EP1465650A2

EP1465650A2 - Sk-3 potassium channels and the treatment of sexual dysfunctions and/or vascular disorders

Info

Publication number: EP1465650A2
Application number: EP02788193A
Authority: EP
Inventors: Mark Chen; Derek Trezise
Original assignee: Glaxo Group Ltd
Current assignee: Glaxo Group Ltd
Priority date: 2001-12-21
Filing date: 2002-12-19
Publication date: 2004-10-13
Also published as: WO2003055505A2; GB0130704D0; AU2002352477A1; AU2002352477A8; JP2005524615A; WO2003055505A3

Abstract

1. The use of a compound selected from: (a) an SK-3 calcium activated potassium channel polypeptide; (b) a compound which acitvates an SK-3 calcium activated potassium channel polypeptide; (c) a compound which inhibits an SK-3 calcium activated potassium channel polypeptide; or (d) a polynucleotide encoding an SK-3 calcium activated potassium channel polypeptide; for the manufacture of a medicament for treating: (i) dysfunctions of male or female sexual organs; or (ii) vascular disorders such as angina, stroke and hypertension. 2. The use according to claim 1 wherein the medicament is used in the treatment of sexual dysfunction and vascular disorders. 3. The use according to claim 1 wherein the medicament comprises a compound which specifically activates or inhibits an SK-3 calcium activated potassium channel polypeptide. 4. A method of screening for compounds that act as agonists or antagonists of SK-3 calcium activated potassium channel polypeptides for use in the preparation of a medicament for treating dysfunctions of male or female sexual organs or vascular disorders such as angina, stroke and hypertension.

Description

New Use

Field of the Invention

This invention relates to new uses for SK-3 calcium activated potassium channel polynucleotides and polypeptides encoded by them, to their use in the therapy of male and female sexual dysfunction and vascular disorders and in identifying compounds which are useful in therapy.

Background of the Invention In a range of cell types, increases in intracellular Ca²⁺ are linked to membrane hyperpolarisation by calcium-activated K⁺ channels. On the basis of unitary conductance and pharmacological properties, and more recently on identification of the relevant genes, three types of calcium-activated potassium (KCa) channels are known. These are BK (big conductance), IK (intermediate conductance) and SK (small conductance). The pore- forming β subunit of BK is encoded by a single gene, hSlo. The hSlo protein shares little sequence similarity to SK and IK. Its activity in different tissues is regulated by at least four β subunits (Meera et al., 2000; Brenner et al. 2000). The IK channel belongs to the same gene family as SK, sharing about 40% sequence identity. The SK channels consist of three members, SK-1 , SK-2 and SK-3, sharing 60-70% sequence identity among them. Each of these channels has 6 transmembrane domains, a pore-forming region, and intracellular N- and C-termini (Kohler et al. 1996). Calmodulin binds constitutively to the proximal section of the C-terminus and acts as the Ca²⁺-sensor (Xia et al., 1998). The SK, but not IK and BK, channels can be blocked by the bee venom apamin. Indeed, this is a defining feature of SK channels since apamin is highly specific and selective and has been shown, where tested, to be inactive against all other channel types. The cloning of the genes that encode SK-1 , SK-2 and SK-3 channels has been described in Terstappen et. al. Neuropharmacology 40:772-783(2001).

Apamin-sensitive K⁺ channels are present in many types of smooth muscle including corpus cavernosum (Prieto et al. 1998; Ayajiki et al. 2001). Opening of SK channels causes membrane hyperpolarisation, leading to smooth muscle relaxation by reducing calcium entry through voltage-gated calcium channels. The SK-3 subtype is expressed in colonic smooth muscle, notably in the interstitial cells of Cajal (Fujita et al. 2001; Ro et al. 2001) but has not been previously observed in vascular smooth muscle. Apamin and scyllatoxin, another SK channel antagonist, inhibit acetycholine induced smooth muscle relaxation in canine corpus cavernosum smooth muscle through a nitric oxide independent mechanism. This suggests an important role for SK channels in this tissue. Apamin also reduces acetycholine induced relaxation of horse corpus covernosum small arteries. The non-selective SK IK channel opener EBIO induces vascular smooth muscle hyperpolarisation and relaxation. Despite these reports, it is still unknown which SK channel subtype is present in male and female genital tissues and vascular smooth muscle.

Summary of the Invention In one aspect, the invention relates to new uses of SK-3 calcium activated potassium channel polynucleotides and polypeptides as disclosed in Terstappen et. al. Neuropharmacology 40:772-783(2001). Such uses include the treatment of erectile dysfunction and vascular disorders, hereinafter referred to as "the Diseases", amongst others. In a further aspect, the invention relates to methods for identifying modulating compounds (agonists and antagonists) using the materials provided by the invention, and treating conditions associated with SK-3 calcium activated potassium channels with the identified compounds.

Brief Description of the Figures Figurel :

Multitissue distribution of human SK-3 by real time PCR.

A panel of RNA was analysed by real time PCR for hSK-3. *RNA from Clontech. # RNA prepared in GlaxoSmithKline from one patient. $ RNA prepared in GlaxoSmithKline from two patients. Relative abundance was calculated as 2^40"n, n = number of PCR cycles.

Figure 2:

Distribution of hSK-3 in human genital tissues by real time PCR.

RNA samples were prepared from human male corpus cavernosum and female clitoris. Real time PCR analysis was carried out for SK-1 , 2, 3 and IK-1. Relative abundance was calculated as 2^40"n, n = number of PCR cycles.

Figure 3:

A. Comparison of multitissue distribution of human SK-3.

Results of real time PCR experiments for hSK-3 from Figure 1 and Figure 2 are combined, in order to illustrate the difference in hSK-3 expression between genital tissues and other tissues.

B. Distribution of mRNA for SK/IK ion channels in human penile corpus cavernosum by real time PCR. RNA samples were prepared from ten human male corpus cavernosum samples. Real time PCR experiment was performed for SK-1 , 2, 3 and IK-1. Calculated copies of channel type mRNA per 50 ng total RNA is shown. SK-4 = IK-1.

Figure 4: Validation of anti-hSK-3 antibody M75 by immunoblotting. Labels on the left indicate position of molecular weight markers. The ladder of bands in the lane of HEK-hSK3 shows specificity of this antibody.

Figure 5: Immunohistochemistry of rabbit IgG on human colon.

Sections from human colon were stained with control rabbit IgG, at relative concentrations to the rabbit anti-SK-3 antibody M75. The nucleus was counter-stained by Mayers haematoxylin. Antibody stain was absent in nearly all cell types of all colon sections, showing lack of non-specific IgG immunoreactivity. Representative result from one colon is shown.

Figure 6:

A. Immunohistochemistry of hSK-3 on human colon.

Sections from human colon were stained with antibody M75. Nucleus was stained by Mayers haematoxylin. Arrows show M75 immunoreactivity. All staining (top panels) was blocked by the corresponding peptide (bottom panels). Representative result from one colon is shown.

B. Immunohistochemistry of hSK-3 on human male corpus cavernosum.

Sections from human male corpus cavernosum were stained with antibody M75. Nuclear staining was with Mayers haematoxylin. Arrows show M75 immunoreactivity.

Figure 7:

Identification of SK-3 channel inhibitors and SK-3 channel activators using fluorescence- membrane potential screening methods. Concentration-response curves for illustrative compounds that either inhibit or activate hSK-3 channels stably expressed in CHO cells. Membrane potential changes were determined by measuring changes in fluorescene intensity of DiBAC, a voltage-sensitive dye.

