AU781940B2 - Methods for identifying modulators of N-type ion channel inactivation - Google Patents

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Class Int. Class Application Number: Lodged: Complete Specification Lodged: Accepted: Published: Priority Related Art: Name of Applicant:

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no 5~ducts Crc,oratio;, -A mrica.n O n Actual Inventor(s): Kathleen H Young, Kenneth J Rhodes Address for Service: PHILLIPS ORMONDE FITZPATRICK Patent and Trade Mark Attorneys 367 Collins Street Melbourne 3000 AUSTRALIA Invention Title: METHODS FOR IDENTIFYING MODULATORS OF N-TYPE ION CHANNEL INACTIVATION Our Ref: 672369 POF Code: 49377/1481 The following statement is a full description of this invention, including the best method of performing it known to applicant(s): -1- 6ooeq r' l' AHP98133P1 METHODS FOR IDENTIFYING MODULATORS OF N-TYPE ION CHANNEL INACTIVATION The present application is a divisional application from Australian patent application number 43639/00, the entire disclosure of which is incorporated herein by reference.

BACKGROUND OF THE INVENTION Ion channels are transmembrane proteins that regulate entry of various ions into cells from the extracellular matrix. Ion channels are physiologically important, playing essential roles in regulating intracellular levels of various ions and in generating action potentials in nerve and muscle cells. Hille, Ionic Channels of Excitable Membranes (Sinauer, Sunderland, MA, 1992). Passage of ions through ion channels is characterized by selective filtering and by a gating-type mechanism which produces a rapid increase in permeability. Angelides, K. J. and T. J. Nuttov, J. Biol.

Chem. 258:11858-11867 (1981). Ion channels may be either voltage-gated, implying that current is gated (or regulated) by membrane potential (voltage), or chemicallygated acetylcholine receptors and y-aminobutyric acid receptors), implying that current is gated primarily by binding of a chemical rather than by the membrane potential. Butterworth, J. F. and G. R. Strichartz, Anesthesiology72:711-734 (1980).

An important characteristic of certain voltage-gated channels is inactivation: soon after opening they close spontaneously, forming an inactive channel that will not reopen until the membrane is repolarized. Miller, Science 252:1092-1096 (1991).

Rapidly inactivating ("A-type") voltage-gated ion channels control the rate at which excitable cells reach the threshold for firing action potentials and thus are key regulators of neuronal excitability. B. Hille, supra.

Many voltage-gated ion channels that generate action potentials have been cloned and sequenced, and all have a remarkably similar structure. A typical potassium channel contains four copies of an approximately 600-amino-acid polypeptide, each of which has six membrane-spanning a-helices. Heginbotham, L., et al., Science 258:1152 (1992). Sodium and calcium channels are single polypeptides of about 2000 amino acids that contain four homologous domains, each AHP 98133 P1 comprised of six transmembrane domains which are similar in sequence and structure to a potassium channel protein. These domains are connected and flanked by shorter stretches of nonhomologous residues. Jessell, T.M. and E.R. Kandel, Neuron 10(Supp):1-3 (1993). It is believed that the a-helical structures provide conformational flexibility for the ion channel which is functionally responsible for the channells gating mechanism. See Heinemann, et al., J. Physiol. 88:173-180 (1994).

In addition to affecting action potentials, ion channels facilitate other important physiological functions such as cardiac pacemaking, neuron bursting, and possibly learning and memory. Crow, Trends Neurosci. 11:136-142 (1988); Hodgkin, A.L.

and Huxley, J. Physiol. 117:500-544 (1952). In addition to their involvement in normal cellular homeostasis, ion channels are associated with a variety of disease states and immune responses. Diseases believed to be associated with dysfunction of ion channels include neurological disorders, metabolic diseases, cardiac diseases, tumor-driven diseases, and autoimmune diseases.

