucleic acid into the male pronuclei of a fertilized oocyte, e.g., by microinjection, retroviral infection, and allowing the oocyte to develop in a pseudopregnant female foster animal. Any of the β-subunit nucleotide sequences can be introduced as a transgene into the genome of a non-human animal, such as a mouse. Any of the regulatory or other sequences useful in expression vectors can form part of the transgenic sequence. This includes intronic sequences and polyadenylation signals, if not already included. A tissue-specific regulatory sequence(s) can be operably linked to the transgene to direct expression of the β- subunit protein to particular cells. Methods for generating transgenic animals via embryo manipulation and microinjection, particularly animals such as mice, have become conventional in the art and are described, for example, in U.S. Patent Nos. 4,736,866 and 4,870,009, both by Leder et al, U.S. Patent No. 4,873,191 by Wagner et al. and in Hogan, B., Manipulating the Mouse Embryo, (Cold Spring Harbor Laboratory Press, Cold Spring Harbor, N.Y., 1986). Similar methods are used for production of other transgenic animals. A transgenic founder animal can be identified based upon the presence of the transgene in its genome and/or expression of transgenic mRNA in tissues or cells of the animals. A transgenic founder animal can then be used to breed additional animals carrying the transgene. Moreover, transgenic animals carrying a transgene can further be bred to other transgenic animals carrying other transgenes. A transgenic animal also includes animals in which the entire animal or tissues in the animal have been produced using the homologously recombinant host cells described herein.

In another embodiment, transgenic non-human animals can be produced which contain selected systems which allow for regulated expression of the transgene. One example of such a system is the cre/loxP recombinase system of bacteriophage PI . For a description of the cre/loxP recombinase system, see, e.g., Lakso et al, PNAS 59:6232-6236 (1992). Another example of a recombinase system is the FLP recombinase system of S. cerevisiae (O'Gorman et al. Science 257:1351- 1355 (1991). lia cre/loxP recombinase system is used to regulate expression of the transgene, animals containing transgenes encoding both the Cre recombinase and a selected protein is required. Such animals can be provided through the construction of "double" transgenic animals, e.g., by mating two transgenic animals, one containing a transgene encoding a selected protein and the other containing a transgene encoding a recombinase. Clones of the non-human transgenic animals described herein can also be produced according to the methods described in Wilmut, I. et αl, Nature 555:810- 813 (1997) and PCT International Publication Nos. WO 97/07668 and WO 97/07669. In brief, a cell, e.g., a somatic cell, from the transgenic animal can be isolated and induced to exit the growth cycle and enter G₀ phase. The quiescent cell can then be fused, e.g., through the use of electrical pulses, to an enucleated oocyte from an animal of the same species from which the quiescent cell is isolated. The reconstructed oocyte is then cultured such that it develops to morula or blastocyst and then transferred to pseudopregnant female foster animal. The offspring borne of this female foster animal will be a clone of the animal from which the cell, e.g., the somatic cell, is isolated.

Transgenic animals containing recombinant cells that express the polypeptides described herein are useful to conduct the assays described herein in an in vivo context. Accordingly, the various physiological factors that are present in vivo and that could effect ligand binding, α-subunit activation, and ability to be phosphorylated may not be evident from in vitro cell-free or cell-based assays.

Accordingly, it is useful to provide non-human transgenic animals to assay in vivo β- subunit function, including ligand and α-subunit interaction, the effect of specific mutant β-subunits on the α-subunit, channel function, and ligand interaction, and the effect of chimeric subunits or channels. It is also possible to assess the effect of null mutations, that is mutations that substantially or completely eliminate one or more β- subunit functions.

Pharmaceutical Compositions

The β-subunit nucleic acid molecules, protein (particularly fragments such as the various domains), modulators of the protein, and antibodies (also referred to herein as "active compounds") can be incoφorated into pharmaceutical compositions suitable for administration to a subject, e.g., a human. Such compositions typically comprise the nucleic acid molecule, protein, modulator, or antibody and a pharmaceutically acceptable carrier.