Brief Description of the sequences

SEQ ID NO:1 is the SK-3 calcium activated potassium channel polynucleotide coding sequence (EMBL accession number AJ251016 (Terstappen et al., 2001)).

SEQ ID NO:2 is the SK-3 calcium activated potassium channel amino acid sequence.

Description of the Invention

In a first aspect, the present invention relates to the use of a compound selected from: (a) an SK-3 calcium activated potassium channel polypeptide; (b) a compound which activates an SK-3 calcium activated potassium channel polypeptide; (c) a compound which inhibits an SK-3 calcium activated potassium channel polypeptide; or

(d) a polynucleotide encoding an SK-3 calcium activated potassium channel polypeptide; for the manufacture of a medicament for treating:

(i) dysfunctions of male or female sexual organs; or

(ii) vascular disorders such as angina, stroke and hypertension.

Such SK-3 calcium activated potassium channel polypeptides include isolated polypeptides comprising an amino acid sequence which has at least 95% identity, preferably at least 97-99% identity, to that of SEQ ID NO:2 over the entire length of SEQ ID NO:2. Such polypeptides include those comprising the amino acid of SEQ ID NO:2.

Further polypeptides include isolated polypeptides in which the amino acid sequence has at least 95% identity, preferably at least 97-99% identity, to the amino acid sequence of SEQ ID NO:2 over the entire length of SEQ ID NO:2. Such polypeptides include the polypeptide of SEQ ID NO:2. In addition polypeptides encoded by a polynucleotide comprising the sequence contained in SEQ ID NO:1 are also included.

The polypeptides for use in the present invention may be in the form of the "mature" protein or may be a part of a larger protein such as a precursor or a fusion protein. It is often advantageous to include an additional amino acid sequence which contains secretory or leader sequences, pro-sequences, sequences which aid in purification such as multiple histidine residues, or an additional sequence for stability during recombinant production.

Polypeptides for use in the present invention can be prepared in any suitable manner. Such polypeptides include isolated naturally occurring polypeptides, recombinantly produced polypeptides, synthetically produced polypeptides, or polypeptides produced by a combination of these methods. Means for preparing such polypeptides are well understood in the art.

In a further aspect, the present invention relates to the use of SK-3 calcium activated potassium channel polynucleotides. Such polynucleotides include isolated polynucleotides comprising a nucleotide sequence encoding a polypeptide which has at least 95% identity to the amino acid sequence of SEQ ID NO:2, over the entire length of SEQ ID NO:2. In this regard, polypeptides which have at least 97% identity are highly preferred, whilst those with at least 98-99% identity are more highly preferred, and those with at least 99% identity are most highly preferred. Such polynucleotides include a polynucleotide comprising the nucleotide sequence contained in SEQ ID NO:1 encoding the polypeptide of SEQ ID NO:2. Further polynucleotides for use in the present invention include isolated polynucleotides comprising a nucleotide sequence that has at least 95% identity to a nucleotide sequence encoding a polypeptide of SEQ ID NO:2, over the entire coding region. In this regard, polynucleotides which have at least 97% identity are highly preferred, whilst those with at least 98-99% identity are more highly preferred, and those with at least 99% identity are most highly preferred.

Further polynucleotides for use in the present invention include isolated polynucleotides comprising a nucleotide sequence which has at least 95% identity to SEQ ID NO:1 over the entire length of SEQ ID NO:1. In this regard, polynucleotides which have at least 97% identity are highly preferred, whilst those with at least 98-99% identiy are more highly preferred, and those with at least 99% identity are most highly preferred. Such polynucleotides include a polynucleotide comprising the polynucleotide of SEQ ID NO:1 as well as the polynucleotide of SEQ ID NO:1.

The nucleotide sequence of SEQ ID NO:1 is a cDNA sequence encoding human SK-3 calcium activated potassium channel (Chandy et. al., Mol. Psychiatry 3:32-37(1998); Terstappen et. al. Neuropharmacology 40:772-783(2001)). The nucleotide sequence of SEQ ID NO:1 comprises a polypeptide encoding sequence (nucleotide 334 to 2544) encoding a polypeptide of 736 amino acids, the SK-3 calcium activated potassium channel polypeptide of SEQ ID NO:2. The nucleotide sequence encoding the polypeptide of SEQ ID NO:2 may be identical to the polypeptide encoding sequence contained in SEQ ID NO:1 or it may be a sequence other than the one contained in SEQ ID NO:1 , which, as a result of the redundancy (degeneracy) of the genetic code, also encodes the polypeptide of SEQ ID NO:2. The polypeptide of the SEQ ID NO:2 is the human SK-3 calcium activated potassium channel protein (Chandy et. al., Mol. Psychiatry 3:32- 37(1998); Terstappen et. al. Neuropharmacology 40:772-783(2001)).

Preferred polypeptides and polynucleotides for use in the present invention are expected to have, inter alia, similar biological functions/properties to their homologous polypeptides and polynucleotides. Furthermore, preferred polypeptides and polynucleotides of the present invention have at least one SK-3 calcium activated potassium channel activity.

Polynucleotides for use in the present invention may be obtained, using standard cloning and screening techniques, from a cDNA library derived from mRNA in cells or tissues that is of mammalian origin (Sambrook et al., Molecular Cloning: A Laboratory Manual, 2nd Ed., Cold Spring Harbor Laboratory Press, Cold Spring Harbor, N.Y. (1989)). Polynucleotides for use in the invention can also be obtained from natural sources such as genomic DNA libraries or can be synthesized using well known and commercially available techniques. The polynucleotides described hereinabove may be used for the recombinant production of SK-3 calcium activated potassium channel polypeptides for use in the present invention. The polynucleotide may include the coding sequence for the mature polypeptide, by itself; or the coding sequence for the mature polypeptide in reading frame with other coding sequences, such as those encoding a leader or secretory sequence, a pre-, or pro- or prepro- protein sequence, or other fusion peptide portions. For example, a marker sequence which facilitates purification of the fused polypeptide can be encoded. In certain preferred embodiments of this aspect of the invention, the marker sequence is a hexa-histidine peptide, as provided in the pQE vector (Qiagen, Inc.) and described in Gentz et al., Proc Natl Acad Sci USA (1989) 86:821-824, or is an HA tag. The polynucleotide may also contain non-coding 5' and 3' sequences, such as transcribed, non-translated sequences, splicing and polyadenylation signals, ribosome binding sites and sequences that stabilize mRNA.

Recombinant polypeptides for use in the present invention may be prepared by processes well known in the art from genetically engineered host cells comprising expression vectors. Cell-free translation systems can also be employed to produce such proteins using RNAs derived from the DNA constructs of the present invention.

For recombinant production, host cells can be genetically engineered to incorporate expression vectors or portions thereof of SK-3 calcium activated potassium channel polynucleotides. Introduction of polynucleotides into host cells can be effected by methods described in many standard laboratory manuals, such as Davis et al., Basic Methods in Molecular Biology (1986) and Sambrook et al., Molecular Cloning: A Laboratory Manual, 2nd Ed., Cold Spring Harbor Laboratory Press, Cold Spring Harbor, N.Y. (1989). Preferred such methods include, for instance, calcium phosphate transfection, DEAE-dextran mediated transfection, microinjection, electroporation, or infection.

