WO2002033124A1

WO2002033124A1 - KChAP-MODULATOR OF POTASSIUM CHANNELS

Info

Publication number: WO2002033124A1
Application number: PCT/US2000/027554
Authority: WO
Inventors: Barbara Wible; Arthur M. Brown; Qing Yang
Original assignee: Case Western Reserve University
Priority date: 2000-10-06
Filing date: 2000-10-06
Publication date: 2002-04-25
Also published as: AU2000278629A1

Abstract

This invention generally relates to a novel gene sequence encoding the KchAP protein, as well as methods for the screening of compounds that are agonistic or antagonistic to K+ channel activity.

Description

KChAP - Modulator of Potassium Channels

This invention was made in part with government support under grants HL-26731 , HL-55404, HL-36930 and NS-23877 from the NIH. The government has certain rights in the invention.

FIELD OF THE INVENTION

This invention generally relates to a novel K⁺ channel chaperone gene named K⁺ Channel Associated Protein (KChAP). Additionally, this invention relates to cells and organisms that are made deficient in expression of this gene or made to express additional copies of this gene. Furthermore, the invention contemplates drugs screens for compounds that are agonistic or antagonistic to K⁺ channel activity. Further still, screens for KCh-AP intra- and interspecific homologs as well as KChAP associated binding molecules are contemplated.

BACKGROUND

The electrical properties of excitable cells are determined in large part by the voltage-gated K⁺ channels (Kv) (Jan, J.Y. and Jan, Y.N., "How might the diversity of potassium channels be generated?" Trends Neurosci 13:415-419, 1990) they possess. Multiple Kv channels control the falling phase of the action potential in excitable cells. Kv channels are also important in many nonexcitable cells, where they may contribute to diverse processes such as volume regulation, hormone secretion and activation by mitogens. The extensive diversity in Kv currents is matched by the multiplicity of genes encoding the pore forming or α-subunit of Kv channels. About 20 mammalian Kvα genes have been cloned, and most have been assigned to one of four major subfamilies based on sequence similarities: Kvl, Kv2, Kv3 and Kv4 (Jan, J.Y. and Jan, Y.N., "How might the diversity of potassium channels be generated?" Trends Neurosci 13:415-419, 1990). Each K⁺ channel gene encodes a single subunit, and functional channels are formed by the tetrameric association of individual subunits apparently mediated by specific binding between the N- terminal domains of subunits within individual subfamilies (Li, M., et al., "Specification of subunit assembly by the hydrophilic amino-terminal domain of the Shaker potassium channel" Science 257:1225-1230, 1992; Xu, J., et al., "Assembly of voltage-gated potassium channels. Conserved hydrophilic motifs determine subfamily-specific interactions between alpha-subunits" J. Biol Chem 270:24761-24768, 1995). With multiple Kv channel genes whose products may assemble as multisubunit heteromeric complexes (Ruppersberg, J.P., et al, "Heteromultimetric channels formed by rat brain potassium-channel proteins" Nature 345:535-537, 1990; Sheng, M., et al, "Presynaptic A-current based on heteromultimeric K⁺ channels detected in vivo" Nature 365:72-75, 1993; Wang, H., et al, "Heteromultimeric K⁺ in terminal and juxtaparoaodal regions of neurons" Nature 365:75-79, 1993), these may be hundreds of functionally distinct K⁺ channels. Given the great diversity and fundamental importance of K⁺ channels, the cellular mechanisms regulating their synthesis, assembly and metabolism are of prime interest but remain largely unknown. The identification and characterization of accessory or modulatory subunits for Kv channels is a new and rapidly expanding area of research. One family of modulatory proteins that interact with Kv channels, the Kvβ subunits, has been cloned and characterized in the past several years. Kvβ subunit genes, cloned from heart (Majumder, K., et al., "Molecular cloning and functional expression of a novel potassium channel beta-subunit from human atrium" EEES ett 361:13-16, 1995; Morales, M.J., et al, "A novel beta subunit increases rate of inactivation of specific voltage-gated potassium channel alpha subunits" J. Biol Chem 210:6212-6211, 1995; England, S.K., et al., "Characterization of a voltage-gated K beta-subunit expressed in human heart" Proc Natl Acad Sci USA 92:6309- 6313, 1995; England, S.K., et al., "A novel K⁺ channel beta-subunit (hKv beta 1.3) is produce via alternate RNA splicing" J Biol Chem 270:28531-28534, 1995) and brain (Scott, V.E., et al., "Primary structure of a beta subunit of alpha-dendrotoxin-sensitive K⁺ channels from bovine brain" Proc Natl Acad Sci USA 91:1637-1641, 1994; Rettig, J., et al, "Inactivation properties of voltage-gated K⁺ channels altered by presence of beta-subunit" Nature 368:289-294, 1994; Heinemann, S.J., et al. "Molecular and functional characteriza- tion of Rat brain Kv beta 3 potassium channel subunit" EERS Lett 377:393-389, 1995), encode cytoplasmic proteins that form stable complexes with Kvα 1 subunits and exert multiple effects on Kvαl currents. The three Kvβl isoforms and Kvβ3 introduce inactivation into Kvαl subunit currents but with variable potency (Rettig, J., et al., "Inactivation properties of voltage-gated K⁺ channels altered by presence of beta-subunit" Nature 368:289-294, 1994; Heinemann, S.J., et al. "Molecular and functional characterization of Rat brain Kv beta 3 potassium channel subunit" FEBS Lett 377:393-389, 1995; Wang, Z., et al., "Comparison of binding and block produced by alternately spliced Kv beta 1 subunits" J. Biol Chem 271 :28311-28317, 1996). A second affect of Kvβ subunits is to increase the surface expression of certain Kvαl channels. This has been demonstrated both as an increase in the number of dendrotoxin-binding sites (for Kvl .2 transient expression) (Shi, G., et al., "Beta subunits promote K⁺ surface expression through effects early in biosynthesis" Neuron 16:843-855, 1996), as well as an increase in the number of functional channels (Accili, E.A., et al., "Separable Kv beta subunit domains alter expression and gating of potassium channels" J Biol Chem 272:25824-25831, 1997). Complexes between Kvαl and Kvβ subunits have been found to form in the endoplasmic reticulum (Shi, G., et al., "Beta subunits promote K⁺ surface expression through effects early in biosynthesis" Neuron 16:843-855, 1996; Nagaya, N. and Papazian, D.M. "Potassium alpha and beta subunits assemble in the endoplasmic reticulum" J Biol Chem 272:3022-3027, 1997), suggesting that Kvβ subunits assist in the folding and assembly of at least some Kvαl subunits. The association of Kvl .2 with Kvβ subunits produces more efficient glycosylation of Kvl.2, increases the stability of Kvl.2 through Kvl .2-Kvβ complex formation and results in an increase in cell surface expression (Shi, G., et al, "Beta subunits promote K⁺ surface expression through effects early in biosynthesis" Neuron 16:843-855, 1996).

Kv channels, either functioning or malfunctioning, are implicated in many disease states including cardiac arrhythmias (e.g. LQTS: long QT syndrome) (Yang, W-P, et al. "KvLQTl, a voltage-gated potassium channel responsible for human cardiac arrhythmias" Proc Natl Acad Sci, USA 94:4017-4021, 1997), hypertension, angina, asthma, diabetes, renal insufficiency, urinary incontinence, irritable colon, epilepsy, cerebrovascular ischemia and autoimmune disease. Such diseases afflict millions of people world wide. For example, cardiac arrhythmias cause about a third of a million deaths each year in the USA alone (Sansom, M.S.P. "Ion channels: Structure of a molecular brake" Current Biology 9:R173-R175, 1999). Accordingly, efforts are underway to identify and characterize pharmacological agents that alter the kinetics, gating or formation of Kv channels. The efficacy of such agents is determined by treating cells with such agents and measuring changes in current across the plasma membrane of the cells. Unfortunately, it is difficult to measure small changes in whether a pharmacological agent alters current flow through a specific Kv channel due to the limited number of native channels on cells. Accordingly, it is desirable to have methods and tools which can be used to regulate the numbers and types of Kv channels on the plasma membrane of cells. It is also desirable to have new research tools that can be used for examining the assembly and synthesis of I v channels. What is needed is an assay capable of screening K⁺ channel agonists and antagonists, both in vivo and in vitro, that eliminates or avoids the limitations of the presently available assays, namely the difficulty associated with the measurement of minute changes in current across plasma membranes.

SUMMARY OF THE INVENTION

The present invention relates to a novel gene sequence (SEQ ID NO:l) that encodes a K⁺ channel chaperone and regulatory protein.

The present invention generally comprises a novel, substantially purified oligonucle- otide sequence that encodes for the newly discovered gene, KChAP. Although the present invention is not limited by any particular mechanism, the expression product of this gene is believed to function as a chaperone protein bringing K⁺ channel Kvα subunits to the cell surface. Unlike other known K⁺ channel chaperone proteins, KChAP does not remain bound to the Kvα subunit where it is believed to function in K⁺ channel activity. The exogenous expression of this gene has been shown to result in a greater number of K⁺ channel Kvα subunits at the plasma membrane and resulting greater K⁺ channel activity. Therefore, this gene and derivative gene products will allow for methods and tools which can be used to regulate the numbers and types of Kv receptors on the plasma membrane of cells and, thus, provide novel reagents and methods for the detection of compounds that are agonistic or antagonistic to Kv receptor function.

Although the present invention is not limited to any particular mechanism, it has been determined that expressing polynucleotides that encode KChAP in host cells, along with polynucleotides that encode the Kvα channels subunit Kv2.1, Kv2.2, Kvl .3 or Kv4.3, increase the number of Kv2.1, Kv2.2, Kvl .3 or Kv4.3 channels, respectively, in the plasma membrane of such cells. Accordingly, KChAP polynucleotides are useful for making cells that have increased numbers of Kv channels on the cellular plasma membrane. Such cells are useful model systems for studying the effect of pharmacological agents on Kv channels, particularly on Kv2.1, Kv2.2, Kvl.3 and Kv4.3 channels. In particular, such cells are useful for screening compounds that modulate K⁺ fluxing by said channels. In this regard, it is noted that KChAP shares regions of conserved DNA and amino acid sequences (KChAP consensus sequences, Figures 18 and 19) with the translation products of several other genes that have been shown to function as potassium channel regulators. As such, it is contemplated that these regions of sequence homology may be utilized in any of the capaci- ties detailed below. An alignment of the nucleotide sequences is shown in Figure 18. Examples of consensus sequences are shown in Figure 19.

The present invention generally relates to compositions and methods of identifying and testing K-⁺ channel pathway agonists and antagonists. The present invention is not limited by the method of the employed screen. In one embodiment, the present invention contemplates screening suspected compounds in a system utilizing transfected cell lines, Xenopus oocytes or microorganisms. In one embodiment, the cells or microorganisms may be transfected transiently. In another embodiment, the cells may be stably transfected. In yet another embodiment, translation products of the invention may be used in a cell-free assay system. For example, providing i) KchAP or a KChAP consensus sequence, ii) a binding partner selected from a group consisting of the N-terminal amino acid sequence of Kvαl or the C-terminal amino acid sequence of Kvβ subunits, and iii) a compound suspected of modulating KChAP binding activity; a) mixing said KChAP or a KChAP consensus sequence with said binding partner and said compound suspected of modulating KChAP binding activity; b) detecting binding by, for example, Western blot. In another example, Kv2.1 and KChAP cRNAs may be translated in vitro either separately or together in a rabbit reticulocyte in the presence of ³⁵S-methionine. Antisera to Kv2.1 may be used to immunoprecipitate Kv2.1 and associated KChAP. Immune complexes may then be analyzed by SDS-PAGE and fluorography. Furthermore, in yet another embodiment, antibodies generated to the translation products of the invention may be used in immunoprecipitation assays. In still another embodiment cell based assays incorporating transfected cells (e.g. transiently or stability transfected cells) may be used to screen for K⁺ channel agonists and antagonists. And in still another embodiment, transgenic animals may be generated with the transgene contained in a vector containing an inducible, tissue specific promoter or a restrictive promoter such as a metallothione promoter. The present invention also relates to the anti-sense sequence of SEQ ID NO:l, as well as the anti-sense sequence of the transcription product of SEQ ID NO: l . In one embodiment, said sequences are transfected into cells to inhibit the expression of the endogenous KChAP gene. The invention also relates to methods to identify other binding partners of the

KChAP or a KChAP consensus sequence gene product. The present invention is not limited to the methods employed to identify KChAP or a KChAP consensus sequence binding partners. In one embodiment, antibodies generated to translation products of the invention may be used in immunoprecipitation experiments to isolate novel KChAP binding partners or natural mutations thereof. In another embodiment, the invention may be used to generate fusion proteins (e.g. KChAP-GST fusion proteins) that could also be used to isolate novel KChAP binding partners or natural mutations thereof. In yet another embodiment, screens may be conducted using the yeast two-hybrid system using KChAP or a KChAP consensus sequence as the bait. In yet another embodiment, screens may be conducted using affinity chromatography using KChAP or a KChAP consensus sequence as the ligand.

The invention also relates to the production of derivatives of the KChAP gene such as, but not limited to, mutated gene sequences (and portions thereof), transcription products (and portions thereof), expression constructs, transfected cells and transgenic animals generated from the nucleotide sequences (and portions thereof). The present invention also contemplates antibodies (both polyclonal and monoclonal) to the gene product or nucleic acid aptamers, including the product of mutated genes or a KChAP consensus sequence.

The present invention contemplates using oligonucleotide probes that are comple- mentary to a portion of the KChAP gene sequence or a KChAP consensus sequence to detect the presence of the KChAP DNA or RNA. Such probes are preferably between approximately 10 and 50 bases and more preferably between approximately 50 and 100 bases. On the other hand, the present invention also contemplates probes complementary to less conserved regions or even unique regions (e.g. a portion of the gene having a sequence unique to the KChAP gene).