As used herein the language "pharmaceutically acceptable carrier" is intended to include any and all solvents, dispersion media, coatings, antibacterial and antifungal agents, isotonic and absoφtion delaying agents, and the like, compatible with pharmaceutical administration. The use of such media and agents for pharmaceutically active substances is well known in the art. Except insofar as any conventional media or agent is incompatible with the active compound, such media can be used in the compositions of the invention. Supplementary active compounds can also be incoφorated into the compositions.

A pharmaceutical composition of the invention is formulated to be compatible with its intended route of administration. Examples of routes of administration include parenteral, e.g., intravenous, intradermal, subcutaneous, oral (e.g., inhalation), transdermal (topical), transmucosal, and rectal administration. Solutions or suspensions used for parenteral, intradermal, or subcutaneous application can include the following components: a sterile diluent such as water for injection, saline solution, fixed oils, polyethylene glycols, glycerine, propylene glycol or other synthetic solvents; antibacterial agents such as benzyl alcohol or methyl parabens; antioxidants such as ascorbic acid or sodium bisulfite; chelating agents such as ethylenediaminetetraacetic acid; buffers such as acetates, citrates or phosphates and agents for the adjustment of tonicity such as sodium chloride or dextrose. pH can be adjusted with acids or bases, such as hydrochloric acid or sodium hydroxide. The parenteral preparation can be enclosed in ampules, disposable syringes or multiple dose vials made of glass or plastic. Pharmaceutical compositions suitable for injectable use include sterile aqueous solutions (where water soluble) or dispersions and sterile powders for the extemporaneous preparation of sterile injectable solutions or dispersion. For intravenous administration, suitable carriers include physiological saline, bacteriostatic water, Cremophor EL™ (BASF, Parsippany, NJ) or phosphate buffered saline (PBS). In all cases, the composition must be sterile and should be fluid to the extent that easy syringability exists. It must be stable under the conditions of manufacture and storage and must be preserved against the contaminating action of microorganisms such as bacteria and fungi. The carrier can be a solvent or dispersion medium containing, for example, water, ethanol, polyol (for example, glycerol, propylene glycol, and liquid polyetheylene glycol, and the like), and suitable mixtures thereof. The proper fluidity can be maintained, for example, by the use of a coating such as lecithin, by the maintenance of the required particle size in the case of dispersion and by the use of surfactants. Prevention of the action of microorganisms can be achieved by various antibacterial and antifungal agents, for example, parabens, chlorobutanol, phenol, ascorbic acid, thimerosal, and the like. In many cases, it will be preferable to include isotonic agents, for example, sugars, polyalcohols such as manitol, sorbitol, sodium chloride in the composition. Prolonged absoφtion of the injectable compositions can be brought about by including in the composition an agent which delays absoφtion, for example, aluminum monostearate and gelatin.

Sterile injectable solutions can be prepared by incoφorating the active compound (e.g., a β-subunit protein or anti- β-subunit antibody) in the required amount in an appropriate solvent with one or a combination of ingredients enumerated above, as required, followed by filtered sterilization. Generally, dispersions are prepared by incoφorating the active compound into a sterile vehicle which contains a basic dispersion medium and the required other ingredients from those enumerated above. In the case of sterile powders for the preparation of sterile injectable solutions, the preferred methods of preparation are vacuum drying and freeze-drying which yields a powder of the active ingredient plus any additional desired ingredient from a previously sterile-filtered solution thereof. Oral compositions generally include an inert diluent or an edible carrier.

They can be enclosed in gelatin capsules or compressed into tablets. For oral administration, the agent can be contained in enteric forms to survive the stomach or further coated or mixed to be released in a particular region of the GI tract by known methods. For the puφose of oral therapeutic administration, the active compound can be incoφorated with excipients and used in the form of tablets, troches, or capsules. Oral compositions can also be prepared using a fluid carrier for use as a mouthwash, wherein the compound in the fluid carrier is applied orally and swished and expectorated or swallowed. Pharmaceutically compatible binding agents, and/or adjuvant materials can be included as part of the composition. The tablets, pills, capsules, troches and the like can contain any of the following ingredients, or compounds of a similar nature: a binder such as microcrystalline cellulose, gum tragacanth or gelatin; an excipient such as starch or lactose, a disintegrating agent such as alginic acid, Primogel, or corn starch; a lubricant such as magnesium stearate or Sterotes; a glidant such as colloidal silicon dioxide; a sweetening agent such as sucrose or saccharin; or a flavoring agent such as peppermint, methyl salicylate, or orange flavoring.