Representative examples of appropriate hosts include bacterial cells, yeast cells; insect cells such as Drosophila S2 and Spodoptera Sf9 cells; or preferably animal cells such as CHO, COS, HeLa or HEK 293.

A great variety of expression vectors can be used, for instance, chromosomal, episomal and virus-derived systems, e.g., vectors derived from bacterial plasmids, from bacteriophage, from transposons, from yeast episomes, from insertion elements, from yeast chromosomal elements, from viruses such as baculoviruses, papova viruses, such as SV40, vaccinia viruses, adenoviruses, fowl pox viruses, pseudorabies viruses and retroviruses, and vectors derived from combinations thereof, such as those derived from plasmid and bacteriophage genetic elements, such as cosmids and phagemids. The expression systems may contain control regions that regulate as well as engender expression. Generally, any system or vector which is able to maintain, propagate or express a polynucleotide to produce a polypeptide in a host may be used. The appropriate nucleotide sequence may be inserted into an expression system by any of a variety of well-known and routine techniques, such as, for example, those set forth in Sambrook et al., Molecular Cloning, A Laboratory Manual (supra). Appropriate secretion signals may be incorporated into the desired polypeptide to allow secretion of the translated protein into the lumen of the endoplasmic reticulum, the periplasmic space or the extracellular environment. These signals may be endogenous to the polypeptide or they may be heterologous signals.

Polypeptides of the present invention can be recovered and purified from recombinant cell cultures by well-known methods including ammonium sulfate or ethanol precipitation, acid extraction, anion or cation exchange chromatography, phosphocellulose chromatography, hydrophobic interaction chromatography, affinity chromatography, hydroxylapatite chromatography and lectin chromatography. Most preferably, high performance liquid chromatography is employed for purification. Well known techniques for refolding proteins may be employed to regenerate active conformation when the polypeptide is denatured during isolation and or purification.

SK-3 calcium activated potassium channel polypeptides are believed to be involved directly or indirectly with disease states such as dysfunctions of male or female sexual organs or vascular disorders such as angina, stroke and hypertension. For example, in such disease states the SK-3 calcium activated potassium channel polypeptides may be underexpressed or inadequately stimulated or overexpressed. Thus it is desirous to devise screening methods to identify compounds which can activate or inhibit the SK-3 calcium activated potassium channel polypeptide. Accordingly, in a further aspect, the present invention provides for a method of screening compounds to identify those which modulate, that is activate (agonists) or inhibit (antagonists) the SK-3 calcium activated potassium channel polypeptide. An SK-3 calcium activated potassium channel modulator is either an SK-3 calcium activated potassium channel agonist or an SK-3 calcium activated potassium channel antagonist.

In general, modulators, which includes both agonists and antagonists may be employed for therapeutic and prophylactic purposes for such Diseases as hereinbefore mentioned.

An SK-3 channel agonist is a compound which activates SK-3 calcium activated potassium channels through either a direct or indirect mechanism. An SK-3 calcium activated potassium channel is activated when it opens to allow potassium ions to flow through the channel. The SK-3 calcium activated potassium channel agonist may activate IK, SK-1 and/or SK-2 channels in addition to SK-3 channels. Preferably the SK-3 calcium activated potassium channel agonist selectively activates SK channels. A compound which selectively activates SK channels will activate SK channels but will not activate or will activate less strongly IK channels. More preferably the SK-3 calcium activated potassium channel agonist selectively activates SK-3 channels. A compound which selectively activates SK-3 channels will activate SK-3 channels but will not activate or will activate only to a lesser extent SK-1 and SK-2 channels. Preferably the SK-3 calcium activated potassium channel agonist will be activity dependent. Either it will enhance the affinity of calcium ions for the SK-3 calcium activated potassium channel or stabilise the channel in the open state (prevent channel closure). A SK-3 calcium activated potassium channel agonist causes reduction in membrane potential, and reduces calcium entry in smooth muscle cells. A calcium activated potassium SK-3 agonist also causes relaxation of the vascular and corpus cavernosum smooth muscle, possibly through these mechanisms.

An SK-3 calcium activated potassium channel antagonist is a compound which inhibits SK-3 channels through either a direct or indirect mechanism. An SK-3 calcium activated potassium channel is inhibited when potassium ions are prevented from flowing through the channel. The SK-3 calcium activated potassium channel antagonist may inhibit IK, SK-1 and/or SK-2 channels in addition to SK-3 channels. Preferably the SK-3 calcium activated potassium channel antagonist selectively inhibits SK channels. A compound which selectively inhibits SK channels will inhibit SK channels but will not inhibit or will inhibit less strongly IK channels. More preferably the SK-3 calcium activated potassium channel antagonist selectively inhibits SK-3 calcium activated potassium channels. A compound which selectively inhibits SK-3 calcium activated potassium channels will inhibit SK-3 calcium activated potassium channels but will not inhibit or will activate only to a lesser extent SK-1 and SK-2 channels. An SK-3 calcium activated potassium channel antagonist will indirectly enhance calcium entry in smooth muscle cells and cause contraction of the vascular and corpus cavernosum smooth muscle.

Modulating compounds may be identified from a variety of sources, for example, cells, cell- free preparations, chemical libraries, and natural product mixtures. Such modulators so- identified may be natural or modified substrates, ligands, receptors, enzymes, etc., as the case may be, of the polypeptide; or may be structural or functional mimetics thereof (see Coligan et al., Current Protocols in Immunology 1(2):Chapter 5 (1991)).

The screening method may simply measure the binding of a candidate compound to the polypeptide, or to cells or membranes bearing the polypeptide, or a fusion protein thereof by means of a label directly or indirectly associated with the candidate compound. Alternatively, the screening method may involve competition with a labeled competitor. The screening method may involve the use of membrane potential sensitive dyes such as bis-oxonol DiBAC or DiSBACn in conjunction with coumarin -tagged phospholipids. With these methods changes in membrane potential resulting from SK-3 Ca2+-activated potassium channels are recorded as changes in fluorescence or fluorescence-resonance energy transfer (FRET). A further alternative screening method may involve patch clamp electrophysiology to detect ionic currents resulting from channel opening. Another method that may be employed is the use of either radioactive or non-radioactive Rb+ measurements to monitor channel opening. Further, these screening methods may test whether the candidate compound results in a signal generated by activation or inhibition of the polypeptide, using detection systems appropriate to the cells bearing the polypeptide. Further, the screening methods may simply comprise the steps of mixing a candidate compound with a solution containing a polypeptide of the present invention, to form a mixture, measuring SK-3 calcium activated potassium channel activity in the mixture, and comparing the SK-3 calcium activated potassium channel activity of the mixture to a standard. Fusion proteins, such as those made from Fc portion and SK-3 calcium activated potassium channel polypeptide, as hereinbefore described, can also be used for high-throughput screening assays to identify antagonists for the polypeptide of the present invention (see D. Bennett et al., J Mol Recognition, 8:52-58 (1995); and K. Johanson et al., J Biol Chem, 270(16):9459-9471 (1995)).

The SK-3 calcium activated potassium channel polynucleotides, polypeptides and antibodies to the SK-3 calcium activated potassium channel polypeptide may also be used to configure screening methods for detecting the effect of added compounds on the production of mRNA and polypeptide in cells. For example, an ELISA assay may be constructed for measuring secreted or cell associated levels of polypeptide using monoclonal and polyclonal antibodies by standard methods known in the art. This can be used to discover agents which may enhance the production of SK-3 calcium activated potassium channel polypeptide from suitably manipulated cells or tissues.