In addition, the present invention contemplates a diagnostic wherein, for example, a sample of the DNA of the KChAP gene sequence or a KChAP consensus sequence is determined (e.g. by sequencing) to identify suspected mutations. In such a method, the present invention contemplates isolating the gene from a mixture of DNA. Such isolation can be done using one or more of the probes describes above. For example, the present invention contemplates utilizing oligonucleotides that are complementary to the gene as primers in PCR (see U.S. Patent Nos. 4,683,195, 4,683,202 and 4,965,188, all of which are hereby incorporated by reference). Such primers can be complementary to internal regions of the gene. More preferably, primers can be designed that will hybridize to each end of the gene so that the entire gene can be amplified and analyzed (e.g. for mutations).

The present invention also relates to the identification of new homologs of KChAP or natural mutations thereof. The present invention is not limited to a particular method to identify FCChAP homologs. The present invention contemplates screening for homologs using a variety of molecular procedures. In one embodiment, screens are conducted using Northern and Southern blotting. In another embodiment, screens are conducted using DNA chip arrays composed of KChAP DNA sequences for binding complementary sequences. The invention contemplates methods for screening for intra- and inter- specific homologs of KChAP, one method comprising (for example): a) providing in any order: i) extracts from cell suspected of containing said homolog, ii) antibodies reactive to KChAP or a KChAP consensus sequence and specific for at least a portion of the peptide of KChAP or a KChAP consensus sequence; and b) mixing said antibody with said extract under conditions such that said homolog is detected. The present invention further contemplates a method to screen for homologs of KChAP comprising: a) extracts from cells suspected of containing said homolog; b) contacting the extract with anti-KChAP antibody; c) detecting said homo- log by techniques known to those practiced in the art, for example Western blotting. Polynucleotides containing the KChAP gene or a KChAP consensus sequence may also be fused in frame to a marker sequence which allows for purification of the KChAP protein, such as the maltose binding protein, which binds to amylose resin, or glutathione, which binds glutathione-S-transferase-coupled resin. Polynucleotides encoding KChAP protein, KChAP peptide fragments or a KChAP consensus sequence may also be fused in frame to a marker sequence, such as c-myc, which encodes an eptitope tag that allows for monitoring the intracellular location of KChAP using commercially available antibodies. The invention also contemplates novel compositions such as the KChAP gene sequence (or portion thereof) or a KChAP consensus sequence inserted into a transfection vector. The invention is not limited to a particular transfection vector. Many commercial vectors are available. Additionally, novel vectors may be made and utilized. The present invention also contemplates a composition comprising said transfection vector transfected into primary cells, a cell line, a microorganism (e.g. paramecium) or embryonic cells (e.g. Xenopus oocytes). The invention is not limited to a particular cell line, cell type or to any particular species from which the cells are derived. The present invention is not limited to a particular transfection method. Many transfection methods are envisioned by the present invention including electroporation, lipofectamine methods, CaCl₂ methods (see, generally, Sambrook et al. Molecular Cloning: A Laboratory Manual, 2d ed. (1989) Cold Spring Harbor Laboratory Press, Cold Spring Harbor, N.Y., and Current Protocols in Molecular Biology (1996) John Wiley and Sons, Inc., N.Y., ) and particle bombardment (Boileau, A.J., et al., "Transformation of Paramecium tetraurelia by electroporation or particle bombardment" J Euk Microbiol 46:56-65, 1999), which is incorporated herein by reference), all of which are known in the art. The present invention contemplates the use of cDNA or cRNA for transfections. Additionally, the transfection of the KChAP or a KChAP consensus sequence protein is also contemplated by the present invention. In embodiments where more than one vector or sequence is transfected, the vectors or sequences may be transfected either simultaneously or sequentially. The present invention is not limited by the number of different expression vectors or sequences that may be transfected simultaneously. In one embodiment, vectors expressing Kvα K⁺ channel subunits are transfected along with the vector encoding the KChAP gene. Preferably, the Kvα subunit is a Kv2.1 (SEQ ID NO:2), Kv2.2 (SEQ ID NO:3), Kvl .3 (SEQ ID NO:4) or Kv4.3 (SEQ ID NO:5) subunit. In another embodiment, the expression vector encodes for both the KChAP gene and the Kvα subunit. The resulting cells, will have on their surface increased numbers of Kv channels formed by the exogenous Kvα subunits and KChAP. Another contemplated composition comprises the KChAP gene sequence in an appropriate vector used to make a transgenic animal or microorganism. Such KChAP gene sequences may be mutated by methods know in the art such that they are loss of function (lof) or gain of function (gof) mutants. Additionally, they may be combined with other gene sequences (the secondary gene sequence) for the purposes of producing a fusion product. The invention is not limited to any specific secondary gene sequence. The secondary gene sequence may be used to permit, for example, the isolation of the gene (e.g., with a His tag), the isolation of transcription product or the isolation of translation product. Likewise, said secondary sequence may serve as a marker for identifying or visualizing the vector, the translated RNA or the transcribed protein.

Furthermore, the present invention also contemplates using the above-named sequences and derived products in screening assays. The invention is not limited to any par- ticular screening method. In one embodiment, the invention contemplates drug screens for compounds that are agonistic or antagonistic for KChAP function. In one embodiment cells (e.g. mammalian, Xenopus oocytes or paramecium) are transfected with vectors containing a KChAP gene, a complementary DNA (cDNA), a complementary RNA (cRNA) or a KChAP consensus sequence. In another embodiment cells are made defective in KChAP expression through homologous recombination (i.e., genetic recombination involving exchange of homologous loci useful in the generation of null alleles (knockouts) in transgenic animals) (See generally, te Riele, H, et al., "Consecutive inactivation of both alleles of the pim-1 protooncogene by homologous recombination in embryonic stem cells" Nature 348:649-651 , 1990). In one embodiment, the expression vectors are under the control of tissue specific promoters (e.g. the metallothione promoter). Cells can be exposed to the compound suspected of altering KChAP function. The culture can then be exposed to metal ions to activate transcription of the KChAP gene and inhibition or enhancement of K⁺ channel activity can measured by techniques known to those practiced in the art. The invention is not limited to any particular measurement technique. Various methods are envisioned. For example, K⁺ channel activity could be measured by the using the conventional two micro electrode voltage-clamp technique. In another embodiment, the transfection and use of paramecium in said screening assay would allow for the large-scale screening of compounds since chemoattractant methods may be used to quantitate the effect of the suspected compound on K⁺ channel activity.

In one embodiment, the present invention contemplates a composition comprising isolated and purified DNA having an ohgonucleotide sequence of SEQ ID NO:l (or portion thereof, e.g. a KChAP consensus sequence). The present invention further contem- plates a composition comprising RNA transcribed from such DNA as well as a composition comprising protein translated from transcribed RNA. The protein (or portion thereof) can be used as an antigen and the present invention specifically contemplates an antibody produced from the protein or portion of the protein.

The present invention contemplates that the isolated and purified DNA (i.e. having an ohgonucleotide sequence of SEQ ID NO: 1) can be used to make transgenic organisms. For example, the present invention contemplates both transgenic animals comprising such DNA sequences as well as transgenic microorganisms (e.g. paramecium) comprising such DNA sequences. Such transgeneic animals and microorganisms will typically be made using such DNA sequences in operable combination with promoters and enhancers in a transfection vector. The present invention also contemplates such vectors and expression constructs comprising such DNA sequences.

While a variety of screening methods are contemplated. In one embodiment, the present invention contemplates a method to detect K⁺ channel agonists and antagonists, comprising: a) providing i) one or more compounds suspected of modulating K⁺ channel activity, ii) a first mammalian, Xenopus oocytes or paramecium cell line comprising the KChAP gene; b) contacting a portion of said cells from said transfected cell line with said one or more compounds under conditions such that said compound can enter said cells, so as to create treated portions and untreated portions of cells; and c) comparing the amount K⁺ channel activity in said treated portion of cells as compared to said untreated portion of cells.

In another embodiment, the present invention contemplates a method to detect K⁺ channel agonists and antagonists, comprising: a) providing i) one or more compounds sus- pected of modulating K⁺ channel activity, ii) a first mammalian, Xenopus oocytes or paramecium cell line comprising the KChAP gene and one gene from the group comprising Kv2.1, Kv2.2, Kvl.3 or Kv4.3; b) contacting a portion of said cells from said transfected cell line with said one or more compounds under conditions such that said compound can enter said cells, so as to create treated portions and untreated portions of cells; and c) com- paring the amount K⁺ channel activity in said treated portion of cells as compared to said untreated portion of cells.

The present invention contemplates transgenic animals and microorganisms that express increased levels of KChAP or have the expression of KChAP diminished or inhibited (i.e. gene knock-out animals and microorganisms). Such animals and microorganisms can be made by methods known to those practiced in the art.

DESCRIPTION OF THE FIGURES

Figure 1. Yeast two-hybrid assay of the interaction of a novel protein, KChAP, with K⁺ channel fragments. Figure 2. Comparison of KChAP peptide sequence with Gu-binding protein (GBP).

Figure 3. Northern blot analysis of KChAP expression in rat tissues.

Figure 4. Effect of KChAP on K⁺ channel functional expression.

Figure 5. KChAP increases the amount of Kv2.1 protein at the oocyte surface.

Figure 6. KChAP increases the total amount of Kv2.1 protein and is localized pri- marily to the nucleus of coinjected Xenopus oocytes.

Figure 7. KChAP increases the functional expression of Kv2.1 in Xenopus oocytes without altering voltage-dependence or channel kinetics.

Figure 8. Coimmunoprecipitation of Kv2.1 and KChAP from in vitro translation reactions. Figure 9. KChAP increases the functional expression of Kvl .3 Xenopus oocytes.

Figure 10. Summary of KChAP effects on the functional expression of K⁺ channels in Xenopus oocytes. Figure 11. KChAP increases functional expression of Kv4.3 in Xenopus oocytes without affecting activation or inactivation properties.

Figure 12. The transcription inhibitor actinomycin-D does not alter KChAP effects on the functional expression of Kvl .3 in Xenopus oocytes. Figure 13. KChAP increases the functional expression of Kv2.1 and Kv4.3 channels in mammalian cells.

Figure 14. Yeast two-hybrid assay of the interaction of KChAP fragments with Kvl .2 N-terminus. (A) Schematic diagram of KChAP fragments. (B) Yeast two-hybrid interaction assay. Figure 15. KChAP and KChAP-M produce comparable increases in Kv4.3 protein and currents in mammalian cells. The stable cell line, L/Kv4.3, was transiently transfected with the following EGFP-tagged constructs: (A) KChAP, (B) KChAP-M, (C) KChAP-N, (D) KChAP-C. Left panels show the localization of the EGFP-tagged proteins while the right panels show costaining of the Kv4.3 antibody in the same cells. (E,F) K⁺ current density in L/Kv4.3 cells transiently transfected with EGFP-tagged constructs. (*) indicates values significantly different from cells transfected with EGFP-C2 alone.

Figure 16. Coimmunoprecipitation of KChAP with Kv channels from native tissue.

Figure 17 shows the amino acid (SEQ ID NO:32) and nucleotide (SEQ ID NO:l) sequences of KChAP. Figure 18 (a & b) show consensous amino acid sequences (KChAP consensus sequences) between KChAP, PIAS3C, GBP, Mizl, Mncl, PlASy and ARIP3.

Figure 19 shows an alignment of amino acid sequences of the peptides listed in figure 18. (KChAP = SEQ ID NO: 54; PIAS3C = SEQ ID NO:55; GBP = SEQ ID NO:56; Mizl = SEQ ID NO: 57; MNC1 - SEQ ID NO:58; PlASy = SEQ ID NO: 59; ARIP3 = SEQ ID NO:60).

DEFINITIONS

To facilitate understanding of the invention, a number of terms are defined below. As used herein "agent", "compound" or "drug" denote an inorganic or organic compound, a mixture of chemical compounds, a biological macromolecule, or an extract made from biological materials such as bacteria, plants, fungi, or animal (particularly mammalian) cells or tissues that are suspected of having therapeutic properties. The compound, agent or drug may be purified, substantially purified or partially purified. As used herein "agonist" refers to molecules or compounds which mimic the action of a "native" or "natural" compound. Agonists may or may not be homologous to these natural compounds in respect to conformation, charge or other characteristics. Thus, agonists may or may not be recognized by, e.g., receptors expressed on cell surfaces. In any event, regardless if the agonist is recognized by a natural compound in a manner similar to a "natural" compound or molecule, the agonist may cause physiologic and/or biochemical changes within the cell, such that the cell reacts to the presence of the agonist in the same manner as if the natural compound was present.

As used herein "antagonist" refers to molecules or compounds which inhibit the action of a "native" or "natural" compound. Antagonists may or may not be homologous to these natural compounds in respect to conformation, charge or other characteristics. Thus, antagonists may be recognized by the same or different receptors or molecules that are recognized by an agonist. Antagonists may have allosteric effects which prevent the action of an agonist (e.g., by modifying a DNA adduct, or antagonists may prevent the function of the agonist (e.g., by blocking a DNA repair molecule).

As used herein "exogenous" means that the gene encoding the protein is not normally expressed in the cell. Additionally, "exogenous" refers to a gene transfected into a cell to augment the normal (i.e. natural) level of expression of that gene.

As used herein "anti-sense" shall be defined as a nucleotide sequence that is comple- mentary to another single strand of DNA or RNA. Said complementation is typically at least 50%, more typically said complementation is greater than 75%, even more typically said complementation is greater than 90%.

"Gain of function" (gof) shall be defined as all modifications to an ohgonucleotide that, when that ohgonucleotide is transfected into a host organism and translated into a peptide, that peptide will function with increased efficiency as compared to the wild type peptide when the gene or gene product is induced to function whether that induction be continuous or non-continuous. It may, in effect, function as an augmenter of the natural gene if the natural gene is present and functional in the host into which the gof ohgonucleotide was transfected, or it may add that function to the host if the natural gene is not pres- ent or is non- functional.