For administration by inhalation, the compounds are delivered in the form of an aerosol spray from pressured container or dispenser which contains a suitable propellant, e.g., a gas such as carbon dioxide, or a nebulizer. Systemic administration can also be by transmucosal or transdermal means.

For transmucosal or transdermal administration, penetrants appropriate to the barrier to be permeated are used in the formulation. Such penetrants are generally known in the art, and include, for example, for transmucosal administration, detergents, bile salts, and fusidic acid derivatives. Transmucosal administration can be accomplished through the use of nasal sprays or suppositories. For transdermal administration, the active compounds are formulated into ointments, salves, gels, or creams as generally known in the art.

The compounds can also be prepared in the form of suppositories (e.g., with conventional suppository bases such as cocoa butter and other glycerides) or retention enemas for rectal delivery.

In one embodiment, the active compounds are prepared with carriers that will protect the compound against rapid elimination from the body, such as a controlled release formulation, including implants and microencapsulated delivery systems. Biodegradable, biocompatible polymers can be used, such as ethylene vinyl acetate, polyanhydrides, polyglycolic acid, collagen, polyorthoesters, and polylactic acid.

Methods for preparation of such formulations will be apparent to those skilled in the art. The materials can also be obtained commercially from Alza Coφoration and Nova Pharmaceuticals, Inc. Liposomal suspensions (including liposomes targeted to infected cells with monoclonal antibodies to viral antigens) can also be used as pharmaceutically acceptable carriers. These can be prepared according to methods known to those skilled in the art, for example, as described in U.S. Patent No. 4,522,811.

It is especially advantageous to formulate oral or parenteral compositions in dosage unit form for ease of administration and uniformity of dosage. Dosage unit form as used herein refers to physically discrete units suited as unitary dosages for the subject to be treated; each unit containing a predetermined quantity of active compound calculated to produce the desired therapeutic effect in association with the required pharmaceutical carrier. The specification for the dosage unit forms of the invention are dictated by and directly dependent on the unique characteristics of the active compound and the particular therapeutic effect to be achieved, and the limitations inherent in the art of compounding such an active compound for the treatment of individuals.

The nucleic acid molecules of the invention can be inserted into vectors and used as gene therapy vectors. Gene therapy vectors can be delivered to a subject by, for example, intravenous injection, local administration (U.S. 5,328,470) or by stereotactic injection (see e.g., Chen et al, PNAS 97:3054-3057 (1994)). The pharmaceutical preparation of the gene therapy vector can include the gene therapy vector in an acceptable diluent, or can comprise a slow release matrix in which the gene delivery vehicle is imbedded. Alternatively, where the complete gene delivery vector can be produced intact from recombinant cells, e.g. retroviral vectors, the pharmaceutical preparation can include one or more cells which produce the gene delivery system.

The pharmaceutical compositions can be included in a container, pack, or dispenser together with instructions for administration.

EXPERIMENTAL

The pore-forming α-subunit of the high conductance calcium-activated potassium channel (maxi-K) has been identified and cloned in human (called hSlo) and mouse (called mSlo). See, for example, Knaus, J Biol. Chem.269: 921-3924 (1994), and Butler et al, Science 261:221-224 (1993). Experiments were conducted to examine the functional role of the novel human calcium-activated potassium channel β-subunit C7F2 (SEQ ID NO 2) in the high conductance calcium-activated potassium channel maxi-K.

These experiments show that a physical interaction of C7F2 with hSlo and mSlo modifies the channel activity of maxi-K, supporting the claim that C7F2 is a functional β-subunit of maxi-K.