Thus, in another aspect, the present invention relates to a screening kit for identifying agonists or antagonists for SK-3 calcium activated potassium channel polypeptides of the present invention; or compounds which enhance or inhibit the production of such polypeptides, which comprises: (a) a SK-3 calcium activated potassium channel polypeptide; (b) a recombinant cell expressing a SK-3 calcium activated potassium channel polypeptide;

(c) a cell membrane expressing a SK-3 calcium activated potassium channel polypeptide; or

(d) antibody to a SK-3 calcium activated potassium channel polypeptide; which SK-3 calcium activated potassium channel polypeptide is preferably that of SEQ ID NO:2.

It will be appreciated that in any such kit, (a), (b), (c) or (d) may comprise a substantial component. It will be readily appreciated by the skilled artisan that a polypeptide of the present invention may also be used in a method for the structure-based design of an agonist or antagonist, by:

(a) determining in the first instance the three-dimensional structure of the polypeptide; (b) deducing the three-dimensional structure for the likely reactive or binding site(s) of an agonist or antagonist;

(c) synthesing candidate compounds that are predicted to bind to or react with the deduced binding or reactive site; and

(d) testing whether the candidate compounds are indeed agonists or antagonists. It will be further appreciated that this will normally be an iterative process.

In a further aspect, the present invention provides methods of treating abnormal conditions such as, for instance, dysfunctions of male or female sexual organs or vascular disorders such as angina, stroke and hypertension , related to SK-3 calcium activated potassium channel polypeptide activity. For treating such abnormal conditions several approaches are also available. One approach comprises administering to a subject a therapeutically effective amount of a compound which activates or inhibits a polypeptide of the present invention, i.e., an agonist or antagonist as described above, in combination with a pharmaceutically acceptable carrier, to thereby alleviate the abnormal condition. Alternatively, gene therapy may be employed to effect the endogenous production of SK-3 calcium activated potassium channels or other calcium activated potassium channels by the relevant cells in the subject. For example, a polynucleotide of the invention may be engineered for expression in a replication defective retroviral vector, as discussed above. The retroviral expression construct may then be isolated and introduced into a packaging cell transduced with a retroviral plasmid vector containing RNA encoding a polypeptide of the present invention such that the packaging cell now produces infectious viral particles containing the gene of interest. These producer cells may be administered to a subject for engineering cells in vivo and expression of the polypeptide in vivo. For an overview of gene therapy, see Chapter 20, Gene Therapy and other Molecular Genetic-based Therapeutic Approaches, (and references cited therein) in Human Molecular Genetics, T Strachan and A P Read, BIOS Scientific Publishers Ltd (1996). Another approach is to administer a therapeutic amount of a polypeptide of the present invention in combination with a suitable pharmaceutical carrier.

In a further aspect, the present invention provides for pharmaceutical compositions comprising a therapeutically effective amount of an agonist or antagonist peptide or small molecule compound, in combination with a pharmaceutically acceptable carrier or excipient. Such carriers include, but are not limited to, saline, buffered saline, dextrose, water, glycerol, ethanol, and combinations thereof. The invention further relates to pharmaceutical packs and kits comprising one or more containers filled with one or more of the ingredients of the aforementioned compositions of the invention. Polypeptides and other compounds of the present invention may be employed alone or in conjunction with other compounds, such as therapeutic compounds.

The composition will be adapted to the route of administration, for instance by a systemic or an oral route. Preferred forms of systemic administration include injection, typically by intravenous injection. Other injection routes, such as subcutaneous, intramuscular, or intraperitoneal, can be used. Alternative means for systemic administration include transmucosal and transdermal administration using penetrants such as bile salts or fusidic acids or other detergents. In addition, if a polypeptide or other compounds of the present invention can be formulated in an enteric or an encapsulated formulation, oral administration may also be possible. Administration of these compounds may also be topical and/or localized, in the form of salves, pastes, gels, and the like.

The dosage range required depends on the choice of peptide or other compounds of the present invention, the route of administration, the nature of the formulation, the nature of the subject's condition, and the judgment of the attending practitioner. Suitable dosages, however, are in the range of 0.1-100 μg/kg of subject. Wide variations in the needed dosage, however, are to be expected in view of the variety of compounds available and the differing efficiencies of various routes of administration. For example, oral administration would be expected to require higher dosages than administration by intravenous injection. Variations in these dosage levels can be adjusted using standard empirical routines for optimization, as is well understood in the art.

Polypeptides used in treatment can also be generated endogenously in the subject, in treatment modalities often referred to as "gene therapy" as described above. Thus, for example, cells from a subject may be engineered with a polynucleotide, such as a DNA or RNA, to encode a polypeptide ex vivo, and for example, by the use of a retroviral plasmid vector. The cells are then introduced into the subject.

The following definitions are provided to facilitate understanding of certain terms used frequently hereinbefore.

"Antibodies" as used herein includes polyclonal and monoclonal antibodies, chimeric, single chain, and humanized antibodies, as well as Fab fragments, including the products of an Fab or other immunoglobulin expression library.

"Isolated" means altered "by the hand of man" from the natural state. If an "isolated" composition or substance occurs in nature, it has been changed or removed from its original environment, or both. For example, a polynucleotide or a polypeptide naturally present in a living animal is not "isolated," but the same polynucleotide or polypeptide separated from the coexisting materials of its natural state is "isolated", as the term is employed herein.

"Polynucleotide" generally refers to any polyribonucleotide or polydeoxribonucleotide, which may be unmodified RNA or DNA or modified RNA or DNA. "Polynucleotides" include, without limitation, single- and double-stranded DNA, DNA that is a mixture of single- and double-stranded regions, single- and double-stranded RNA, and RNA that is mixture of single- and double-stranded regions, hybrid molecules comprising DNA and RNA that may be single-stranded or, more typically, double-stranded or a mixture of single- and double-stranded regions. In addition, "polynucleotide" refers to triple-stranded regions comprising RNA or DNA or both RNA and DNA. The term "polynucleotide" also includes DNAs or RNAs containing one or more modified bases and DNAs or RNAs with backbones modified for stability or for other reasons. "Modified" bases include, for example, tritylated bases and unusual bases such as inosine. A variety of modifications may be made to DNA and RNA; thus, "polynucleotide" embraces chemically, enzymatically or metabolically modified forms of polynucleotides as typically found in nature, as well as the chemical forms of DNA and RNA characteristic of viruses and cells. "Polynucleotide" also embraces relatively short polynucleotides, often referred to as oligonucleotides.