Due to the importance of ion channels in both normal cellular homeostasis and disease, considerable research effort has focused on ion channels, and particularly on identifying compounds which affect their function. Thus, several techniques have been developed to evaluate the gating mechanism of ion channels and the mode of action of channel-drug interaction. Electrophysiological recording has been used to define the roles of ion currents, and especially potassium and sodium currents, in generating action potentials in excised nerves. Hodgkin, A. L.

and A. F. Huxley, supra. This technique, however, is not suitable for mass screening of compounds due to its technical complexity and the requirement of a high degree of sophistication to generate reproducible results. Radioligand binding assays have been used to characterize the site of action of various classes of ion channel blockers. However, the availability of radiolabelled ligands, the level of nonspecific binding, and the physico-chemical property of the molecules have limited the application of this technique. Strichartz, et al., Ann. Rev. Neurosci. 10:239-67 (1987).

Fluorescent-labelled neurotoxin probes have also been used to map the molecular structure of the functional site of the channel, but have not gained general popularity for broader use. Angelides, K. A. and T. J. Nuttov, J. Biol. Chem. 256:11958-11967 (1983).

AHP 98133 P1 Recently, a modified yeast "two-hybrid" system has been developed to identify compounds that bind to either the NH 2 -terminal multimerization domain (commonly referred to as the "NAB" or "T1" domain) on the a-subunit of a Shaker-like potassium channel or to the "core" domain of the P-subunit of the potassium channel, thereby preventing the a/p subunit interaction. See U.S. Pat. No. 5,856,155 Li), issued January 5, 1999; and PCT App. No. PCT/US97/02292, published August 28, 1997 (WO 97/31112). Unfortunately, significant inherent limitations in this system may prevent or limit its practical application. Such limitations include, for example, the extraordinarily tight nature of the a-NAB/P-core interaction (which survives such harsh treatments as detergent extraction and affinity chromatography), the limited applicability to potassium channels whose activity requires interaction between the NAB domain of the a-subunit and the core domain of the P-subunit, and, most importantly, the potentially significant inhibitory effect such compounds would have on potassium channel surface expression. [Regarding the tight association of a-and 13subunits, see Parcej, and J.O. Dolly, Biochem. J. 257:899-903 (1989) and Muniz, et al., Biochemistry 31:12297-12303 (1992).] With respect to the latter limitation, P-subunits have been shown to promote N-linked glycosylation and surface expression of a-subunits. Shi, et al. Neuron 16:843-852 (1996). Thus, one would expect compounds that bind to the core domain of the 1-subunit to block these chaperone-like effects, thereby reducing, if not preventing, the biosynthesis of functional potassium channels. By affecting the abundance or distribution of potassium channels in excitable membranes, such compounds would essentially act as ion channel blockers, and thus would likely have adverse neurophysiological effects. Finally, any compound that can effectively block the strong a-NAB/13-core binding interaction compounds identified using this modified yeast two-hybrid system) must themselves have extremely high binding affinity for potassium channel subunits, and thus would likely be toxic to a mammalian host.

In view of the complexity of ion channel pharmacology and its attractiveness as a target site for the discovery of novel therapeutic compounds, there exists a need for an alternative technique which will enable the large-scale screening of compounds for ion channel modulatory activity in a simple and reliable manner. The present invention fulfills these and other needs.

Throughout the description and the claims of this specification the word "comprise" and variations of the word, such as "comprising" and "comprises" is not intended to exclude other additives, components, integers or steps.

The discussion of documents, acts, materials, devices, articles and the like is included in this specification solely for the purpose of providing a context for the present invention. It is not suggested or represented that any or all of these matters formed part of the prior art base or were common general knowledge in the field relevant to the present invention before the priority date of each claim of this application.

X \F deURN6772872389_ sped 270405 doc SUMMARY OF THE INVENTION The present invention provides several novel methods and compositions for identifying compounds which affect binding between two key regulatory regions of voltage-gated ion channels. More specifically, the invention relates to methods and compositions for identifying compounds which affect the binding of an intracellular receptor region of an a-subunit of a voltage-gated ion channel or a biologically active fragment thereof and an amino-terminal inactivation region off an ion channel protein. Compounds that disrupt or facilitate the functional or binding interaction of these two key regulatory regions have significant modulatory effects on ion channel activity, and thus are likely to be useful for treating and/or preventing as wide variety of diseases and pathological conditions associated with ion channel dysfunction. Such conditions include, for example, neurological disorders, cardiac diseases, metabolic diseases, tumor-driven diseases, and autoimmune diseases.