"Loss of function" (lof) shall be defined as all modifications to an ohgonucleotide that, when that ohgonucleotide is transfected into a host organism and translated into a peptide, that peptide will function with decreased efficiency as compared to the wild type peptide when the gene or gene product is induced to function whether that induction be continuous or non-continuous. It may, in effect, function as a diminisher of natural gene function if the natural gene is present and functional in the host into which the lof ohgonucleotide was transfected, or may negatively interfere with processes in the host if the natural gene is not present or is non- functional.

As used herein, the term "purified" or "to purify" refers to the removal of contaminants from a sample. The present invention contemplates purified compositions (discussed above).

As used herein, the term "partially purified" refers to the removal of a moderate portion of the contaminants of a sample to the extent that the substance of interest is recognizable by techniques known to those skilled in the art as accounting for an amount of the mixture greater than approximately 5% of the total.

As used herein, the term "substantially purified" refers to the removal of a significant portion of the contaminants of a sample to the extent that the substance of interest is recognizable by techniques known to those skilled in the art as the most abundant substance in the mixture.

As used herein the term "portion" when in reference to a protein (as in "a portion of a given protein") refers to fragments of that protein. The fragments may range in size from four amino acid residues to the entire amino acid sequence minus one amino acid. In one embodiment, the present invention contemplates "functional portions" of a protein. Such portions are "functional" if they contain a binding region (i.e. a region having affinity for another molecule) and such binding can take place (i.e. the binding region functions, albeit with perhaps lower affinity than that observed for the full-length protein). Such "functional portions" of the gene product are typically greater than approximately 10 amino acids in length, and more typically greater than approximately 50 amino acids in length, and even more typically greater than approximately 100 amino acids in length. "Functional portions" may also be "conserved portions" of the protein. The alignment of the various gene products permit one skilled in the art to select conserved portions of the protein (i.e. those portions in common between two or more species) as well as unconserved portions (i.e. those portions unique to two or more species). The present invention contemplates conserved portions approximately 10 amino acids in length or greater, and more typically greater than approximately 50 amino acids in length. "In operable combination", "in operable order" and "operably linked" as used herein refer to the linkage of nucleic acid sequences in such a manner that a nucleic acid molecule capable of directing the transcription of a given gene and/or the synthesis of a desired protein molecule is produced. The term also refers to the linkage of amino acid sequences in such a manner so that a functional protein is produced.

"Expression vector" shall be defined as a sequence of DNA or RNA, in operable combination that is used to transfect a cell or cells. The sequence may be single or double stranded.

"Patient" shall be defined as a human or other animal, such as a farm animal, guinea pig or mouse and the like.

"Consensus sequence" shall be defined as a sequence of amino acids or nucleotides that contain identical amino acids or nucleotides or functionally equivalent amino acids or nucleotides for at least 25 % of the sequence. The identical or functionally equivalent amino acids or nucleotides need not be contiguous.

GENERAL DESCRIPTION OF THE INVENTION

Voltage-gated K (Kv) channels are important in the physiology of both excitable and nonexcitable cells. The diversity in Kv currents is reflected in multiple Kv channel genes whose products may assemble as multisubunit heteromeric complexes. Given the funda- mental importance and diversity of Kv channels, surprisingly little is known regarding the cellular mechanisms regulating their synthesis, assembly and metabolism. To begin to dissect these processes, we have used the yeast two-hybrid system to identify cytoplasmic regulatory molecules that interact with Kv channel proteins. Here we report the cloning of a novel gene encoding a Kv channel binding protein (KChAP, for K⁺ Channel-Associated Protein), which modulates the expression of Kv2 channels in heterologous expression system assays. KChAP interacts with the N terminal of Kvαl and the C termini of Kvβ sub- units. Kv2.1 and KChAP were coimmunoprecipitated from in vitro translation reactions supporting a direct interaction between the two proteins. The amplitudes of Kv2.1 and Kv2.2 currents are enhanced dramatically in Xenopus oocytes coexpressing KChAP, but channel kinetics and gating are unaffected. Although KChAP binds to Kvl .5, it has no effect on Kvl .5 currents. Although the present invention is not limited by any particular mechanism, we suggest that KChAP may act as a novel type of chaperone protein to facilitate the cell surface expression of Kv2 channels. In this regard, our results demonstrate that the protein KChAP modulates the functional expression of specific Kv channels without changing channel prop- erties such as gating or voltage-dependence in both Xenopus oocyte and mammalian expression systems. Following our initial characterization of KChAP and Kv2.1, we hypothesized that KChAP was a novel type of chaperone protein which interacted transiently with the channel but did not remain attached to the mature channel complex at the cell surface (Wible, B.A., et al. "Cloning and expression of a novel K⁺ channel regulatory protein, KChAP" JBiol Chem 273:11745-11751, 1998). Considered together with our previous data, the results reported here support the view that interaction of KChAP with Kv channels is responsible for the observed increase in current and protein levels. First, the coimmunoprecipitation of complexes of KChAP with Kv2.1 and Kv4.3 from rat heart lysates demonstrates that the proteins do interact in vivo. Secondly, yeast two-hybrid data suggests a direct interaction between KChAP and the N-termini of Kvl and Kv2 channels. This we had reported previously (Wible, B.A., et al. "Cloning and expression of a novel K⁺ channel regulatory protein, KChAP" J Biol Chem 273:11745-11751, 1998) but confirmed here for Kvl .3-N. Unfortunately, Kv3 and Kv4 N-termini produced non-specific transactivation of reporter genes in yeast and could not be assayed.

DETAILED DESCRIPTION OF PREFERRED EMBODIMENTS

Generally, the nomenclature used hereafter and the laboratory procedures in cell culture, molecular genetics, and nucleic acid chemistry and hybridization described below are those well known and commonly employed in the art. Standard techniques are used for recombinant nucleic acid methods, polynucleotide synthesis, and microbial culture and transformation (e.g., electroporation, lipofection). Generally enzymatic reactions and purification steps are performed according to the manufacturer's specifications. The techniques and procedures are generally performed according to conventional methods in the art and various general references (see, generally, Sambrook et al. Molecular Cloning: A Laborato- ry Manual, 2d ed. (1989) Cold Spring Harbor Laboratory Press, Cold Spring Harbor, N.Y., and Current Protocols in Molecular Biology (1996) John Wiley and Sons, Inc., N.Y., which are incorporated herein by reference) which are provided throughout this document. Oligonucleotides can be synthesized on an Applied BioSystems ohgonucleotide synthesizer [for details see Sinha et al., Nucleic Acids Res. 12:4539 (1984)], according to specifications provided by the manufacturer. Complementary oligonucleotides are annealed by heating them to 90°C in a solution of 10 mM Tris-HCl buffer (pH 8.0) containing NaCI (200 mM) and then allowing them to cool slowly to room temperature. For binding and turnover assays, duplex DNA is purified from native polyacrylamide (15% w/v) gels. The band corresponding to double-stranded DNA is excised and soaked overnight in 0.30 M sodium acetate buffer (pH 5.0) containing EDTA (1 mM). After soaking, the supernatant is extracted with phenol/chloroform (1/1 v/v) and precipitated with ethanol. DNA substrates are radiolabeled on their 5'-OH group by treatment with [g-³²P]ATP and T4 polynucleotide kinase. Salts and unincorporated nucleotides are removed by chromatography on Sephadex G columns.

Assays for detecting the ability of agents to inhibit or enhance K⁺ channel activity provide for facile high-throughput screening of agent banks (e.g., compound libraries, pep- tide libraries, and the like) to identify antagonists or agonists. Such K⁺ antagonists and agonists may be further developed as potential therapeutics and diagnostic or prognostic tools for diverse types of neurological and muscular diseases, as well as cardiac arrhythmias (e.g. LQTS: long QT syndrome), hypertension, angina, asthma, diabetes, renal insufficiency, urinary incontinence, irritable colon, epilepsy, cerebrovascular ischemia and autoimmune disease. Likewise, the KChAP gene, and modifications thereof, may be useful in gene therapy. For example, the incorporation of the KChAP gene sequence into cells in context of tissue specific or inducible promoters might be useful in the treatment of hereditary diseases.

1. Screens to identify Agonists of Antagonists of K⁺ Channel Activity

There are several different approaches contemplated by the present invention to look for small molecules that specifically inhibit or enhance K⁺ channel activity. One approach is to transfect expression constructs (vectors) comprising the KChAP gene into cells and measure changes in the rate of K⁺ as compared to controls after the cells have been exposed to the compound suspected of modulating KChAP activity. Cells may be transiently transfected or stably transfected with the construct under control of an inducible or temperature sensitive promoter. Another embodiment is to transfect cRNA for the KChAP protein. In yet another embodiment, cRNA is transfected simultaneously with cRNA encoding a Kv channel protein. Other embodiments would include translation of the invention and purification of the peptide. The purified peptide could then be used as a substrate in a cell-free assay. Furthermore, transgenic animals and stably transfected cell lines could be produced allowing for in vivo assays to be conducted. A. In vitro Assays i. Transfection Assays

Transfection assays allow for a great deal of flexibility in assay development. The wide range of commercially available transfection vectors will permit the expression of the invention in a extensive number of cell types. In one embodiment, cells are transiently transfected with an expression construct comprising, in operable combination, the KChAP gene and an inducible promoter allowing for the initiation of translation and transcription when needed. Cells are exposed to the agent suspected of modulating K⁺ activity, KChAP expression is initiated and K⁺ channel activity is measured. Rates of K⁺ flux in cells treated with said compound are compared to rates in cells that are untreated. Rates of K⁺ fluxing are quantitated by any of a number of ways reported in the literature and known to those practiced in the art.

In another embodiment, stably transfected cells lines are employed. The use of an inducible promoter or temperature sensitive promoter can be utilized in these systems. Screening assays for compounds suspected of modulating K⁺ channel activity are conducted in the same manner as with the transient transfection assays. Using stably transfected cell lines, however, allows for greater consistency between experiments and allow for inter-experimental comparisons.

In order to test the stimulatory or inhibitory effect of a compound, particularly a pharmacological agent, on the flow of a current through Kv channels, it is desirable to have a model system comprising a population of cells that have increased numbers of Kv channels on their cellular plasma membrane. Such a model system is especially suitable for measuring small changes in current flow. Such model systems are prepared by coinjecting into host cells cDNA or cRNA molecules encoding KChAP and for the Kvα subunit. The encoding regions for KChAP and for the Kvα subunit may be on separate GDNA or cRNA molecules. Preferably, the Kvα subunit is an exogenous Kvα subunit, i.e., the Kvα subunit is not normally expressed in the cell. Such model systems are especially useful for monitoring the effect of a compound on a particular Kv channel, i.e., the Kv channel formed by assembly of a plurality of the exogenous Kvα subunits. Thereafter, the cells are cultured for a time and under conditions which permit transfoπnation of the host cells, i.e., expression of the coinjected cDNA or cRNA molecules and assembly of Kv channels comprising the corresponding Kvα subunits.

The compound (which, depending on the compound, may be dissolved in a suitable carrier) is added to the culture medium of a test population of transformed host cells. Preferably, a plurality of concentrations of the compound are added to a corresponding plurality of test populations. The compound is also added to the culture medium of a control population of cells that have not been transformed, i.e., cRNA or cDNA molecules encoding KChAP and the Kvα subunit are not transfected into the cell. Thereafter, whole cell cur- rents are measured using conventional techniques, such as, for example, using a two microelectrode voltage-clamp technique and the gigaseal patch clamp technique. A difference between whole cell currents in the control population and the test population is indicative of a stimulatory or inhibitory effect of the compound on the Kv channels formed by the exogenous Kvα subunit. Such measurements are also used to determine the effective compound dosage. ii. Cell-free Assays

KChAP protein may be utilized in cell-free assays. For example, a compound suspected of modulating the binding of KChAP to the N-terminal of Kvαl or the C-terminal of Kvβ subunits could be added to a reaction mixture including the appropriate Kv subunit and KChAP. Modulation of binding activity could be measured by changes in electrophoresis mobility. Such assays would allow for high-through put screening assays. B. In Vivo Assays i. Transgenic Animal Assays

In one embodiment, transgenic animals are constructed using standard protocols, including homologous recombination (i.e., genetic recombination involving exchange of homologous loci useful in the generation of null alleles (knockouts) in transgenic animals) (See generally, te Riele, H, et al, "Consecutive inactivation of both alleles of the pim-1 protooncogene by homologous recombination in embryonic stem cells" Nature 348:649-651, 1990). The KChAP gene may be placed under the control of a tissue specific promoter or inducible promoter. The generation of transgenic animals will allow for the creation of model systems to investigate the numerous diseases associated with aberrant K⁺ channel activity which may provide the means for determining the physiology of the disease or its treatment. ii. Paramecium Based Assays In yet another embodiment, paramecium are transfected with the KChAP gene by methods known to those in the art (e.g. electroporation or particle bombardment; Boileau, A.J., et al., "Transformation of Paramecium tetraurelia by electroporation or particle bom- bardment" J Euk Microbiol 46:56-65, 1999), which is incorporated herein by reference). Said transfected paramecium are then exposed to compounds suspected of modulating K⁺ channel activity. Rates of K⁺ flux in the paramecium are measured by chemosensory assays known in the art (Fraga, D., et al, "Introducing antisense oligodeoxynucleotides into Paramecium via electroporation" J Euk Microbiol 45:582-588, 1998) and compared to rates of K⁺ flux in untreated paramecium.