"Polypeptide" refers to any peptide or protein comprising two or more amino acids joined to each other by peptide bonds or modified peptide bonds, i.e., peptide isosteres. "Polypeptide" refers to both short chains, commonly referred to as peptides, oligopeptides or oligomers, and to longer chains, generally referred to as proteins. Polypeptides may contain amino acids other than the 20 gene-encoded amino acids. "Polypeptides" include amino acid sequences modified either by natural processes, such as post-translational processing, or by chemical modification techniques which are well known in the art. Such modifications are well described in basic texts and in more detailed monographs, as well as in a voluminous research literature. Modifications may occur anywhere in a polypeptide, including the peptide backbone, the amino acid side-chains and the amino or carboxyl termini. It will be appreciated that the same type of modification may be present to the same or varying degrees at several sites in a given polypeptide. Also, a given polypeptide may contain many types of modifications. Polypeptides may be branched as a result of ubiquitination, and they may be cyclic, with or without branching. Cyclic, branched and branched cyclic polypeptides may result from post-translation natural processes or may be made by synthetic methods. Modifications include acetylation, acylation, ADP-ribosylation, amidation, biotinylation, covalent attachment of flavin, covalent attachment of a heme moiety, covalent attachment of a nucleotide or nucleotide derivative, covalent attachment of a lipid or lipid derivative, covalent attachment of phosphotidylinositol, cross-linking, cyclization, disulfide bond formation, demethylation, formation of covalent cross-links, formation of cystine, formation of pyroglutamate, formylation, gamma-carboxylation, glycosylation, GPI anchor formation, hydroxylation, iodination, methylation, myristoylation, oxidation, proteolytic processing, phosphorylation, prenylation, racemization, selenoylation, sulfation, transfer-RNA mediated addition of amino acids to proteins such as arginylation, and ubiquitination (see, for instance, Proteins - Structure and Molecular Properties, 2nd Ed., T. E. Creighton, W. H. Freeman and Company, New York, 1993; Wold, F., Post-translational Protein Modifications: Perspectives and Prospects, pgs. 1-12 in Post-translational Covalent Modification of Proteins, B. C. Johnson, Ed., Academic Press, New York, 1983; Seifter et al., "Analysis for protein modifications and nonprotein cofactors", Meth Enzymol (1990) 182:626-646 and Rattan et al., "Protein Synthesis: Post-translational Modifications and Aging", Ann NY Acad Sci (1992) 663:48-62).

"Identity," as known in the art, is a relationship between two or more polypeptide sequences or two or more polynucleotide sequences, as determined by comparing the sequences. In the art, "identity" also means the degree of sequence relatedness between polypeptide or polynucleotide sequences, as the case may be, as determined by the match between strings of such sequences. "Identity" and "similarity" can be readily calculated by known methods, including but not limited to those described in {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 I, 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 ; and Carillo, H., and Lipman, D., SIAM J. Applied Math., 48: 1073 (1988). Preferred methods to determine identity are designed to give the largest match between the sequences tested. Methods to determine identity and similarity are codified in publicly available computer programs. Preferred computer program methods to determine identity and similarity between two sequences include, but are not limited to, the GCG program package (Devereux, J., et al., Nucleic Acids Research 12(1): 387 (1984)), BLASTP, BLASTN, and FASTA (Atschul, S.F. et al., J. Molec. Biol. 215: 403-410 (1990). The BLAST X program is publicly available from NCBI and other sources {BLAST Manual, Altschul, S., et al., NCBI NLM NIH Bethesda, MD 20894; Altschul, S., et al., J. Mol. Biol. 215: 403-410 (1990). The well known Smith Waterman algorithm may also be used to determine identity.

Preferred parameters for polypeptide sequence comparison include the following:

1) Algorithm: Needleman and W.unsch, J. Mol Biol. 48: 443-453 (1970)

Comparison matrix: BLOSSUM62 from Hentikoff and Hentikoff, Proc. Natl. Acad. Sci.

USA. 89:10915-10919 (1992) Gap Penalty: 12

Gap Length Penalty: 4 A program useful with these parameters is publicly available as the "gap" program from Genetics Computer Group, Madison Wl. The aforementioned parameters are the default parameters for peptide comparisons (along with no penalty for end gaps).

Preferred parameters for polynucleotide comparison include the following: 1) Algorithm: Needleman and Wunsch, J. Mol Biol. 48: 443-453 (1970) Comparison matrix: matches = +10, mismatch = 0 Gap Penalty: 50 Gap Length Penalty: 3

Available as: The "gap" program from Genetics Computer Group, Madison Wl. These are the default parameters for nucleic acid comparisons.

By way of example, a polynucleotide sequence of the present invention may be identical to the reference sequence of SEQ ID NO:1 , that is be 100% identical, or it may include up to a certain integer number of nucleotide alterations as compared to the reference sequence. Such alterations are selected from the group consisting of at least one nucleotide deletion, substitution, including transition and transversion, or insertion, and wherein said alterations may occur at the 5' or 3' terminal positions of the reference nucleotide sequence or anywhere between those terminal positions, interspersed either individually among the nucleotides in the reference sequence or in one or more contiguous groups within the reference sequence. The number of nucleotide alterations is determined by multiplying the total number of nucleotides in SEQ ID NO:1 by the numerical percent of the respective percent identity(divided by 100) and subtracting that product from said total number of nucleotides in SEQ ID NO:1 , or: n_n < x_n - (x_n • y), wherein n_n is the number of nucleotide alterations, x_n is the total number of nucleotides in SEQ ID NO:1 , and y is, for instance, 0.70 for 70%, 0.80 for 80%, 0.85 for 85%, 0.90 for 90%, 0.95 for 95%,etα, and wherein any non-integer product of x_n and y is rounded down to the nearest integer prior to subtracting it from x_n. Alterations of a polynucleotide sequence encoding the polypeptide of SEQ ID NO:2 may create nonsense, missense or frameshift mutations in this coding sequence and thereby alter the polypeptide encoded by the polynucleotide following such alterations.

Similarly, a polypeptide sequence of the present invention may be identical to the reference sequence of SEQ ID NO:2, that is be 100% identical, or it may include up to a certain integer number of amino acid alterations as compared to the reference sequence such that the % identity is less than 100%. Such alterations are selected from the group consisting of at least one amino acid deletion, substitution, including conservative and non-conservative substitution, or insertion, and wherein said alterations may occur at the amino- or carboxy-terminal positions of the reference polypeptide sequence or anywhere between those terminal positions, interspersed either individually among the amino acids in the reference sequence or in one or more contiguous groups within the reference sequence. The number of amino acid alterations for a given % identity is determined by multiplying the total number of amino acids in SEQ ID NO:2 by the numerical percent of the respective percent identity(divided by 100) and then subtracting that product from said total number of amino acids in SEQ ID NO:2, or: n_a<x_a - (x_a • y), wherein n_a is the number of amino acid alterations, x_a is the total number of amino acids in SEQ ID NO:2, and y is, for instance 0.70 for 70%, 0.80 for 80%, 0.85 for 85% etc., and wherein any non-integer product of x_a and y is rounded down to the nearest integer prior to subtracting it from x_a.

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

Examples

Example 1 : Human multitissue distribution of SK-3

SK-3 mRNA is widely expressed in a range of neuronal and non-neuronal tissues (Figure 1 ). It is most abundant in regions of the brain but is also present at significant levels in many smooth muscle-rich tissues such as myometrium, omentum, rectum, small intestine and urinary bladder. Other tissues expressing SK-3 mRNA are skeletal muscle, thyroid, uterus, cervix, tonsil, thymus, lung, adenoid, kidney, esophagus, heart, ovary, testis, trachea, adrenal gland, spleen, salivary gland, parotid gland, mammary gland and stomach. Little or no SK-3 mRNA was present in pancreas, adipose, liver, fetal liver, placenta and prostate.