Example 1 : Physical Association of C7F2 with mSlo

The open reading frame of C7F2 (nucleotides 502-1131 of SEQ ID NO 2) was cloned into the pcDNA3.1/V5/His-TOPO vector (Invitrogen) to provide a V5 epitope tag. This vector and a vector containing mSlo were transiently co-transfected into HEK293 cells with lipofectamine or Fugene. mSlo was immunoprecipitated with antibodies directed against the α-subunit. The immunoprecipitates were subjected to Western blotting with monoclonal antibody directed against the V5 epitope tag to reveal the presence of the V5 -tagged C7F2.

These experiments demonstrate that human C7F2 can associate with mSlo (data not shown), suggesting a physical interaction of C7F2 with the pore-forming α- subunit of maxi-K. These results confirm the claim that C7F2 is a β-subunit for maxi-K.

Example 2: Electrophysiological Consequences of Association of C7F2 with hSlo and mSlo on the Channel Activity of Maxi-K

The open reading frame of C7F2 was cloned into the pIRES-EGFP vector (Clontech) to express both C7F2 and green fluorescent protein (GFP) in transfected cells. This vector and vectors containing either hSlo or mSlo were transiently co- transfected into HEK293 cells with lipofectamine or Fugene. Cells were selected for recording based on the expression of GFP.

Activation and deactivation kinetics of the mouse maxi-K channel (mSlo) were dramatically different when expressed alone or when co-expressed with C7F2 (Figure 6). These inside-out patch-clamp experiments revealed that co-expression of C7F2 with mSlo (horizontal bars labeled mSlo + C7F2) dramatically increases the time constants of activation (mSlo + C7F2 activation) and deactivation (mSlo + C7F2 deactivation) of the mouse maxi-K when compared to expression of mSlo alone (horizontal bars labeled mSlo activation and mSlo deactivation, respectfully). Similar effects were seen for the human maxi-K hSlo. In the presence of 3 μM Ca⁺⁺, C7F2 co-expression with mSlo causes a hypeφolarizing (leftward) shift of 20 mV of half-maximal channel activation, suggesting increased sensitivity of the mouse maxi-K channel to calcium ions (Figure 7). This is the typical behavior of the previously characterized β-subunit. However, when C7F2 is co-expressed with hSlo, there is a 20-50 mV depolarizing (rightward) shift of half-maximal channel activation, suggesting decreased sensitivity of the human maxi-K channel to calcium ions (Figure 8). This is a unique, novel behavior of C7F2 compared to the previously characterized β-subunit.

Functional interaction of C7F2 with mSlo and hSlo, leading to changes in activation and deactivation kinetics of the maxi-K channel along with shifts in the half-maximal channel activation in response to calcium, confirms the claim that C7F2 is a β-subunit for maxi-K. This invention may be embodied in many different forms and should not be construed as limited to the embodiments set forth herein; rather, these embodiments are provided so that this disclosure will fully convey the invention to those skilled in the art. Many modifications and other embodiments of the invention will come to mind in one skilled in the art to which this invention pertains having the benefit of the teachings presented in the foregoing description. Although specific terms are employed, they are used as in the art unless otherwise indicated.

All publications and patent applications mentioned in the specification are indicative of the level of those skilled in the art to which this invention pertains. All publications and patent applications are herein incoφorated by reference to the same extent as if each individual publication or patent application was specifically and individually indicated to be incoφorated by reference.

Although the foregoing invention has been described in some detail by way of illustration and example for puφoses of clarity of understanding, it will be obvious that certain changes and modifications may be practiced within the scope of the appended claims.