Many of these compounds are expected to have potent anticonvulsant and neuroprotective properties which will prove especially useful for the prevention and/or treatment of neurodegenerative disorders such as epilepsy, stroke, cerebral ischemia, cerebral palsy, hypoglycemia, Alzheimer's disease, Huntington's disease, asphyxia and anoxia, as well as for the treatment of neuropathic pain, spinal cord trauma, and traumatic brain injury.

In one aspect, the invention provides methods of evaluating and screening candidate compounds for the ability to affect binding of an intracellular receptor region of an a-subunit and an amino-terminal inactivation region of an ion channel protein.

The methods comprise contacting the compound with the intracellular receptor region and the amino-terminal inactivation region, and determining the ability of the compound to interfere with or facilitate the functional interaction or binding of these two regions. A decrease in binding in the presence of the compound compared to the binding in the absence of the compound indicates that the compound inhibits functional interaction or direct binding between these two regulatory regions. Similarly, an increase in binding in the presence of the compound compared to the binding in the absence of the compound indicates that the compound facilitates functional interaction between these two regulatory regions.

In another aspect, the invention provides methods for evaluating or screening candidate compounds comprising adding a candidate compound to a modified host cell and comparing the expression of a reporter gene in the presence and absence of the compound. A decrease (or increase) in expression of the reporter genes is an X:TlesRN672369k672369_Spe 270405 Doc AHP 98133 P1 indication that the compound inhibits (or promotes) functional or binding interaction between the intracellular receptor region and the amino-terminal inactivation region.

In yet another aspect, the invention provides modified host cells and methods for evaluating or screening candidate compounds for ion channel modulatory activity.

The modified host cells contain a first hybrid protein comprising a DNA-binding domain of a transcriptional activator in polypeptide linkage to either an intracellular receptor region of an a-subunit of a voltage-gated ion channel or (ii) an aminoterminal inactivation region of an ion channel protein, and a second hybrid protein comprising an activation domain of a transcriptional activator in polypeptide linkage to the intracellular receptor region if the DNA-binding domain is in polypeptide linkage to the amino-terminal inactivation region or to the amino-terminal inactivation region if the DNA-binding domain is in polypeptide linkage to the intracellular receptor region.

The modified host cell may optionally comprise a reporter gene whose expression is inhibited in the presence of an inhibitor of N-type inactivation.

In still another aspect, the modified host cell contains a first hybrid protein comprising an intracellular receptor region of an a-subunit of a voltage-gated ion channel in polypeptide linkage to a first peptide of a peptide binding pair, and a second hybrid protein comprising an amino-terminal inactivation region of an ion channel protein in polypeptide linkage to a second peptide of the peptide binding pair, wherein binding interaction between the two peptides causes activation of a signal transduction pathway in the modified host cell. Activation of the signal transduction pathway does not occur in the presence of a molecule which inhibits binding of the intracellular receptor region and the amino-terminal inactivation region of an ion channel protein.

In other aspects, the invention provides polynucleotides, expression vectors, and host cells transfected or transformed with expression vectors containing nucleotide sequences which encode an intracellular receptor region of an a-subunit of a voltage-gated ion channel and an amino-terminal inactivation region of an ion channel protein, or biologically active fragments thereof.

BRIEF DESCRIPTION OF THE FIGURES Figure 1. Amino acid comparison of the intracellular receptor regions ("loops") of the human Kv1.2 human Kv1.3 human Kv1.4 human Kv1.5 human Kv1.6 ("hKvl.6") and human Kv3.4 AHP 98133 P1 ("hKv3.4") are shown in alignment with human Kv1.1 The black boxes indicate sequence identity; shaded boxes indicate conservative amino acid substitutions.