Example 2: Localisation of SK-3 in corpus cavanosum and clitoris

TaqMan analysis showed that SK-3 is the predominant channel type of the SK/IK family of calcium-activated potassium channels in the female clitoris and male corpus cavernosum (Figure 2). Expression of SK-3 in the clitoris (Figure 3A) is at levels similar to other types of smooth muscle tissues. However, in corpus cavernosum (B) SK-3 expression is apparently greater than 30 times higher than the brain and other types of smooth muscle tissues (Figure 3A). In a separate experiment (Figure 3B), SK-3 was found also to be very highly expressed in other human male penis tissue samples, and is by far the most abundant channel of the SK/IK family of ion channels in 9 out of 10 separate tissues.

Example 3: Specificity of the anti-hSK-3 antibody M75

Rabbit anti-hSK-3 antibody M75 was raised against a N-terminal hSK-1 peptide. The specificity of antibody M75 was analysed by immunoblotting using cell extracts from HEK293 stable cell lines expressing hSK-1 , hSK-2, hSK-3 and hlK-1 , as well as untransfected HEK293 cells. Antibody M75 recognised high molecular weight protein complexes from total cell extracts from HEK-hSK-3 stable cell line, but not from HEK- hSK-1, HEK-hSK-2, HEK-hlK-1 or untransfected HEK293 cells (Figure 4). The SK-3 immunoreactivity was blocked by the corresponding immunopeptide.

Example 4: Localisation of SK-3 protein in colonic and cavernosal smooth muscle and blood vessels

Immunohistochemistry was performed on 5 human colon biopsy samples (Table 1). 'Specific' staining was defined as that obtained at low antibody concentrations (1-8 μg ml^" ¹) that could be blocked by the presence of 5 times concentration of the corresponding peptide. Control experiments with rabbit IgG (Figure 5) used at the same concentration illustrated the absence of non-specific immunoreactivity on the sections, with the exception of a very low level of background staining in smooth muscle.

Table 1. Colons used in this study.

SK-3-staining (Figure 6A) was strong in both the vascular and visceral smooth muscle of 5 of 5 colons. In 3 of 5 colons a small subset of monouncleated cells, likely to be monocytes or macrophages, were strongly stained. The myenteric plexus and submucus plexus neurones neurons were weakly stained.

In a separate experiment with human male corpus cavernosum, SK-3-staining (Figure 6B) was also very strong in both cavernosal and vascular smooth smooth muscle as well as vascular endothelial cells.

Example 5 Identification of SK-3 channel inhibitors and activators using fluorescence-membrane potential based screening methods.

Compounds were assessed for hSK-3 channel activating and inhibiting effects using the membrane-potential sensitive dye DiBAC₄ in a cell based fluorescence assay. Chinese Hamster Ovary cells stably expressing hSK-3 channels were pre-plated at a density of 7.5K cells/ well on 384-well microtitre plates and left for 4 days. Cells were then loaded with DiBAC4 (5μM) for 30min and then transferred to a fluorimetric imaging plate reader (FLIPR384, Molecular devices). Test compounds were added online and changes in fluorescence were recorded temporally. Channel activators reduce the intensity of fluorescence, whilst inhibitors prevent responses to ionomycin (5μM), a Ca²⁺-ionophore. Figure 7 shows representative concentration-response curves for 2 compounds in activator and inhibitor screen formats (up triangle and circle symbols, respectively). The down triangle symbols indicate the lack of activity of the channel opener when the extracellular incubation solution contains a high concentration of K⁺ (145mM). Under these conditions there is no concentration gradient for K⁺ and thus no change in membrane potential should occur for a specific opener.

Methods Real time PCR (TaαMan assay)

Expression of each of the four genes (hSK-1 , hSK-2, hSK-3, hlK-1) was assessed in five different templates. hSK-1 , hSK-2, hSK-3, and hlK-1 plasmid DNAs were provided as templates. The total number of copies contained within 1 μg of each plasmid DNA was calculated using Copy Calculator (John Pfeifer, Applied Biosystems, Foster City, CA). A stock solution for each sample was made which contained 1e⁶ copies of plasmids per 5 μl DNA. For an additional measure of specificity, a mixture of all 4 plasmids was also created where each plasmid was represented by 2e⁵ copies. A total of 5 μl of DNA was used per PCR reaction. PCR results were generated using a 5' nuclease assay (Taq an) and the ABI Prizm 7700 Sequence Detection System (Applied Biosystems, Foster City, CA). In the 5' nuclease assay, two traditional PCR primers are used in conjunction with a fluorogenic probe. The probe is a nonextendable, oligonucleotide labelled with a FAM fluorescent dye at the 5' end and TAMRA quencher dye at the 3' end. The 5' nuclease activity of the Taq DNA polymerase results in the hydrolysis and release of the fluorescent FAM label. The level of fluorescence was monitored and recorded at each cycle of the PCR. The increase in fluorescence is proportional to the amount of starting template.

The sequences of the forward and reverse primers and the probes are as follows:

HSK-1 : forward primer ACGCTTACCGACCTAGCCAAG (SEQ ID NO: 3), reverse primer CTCCAGCTCCTCGTGCTGAG (SEQ ID NO:4 ), and hSK-1 probe FAM-

CAGACCGTCATGTACGACCTTGTATCGGA-TAMRA (SEQ ID NO: 5 ).

HSK-2: forward primer TTACCCTGGAAACAAAACTAGAGACTT (SEQ ID NO:6 ), reverse primer TGCCTGATGGTCTGGCTTATG (SEQ ID NO: 7 ), and hSK-2 probe FAM-

CATCCACGCCCTCCCTGGGC-TAMRA (SEQ ID NO: 8). HSK-3: Forward primer TCCAAGATGCAGAATGTCATGTATG(SEQ ID NO: 9 ), reverse primer GGTGAGATGCTCCAGCTTCG(SEQ ID NO: 10 ), and hSK-3 probe FAM-

TCCAGGTCTTCGCTCCGGTCATTG -TAMRA(SEQ ID NO: 11 ).

HIK-1 : forward primer CTCCAAGATGCACATGATCCTGTA(SEQ ID NO:12 ), reverse primer GCAGTGCTAAGCAGCTCAGTCA(SEQ ID NO:13 ), and hlK-1 probe FAM- CAGCAGAATCTGAGCAGCTCACACCG -TAMRA(SEQ ID NO: 14 ).

A master mix was utilized which included 900 nM each of the forward and reverse primers, 150 nM probe, and 1X PCR master mix (Applied Biosystems, Foster City, CA). The total volume per reaction was 25 μl with 20 μl of the master mix and 5 μl of the cDNA. All samples were assayed in duplicate. The cycling conditions for cDNA samples were 95°C for 10 min, and 40 cycles of 95°C for 15 seconds and 60°C for 1 minute.