Applicant's or agent's International application No file reference 5800-5-1 PCT/US99/

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Form PCT/RO/134 (July 1998)

Claims

THAT WHICH IS CLAIMED:

1. An isolated polypeptide having an amino acid sequence selected from the group consisting of: (a) the amino acid sequence shown in SEQ ID NO 1 ;

(b) the amino acid sequence encoded by the cDNA contained in ATCC Deposit No. ;

(c) the amino acid sequence of an allelic variant of the amino acid sequence shown in SEQ ID NO 1 ; (d) the amino acid sequence of an allelic variant of the amino acid sequence encoded by the cDNA contained in ATCC Deposit No. ;

(e) the amino acid sequence of a sequence variant of the amino acid sequence shown in SEQ ID NO 1 , wherein the sequence variant is encoded by a nucleic acid molecule hybridizing to the nucleic acid molecule shown in SEQ ID NO 2 under stringent conditions;

(f) the amino acid sequence of a sequence variant of the amino acid sequence encoded by the cDNA clone contained in ATCC Deposit No. , wherein the sequence variant is encoded by a nucleic acid molecule hybridizing under stringent conditions to the cDNA contained in ATCC Deposit No. ;

(g) a fragment of the amino acid sequence shown in SEQ ID NO 1 , wherein the fragment comprises at least 216 contiguous amino acids; (h) a fragment of the amino acid sequence encoded by the cDNA contained in ATCC Deposit No. , wherein the fragment comprises at least 216 contiguous amino acids;

(i) the amino acid sequence of the mature subunit polypeptide from about amino acid 7 to about amino acid 210, shown in SEQ ID NO 1 ; (j) the amino acid sequence of the mature polypeptide from about amino acid 7 to about amino acid 210, encoded by the cDNA clone contained in ATCC Deposit No. ;

68 (k) the amino acid sequence of the region spanning the transmembrane domain of the polypeptide shown in SEQ ID NO 1, from about amino acid 20 to about amino acid 40;

(1) the amino acid sequence of the region spanning the transmembrane domain from about amino acid 168 to about amino acid 192 in the polypeptide encoded by the cDNA contained in ATCC Deposit No. ;

(m) the amino acid sequence of an epitope bearing region of any one of the polypeptides of (a)-(l);

(n) the amino acid sequence of the amino terminal intracellular region shown in SEQ ID NO 1 from amino acid 1 to about amino acid 19;

(o) the amino acid sequence of the carboxyl terminal intracellular region of the polypeptide shown in SEQ ID NO 1 from about amino acid 193 to amino acid 210; and

(p) the amino acid sequence of the extracellular loop region of the polypeptide shown in SEQ ID NO 1 from about amino acid 41 to about amino acid 167.

2. An isolated antibody that selectively binds to a polypeptide of claim l, (a)-(p).

3. An isolated nucleic acid molecule having a nucleotide sequence selected from the group consisting of:

(a) the nucleotide sequence shown in SEQ ID NO 2;

(b) the nucleotide sequence in the cDNA contained in ATCC Deposit No.

(c) a nucleotide sequence encoding the amino acid sequence shown in SEQ ID NO 1;

69 (d) a nucleotide sequence encoding the amino acid sequence encoded by the cDNA contained in ATCC Deposit No. ; and

(e) a nucleotide sequence complementary to any of the nucleotide sequences in (a), (b), (c), or (d).

4. An isolated nucleic acid molecule having a nucleotide sequence selected from the group consisting of:

(a) a nucleotide sequence encoding an amino acid sequence of a sequence variant of the amino acid sequence shown in SEQ ID NO 1 that hybridizes to the nucleotide sequence shown in SEQ ID NO 2 under stringent conditions;

(b) a nucleotide sequence encoding the amino acid sequence of a sequence variant of the amino acid sequence encoded by the cDNA contained in ATCC Deposit No. , the nucleic acid sequence of the sequence variant hybridizing to the cDNA contained in ATCC Deposit No. under stringent conditions; and

(c) a nucleotide sequence complementary to either of the nucleotide sequences in (a) or (b).