Figure 2. Amino acid comparison of the amino-terminal inactivation regions of the human Kvp1b ("hKvplb"; also known as "hKvpl.2"), human KvS1 c ("hKvplc"; also known as "hKvpl.3"), human Kvp3 ("hKvp3"), human Kv1.4 and human Kv3.4 ("hKv3.4") are shown in alignment with human KvP1 ("hKvpl"). The black boxes indicate sequence identity; shaded boxes indicate conservative amino acid substitutions.

Figure 3. (Fig. 3a) Electrophysiological current recordings of inactivating channels expressed in Xenopus oocytes. Xenopus oocytes were injected with 0.5 ng of hKv1.1:10 ng of hKvp1 mRNA transcribed in vitro using standard procedures (Sambrook et al, (1989) Molecular Cloning: A Laboratory Manual). Cells were challenged with families of voltage pulses of 200 ms duration ranging from -60 mV to mV once every 2 min. Cells were exposed to each dose of "Wy-8340" (CloHs 1

NO;

6-aminothymol or 4-amino-2-isopropyl-5-methylphenol) for 6 min and cumulative dose-response curves were performed. Relative inactivation was calculated by measuring the amplitude of the peak and steady-state currents, setting the inactivation (without compound) for each cell to 100% maximum) and measuring the percent disinactivation.with each dose of compound. (Fig. 3b) Concentrationresponse curves showing the effect of Wy-8340 on inactivation of hKv1.1 and hKv31 channels expressed in Xenopus Oocytes.

Figure 4. Protection against pentelenetetrazol-induced seizures in the mouse. Adult male mice were treated with valproic acid (Fig. 4a), Compound A (Fig.

4b), and Compound B (Fig. 4c) at doses from 30-178 mg/kg i.p. (n 8/dose). Thirty minutes later, these animals were challenged with pentelenetetrazol (85 mg/kg, SC) and observed for onset of seizures during a 30 minute test period. The number of animals protected from seizures was plotted versus dose of test compound and were estimated from this dose-response data.

AHP 98133 P1 DESCRIPTION OF THE SEQUENCES SEQ ID NO:1 is an amino acid sequence containing the intracellular receptor region of the a-subunit of the hKvl.1 protein.

SEQ ID NO:2 is an amino acid sequence containing the intracellular receptor region of the a-subunit of the hKvl1.4 protein.

SEQ ID NO:3 is a nucleotide sequence containing the nucleotide sequence encoding the intracellular receptor region of the a-subunit of the hKv1.1 protein.

SEQ ID NO:4 is a nucleotide sequence containing the nucleotide sequence encoding the intracellular receptor region of the a-subunit of the hKvl1.4 protein.

SEQ ID NO:5 is an amino acid sequence containing the amino-terminal inactivation region of the hKvP1 protein.

SEQ ID NO:6 is an amino acid sequence containing the amino-terminal inactivation region of the hKvl1.4 protein.

SEQ ID NO:7 is a nucleotide sequence containing the nucleotide sequence encoding the amino-terminal inactivation region of the hKvP1 protein.

SEQ ID NO:8 is a nucleotide sequence containing the nucleotide sequence encoding the amino-terminal inactivation region of the hKvl1.4 protein.

DETAILED DESCRIPTION OF THE INVENTION Unless defined otherwise, all technical and scientific terms used herein have the same meaning as commonly understood by one of ordinary skill in the art to which this invention belongs. Although any methods and materials similar or equivalent to those described herein can be used in the practice or testing of the present invention, the preferred methods and materials are described. For purposes of the present invention, the following terms are defined below.

As used herein, the term "ion channel protein" refers generally to voltagegated ion channels, including the pore-forming a-subunit proteins ("a-subunits") and the cytoplasmic P-subunit proteins (also known in the art as "auxiliary subunits"or "13subunits").

The term "intracellular receptor region" means a portion of an a-subunit of a voltage-gated ion channel which can form a specific binding interaction with an amino-terminal portion an amino-terminal inactivation region) of an ion channel protein. The term "S4-S5 cytoplasmic receptor domain" refers to the stretch of AHP 98133 P1 hydrophilic amino acid residues between the membrane-spanning segments S4 and (also known as of a pore-forming a-subunit.