Gene expression was analyzed in cDNAs obtained from a range of tissues including normal and diseased lung, normal and diseased brain, CHO stable cell lines, normal and multiple sclerosis cerebellum, normal and motoneuron diseased motor cortex, dorsal root and nodose ganglia, dorsal and ventral spinal cord and colon muscle and mucosa. All RNA samples were DNased in a total volume of 120 μl (102 μl RNA + H₂O sample, 12 μl 10X transcription buffer, and 6 μl DNase 1 at 2 U/μl). The samples were incubated at 37°C for 1 h and then at 75°C for 5 min. The samples were then quantitated by Ribogreen. GAPDH +/- RT was analyzed in all samples using 25 ng tissue per well. Samples were concentrated when necessary by speed vac centrifugation for approximately 15 min on low heat. The remaining volume was assessed and water was added to a total volume of 56.5 μl. The samples were then converted to cDNA using random hexamers and then normalized. The samples were assessed for normalization by analysis of 18S rRNA expression. Because 18S is high in abundance, all samples were diluted 1:100. A master mix was utilized which included 40 nM forward primer, 20 nM reverse primer, 50 nM probe, 5.5 mM MgCI₂, 1X Taqman Buffer A, 300 μM dNTPs, 10 U RNase inhibitor, 12.5 U MuLV reverse transcriptase, and 1.25 U Amplitaq Gold. The total volume per reaction was 25 μl with 20 μl of the master mix and 5 μl or each diluted sample. All samples were assayed in duplicate. The cycling conditions were 48°C for 30 min, 95°C for 10 min, and 40 cycles of 95°C for 15 s and 60°C for 1 min.

Real time PCR was then performed using the conditions and probe and primer sets described above.

Immunoblotting: Immunoblotting was using total cell extracts from the panel of hSK/IK stable cell lines, including HEK-hSK-1 , HEK-hSK-2, HEK-hSK-3, HEK-hlK-1 and HEK293 wild type cells. Cells were lysed in buffer containing 50mM Tris-HCI pH 7.6, 2mM EDTA, 150mM NaCI and 1% NP40. Samples were then boiled for 5 min in sample buffer containing 0.25M Tris-HCI, 10% glycerol, 5% SDS, 0.05% bromophenol blue, and 0.1 M DTT. Total cell protein extract (10 μg) was loaded in each well on 4-20% gradient Tris-glycine gels (Novex) for electrophoresis. Resolved proteins were then electroblotted onto nitrocellulose membranes. Remaining protein binding sites were blocked by PBS + 5% fat-free milk powder for more than 1 hour and incubated in 1 μg/ml immunoglobulins in blocking solution at 4°C overnight. Following a washing step: 6X15 min in PBS + 0.1 % Tween20 immunoreactive bands were localised using HRP conjugated goat anti-rabbit IgG (Dako) and Pierce SuperSignal reagents.

Immunohistochemistry

Human colon was removed at surgery and placed into 4% buffered neutral formalin for 24 hours. After fixation the tissue was vacuum processed through to paraffin wax blocks. Wax sections were cut at 3-5 μm and mounted onto Vectabond coated slides. For immunohistochemistry, sections were deparaffinised and brought to water. The sections were microwaved in antigen retrieval solution pH 6.0 (HD supplies Aylesbury, Buck.) for 20 minutes, followed by distilled water wash, and then placed into PBS. Endogenous peroxidase activity was quenched by incubating the sections in 0.3% hydrogen peroxide for 20 minutes. Sections were then rinsed with PBS 3x 5 minutes, and placed in PBS with 5% milk protein for 30 minutes to block any non-specific binding. Sections were incubated with the primary antiserum (rabbit) in PBS containing 0.3%Triton X-100, 3% normal goat serum for 1 hour at room temperature. A negative control sections was also used, replacing the primary antibody with rabbit IgG (Vector Inc.Burlingame. CA). Sections were rinsed in PBS 3x5 minutes, and then incubated in secondary antibody, biotinylated goat anti-rabbit 1 :20 dilution (BioGenex, San Ramon, CA) in PBS for 20 minutes at room temperature. Sections were rinsed in PBS 3x5 minutes and incubated in Strepavidin Peroxidase solution (BioGenex, San Ramon, CA), for 20 minutes, at room temperature. Sections were rinsed in PBS 3x5 minutes. Colour chromagen development of the sections was with a solution of 3,3'-diaminobenzidine (DAB) for 2-5 minutes. Sections were rinsed with distilled water and counterstained with Mayers haematoxylin (Pioneer Research Chemicals, Colchester, Essex) for 2 minutes and then rinsed in running tap water, for 5 minutes. Finally sections were dehydrated in ascending series of ethanol concentrations (70%, 90% 100%, 5 minutes in each), delipidated in xylene (5 minutes) and then cover slipped.

SEQUENCE INFORMATION

SEQ ID NO:l EMBL accession number AJ251016 (Terstappen et al, 2001)