5. An isolated nucleic acid molecule a polynucleotide having a nucleotide sequence selected from the group consisting of:

(a) a nucleotide sequence encoding a fragment of the amino acid sequence shown in SEQ ID NO 1 , wherein the fragment comprises at least 216 contiguous amino acids;

(b) a nucleotide sequence encoding a fragment of the amino acid sequence encoded by the cDNA contained in ATCC Deposit No. , wherein the fragment comprises at least 216 contiguous amino acids;

(c) a nucleotide sequence encoding a fragment of the amino acid sequence shown in SEQ ID NO 1 , wherein the fragment is from amino acid 1 to about amino acid 19;

70 (d) a nucleotide sequence encoding a fragment of the amino acid sequence shown in SEQ ID NO 1 , wherein the fragment is from about amino acid 20 to about amino acid 40;

(e) a nucleotide sequence encoding a fragment of the amino acid sequence shown in SEQ ID NO 1 , wherein the fragment is from about amino acid 41 to about amino acid 167;

(f) a nucleotide sequence encoding a fragment of the amino acid sequence shown in SEQ ID NO 1 , wherein the fragment is from about amino acid 168 to about amino acid 192; (g) a nucleotide sequence encoding a fragment of the amino acid sequence shown in SEQ ID NO 1 , wherein the fragment is from about amino acid 193 to amino acid 210; and

(h) a nucleotide sequence complementary to any of the nucleotide sequences in (a)-(g).

6. A nucleic acid vector comprising the nucleic acid sequences in any of claims 3-5.

7. A host cell containing the vector of claim 6.

8. A method for producing any of the polypeptides in claim 1 comprising introducing a nucleotide sequence encoding any of the polypeptide sequences in (a)-(p) into a host cell, and culturing the host cell under conditions in which the proteins are expressed from the nucleic acid.

9. A method for detecting the presence of any of the polypeptides in claim 1 in a sample, said method comprising contacting said sample with an agent that specifically allows detection of the presence of the polypeptide in the sample and then detecting the presence of the polypeptide.

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10. The method of claim 9, wherein said agent is capable of selective physical association with said polypeptide.

11. The method of claim 10, wherein said agent binds to said polypeptide.

12. The method of claim 11 , wherein said agent is an antibody.

13. The method of claim 11 , wherein said agent is a ligand.

14. A kit comprising reagents used for the method of claim 9, wherein the reagents comprise an agent that specifically binds to said polypeptide.

15. A method for detecting the presence of any of the nucleic acid sequences in any of claims 3-5 in a sample, the method comprising contacting the sample with an oligonucleotide that hybridizes to the nucleic acid sequences under stringent conditions and determining whether the oligonucleotide binds to the nucleic acid sequence in the sample.

16. The method of claim 15, wherein the nucleic acid, whose presence is detected, is mRNA.

17. A kit comprising reagents used for the method of claim 15, wherein the reagents comprise a compound that hybridizes under stringent conditions to any of the nucleic acid molecules.

18. A method for identifying an agent that binds to any of the polypeptides in claim 1 , said method comprising contacting the polypeptide with an agent that binds to the polypeptide and assaying the complex formed with the agent bound to the polypeptide.

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19. A method for modulating the activity of a polypeptide of claim 1 , said method comprising contacting said polypeptide, or a cell expressing said polypeptide, with an agent which binds to said polypeptide in a sufficient concentration to modulate the activity of said polypeptide.

20. The method of claim 19, wherein said activity of said polypeptide is activation of a pore-forming ╬▒-subunit of a potassium channel.

21. A method for identifying an agent which inhibits formation of a complex between a polypeptide of claim 1 and a pore-forming ╬▒-subunit of a potassium channel, said method comprising the steps of: a) contacting said polypeptide of claim 1 , or a cell expressing said polypeptide of claim 1, with a test agent; and b) assaying the complex formed between said polypeptide of claim 1 and said ╬▒-subunit to determine whether said test agent inhibits formation of said complex.

22. A method for identifying an agent which modulates a regulatory effect of a polypeptide of claim 1 on a pore-forming ╬▒-subunit of a potassium channel, said method comprising the steps of: a) contacting said polypeptide, or a cell expressing said polypeptide, with a test agent; and b) determining whether said test agent modulates said regulatory effect.