2. Screens to Identify KChAP Binding Partners

A. In vitro Assays

There are several different approaches to identifying KChAP interactive molecules or binding partners. Techniques that may be used are, but not limited to, immunoprecipitation of KChAP with antibodies generated to the translation product of the invention. This would also bring down any associated bound proteins, i.e. proteins in the cell with affinity for the KChAP polypeptide. Another method is to generate fusion proteins comprising KChAP connected to a generally recognized pull-down protein such as glutathione S-trans- ferase (GST). Bound proteins can then be eluted and analyzed. Yet another method is to bind KChAP to a solid support and expose the bound KChAP to cell extracts suspected of containing an KChAP interactive molecule or binding partner. i. Immunoprecipitation

After the generation of antibodies to KChAP, cells expressing transfected KChAP are lysed and then incubated with one of the antibodies. Antibodies interact with the bound KChAP and any associated proteins can then be pulled down with protein-A Sepharose or protein-G Sepharose beads, using standard techniques. Where yeast binding partners are sought, yeast cells are lysed. ii. Fusion Protein Pull-down A method similar to immunoprecipitation is to construct fusion proteins of the mutant and wild type KChAP and glutathione S-transferase (GST). The GST-KChAP fusion proteins are then incubated with cell extracts and then removed with glutathione Sepharose beads. Any bound, KChAP-associated proteins are then characterized. B. In Vivo Assays i. Yeast Two-hybrid System

The yeast two-hybrid system identifies the interaction between two proteins by reconstructing active transcription factor dimers (Chien, C.T., et al. "The two-hybrid system: a method to identify and clone genes for proteins that interact with a protein of interest" Proc Natl Acad Sci, USA 88:9578-9582, 1991). The dimers are formed between two fusion proteins, one of which contains a DNA-binding domain (DB) fused to the first protein of interest (DB-X, where X will be MinK2) and the other, an activation domain (AD) fused to the second protein of interest (AD-Y, where Y will be a candidate KChAP-binding protein encoded by cDNA from a commercially available library). The DB-X: AD-Y interaction reconstitutes a functional transcription factor that activates chromosomally-integrated reporter genes driven by promoters containing the relevant DB binding sites. Large cDNA libraries can be easily screened with the yeast-two hybrid system. Yeast cDNA libraries are commercially available. Standard molecular biological techniques can be employed to iso- late and characterize the interacting protein.

3. Screens to Identify KChAP Homologs

Standard molecular biological techniques can be used along with the reagents of the present invention to identify KChAP homologs in various species. For example, preferred embodiments may included, but are not limited to, DNA-DNA hybridization techniques (e.g. Southern blots) and DNA-RNA hybridization techniques (e.g. Northern blots). Additional techniques may include, for example, immunoscreening of proteins made from library stocks by antibodies generated from the invention. The present invention also contemplates a number of approaches including, but not limited to, immunoprecipitation and affinity purification of cell and tissue extracts and immunoscreening of proteins and glycoproteins translated from DNA and RNA library stocks. Furthermore, hybridization screens of RNA and DNA library stocks could be accomplished using RNA and DNA sequences reverse engineered from isolated KChAP protein or by using anti-sense DNA or amino RNA sequences. EXPERIMENTAL

Materials and Methods

Constructs and yeast two-hybrid assay. The entire Kvβ 1.2 coding sequence (amino acids 1-408, SEQ ID NO;6) was subcloned in frame into the GAL4 DNA binding domain vector, pGBT9 (Clontech) after PCR-mediated addition of a 5' EcoRI site and a 3' Sail site and used to screen a pGADIO rat brain cDNA library (Clontech). Transformants in the yeast Y190 strain were plated on synthetic medium lacking tryptophan (trp), leucine (leu), and histidine (his) but containing 3-aminotriazole (25 mm). After incubation for 8 days at 30°C, His⁺ colonies were screened for β-galactosidase activity by a filter lift assay. Individ- ual pGADIO recombinant plasmids were screened for interaction with Kvβl .2 by repeating the yeast two-hybrid assay. Full-length KChAP (residues 1-619, SEQ ID NO:7) and the following KChAP fragments were tagged with EGFP at their N-termini by subcloning into the EcoRI and Sail sites of EGFP-C2 (Clontech): KChAP-N (residues 46-354, SEQ ID NO:8), KChAP-M (residues 355-452, SEQ ID NO:9), and KChAP-C (residues 453-619, SEQ ID NO:10). The same fragments were also subcloned into the GAL4 yeast two-hybrid vectors, pGBT9 and pGAD424. Fragments encoding Kvl.2-N (residues 1-164, SEQ ID NO:l l) and Kvl .3-N (residues 1-186, SEQ ID NO: 12) were also subcloned into pGBT9. Protein-protein interactions were tested in the yeast two-hybrid system by cotransformation of host strain HF7C with pairs of pGBT9 and pGAD424 fusion constructs as previously described (Wang, Z., et al. "Comparison of binding and block produced by alternatively spliced Kvbetal subunits " JBiol Chem 271 :28311-29317, 1996). Cotransformants were selected and spotted on media with and without histidine to follow the activation of the HIS3 reporter gene.

Cloning of Full-Length cDNA. The KChAP-Y/pGADlO plasmid contained a 1.78 kb insert with an open reading frame of 264 amino acids. To obtain the full-length clone, ³ P labeled KChAP-Y insert was used to screen a rat brain cDNA library in λgtlO (Clontech). One of the hybridizing clones contained an insert of 3.2 kb with a single open reading frame of 574 amino acids. KChAP cDNA without 5' or 3' untranslated sequences for subcloning in frame into the yeast two-hybrid vector pGBT9 was prepared by PCR to include a 5' EcoRI site and a 3' Sail site. Analysis of Protein-Protein Interactions by the Yeast-Two Hybrid System. Protein- protein interactions were monitored with the yeast Matchmaker Two-Hybrid System (Clontech). The following fragments were tested for interaction with KChAP: Kvβl .2 (amino acids 1-408, SEQ ID NO:6), Kvβl .2-N terminus (amino acids 1-79, SEQ ID NO.T 3), KvβlC (carboxyl terminal 329 amino acids of the Kvβl subfamily, SEQ ID

NO:14), Kvβ2 (amino acids 1-367 SEQ ID NO:15), Kvl .l-N terminus (amino acids 1-168, SEQ ID NO: 16), Kvl .2 N-terminus (amino acids 1-124, SEQ ID NO: 17), Kvl.4 N-termi- nus (amino acids 1-305, SEQ ID NO: 18), Kvl.4 C-terminus (amino acids 562-654, SEQ ID NO:19), Kvl.5 N-terminus (amino acids 1-248, SEQ ID NO:20), Kv2.1 N-terminus (amino acids 1-168, SEQ ID NO:21), Kv2.2 N-terminus (amino acids 1-185, SEQ ID NO:22),

Kv6.1 N-terminus (amino acids 1-209, SEQ ID NO:23), Kir2.2 N-terminus (amino acids 1- 86, SEQ ID NO:24), and HERG N-terminus (amino acids 1-396, SEQ ID NO:25). Human Gu-binding protein (GBP) cDNA encoding the region from residues M49 to D645 was obtained by RT-PCR from human brain poly A⁺ RNA. M49 corresponds to the start methi- onine residue in KChAP. Protein-protein interactions were tested by cotransformation of plasmid pairs into the yeast host strain Y190 as described previously (Accili, E.A., et al. "Separable Kvbeta subunit domains alter expression and gating of potassium channels" J Biol Chem 272:25824-25832, 1997). Appearance of blue color within 8 hours was scored as a positive interaction, Northern Blot Analysis. A rat multiple tissue Northern blot (Clontech) was probed with a ³²P-riboprobe spanning the region encoding the C-terminal 167 amino acids of KCHAP. A T7 promoter sequence was engineered directly onto the end of the 501 bp coding fragment using the Lig'nScribe kit from Ambion and riboprobe synthesized with the Maxiscript T7 kit (Ambion). The blot was hybridized with probe (10⁶ cpm/ml) overnight at 68°C in NorthemMax hybridization buffer (Ambion). Two room temperature washes in 2 x SSC/0.1% SDS (15 minutes each) were followed by two washes at 70°C in 0.1 x SSC/0.1% SDS (20 minutes each).

In Vitro Translation. Full-length KChAP cDNA was removed from pGBT9 with EcoRI and Sail and subcloned into a pCR3 vector which we modified to allow the cloning of EcoRI/Sall fragments in frame behind a c-myc tag. cRNA for c-myc-KChXP was prepared with the T7 mMESSAGE mMACHINE kit (Ambion). cRNAs for c-myc-KChAP and Kv2.1 were translated in vitro either separately or together in rabbit reticulocyte lysates in the presence of ³⁵S-methionine using the Retic Lysate IVT kit (Ambion). A maximum of 500 ng of cRNA was used in each 25 μl translation reaction. Canine pancreatic microsomes (Boehringer-Mannheim) (1 μl/25 μl translation reaction) were included in reactions in which Kv2.1 was translated.

Immunoprecipitation. For immunoprecipitation (IP), 10 μl aliquots of each transla- tion were diluted into 1 ml IP buffer (1% Triton X-100, 150 mM NaCI, 50 M Tris pH 7.5, 1 mM EDTA). To monitor the ability of the two proteins to associate after translation, 10 μl aliquots of individual translates of Kv2.1 and c-myc-KChAP were mixed in 1 ml IP buffer prior to addition of antibody. IP was performed with two primary antibodies: anti- Kv2.1 polyclonal (1 : 100; Upstate Biotechnology, Inc) or anti-c-myc monoclonal (1:400; Boehringer Mannheim). After addition of the primary antibody, the reactions were mixed gently overnight at 4°C. Immune complexes were collected on magnetic beads coupled to either anti-rabbit or anti-mouse secondary antibodies (Dynal, Inc). After four washes in IP buffer, bound protein was eluted by boiling in SDS sample buffer, and analyzed on 10% polyacrylamide/SDS gels. The gel was fixed, soaked in Amplify (Amersham), and radiolabeled protein detected by fluorography. Coimmunoprecipitation experiments of

KChAP and Kv channels were performed as follows. Immunoprecipitation reactions were performed at 4°C using rat heart lysates. For each experiment, the lysate (2-4 mg/ml) was equally divided into 2 tubes. Lysates were pre-cleared by incubation with anti-rabbit IgG conjugated magnetic beads (Dynabeads; Dynal, Inc.) after which affinity purified anti- KChAP was added to one tube (1:100 dilution). Both tubes were incubated overnight with gentle mixing at 4°C. Antigen-antibody complexes were captured on Dynabeads by gentle mixing for 1 h at 4°C. The Dynabeads were washed three times with lysis buffer and immunoprecipitates were eluted by boiling in reducing SDS sample buffer.

Immiinofluorescence microscopy. For Xenopus oocytes: Five days post-injection, two microelectrode voltage-clamp recordings were made from Xenopus oocytes injected with either Kv2.1 cRNA alone or coinjected with Kv2.1 plus c-w^c-KChAP cRNAs. Several hours after recording the same oocytes were fixed and sectioned as described previously (Chan, K.W., et al. "Specific regions of heteromeric subunits involved in enhancement of G protein-gated K+ channel activity" J Biol Chem 272:6548-6555, 1997). After incubation for two hours in 1% BS A/PBS to block nonspecific binding sites, the oocyte sections were incubated at 4°C overnight with primary antibody (anti-Kv2.1 polyclonal, 1 :100) in 1% BSA/PBS. The secondary antibody (FITC (fluorescein isothiocyanate)-conjugated anti- rabbit, 1 : 100; Cappel Labs) was added for two hours at room temperature. For oocytes coinjected with c-wyc-KChAP, the same protocol was followed with anti-c-myc monoclonal antibody (1 :400) and TRITC (tetramethylrhodamine isothiocyanate)-conjugated anti-mouse secondary antibody (1 :125; Sigma). The sections were examined with an Olympus BH-2 microscope. For L/Kv4.3 stably transfected cell line: About 48 hours post-transfection, cells were fixed in 4% paraformaldehyde in phosphate-buffered saline (PBS) for 15 min at room temperature and permeabilized for 5 min with 0.1% Triton X-100 in PBS. After blocking for 1 h in 5% nonfat milk/PBS, the cells were incubated overnight at 4°C with Kv4.3 polyclonal antibody (Alomone Labs; 1:100 dilution) in 5% nonfat milk/PBS. The second- ary antibody, tetramethylrhodamine B isothiocyanate (TRITC)-conjugated anti-rabbit IgG, (1 : 100) (Jackson Labs), was added for 1 h at room temperature. Coverslips were mounted with Vectashield (Vector Labs) and examined with an Olympus BH-2 microscope. Images were obtained with a Spot 2.1 digital camera (Diagnostic Instruments).