GGGTCGACCCACGCGTCCGGAGCCAGCGAGGAGTGAAGCTGAGCCTGGCCTCACACGCTCCTAGAGGACCACCTCCTGAGA GAGTTCTTTCACCCCCTCTTCTTTCTCCAAGCTCCCCTCCTGCTCTCCCTCCCTGCCCAATACAATGCATTCTTGAGTGGC AGCGTCTGGACTCCAGGCAGCCCCAGAGAACCGAAGCAAGCCAAAGAGAGGACTGGAGCCAAGATACTGGTGGGGGAGATT GGATGCCTGGCTTTCTTTGAGGACATCTTTGGAGCGAGGGTGGCTTTGGGGTGGGGGCTTGTGCTGCAGGGAATACAGCCA GGCCCCAAGATGGACACTTCTGGGCACTTCCATGACTCGGGGGTGGGGGACTTGGATGAAGACCCCAAGTGCCCCTGTCCA TCCTCTGGGGATGAGCAGCAGCAGCAGCAGCAGCAGCAACAGCAGCAGCAGCCACCACCGCCAGCGCCACCAGCAGCCCCC CAGCAGCCCCTGGGACCCTCGCTGCAGCCTCAGCCTCCGCAGCTTCAGCAGCAGCAGCAGCAGCAGCAGCAGCAGCAGCAG CAGCAGCAGCAGCAGCAGCAGCCACCGCATCCCCTGTCTCAGCTCGCCCAACTCCAGAGCCAGCCCGTCCACCCTGGCCTG CTGCACTCCTCTCCCACCGCTTTCAGGGCCCCCCCTTCGTCCAACTCCACCGCCATCCTCCACCCTTCCTCCAGGCAAGGC AGCCAGCTCAATCTCAATGACCACTTGCTTGGCCACTCTCCAAGTTCCACAGCTACAAGTGGGCCTGGCGGAGGCAGCCGG CACCGACAGGCCAGCCCCCTGGTGCACCGGCGGGACAGCAACCCCTTCACGGAGATCGCCATGAGCTCCTGCAAGTATAGC GGTGGGGTCATGAAGCCCCTCAGCCGCCTCAGCGCCTCCCGGAGGAACCTCATCGAGGCCGAGACTGAGGGCCAACCCCTC CAGCTTTTCAGCCCTAGCAACCCCCCGGAGATCGTCATCTCCTCCCGGGAGGACAACCATGCCCACCAGACCCTGCTCCAT CACCCTAATGCCACCCACAACCACCAGCATGCCGGCACCACCGCCAGCAGCACCACCTTCCCCAAAGCCAACAAGCGGAAA AACCAAAACATTGGCTATAAGCTGGGACACAGGAGGGCCCTGTTTGAAAAGAGAAAGCGACTGAGTGACTATGCTCTGATT TTTGGGATGTTTGGAATTGTTGTTATGGTGATAGAGACCGAGCTCTCTTGGGGTTTGTACTCAAAGGACTCCATGTTTTCG TTGGCCCTGAAATGCCTTATCAGTCTGTCCACCATCATCCTTTTGGGCTTGATCATCGCCTACCACACACGTGAAGTCCAG CTCTTCGTGATCGACAACGGCGCGGATGACTGGCGGATAGCCATGACCTACGAGCGCATCCTGTACATCAGCCTGGAGATG CTGGTGTGCGCCATCCACCCCATTCCTGGCGAGTACAAGTTCTTCTGGACGGCACGCCTGGCCTTCTCCTACACACCCTCC CGGGCGGAGGCCGATGTGGACATCATCCTGTCTATCCCCATGTTCCTGCGCCTGTACCTGATCGCCCGAGTCATGCTGCTG CACAGCAAGCTCTTCACCGATGCCTCGTCCCGCAGCATCGGGGCCCTCAACAAGATCAACTTCAACACCCGCTTTGTCATG AAGACGCTCATGACCATCTGCCCTGGCACTGTGCTGCTCGTGTTCAGCATCTCTCTGTGGATCATTGCTGCCTGGACCGTC CGTGTCTGTGAAAGGTACCATGACCAGCAGGACGTAACTAGTAACTTTCTGGGTGCCATGTGGCTCATCTCCATCACATTC CTTTCCATTGGTTATGGGGACATGGTGCCCCACACATACTGTGGGAAAGGTGTCTGTCTCCTCACTGGCATCATGGGTGCA GGCTGCACTGCCCTTGTGGTGGCCGTGGTGGCCCGAAAGCTGGAACTCACCAAAGCGGAGAAGCACGTTCATAACTTCATG ATGGACACTCAGCTCACCAAGCGGATCAAGAATGCTGCAGCCAATGTCCTTCGGGAAACATGGTTAATCTATAAACACACA AAGCTGCTAAAGAAGATTGACCATGCCAAAGTGAGGAAACACCAGAGGAAGTTCCTCCAAGCTATCCACCAGTTGAGGAGC GTCAAGATGGAACAGAGGAAGCTGAGTGACCAAGCCAACACTCTGGTGGACCTTTCCAAGATGCAGAATGTCATGTATGAC TTAATCACAGAACTCAATGACCGGAGCGAAGACCTGGAGAAGCAGATTGGCAGCCTGGAGTCGAAGCTGGAGCATCTCACC GCCAGCTTCAACTCCCTGCCGCTGCTCATCGCCGACACCCTGCGCCAGCAGCAGCAGCAGCTCCTGTCTGCCATCATCGAG GCCCGGGGTGTCAGCGTGGCAGTGGGCACCACCCACACCCCAATCTCCGATAGCCCCATTGGGGTCAGCTCCACCTCCTTC CCGACCCCGTACACAAGTTCAAGCAGTTGCTAAATAAATCTCCCCACTCCAGAAGCATTACCCATAGGTCTTAAGATGCAA ATCAACTCTCTCCTGGTCGCTTTGCCATCAAGAAACATTCAGACCAGGGAACGGAAAGAAGAGAGACCGAGCTAATTAACT AACTCATGTTCATTCAGCGTGCTTGGTCCGACATGCCTTGAAACCAGAAATCTAATCTCTGTTTAGGTGCCTCTACTTGGG AGCGGGAAGAGGAGATGACAGGAAGCGACGCCTCTGGCAGGGCCCTTGCTGCAGAGTTGGTGGAGAACAGAAATCCACGCT CAATCTCAGGTCTTCACGCGGGGGGTGGGGGTCAGATGCACTGAAGTAGCCAACAGCGAAACCAGTCCAGAAGAGGGGTCC GCTGGGAGGGAGGGTTGTGTCAGGCTTGGGGGATGGGCTCTTCGCCATGGGGGTCTTTGAACACACCTCTCTCCTTTCCTT TTGTCTACGGAAGCCTCTGGGTGACAAAAGTAAAAGAGAGCTGCCCACAACTTGCCAAAACAGATATACTCGAATCAGACT GAAAAAAAAAAAAAAAA

SEQ ID NO:2: translation of positions 334. .2544 from SEQ ID Nθ:l.

DTSGHFHDSGVGDLDEDPKCPCPSSGDEQQQQQQQQQQQQPPPPAPPAAPQQPLGPSLQPQPPQLQQQQQQQQQQQQQQQ QQQQPPHP SQLAQLQSQPVHPGL HSSPTAFRAPPSSNSTAILHPSSRQGSQLNLNDHLLGHSPSSTATSGPGGGSRHRQ ASPLVHRRDSNPFTEIAMSSCKYSGGVMKPLSR SASRRNLIEAETEGQPLQLFSPSNPPEIVISSREDNHAHQT H HPNAT--NHQHAGTTASSTTFPKA -O.K QNIGYKLGHRRA FEKRKRLSDYALIFGMFGIVVMVIETE SWGLYSKDSMFS LALKCLISLSTIILLGLIIAYHTREVQ FVIDNGADD RIAMTYERI-YISLE LVCAIHPIPGEYKFF TAR AFSYTPS RAEADVDIILSIPMFLR YLIARVM LHSK FTDASSRSIGALNKINFNTRFVMKT MTICPGTVL VFSISL IIAA TV RVCERYHDQQDVTSNFLGAM LISITFLSIGYGDMVPHTYCGKGVCLLTGIMGAGCTALVVAVVARKLELTKAEKHVH FM MDTQLTKRI NAAANVLRET IY-HTKLLKKIDHA-VR--HQRKFLQAIHQLRSVKMEQRK SDQA TLVD SK QNVMYD ITELNDRSEDLEKQIGS ESKLEHLTASFNS PL IADT RQQQQQLLSAIIEARGVSVAVGTTHTPISDSPIGVSSTSF PTPYTSSSSC

SEQ ID NO: 3 ACGCTTACCGACCTAGCCAAG

SEQ ID NO: 4 CTCCAGCTCCTCGTGCTGAG

SEQ ID NO: 5 CAGACCGTCATGTACGACCTTGTATCGGA

SEQ ID NO: 6 TTACCCTGGAAACAAAACTAGAGACTT SEQ ID NO: 7 TGCCTGATGGTCTGGCTTATG

SEQ ID NO: 8 CATCCACGCCCTCCCTGGGC

SEQ ID NO: 9 TCCAAGATGCAGAATGTCATGTATG

SEQ ID NO: 10 GGTGAGATGCTCCAGCTTCG

SEQ ID NO: 11 TCCAGGTCTTCGCTCCGGTCATTG SEQ ID NO: 12 CTCCAAGATGCACATGATCCTGTA

SEQ ID NO: 13 GCAGTGCTAAGCAGCTCAGTCA

SEQ ID NO: 14 CAGCAGAATCTGAGCAGCTCACACCG

Claims

1. The use of a compound selected from:

(a) an SK-3 calcium activated potassium channel polypeptide; (b) a compound which acitvates an SK-3 calcium activated potassium channel polypeptide;

(c) a compound which inhibits an SK-3 calcium activated potassium channel polypeptide; or

(i) dysfunctions of male or female sexual organs; or

(ii) vascular disorders such as angina, stroke and hypertension.

2. The use according to claim 1 wherein the medicament is used in the treatment of sexual dysfunction and vascular disorders.

3. The use according to claim 1 wherein the medicament comprises a compound which specifically activates or inhibits an SK-3 calcium activated potassium channel polypeptide.

4. A method of screening for compounds that act as agonists or antagonists of SK-3 calcium activated potassium channel polypeptides for use in the preparation of a medicament for treating dysfunctions of male or female sexual organs or vascular disorders such as angina, stroke and hypertension.

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