23. A method of treating a patient afflicted with a disorder associated with aberrant activity or expression of a protein, the method comprising administering to the patient a compound which modulates the activity of said protein in an amount effective to modulate the activity of the protein in the patient, whereby at least one symptom of the disorder is alleviated, wherein said protein has an amino acid sequence selected from the group consisting of:

(a) the amino acid sequence shown in SEQ ID NO 1 ;

73 (b) the amino acid sequence encoded by the cDNA contained in ATCC Deposit No. ;

(c) the amino acid sequence of an allelic variant of the amino acid sequence shown in SEQ ID NO 1 ;

(d) the amino acid sequence of an allelic variant of the amino acid sequence encoded by the cDNA contained in ATCC Deposit No. ;

(e) the amino acid sequence of a sequence variant of the amino acid sequence shown in SEQ ID NO 1 , wherein the sequence variant is encoded by a nucleic acid molecule hybridizing to the nucleic acid molecule shown in SEQ ID NO 2 under stringent conditions;

(f) the amino acid sequence of a sequence variant of the amino acid sequence encoded by the cDNA clone contained in ATCC Deposit No. , wherein the sequence variant is encoded by a nucleic acid molecule hybridizing under stringent conditions to the cDNA contained in ATCC Deposit No. .

24. A method of treating a patient afflicted with a disorder associated with aberrant activity or expression of a protein, the method comprising administering to the patient, in an amount effective to modulate the activity of the protein in the patient, a compound selected from the group consisting of the protein, a nucleic acid encoding the protein, and an antisense nucleic acid which is capable of annealing with either of an mRNA encoding the protein and a portion of a genomic DNA encoding the protein, whereby at least one symptom of the disorder is alleviated, wherein said protein has an amino acid sequence selected from the group consisting of:

(a) the amino acid sequence shown in SEQ ID NO 1 ;

(b) the amino acid sequence encoded by the cDNA contained in ATCC Deposit No. ;

(c) the amino acid sequence of an allelic variant of the amino acid sequence shown in SEQ ID NO 1 ;

74 (d) the amino acid sequence of an allelic variant of the amino acid sequence encoded by the cDNA contained in ATCC Deposit No.

(e) the amino acid sequence of a sequence variant of the amino acid sequence shown in SEQ ID NO 1 , wherein the sequence variant is encoded by a nucleic acid molecule hybridizing to the nucleic acid molecule shown in

SEQ ID NO 2 under stringent conditions; (i) the amino acid sequence of a sequence variant of the amino acid sequence encoded by the cDNA clone contained in ATCC Deposit No. , wherein the sequence variant is encoded by a nucleic acid molecule hybridizing under stringent conditions to the cDNA contained in ATCC Deposit No. .

25. A method of diagnosing a disorder associated with aberrant activity or expression of a protein in a patient, the method comprising assessing the level of expression of a gene encoding said protein in the patient and comparing the level of expression of said gene with the normal level of expression of said gene in a human not afflicted with the disorder, whereby a difference between the level of expression of said gene in the patient and the normal level of expression is an indication that the patient is afflicted with the disorder, wherein said protein has an amino acid sequence selected from the group consisting of:

(a) the amino acid sequence shown in SEQ ID NO 1 ;

(b) the amino acid sequence encoded by the cDNA contained in ATCC Deposit No. ;

(c) the amino acid sequence of an allelic variant of the amino acid sequence shown in SEQ ID NO 1 ;

(d) the amino acid sequence of an allelic variant of the amino acid sequence encoded by the cDNA contained in ATCC Deposit No. ;

(e) the amino acid sequence of a sequence variant of the amino acid sequence shown in SEQ ID NO 1 , wherein the sequence variant is encoded by a nucleic acid molecule hybridizing to the nucleic acid molecule shown in

SEQ ID NO 2 under stringent conditions;

75 (i) the amino acid sequence of a sequence variant of the amino acid sequence encoded by the cDNA clone contained in ATCC Deposit No. , wherein the sequence variant is encoded by a nucleic acid molecule hybridizing under stringent conditions to the cDNA contained in ATCC Deposit No. .