Oocyte Fraciionation and Western blotting. To prepare microsomal proteins, oocytes were homogenized in 0.3 M sucrose/10 mM NaPO₄ pH 7.4 (20 μl per oocyte) containing a protease inhibitor cocktail (Complete, Boehringer Mannheim). After removal of nuclei and debris by centrifugation at 3,000 x g for 10 minutes, the supernatant was spun at 48,000 x g for one hour to pellet microsomes. Microsomes from adult rat brain were prepared using the same protocol. In some experiments, oocyte nuclei were removed manu- ally (Feldherr, CM., et al. "Manual enucleation of Xenopus oocytes" Methods Cell Biol 17:75-79, 1978). Enucleated oocytes were processed as described above while the nuclei were extracted for one hour in homogenization buffer plus 1% Triton X-100. Following a 20 minute spin at 10,000 x g, the supernatant containing solubilized nuclear proteins was collected. Protein concentrations were determined by the BCA method (Pierce). For West- ern blotting, proteins were separated on SDS-PAGE and blotted to PVDF membranes. After blocking with 5% nonfat dry milk in PBS + 0.1% Tween-20 (PBS-T), blots were incubated with primary antibodies, either a monoclonal Kv2.1 antibody (Upstate Biotechnology, Inc; 1 : 1000), monoclonal anti-c-/// 'c antibody (1 :400) or KChAP antibody (1 :100 dilution) for one hour at room temperature. The blots were then washed and then incubated with secondary antibody (anti-mouse HRP conjugate, Amersham; 1 :3000) and developed with the ECL+Plus detection system (Amersham). Expression in Xenopus Oocytes and Electrophysiology. For cRNA synthesis and expression in Xenopus oocytes, full-length KChAP coding sequence was subcloned into the vector, pCR3 (Invitrogen). KChAP cRNA was prepared using the T7 mMESSAGE mMACHINE kit (Ambion) following linearization of the construct with Noil. GBP cDNA was subcloned into a modified pSP64 vector (Nrul site for linearization incorporated past the poly A⁺ tail) for in vitro transcription with SP6 polymerase. cRNAs for Kvlα subunits were prepared as previously described (Majumder, K., et al. "Molecular cloning and functional expression of a novel potassium channel beta-subunit from human atrium" FEBS Lett 361 :13-16, 1995; Wang, Z, et al. "Comparison of binding and block produced by alterna- tively spliced Kvbetal subunits" J Biol Chem 271:28311-28317, 1996). Rat Kv2.1 in pBluescript was linearized with Notl and cRNA was prepared with T7 polymerase. cRNAs were mixed and injected into Xenopus oocytes as previously described (Majumder, K., et al. "Molecular cloning and functional expression of a novel potassium channel beta-subunit from human atrium" FEBS Lett 361 :13-16, 1995). HERG cDNA (SEQ ID NO:26) was kindly provided by Dr. M. Keating. Kv2.2 (SEQ ID NO:3) was a gift from Drs. S. Snyder and J. Trimmer. Kir2.2 cRNA (SEQ ID NO:27) was prepared as previously described (Wible, B.A., et al. "Cloning and functional expression of an inwardly rectifying K+ channel from human atrium" Circ Res 76:343-350, 1995). We also used a cRNA encoding Kv2.1ΔN (SEQ ID NO:28) in which the N-terminal 139 amino acids had been deleted (VanDongen, A.M.J., et al. "Alteration and restoration of K+ channel function by deletions at the - and C-termini" Neuron 5:433-443, 1990).

Measurements of Xenopus oocyte whole-cell currents were performed using the standard two-microelectrode voltage clamp technique as described previously (Heinemann, S.J., et al. "Molecular and functional characterization of a rat brain Kv beta 3 potassium channel subunit" EEES Lett 377:383-389, 1995). In mouse L cells, K⁺ currents were measured using the whole-cell configuration of the giga-seal recording technique (Wible, B.A., et al. "Cloning and functional expression of an inwardly rectifying K+ channel from human atrium" Circ Res 76:343-350, 1995). The pipette solution contained (in mM): 140 KAspartate, 5 MgCl₂, 10 HΕPΕS, 10 ΕGTA, 10 glucose, and 2 Na₂ATP at pH 7.2. The bath solution contained (in mM): 140 NaCI, 5.4 KCl, 1 MgCl₂, 2 CaCl₂, 10 HΕPΕS and 10 glucose at pH 7.4. Data acquisition and analyses were performed with pClamp 5.5.1 software (Axon Instruments, CA). In oocytes, linear leakage and capacity transient currents were subtracted using a P/4 prepulse protocol. Records were low-pass filtered at 2 or 5 kHz and digitized at 10 kHz. Experiments were conducted at room temperature (20-22 °C). Data are reported as mean ± S.E.M. Comparisons between two groups of cells were performed by t-test. Comparisons between multiple groups of cells were performed by oneway analysis of variance test and Student-Newman- euls post hoc test. Means are consid- ered to be significantly different when p<0.05.

In vitro transcription of cRNAs and oocyte injection. Kv4.3 was obtained by RT- PCR from rat brain RNA using primers A (SEQ ID NO:29) and B (SEQ ID NO:30), and subcloned into pSP64 for cRNA synthesis. Mouse Kvl .3 cDNA in pSP64 was purchased from ATCC. KChAP with an N-terminal extension of 45 amino acids (MVMSFRVSELQVLLGFAGRNKSGRKHELLAKALHLLKSSCAPSVQ, SEQ ID NO:31) was amplified by PCR from our original rat brain cDNA clone, and subcloned into pSP64. We had originally assigned KChAP methioninc 46 as the initiating residue based on the presence of an in-frame upstream stop codon (Wible, B.A., et al. "Cloning and expression of a novel lC channel regulatory protein, KChAP" J Biol Chem 273: 1 1745-1 1751, 1998). After a DNA sequencing rror upstream of this position was discovered which removes this stop codon, it now appears likely that the start site is 45 residues upstream. The original KChAP as well as KChAP with the N-tεrminal extension at present exhibit no detectable differences in their binding in yeast two-hybrid assays or effect on Kv channels in heterologous expression assays. cRNAs were prepared and injected as previously described (Heinemann. S.J., et al. "Molecular and functional characterization of a rat brain Kv beta 3 potassium channel subunit" FEBS Lett 377:383-389, 1995; Accili, E.A., et al. "Separable Kvbeta subunit domains alter expression and gating of potassium channels" Biol Chem 272:25824-25832, 1997).

Cell lines and injection. Mouse L cells were grown in minimum essential medium with 10% fetal bovine serum. 100 u/ml penicillin and 100 μg/ml streptomycin (Life Technologies, Inc.). L cells stably transfected with cither Kvl .l, Kvl .5. Kv4.3, or HERG cDNAs were isolated using methods as previously described (Nagaya, N. and Papazian, D.M. "Potassium channel alpha and beta subunits assemble in the endoplasmic reticulum" J Biol Chem 272.3022-3027, 1997), and maintained in media containing 0.5 mg/iril G418 (Life Technologies Inc). L cells .vere injected with an Eppendorf 5242 niicroinjector and 5171 micromanipulator (Madison, Wl) as described previously (Chan, tv.W., ei al. "Specific regions of heteromeric subunits involved in enhancement of G protein-gated K+ channel activity" J Bϊoi Chem 272:6548-6555, 1997). Bottoms of 35 mm plastic dishes were la- beled with circles (1 mm diameter) and cells were plated at a density of about 20 cells per circle.

Transient Transfection. L cells stably expressing Kv4.3 (L/Kv4.3) were plated one day prior to transfection on poly-L-lysine coated coverslips. For transient transfection with EGFP-C2, EGFP tagged full-length KChAP or KChAP fragments, cDNAs were mixed in a 1 :5 ratio with lipofectin (Life Technologies, Inc) and incubated with the cells for four hours at 37°C. In some experiments, polyethylenimine (PEI) (Fluka) was used as the lipid carrier (Feldherr, CM., et al. "Manual enucleation of Xenopus oocytes" Methods Cell Biol 17:75- 79, 1978). Tissue and oocyte lysate preparation. Freshly dissected adult rat hearts (Sprague-

Dawley) were minced and placed into ice-cold lysis buffer (1:7, w/v) containing (in mM) 150 NaCI, 50 Tris, 1 EDTA, 0.2% BSA, 1% Triton X-100, pH 7.5, supplemented with a protease and phosphatase inhibitor cocktail (Complete, Boehringer-Mannheim plus 50 mM sodium fluoride and 0.2 mM sodium vanadate). Samples were homogenized with a Polytron at setting 6 for 5 sec. After a 1 h incubation on ice, the lysates were centrifuged at 900 g for 10 min to remove insoluble material. Xenopus oocytes were homogenized with 20 strokes in a glass homogenizer in lysis buffer (20 μl/oocyte), incubated on ice for 1 h, and insoluble debris removed by centrifugation at 3000 x g. Protein concentrations were determined by the BCA method (Pierce). Antibody Production. A bacterial fusion protein consisting of maltose-binding protein (MBP; New England Biolabs) and the C-terminus of KChAP (residues 453-619, SEQ ID NO: 10) was purified on amylose resin and sent to Research Genetics for polyclonal antibody production. IgG was purified on a Protein-G Sepharose column (Pharmacia), passed over an MBP affinity column, and anti-KChAP reactivity immunopurified on a KChAP:MBP affinity column.

Example 1 Isolation of a novel Kvβ and Kv -subunit binding protein with the yeast two-hybrid system. Full-length Kvβl .2 (SEQ ID NO:6) was used as bait to screen a rat brain cDNA library in the GAL4 activation domain vector, pGADIO. We isolated one clone which exhibited a strong positive signal in the β-galactosidase assay. pGADIO plasmid DNA containing a 1.78 kb insert was isolated from this clone and tested positive for interaction with Kvβl .2. Sequence analysis of the clone which we termed KChAP-Y, for K Channel Associated Protein (Y refers to the fragment isolated in the yeast two-hybrid screen), revealed a novel clone with no similarity to Kvα or Kvβ subunits.

We tested the specificity of interaction of KChAP-Y with a panel of Kvβ, Kvα, and other KA channel subunit fragments with the yeast two-hybrid assay. As shown in Figure 1, KChAP-Y interacted with both Kvβ 1.2 and Kvβ2 subunits. KChAP-Y interacted with the conserved Kvβl C-terminus but not the unique N-terminus of Kvβ 1.2 suggesting that the protein may recognize conserved sequences among Kvβ subunits. Kvβ subunits interact specifically with the N-terminus of Kvlα subunits so we tested these fragments for binding to KChAP-Y as well. Surprisingly, a positive signal was observed between the N-termini of Kvl.1, Kvl.2, Kvl.4, and Kvl.5 and KChAP-Y. Just as with the Kvβ subunits, however, no interaction was evident between the Kvl.4 C-terminus and KChAP-Y. KChAP-Y also interacted with the N-termini of Kv2.1 and Kv2.2, but not with the N-terminus of the electrically silent Kv2 partner, Kv6.1 (Post, M., et al. "Kv2.1 and electrically silent Kv6.1 potassium channel subunits combine and express a novel current" FEBS Lett 399:177-182, 1996). Further specificity for a subset of Kv channels was apparent from the lack of interaction with the N-terminus of the inward rectifier K⁺ channel, Kir2.2, and the N-terminus of the delayed rectifier K⁺ channel, HERG. Thus, KChAP-Y apparently interacts with both the C-terminus of Kvβ subunits as well as the N-termini of Kvl and Kv2 α-subunits.

Example 2

Cloning and sequence analysis of full-length KChAP. Screening of a rat brain cDNA library with the KChAP-Y coding sequence produced a 3.2 kb insert which overlapped KChAP-Y and contained a single open reading frame of 574 amino acids. The initiating methionine was assigned as the first ATG downstream from three in frame stop codons. Hydropathy analysis indicated no potential membrane spanning domains in KChAP suggesting that the protein was cytoplasmic (not shown).

Search of the GenBank nonredundant database revealed significant homology with the mammalian gene encoding Gu binding protein (GBP) (Naldez, B.C., et al. "Cloning and characterization of Gu/RH-II binding protein" Biochen Biophys Res Comrnun 234:335-340, 1997). GBP was isolated originally in a yeast two-hybrid screen as a protein which binds to the Gu/RΝA helicase II subunit. Alignment of KChAP with GBP is presented in Figure 2. GBP has an Ν-terminal extension of 55 to 57 amino acids compared to KChAP, but over the 574 amino acid open reading frame of KChAP, the two proteins are 50% identical. We tested the binding of both full-length KChAP (amino acids 1-574) and human GBP (amino acids 49-645) with K⁺ channel fragments in the yeast two-hybrid assay as was described for KChAP-Y in Figure 1. The first ATG after three in frame stop codons was chosen as the initiating methionine in KChAP. The start codon in GBP has not been deter- mined but may be one of the two methionines (residue number 4 or 6) marked in bold and indicated with asterisks (Valdez et al, 1997). The arrow above KChAP tryptophan residue 310 (W310) indicates where the KChAP-Y fragment begins. The symbol (^e) above KChAP leucine 407 (L407) marks the start of the coding sequence used for construction of a riboprobe for Northern blot analysis. KChAP and GBP share two putative protein kinase A phosphorylation sites at KChAP positions SI 85 and T309 which are bolded and underlined. Full-length KChAP was identical to KChAP-Y in its interaction with protein partners in the yeast two hybrid assay, while GBP did not interact with any of the tested fragments including Kvβ and Kvα subunits (data not shown). Thus, although KChAP shares significant homology with GBP, interaction with Kvβ and Kvα subunits appears to be a unique feature of KChAP.

Example 3 Northern blot analysis. The expression of KChAP mRNA was examined in a panel of rat tissues. A rat Multiple Tissue Northern blot (2 μg poly A⁺ RNA per lane) from Clontech was probed with a ³²P-labeled riboprobe prepared from a fragment of KChAP cDNA encoding the C-terminal 167 amino acids. Hybridization was done overnight in NorthemMax hybridization buffer (Ambion) at 68°C. Washes in 0.1 x SSC/0.1% SDS were done at 70°C. Autoradiography was for five hours at -70°C with Kodak Biomax MS film and intensifying screen. RNA size markers are indicated on the left. The blot was probed with a fragment of KChAP encoding amino acids L407-D574, a region with minimal homology to GBP to avoid detecting GBP transcripts as well. As shown in Figure 3, a single band of -3.2 kb was detected in a variety of tissues including heart and brain with especially high levels in lung and kidney. Example 4 Functional characterization of KChAP -Kv interactions. The surprising finding that KChAP associated with Kvαl and Kvα2 subunits as well as Kvβ subunits led us to examine the functional consequences of KChAP:K⁺ channel interaction upon heterologous ex- pression in Xenopus oocytes. Whole-oocyte currents were recorded by two electrode voltage-clamp from eggs injected with cRNAs encoding different Kvα-subunits alone or with saturating concentrations of KChAP. Coexpression with KChAP produced a dramatic three-fold increase in the amplitude of Kv2.1 currents. Figure 4A shows the averaged macroscopic currents from 8 oocytes in one injection series measured on day 6 post-injection in oocytes injected with Kv2.1 cRNA (0.62 ng/μl) alone (left) or one coinjected with Kv2.1 and KChAP cRNAs (0.62 ng/μl and 250 ng/μl, respectively) (right). Holding potential was -80 mV and pulses were from -70 mV to +80 mV in 10 mV steps; 50 mM K⁺ in bath solution. No change in Kv2.1 currents was apparent when the channel was coexpressed with GBP (data not shown). At more depolarized potentials Kv2.1 has an opening proba- bility of about 0.9 (Shieh, C.C, et al. "Role of transmembrane segment S5 on gating of voltage-dependent K+ channels" J Gen Physiol 109:767-778, 1997), suggesting that the increased currents recorded when KChAP was coexpressed were probably due to an increase in the number of functional channels. KChAP also interacted with the N-terminus of Kvl.5, but, in contrast to Kv2.1, produced no significant change in Kvl.5 currents when coexpressed in oocytes. Figure 4B shows the averaged macroscopic currents from 10 oocytes in one injection series measured five days post-injection from oocytes injected with Kvl.5 cRNA (50 ng/μl) alone (left) or Kvl .5 plus KChAP cRNAs (50 ng/μl and 500 ng/μl, respectively) (right). Holding potential was -80 mV and pulses were from -70 mV to +70 mV in 10 mV steps; 5 mM K⁺ in bath solution. The experiments were done so that Kvl .5 expressed whole-cell currents at +70 mV in the range of 0.5 to 5 μA. This greatly reduced the possibility that amplitude changes might be missed as a result of voltage-clamp difficulties. Thus, while KChAP interacted with the N-termini of both Kv2.1 and Kvl .5 in the yeast two-hybrid assay, KChAP only produced amplitude increases in Kv2.1 currents in oocyte expression assays. Figure 4C summarizes the effects of KChAP on the current amplitudes of a variety of K⁺ channels. In 13 batches of oocytes coinjected with both Kv2.1 and KChAP, we observed an average increase in whole-oocyte currents of about 2.5 fold compared to oocytes expressing Kv2.1 alone. KChAP also produced comparable increases in Kv2.2 currents. We also examined the functional expression of a deleted Kv2.1 in which the N-terminal 139 residues were removed (VanDongen, A.M.J., et al. "Alteration and restoration of K+ channel function by deletions at the - and C-termini" Neuron 5:433-443, 1990). As shown in Figure 4C, coexpression with KChAP did not significantly alter current amplitudes in oocytes expressing Kv2.lΔN. Bar plot shows averaged current levels in the presence of KChAP as fractions of currents in the absence of KChAP (control current). The numbers above eadi bar indicate the number of batches of oocytes examined for each K⁺ channel. Oocytes were injected with either K⁺ channel cRNAs or K⁺ channel plus KChAP cRNAs, and currents were recorded from 8-12 oocytes in each batch. Whole-oocyte currents were measured two days after injection (Kir2.2 and Kv2.lΔN) or five to six days after injection (Kv2.1 , Kv2.2, Kvl .5, and HERG), and the ratio of means (I_coinjectec/I-_ontro-) calculated. For Kv2.1 , Kv2.2, and Kvl.5, the holding potential was -80 mV. Steady-state currents were measured at a test potential of +70 mV (5 or 50 K⁺ in bath). Kir2.2 steady-state and HERG tail currents were recorded with 50 K⁺ in the bath at test potentials to -100 mV with a pre- pulse to +20 mV. (*) indicates that in all injection series, current amplitudes in oocytes coinjected with KChAP were significantly higher than in oocytes without KChAP (t-test, p<0.05). This suggests that binding between the Kv2.1 N-terminus and KChAP is critical for current enhancement.

Two K⁺ channels which did not exhibit N-terminal binding to KChAP, Kir2.2 and HERG, were also tested. For each channel, experiments were done with whole-cell control inward currents not exceeding 10 μA at -100 mV. As shown in Figure 4C, neither channel exhibited altered current amplitudes in the presence of KChAP.

Example 5 KChAP increased functional expression of Kv2.1 without altering channel kinetics or gating. The expression enhancement of Kv2.1 currents in the presence of KChAP could be due to an increase in the number of functional channels at the cell surface or an alteration in the kinetics or gating of individual channels. To distinguish between these mechanisms, we used both immunocytochemical and electrophysiological methods. We examined the surface expression of Kv2.1 protein in oocytes expressing either Kv2.1 alone or Kv2.1 plus c-/?;yc-KChAP by immunocytochemistry. Figure 5 shows the whole cell currents recorded from a single oocyte injected with Kv2.1 alone (panel A) or Kv2.1 plus c-myc- KChAP (panel B). Currents were increased about three-fold in the c-myc-KChAP coinjected egg. Macroscopic currents recorded five days after cRNA injection from an oocyte expressing Kv2.1 (1.25 ng/μl cRNA; panel A) or one expressing Kv2.1 (1.25 ng/μl) plus c-/»yc-KChAP (250 ng/μl; panel B). Recordings were obtained by stepping from a holding potential of -80 mV with 10 mV steps from -70 to +80 mV. The same two oocytes were fixed, sectioned, and co-stained with Kv2.1 and c-myc antibodies (Figure 5, panels C and D). Fluorescence at the oocyte surface was much brighter in the egg expressing both Kv2.1 and KChAP (Figure 5, panel D) compared to the one expressing Kv2.1 alone (Figure 5, panel C) suggesting that the amount of Kv2.1 protein at the cell surface was increased when the channel was coexpressed with KChAP. The oocyte section in panel D was co-stained with anti-c-myc and a TRITC-conjugated secondary antibody. No TRITC fluorescence was visible at the cell surface suggesting that c-myc- KChAP is not present there with Kv2.1 (Figure 5, panel E).

To determine whether KChAP increased the total expression of Kv2.1 protein or only altered the subcellular distribution of the channel, we examined microsomal fractions from oocytes injected with Kv2.1 alone or Kv2.1 plus KChAP by Western blotting. As shown in Figure 6A, the amount of Kv2.1 protein in oocyte microsomes was increased in oocytes coinjected with KChAP (compare lanes 1 and 2). Densitometry of the blots indicated about a 2.5 fold increase in Kv2.1 protein in the presence of KChAP. Microsomal protein (10 μg) prepared five-days post injection from oocytes injected with cRNAs for KChAP (250 ng/μl) plus Kv2.1 (5 ng/μl) (lane 1), Kv2.1 cRNA (5 ng/μl) alone (lane 2), uninjected oocytes (lane 3), or adult rat brain (lane 4) was separated by SDS-PAGE, blotted to PVDF, and Western blotted with a monoclonal antibody to Kv2.1. Immunoreactive bands were visualized with ECL-Plus (Amersham). Molecular weight markers (kD) are indicated on the left, and the position of Kv2.1 is marked on the right. As a control for the antibody, two bands are visible in the rat brain preparation. The upper band is thought to be a phosphorylated form of the channel. No Kv2.1 is detected in uninjected oocytes. Note that there is significantly more Kv2.1 detected in oocytes expressing both Kv2.1 and KChAP. Similar results were obtained when Kv2.1 was immunoprecipitated from homogenates of total oocyte protein (data not shown). This value is comparable to the increase observed in Kv2.1 currents with KChAP.

Since no KChAP was detected at the cell surface of co-expressing oocytes, we examined the cellular distribution of KChAP in oocytes by Western blotting. At 3 days post- injection we manually removed the nuclei from pools of oocytes expressing only Kv2.1 or Kv2.1 plus c-m c-KChAP, and probed the nuclear fraction as well as the soluble and microsomal fractions prepared from the enucleated oocytes with an anti-c-.?zyc antibody to detect tagged KChAP (Figure 6B). Equal amounts (10 μg) of solubilized nuclear protein from Kv2.1 oocytes (lane 1) or Kv2.1 + KChAP oocytes (lane 2) was compared to soluble protein (Kv2.1 oocytes, lane 3; Kv2.1 + KChAP oocytes, lane 4) and microsomal protein fractions (Kv2.1 oocytes, lane 5; Kv2.1 + KChAP oocytes, lane 6) prepared from enucleated oocytes. Anti-c-myc antibody detected a major band of c-myc-KChAP (-68 kD; indicated to right of blot) only in oocytes coinjected with c-rnvc-KChAP cRNA. Molecular weight markers (kD) are marked on the left. Most of the KChAP protein was present in the nucle- ar fraction with smaller amounts detectable in the soluble as well as the microsomal fractions. The signal was so strong in the nuclear material compared to the other two fractions, however, that we were not able to estimate the relative amounts in each fraction. No Kv2.1 was detected in the nuclear fraction indicating that contamination with non-nuclear membranes was minimal (data not shown). The kinetics and gating of Kv2.1 channels were not altered in the presence of

KChAP. In Figure 7A, normalized and averaged values of steady-state currents plotted as a function of test potential in oocytes injected with the following cRNAs: Kv2.1 alone (0.62 ng/μl), filled circles (n=10); Kv2.1 plus KChAP (125 ng/μl), filled triangles (n=9). Inset: superimposition of averaged and scaled currents (at +70 mV test potential) from oocytes injected with Kv2.1 alone and Kv2.1 plus KChAP. Holding potential was -80 mV. 100 ms pulses were given in 10 mV steps from -70 mV to +80 mV. 50 mM K⁺ in bath solution. Recordings were performed six days after injection, and are from one batch of oocytes. Values of τ_{actl alion} and τ_deactivation when pulsing to +70 mV and then back to -80 mV were 16.7 + 0.2 and 5.8 + 0.1 ms (n=10), respectively, for Kv2.1 alone, and 15.8 + 0.5 and 6.2 + 0.2 ms (n=9), respectively, for Kv2.1 plus KChAP. As shown in Figure 7A, the voltage- dependence of activation and the kinetics of activation and deactivation of Kv2.1 channels were not changed. Thus, coexpression with KChAP did not alter the sensitivity of Kv2.1 channels to TEA either (data not shown). Therefore, KChAP increased the number of functional channels at the cell surface without altering individual Kv2.1 channel kinetics. The effect of KChAP on Kv2.1 currents was saturable as shown in Figure 7B.

Current amplitudes were measured at the end of the pulse to +70 mV. In oocytes injected with Kv2.1 cRNA alone (0.62 ng/μl), the current was 10.7 ± 0.9 μA; in oocytes coinjected with KChAP cRNA, the currents were 23.2 + 2.5, 39.5 + 6.8, 43.7 + 4.4, and 49.7 + 6.2 μA at KChAP cRNA concentrations of 15, 31 , 62, and 125 ng/μl, respectively; numbers of oocytes are indicated (the same batch of oocytes as in A). (*) indicates a significant difference from control Kv2.1 (p<0.05; one-way ANOVA/SNK test). Thus, coexpression of a constant amount of Kv2.1 cRNA with increasing amounts of KChAP cRNA resulted in increased steady state Kv2.1 currents until saturation was reached.

The influence of KChAP on the time course of Kv2.1 expression in oocytes over a period of nine days post-injection is shown in Figure 7C. Currents were measured at the end of a 200 ms pulse to +70 mV from oocytes injected with cRNAs for: Kv2.1 alone (0.62 ng/μl, filled circles); Kv2.1 plus KChAP (125 ng/μl, filled triangles). Numbers of oocytes are indicated in parentheses above the points. Average currents in oocytes injected with Kv2.1 cRNA alone were 2.8 + 0.7, 6.1 + 1.9, and 5.3 + 1.5 μA measured at 3, 6, or 9 days post-injection, respectively. Oocytes coinjected with Kv2.1 and KChAP cRNAs had average currents of 6.8 + 2.8, 15.7 + 4.4, and 19.9 + 5.6 mA measured at 3, 6, or 9 days postinjection, respectively. (**) indicates a significant difference from Kv2.1 tested on the same day after injection (p<0.05; t-test). Therefore, in all of the electrophysiological experiments presented thus far, KChAP and Kv2.1 cRNAs were mixed prior to injection into oocytes in the same pipette. Importantly, when we injected the two cRNAs separately into the same oocyte with different pipettes, no enhancement of Kv2.1 currents was observed (data not shown). This result suggests that KChAP exerts its effect on Kv2.1 in oocytes by a direct physical association with the channel which is facilitated by the coinjection of both cRNAs into the same space inside the oocyte.

Example 6 In vitro association of KChAP and Kv2.1. The yeast two-hybrid and electrophysiological data suggest that a direct interaction between Kv2.1 and KChAP occurs and is responsible for the enhancement in Kv2.1 currents observed in oocytes. We used an in vitro binding assay to demonstrate the ability of the two proteins to associate. Kv2.1 and KChAP cRNAs were translated in vitro either separately or together in a rabbit reticulocyte lysate in the presence of ³⁵S-methionine. Immune complexes were analyzed by SDS-PAGE and fluorography. We used a commercially available anti-Kv2.1 polyclonal antisera to immunoprecipitate Kv2.1, and analyzed the immunoprecipitated material with SDS-PAGE and fluorography to detect the presence of associated KChAP. Since an antibody to KChAP was not available, we used an epitope tag fused to the N-terminus of KChAP (c- myc) to allow detection. As we had previously shown in Figure 5, the c-myc tag did not interfere with the functional interaction of KChAP and Kv2.1. The following in vitro translation reactions (Figure 8) were used for immunoprecipitation: Kv2.1 translated alone (lanes 1 ,2), c-myc- ChAP translated alone (lanes 3,4), Kv2.1 and c-myc-KChAP translated in the same reaction (lane 5), and Kv2.1 and c-.-zyc- KChAP translated separately but mixed together prior to immunoprecipitation (lane 6). Control reactions with Kv2.1 translated alone showed that in vitro translated Kv2.1 was immunoprecipitated with anti-Kv2.1 antibody but not anti-c-/7-yc antisera (Figure 8, lanes 1 and 2). Similarly, anti-c-myc antisera immunoprecipitated c-m c-KChAP but not Kv2.1 (Figure 8, lanes 3 and 4). Kv2.1 anti- body coimmunoprecipitated complexes of Kv2.1 and KChAP when the two cRNAs were cotranslated (Figure 8, lane 5) but not when the two cRNAs were translated in separate reactions and mixed together prior to the addition of primary antibody (Figure 8, lane 6). Without restricting the invention to any mechanism, this result suggests that the association of KChAP with Kv2.1 occurs cotranslationally since the mature proteins added after transla- tion did not coimmunoprecipitate. All reactions involving translation of Kv2.1 cRNA shown here included canine pancreatic microsomes to allow the channel to insert into membrane as it was synthesized. When microsomes were omitted from cotranslation reactions, no coimmunoprecipitation of the two proteins was observed (data not shown).