26. A method of treating a patient afflicted with a disorder related to a protein, the method comprising administering to the patient a compound which modulates the activity of said protein in an amount effective to modulate the activity of the protein in the patient, whereby at least one symptom of the disorder is alleviated, wherein said protein has an amino acid sequence selected from the group consisting of:

(a) the amino acid sequence shown in SEQ ID NO 1 ;

(b) the amino acid sequence encoded by the cDNA contained in ATCC Deposit No. ;

(c) the amino acid sequence of an allelic variant of the amino acid sequence shown in SEQ ID NO 1 ;

(d) the amino acid sequence of an allelic variant of the amino acid sequence encoded by the cDNA contained in ATCC Deposit No. ; (e) the amino acid sequence of a sequence variant of the amino acid sequence shown in SEQ ID NO 1 , wherein the sequence variant is encoded by a nucleic acid molecule hybridizing to the nucleic acid molecule shown in SEQ ID NO 2 under stringent conditions; (i) the amino acid sequence of a sequence variant of the amino acid sequence encoded by the cDNA clone contained in ATCC Deposit No. , wherein the sequence variant is encoded by a nucleic acid molecule hybridizing under stringent conditions to the cDNA contained in ATCC Deposit No. .

27. A method of treating a patient afflicted with a disorder related to a protein, the method comprising administering to the patient, in an amount effective to

76 modulate the activity of the protein in the patient, a compound selected from the group consisting of the protein, a nucleic acid encoding the protein, and an antisense nucleic acid which is capable of annealing with either of an mRNA encoding the protein and a portion of a genomic DNA encoding the protein, whereby at least one symptom of the disorder is alleviated, wherein said protein has an amino acid sequence selected from the group consisting of:

(a) the amino acid sequence shown in SEQ ID NO 1 ;

(b) the amino acid sequence encoded by the cDNA contained in ATCC Deposit No. ; (c) the amino acid sequence of an allelic variant of the amino acid sequence shown in SEQ ID NO 1 ;

(d) the amino acid sequence of an allelic variant of the amino acid sequence encoded by the cDNA contained in ATCC Deposit No. ;

(e) the amino acid sequence of a sequence variant of the amino acid sequence shown in SEQ ID NO 1 , wherein the sequence variant is encoded by a nucleic acid molecule hybridizing to the nucleic acid molecule shown in SEQ ID NO 2 under stringent conditions;

(f) the amino acid sequence of a sequence variant of the amino acid sequence encoded by the cDNA clone contained in ATCC Deposit No. , wherein the sequence variant is encoded by a nucleic acid molecule hybridizing under stringent conditions to the cDNA contained in ATCC Deposit No. .

28. A method of diagnosing a disorder related to a protein in a patient, the method comprising assessing the level of expression of a gene encoding said protein in the patient and comparing the level of expression of said gene with the normal level of expression of said gene in a human not afflicted with the disorder, whereby a difference between the level of expression of said gene in the patient and the normal level of expression is an indication that the patient is afflicted with the disorder, wherein said protein has an amino acid sequence selected from the group consisting of:

77 (a) the amino acid sequence shown in SEQ ID NO 1 ;

(b) the amino acid sequence encoded by the cDNA contained in ATCC Deposit No. ;

(c) the amino acid sequence of an allelic variant of the amino acid sequence shown in SEQ ID NO 1 ;

(d) the amino acid sequence of an allelic variant of the amino acid sequence encoded by the cDNA contained in ATCC Deposit No. ;

(e) the amino acid sequence of a sequence variant of the amino acid sequence shown in SEQ ID NO 1 , wherein the sequence variant is encoded by a nucleic acid molecule hybridizing to the nucleic acid molecule shown in

SEQ ID NO 2 under stringent conditions;

(f) the amino acid sequence of a sequence variant of the amino acid sequence encoded by the cDNA clone contained in ATCC Deposit No. , wherein the sequence variant is encoded by a nucleic acid molecule hybridizing under stringent conditions to the cDNA contained in ATCC Deposit No. .

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