Example 7

KChAP increases the functional expression of Kvl.3 and Kv4.3 in Xenopus oocytes. We showed previously that KChAP produced significant increases in Kv2.1, but not Kvl .5, currents when coexpressed in Xenopus oocytes (Wible, B.A., et al. "Cloning and expression of a novel K^" channel regulatory protein, KChAP" J Biol Chem 273: 11745- 11751, 1998). Here we examined the effect of KChAP on other Kv channels when both cRNAs were expressed in Xenopus oocytes. Kvl.3 current amplitude was increased about two-fold when the channel was coexpressed with KChAP. Figure 9A shows whole-cell currents measured 24 hours after injection in oocytes injected with Kvl .3 cRNA (0.5 ng/μl) alone (left) or coinjected with KChAP cRNA (125 ng/μl) (right). Holding potential was -80 mV and 100 ms pulses were from -70 mV to +80 mV in 10 mV steps; 50 mM K ^" in bath solution. As shown in Figure 9B, this increase occurred in the absence of changes in channel gating or voltage-dependence of activation. Figure 9B shows normalized and averaged peak currents plotted as a function of test potential in oocytes (same injection series as in A) injected with Kvl.3 cRNA alone (0.5 ng/μl; filled circles) or plus KChAP cRNA (125 ng/μl; filled triangles). Activation (from -80 to +70 mV) and deactivation (from +70 to -80 mV) time constants of currents in oocytes injected with Kvl .3 cRNA alone were 4.0 + 0.1 and 13.6 + 0.2 ms (n=16), respectively, while in oocytes coinjected with Kvl .3 and KChAP cRNAs, the time constants were 4.1 + 0.1 and 13.3 + 0.3 ms (n=15), respectively. The effect of KChAP on Kvl .3 current amplitude was dose dependent with increasing concentrations of KChAP cRNA producing increasing currents until saturation was reached. Figure 9C shows dose dependence of KChAP cRNA on Kvl.3 expression. Current amplitudes were measured at pulses to +70 mV. Oocytes were injected with Kvl .3 cRNA (0.5 ng/μl) alone or conjugated with increasing amounts of KChAP cRNA (15, 62 and 250 ng/μl; n=l 0 for each concentration. We reported previously that KChAP had no effect on Kir2.2 currents (an inwardly rectifying K⁺ channel) (Wible, B.A., et al. "Cloning and expression of a novel K⁺ channel regulatory protein, KChAP" J Biol Chem 273:11745-11751, 1998). As an additional control of KChAP specificity, we coinjected oocytes with a mix- ture of Kvl .3 and Kir2.2 cRNAs with or without KChAP. In the presence of KChAP, outward current from Kvl.3 channels was significantly increased, while, in the same oocytes, the amplitude of the inward current from Kir2.2 channels was unchanged. Figure 9D shows whole-cell currents measured 24 hours after injection in oocytes coinjected with Kvl.3 cRNA (0.5 ng/μl) and Kir2.2 cRNA (10 ng/μl) (left) or coinjected with KChAP cRNA (125 ng/μl) (right). Holding potential was -80 mV and 100 ms pulses were from -90 mV to +90 mV in 20 mV steps; 50 mM K⁺ in bath solution (same batch of oocytes as in A). Figure 9E shows a bar plot of averaged currents shown in A and D. Currents through Kvl .3 channels (filled bars) were measured at +70 mV and currents through Kir2.2 channels (open bars) were measured at -70 mV. Numbers of oocytes are indicated in pa- rentheses above the bars. (*) indicates a significant difference from currents in oocytes without KChAP.

We demonstrated previously that KChAP was able to bind to the N-terminus of both Kv2.1 and Kvl .5 channels (Wible, B.A., et al, "Cloning and expression of a novel K⁺ channel regulatory protein, KChAP" J Biol Chem 273:1 1745-11751, 1998). Since we saw a significant increase in Kvl .3 currents in the presence of KChAP, we tested the N-terminus of Kvl .3 for its ability to bind to KChAP. A yeast two-hybrid interaction assay indicated that Kvl .3-N was able to interact with KChAP. As shown in Figure 9F, growth on minus histidine selection media as a result of the activation of the HIS3 reporter gene was indicative of interaction between the two proteins.

Kvl .3 is unique among the Kvl subfamily members that we have studied in its sensitivity to KChAP. A thorough examination by heterologous expression in Xenopus oocytes revealed no enhancement of Kvl .2, Kvl .4, Kvl .5, or Kvl .6 current amplitudes by KChAP (Figure 10), even though the N-termini of Kvl.2, Kvl.4, and Kvl.5 interacted with KChAP in yeast two-hybrid assays (Wible, B.A., et al. "Cloning and expression of a novel K⁺ channel regulatory protein, KChAP" J Biol Chem 273:11745-11751, 1998). We expanded our analysis to additional Kv subfamilies and found that Kv3.1 currents were unaffected by KChAP, but that the amplitude of Kv4.3 currents increased significantly. Figure 10 shows a bar plot showing average results from independent injection series. The numbers above bars indicate the number of injection series (different batches of oocytes) for each K⁺ channel. Oocytes were injected either with K⁺ channel cRNA alone (control) or plus KChAP cRNA. In each series, currents from 6-12 oocytes were measured, and the ratio of means (I_C01-_njected / I_controi) ^was calculated. Peak (Kv4.3 and Kvl .4) or steady-state (other Kv channels) currents were measured at a test potential of +70 mV (5 or 50 mM K⁺ in bath). Kir2.2 steady-state and HERG tail currents were recorded at test potentials to -100 mV with a pre-pulse to +20 mV (50 mM K⁺ in bath). Measurements were made on post-injection day 1 for Kvl .3 and Kvl.6; day 2 for Kvl .2, Kv3.1, Kv4.3 and Kir2.2; and day 5 or 6 for Kvl.4, Kvl .5, Kv2.1 and HERG. Coexpression of KChAP with Kvl.3 produced significant increased in current amplitude in 10 of 14 batches of oocytes (in every batch, currents were measured in 6-10 control and coinjected oocytes). Significant increases were obtained in 7 of 12 batches of oocytes (for Kv4.3) and 18 of 22 batches (for Kv2.1). Average increases in all tested batches was 2.04 ± 0.29, 2.27 + 0.20 and 1.87 + 0.45 times for Kvl.3, Kv2.1 and Kv4.3, respectively. The magnitude of the KChAP effect was dependent upon the particular batch of oocytes, but all three channels behaved similarly when examined in the same batch of oocytes. Current increases were never observed when KChAP was tested with Kvl .2, Kvl .4, Kvl .5, Kvl .6, Kv3.1 , Kir2.2, or HERG. In fact, coinjection with KChAP resulted in significant current suppression for Kvl.2 (in 7 of 8 oocyte batches) and Kvl .4 (2 of 2). Due to nonspecific transactivation of the reporter genes by Kv3.1 -N and Kv4.3-N in yeast two-hybrid assays, we were not able to use this method to determine whether KChAP interacted directly with either channel. A detailed examination of the effects of KChAP on Kv4.3 currents in Xenopus oocytes is presented in Figure 1 1. In the presence of KChAP, Kv4.3 currents were increased about two-fold (Fig. 11 A; whole-cell currents measured post-injection day 2 in oocytes injected with Kv4.3 cRNA (10 ng/μl) alone (left) or coinjected with KChAP cRNA (500 ng/μl) (right). Holding potential was -90 mV and 200 ms pulses were from -70 mV to +80 mV in 10 mV steps; 5 mM K⁺ in bath solution), while the kinetics of activation and inactivation were unchanged (Fig. 11B; normalized and averaged peak currents plotted as a function of test potential in oocytes (same injection series as in A) injected with Kv4.3 cRNA alone (10 ng/μl; filled circles) or plus KChAP cRNA (500 ng/μl; filled triangles). Inset: superimposition of averaged and normalized currents from oocytes injected with Kv4.3 alone and Kv4.3 plus KChAP at +70 mV test potential. Figure 1 IC; steady-state inactivation curves of currents from oocytes injected with Kv4.3 cRNA alone (n = 5; filled circles) or plus KChAP cRNA (n = 5; filled triangles ). At depolarizing test potentials from -80 to +70 mV, activation constants were 1.6 + 0.3 and 1.6 + 0.2 ms for currents recorded from oocytes injected with Kv4.3 cRNA alone (n=24) and Kv4.3 plus KChAP cRNAs (n=24), respectively. Inactivation constants were 40.1 + 0.3 ms (Kv4.3 alone; n=24) and 40.8 + 0.4 ms (Kv4.3 plus KChAP; n=24). The values of half-maximal inactivation (V₀₅) were -45.6 + 0.6 mV and -46.8 + 0.2 mV and the slope factors (k) were -5.8 + 0.1 mV and -6.0 ± 0.1 mV for currents from oocytes injected with Kv4.3 cRNA alone (n=5) and Kv4.3 plus KChAP cRNAs (n=5), respectively. As with Kvl.3, KChAP expression enhancement of Kv4.3 currents was dose-dependent and saturable with increasing amounts of KChAP cRNA (Fig. 11D; peak current amplitudes were measured at pulses to +70 mV. Oocytes were injected with Kv4.3 cRNA (10 ng/μl) alone or with increasing amounts of KChAP cRNA (31, 62, 125 and 500 ng/μl ; n = 12 for each concentration; same injection series as in A and B). (*) indicates a significant difference from the value for Kv4.3 alone.). Coexpression with KChAP did not alter the time course of Kv4.3 expression in oocytes which peaked at day 2 and decreased thereafter (Fig. HE; peak currents were measured at a pulse to +70 mV from oocytes injected with Kv4.3 cRNA alone (10 ng/μl; filled circles, n=10 for each point) or plus KChAP cRNA (250 ng/μl; filled triangles, n=10 for each point). (**) indicates a significant difference from Kv4.3 alone on the same day after injection ) suggesting that, if there were more Kv4.3 channels at the cell surface as for Kv2.1 (16), their stability at the surface was not altered. Example 8 Transcription is not required for KChAP modulation of Kv channels in Xenopus oocytes. KChAP is localized primarily to the nucleus when expressed in Xenopus oocytes (Wible, B.A., et al. "Cloning and expression of a novel K⁺ channel regulatory protein, KChAP" J Biol Chem 273:11745-11751, 1998). In spite of our evidence that KChAP and certain Kv channels interact directly (Wible, B.A., et al. "Cloning and expression of a novel K⁺ channel regulatory protein, KChAP" J Biol Chem 273:11745-1 1751, 1998), we had to consider the possibility that nuclear KChAP was modulating Kv channels in oocytes indirectly through a mechanism which involved transcription. To test this possibility, we exam- ined KChAP effects in oocytes incubated with actinomycin D, an inhibitor of transcription. Xenopus oocytes were injected with cRNAs: Kvl.3 (0.5 ng/μl) alone or plus KChAP cRNA (125 ng/μl) (Fig. 12A) or with Kvl .3 cDNA (1.6 ng/μl) (Fig. 12B). After injection oocytes were divided into two groups and incubated for 24 hrs in media with or without actinomycin D (50 μg/ml). Currents were measured as described above (see Fig. 9). (*) indicates significant suppression of Kvl.3 current by actinomycin D in oocytes injected with Kvl.3 cDNA. Similar results were obtained on 4 batches of oocytes with Kvl.3. Figure 12A shows that KChAP increased Kvl .3 currents irrespective of whether the oocytes were incubated with actinomycin-D after injection or not. To show that actinomycin-D was working, we injected Kvl.3 cDNA into oocyte nuclei. Figure 12B shows, as expected, actinomycin- D significantly reduced Kvl.3 currents in comparison to control. We observed similar results with Kv2.1 cRNA, cDNA, and KChAP (not shown). Thus, transcription is not required for KChAP to increase Kv channel amplitudes.

Example 9 KChAP increases Kv2.1 and Kv4.3 currents in transfected mammalian cells. Thus far, all of our functional assays of KChAP have been in Xenopus oocytes. We wanted to determine whether the oocyte observations could be replicated in mammalian cells. In the first set of experiments, KChAP and Kv2.1 cRNAs were microinjected into mouse L cells. Non-injected cells or cells injected with 100 mM KCl had very small outward currents whereas cells injected with Kv2.1 cRNA exhibited voltage-dependent outwardly rectifying current (Fig. 13A). In Figure 13A whole-cell currents in L cells injected with either 100 mM KCl, Kv2.1 cRNA (12.5 ng/μl), or Kv2.1 plus KChAP cRNAs (12.5 and 250 ng/μl, respectively). Holding potential was -80 mV, and 300 ms pulses were from -70 to +70 mV with 10 mV steps. Recordings were made one day after injection. In Figure 13B the bar plot showing the effect of KChAP on Kv2.1 current density at +70 mV. (*) indicates a significant difference from cells injected with Kv2.1 cRNA alone. Numbers of cells are indicated above the bars. Thus, the current density in cells coinjected with a mixture of Kv2.1 and KChAP cRNAs was significantly higher then in cells injected with Kv2.1 cRNA alone (Fig. 13 A, B).

To examine the effect of KChAP on Kv4.3 in mouse L cells, we transiently transfected a cell line stably expressing Kv4.3 (L/Kv4.3) with a plasmid encoding a chimeric protein consisting of KChAP fused to the C-terminus of EGFP (EGFP-KChAP). As a control, the plasmid EGFP-C2 was transfected. Cells exhibiting green fluorescence were chosen for electrophysiological recording. In Figure 13C whole-cell currents in untransfected L cells, in L cells stably transfected with Kv4.3 channels (L/Kv4.3 cells), in L/Kv4.3 cells transiently transfected with EGFP-C2, and in L/Kv4.3 cells transiently transfected with EGFP-KChAP. Recordings were made two days after transfection. Hold- ing potential was -90 mV; 400 ms pulses were from -70 to +40 mV in 10 mV steps.

L/Kv4.3 cells exhibited a relatively small transient outward current (Fig. 13C). Kv4.3 currents were not altered in cells coexpressing EGFP (Fig. 13C). Figure 13D shows the effect of EGFP-KChAP on Kv4.3 current density at +40 V. (*) indicates a significant difference from current density in L/Kv4.3 cells and L/Kv4.3 cells transiently transfected with EGFP-C2. Thus, cells transfected with the chimeric EGFP-KChAP construct exhibited dramatically increased (8-10 fold) currents with no apparent changes in voltage-dependence or channel gating (Fig. 13C, D). The Kv4.3 currents in cells coexpressing EGFP or EGFP- KChAP had similar kinetics of activation and inactivation. With pulses from -90 to +40 mV, activation time constants were: 4.09 + 0.21 ms (EGFP; n=16) and 3.92 + 0.36 ms (EGFP-KChAP; n=16). Inactivation time constants were: 79.3 + 5.4 ms (EGFP; n=16) and 72.8 + 4.0 (EGFP-KChAP; n=16) Parameters of steady- state inactivation were also similar: V₀₅ = -49.4 + 0.8 mV, k = -6.7 + 0.2 mV (n = 5) and V_{0 5} = -51.5 ± 3.0 mV, k - -7.2 + 0.4 V (n = 5) for EGFP and EGFP-KChAP, respectively. Recovery from inactivation did not differ in cells coexpressing EGFP (recovery constant at -100 mV; 217 + 18 ms; n=6) or EGFP-KChAP (194 + 25 ms; n=4).

Since the mammalian cell environment supported the action of KChAP on Kv2.1 and Kv4.3 currents as well or better than Xenopus oocytes, we tested the effect of KChAP on Kvl .5 in L cells, a channel whose amplitude is not modulated by KChAP in oocytes. An L cell line stably expressing Kvl.5 (L/Kvl .5) was transiently transfected with either EGFP-C2 or EGFP-KChAP. Figure 13E shows whole-cell currents in L/Kvl .5 cells transiently transfected with EGFP-C2 or EGFP-KChAP. Voltage protocol was the same as for Kv4.3. Figure 13F shows current density in L/Kvl .5 cells and L/Kvl .5 cells expressing EGFP or EGFP-KChAP at +40 mV. Thus, there was no significant alteration in the amplitude of Kvl .5 currents in any of the transiently transfected cells. Therefore, KChAP did not modulate Kvl .5 in either oocytes or L cells.

A summary of the effects of KChAP on K⁺ channels in L cells is presented in Figure 13G. In addition to Kv2.1, Kv4.3, and Kvl.5, we also examined the effect of KChAP on two other stable L cell lines: L/Kvl.l and L/HERG. As with Kvl.5, Kvl.l currents were unaffected by the coexpression of KChAP. HERG currents were not altered by KChAP in either L cells or oocytes. In case of Kv2.1 channels, the L cells were coinjected with both cRNAs. For Kvl .l , Kvl.5, Kv4.3 and HERG, stably transfected cell lines were used and cells were transiently transfected either with EGFP-C2 (control) or EGFP-KChAP plasmids. Whole-cell currents were averaged and ratio of means was calculated for each injection/transfection series. The numbers above the bars indicate the number of series. HERG tail currents were recorded at test potentials to -120 mV with a pre-pulse to +40 mV. For all transfected cells, recordings were made 2 days after transfection.

Example 10

Kvol subunit binding region is localized to a stretch of 98 residues in KChAP.

KChAP was divided into three fragments and each was tested in the yeast two hybrid system for interaction with the N-terminus of Kvl .2 (Kvl.2-N). The KChAP fragments are diagramed in Figure 14A, and the results of the yeast two-hybrid assay are presented in Figure 11B. The yeast host strain HF7C was cotransformed with GAL4 binding domain (pGBT9) and activation domain (pGAD424) fusion plasmids as indicated, and initially plated on media lacking tryptophan and leucine (-t,-l). Three individual colonies from each cotransformation were respotted on media with (-t,-l) and without histidine (-t,-l,-h) to follow the activation of the HIS 3 reporter gene as shown on the right. Growth on media with- out histidine is indicative of an interaction. All constructs were negative for autonomous activation of transcription prior to use in this assay (not shown). Full-length KChAP as well as KChAP-M, consisting of a stretch of 98 residues in the middle of the protein (AA 355-452), interacted with Kvl .2-N as evidenced by activation of the HIS 3 reporter gene and growth on minus histidine media (Fig. 14B). Neither KChAP-N, an N-terminal KChAP fragment consisting of residues 46-354, nor KChAP-C (the carboxy terminal portion of the protein from residues 453-619) gave a positive result in the yeast two-hybrid assay. Similar results were obtained when the panel of fragments was tested for interaction with Kv2.1-N and Kvβl .2 (not shown). The assignment of the Kvα and β binding region of KChAP to this stretch of 98 residues is consistent with the KChAP fragment that was initially isolated in the yeast two-hybrid screen, KChAP-Y (Wible, B.A., et al. "Cloning and expression of a novel K⁺ channel regulatory protein, KChAP" J Biol Chem 273: 11745-11751, 1998). KChAP-Y consisted of W355 through D619 (Fig. 14A).

Example 11 KChAP and KChAP-M increase Kv4.3 current amplitude and total protein in L cells. In Xenopus oocytes, increased functional expression of Kv2.1 with KChAP was reflected in an increase in the total amount of Kv2.1 protein (Wible, B.A., et al. "Cloning and expression of a novel K⁺ channel regulatory protein, KChAP" J Biol Chem 273: 1 1745- 1 1751, 1998), and not redirection of the same amount of protein to the cell surface. To determine whether KChAP behaved similarly in mammalian cells, we examined L/Kv4.3 cells that were transfected with EGFP-KChAP with an anti-Kv4.3 polyclonal antibody and immunofluorescence microscopy. The cells were fixed 48 hours after transfection, permeabilized, and stained with Kv4.3 antibodies (polyclonal; Alomone Labs; 1 :200 dilution). Kv4.3 staining was visualized with a TRITC-conjugated anti-rabbit secondary antibody. Left panels show the localization of the EGFP-tagged proteins while the right panels show costaining of the Kv4.3 antibody in the same cells. For comparison, cells from the same transfection experiment were recorded and electrophysiological data compiled. L/Kv4.3 cells transfected with EGFP-KChAP are shown in Figure 15 A. EGFP-KChAP

(left panel) was localized primarily in the nuclei of transiently transfected cells. These cells exhibited dramatically increased staining with the Kv4.3 antibody compared to non-tran- siently transfected L/Kv4.3 cells (right panel). Kv4.3 staining was not restricted to the cell surface but was especially bright in the perinuclear region consistent with endoplasmic reticulum staining. Thus, KChAP increased Kv4.3 total protein in L cells in a similar fashion as observed with Kv2.1 in Xenopus oocytes.

Having localized the Kvα binding domain of KChAP to residues 355-452 (KChAP - M), we wanted to determine whether this fragment would also increase Kv4.3 protein in this assay. L/Kv4.3 cells were transiently transfected with EGFP tagged KChAP-M, and the cells stained with anti-Kv4.3 antibody. KChAP-M had a strikingly different cellular localization than KChAP (Fig. 15B, left). Although some KChAP-M appeared to be in the nucleus, cytoplasmic staining, especially in the perinuclear region, was also seen. In the same cell, we saw a dramatic increase in anti-Kv4.3 reactivity comparable to what was observed with KChAP (Fig. 15B, right). Figure 15E presents a summary of the K⁺ currents that were recorded from transiently transfected L/Kv4.3 cells. Both KChAP and KChAP-M produced increased currents of comparable magnitude relative to EGFP-C2 alone. We also examined the effects of EGFP-tagged fusions of KChAP-N and KChAP-C on Kv4.3 protein and current levels. KChAP -N (Fig. 15C, left) showed a diffuse cytoplasmic localization with no concomitant increase in Kv4.3 staining (Fig. 15C, right). KChAP-C (Fig. 15D, left) appeared to be present in both the nucleus and cytoplasm, but again produced no increase in the amount of Kv4.3 protein (Fig. 15D, right). Figures 15E and F show the K⁺ current density in L/Kv4.3 cells transiently transfected with EGFP-tagged constructs. Currents were recorded 48 hours post transfection. Note that both KChAP and the Kv channel binding fragment, KChAP-M, increase Kv4.3 currents whereas KChAP-N and KChAP-C do not. (*) indicates values significantly different from cells transfected with EGFP-C2 alone. The lack of increased Kv4.3 immunoreactivity with these two fragments was reflected in the lack of increase in Kv4.3 current density in cells overexpressing them as well (Fig. 15F). Thus, the small fragment of KChAP that was identified as binding to Kvα N-termini was sufficient to produce increases in Kv4.3 in mammalian cells.

Example 12 Association of KChAP with Kv2.1 and Kv4.3 in rat heart. To begin to analyze the physiological relevance of KChAP interactions with Kv channels, we raised a polyclonal antisera to KChAP and used it to probe for association with Kv channels in native tissue. The anti-KChAP antibody was raised against KChAP-C (residues 453-619), affinity purified, and initially tested for its ability to detect overexpressed KChAP in Xenopus oocytes. A single band of about 68 kD was detected in lysates of oocytes injected with KChAP cRNA consistent with the predicted molecular weight of KChAP (Fig. 16A). In lysates from adult rat heart, the anti-KChAP antibody detected a band greater than 75 kD (Fig. 16B). This suggests that KChAP may be post-translationally modified in heart leading to an increased apparent molecular weight. Since Kv2.1 and Kv4.3 are both sensitive to the effects of KChAP in heterologous expression assays and are expressed in adult rat heart, we searched for complexes of KChAP and Kv channel proteins by coimmunoprecipitation with anti-KChAP. Figures 16C and D show adult rat heart lysates were incubated with or without (lanes labeled + or - ) KChAP antibody (1 :100 dilution). Immunoprecipitates (IP) were collected on Dynabeads and the presence of Kv channels probed by Western blotting. Panel C is a Western blot with Kv2.1 polyclonal antibody (Upstate Biotech; 1 :200 dilution), and panel D is a Western blot with anti-Kv4.3 polyclonal antibody (Alomone Labs; 1 :150 dilution). Lysate (40 μg protein loaded) and IP (- and + anti-KChAP) are shown. Adult rat heart lysate immunoblotted with anti-Kv2.1 revealed two bands of about 105 and 130 kD (Fig. 16C). These observations are consistent with the Western blotting pattern of Kv2.1 in rat heart (Xu, H., et al, "Developmental analysis reveals mismatches in the expression of K+ channel alpha subunits and voltage-gated K+ channel currents in rat ventricular myocytes" J Gen Physiol 108:405-419, 1996) and brain (Shi, G., et al, "Properties of Kv2.1 K+ chan- nels expressed in transfected mammalian cells" J Biol Chem 269:23204-23211 , 1994). The 105 kD Kv2.1 band coimmunoprecipitated with KChAP as shown in Figure 16C Interestingly, the larger 130 kD Kv2.1 band which is thought to be hyperphosphorylated (Murakoshi, H., et al, "Phosphorylation of the Kv2.1 K+ channel alters voltage-dependent activation" Mol P harm 52:821-828, 1997) was not detected in KChAP immunoprecipitates. A polyclonal antibody to Kv4.3 detected a single band of about 75 kD in adult rat heart lysates which was present in complexes coimmunoprecipitated with the KChAP antibody (Fig. 16D). These data indicate that KChAP interacts with Kv channels in native tissue.

It should be clear from the above that the reagents and methods detailed here will allow for the screening of compounds the are agonistic or antagonistic to functioning of K⁺ channels.

Claims

1. Purified DNA having the sequence of SEQ ID NOT .

2. RNA transcribed from the DNA of claim 1.

3. Protein translated from the RNA of claim 2.

4. Antibodies produced from the protein of claim 3.

5. Expression constructs comprising the DNA of claim 1.

6. A transgenic animal comprising DNA of claim 1.

7. Purified DNA having the sequence of SEQ ID NO:9.

8. A method to detect KChAP agonists and antagonists, comprising: a) providing i) one or more compounds suspected of modulating KChAP activity, ii) a cell line or transfected cells comprising the KChAP gene; b) contacting a portion of said cells from said cell line with said one or more compounds under conditions such that said compound can enter said cells, so as to create treated portions and untreated portions of cells; and c) comparing the amount of potassium channel activity of said treated cells with the amount of potassium channel activity of said untreated cells.

9. A method to detect KChAP agonists and antagonists, comprising: a) providing i) one or more compounds suspected of modulating KChAP activity, ii) a cell line or transfected cells comprising the KChAP gene and one gene from the group comprising Kv2.1, Kv2.2, Kvl.3 or Kv4.3; b) contacting a portion of said cells from with said one or more compounds under conditions such that said compound can enter said cells, so as to create treated portions and untreated portions of cells; and c) comparing the amount of potassium channel activity of said treated cells with the amount of potassium channel activity of said untreated cells.

10. A purified peptide selected from the group consisting of SEQ ID NOS:33 - 53.