The term "amino-terminal inactivation region" means a portion of an ion channel protein which can form a specific binding interaction with an intracellular receptor region of an a-subunit. The amino-term.ina! inactivation region (also known in the art as the "inactivation gate," "inactivating ball," or "ball peptide") is a globular domain on the amino-terminus of an ion channel protein, including, for example, the globular domain on the amino-terminus of an a-subunit linked to the first membrane-spanning segment of an a-subunit) or the amino-terminus of a P-subunit.

As used herein, the term "biologically active fragment" means a portion of an intracellular receptor region or an amino-terminal inactivation region capable of binding to an amino-terminal inactivation region or an intracellular receptor region, respectively. The term "fragment," as applied in this context, will typically be at least about 6 amino acids, usually at least about 8 contiguous amino acids, preferably at least about 10 contiguous amino acids, more preferably at least about 12 contiguous amino acids, and most preferably at least about 14 or more contiguous amino acids in length. Such fragments can be generated by methods known to those skilled in the art, including proteolytic cleavage of the polypeptide, de novo synthesis of the fragment, or genetic engineering.

As used herein, the term "peptide binding pair" means a pair of proteins or polypeptides whose binding interaction results in activation of a signal transduction pathway in a cell or organism. The term "effector molecule" means a peptide or polypeptide that can be expressed as a fusion protein and, when so expressed, can activate an "indicator molecule," provided the effector molecule is translocated to the cell compartment containing the indicator molecule. As used herein, the term "indicator molecule" means a molecule acted upon by the effector molecule, either directly or indirectly through an intermediate molecule, such that activation of the indicator molecule produces a detectable signal. The term "activate" or "activation," when used in reference to an indicator molecule, means that the effector molecule has changed the indicator molecule such that the effector function can be detected as a signal generated by the changed indicator molecule or by a molecule subsequently acted upon by the changed indicator molecule. Various effector molecules and indicator molecules are known in the art, including, for example, the "effector AHP 98133 P1 proteins" and "reporter molecules," respectively, described in U.S. Pat. No. 5,776,689 (Karin et which is incorporated by reference in its entirety herein.

The term "cell compartment localization domain" means a peptide or polypeptide sequence that directs translocation of a fusion protein containing the effector molecule to a particular cell compartment. Various cell compartment localization domains are known in the art, including, for example, plasma membrane localization sequences, nuclear localization signal sequences, mitochondrial membrane localization sequences, and the like. See, Karin et al., supra.

Provided by the present invention are methods and compositions for identifying compounds which affect the binding interaction between two key regulatory regions of voltage-gated ion channels, namely an intracellular receptor region of an a-subunit and an amino-terminal inactivation region of an ion channel protein. The present inventors have discovered that compounds that disrupt binding of these two key regulatory regions have significant modulatory effects on ion channel activity, and thus are expected to be clinically significant therapeutic agents for treating and/or preventing a wide variety of diseases and pathological conditions associated with ion channel dysfunction. Such compounds may also be useful as commercial or biological research reagents, for example, to further define interaction domains of ion channel proteins. Surprisingly, compounds identified using the methods of the invention have been found to selectively and dose-dependently eliminate "N-type" ion channel inactivation (discussed below) in modified host cells expressing these heterologous regulatory regions. Also surprisingly, these compounds exhibit potent anti-seizure activity both in vitro and in vivo. Thus, the present invention represents a significant advance in the pharmacological and pharmaceutical arts, by providing a reliable high-throughput screen which can identify potent and selective modulators of N-type inactivation of voltage-gated ion channels.

As discussed above, voltage-gated ion channels, and particularly potassium and sodium channels, are important determinants of membrane excitability. Each of these families of ion channels comprise several classes of proteins, including the pore-forming a-subunits and the auxiliary 1-subunits. The a-subunits comprise six transmembrane-spanning regions, usually referred to sequentially as S1 through S6.

The sequences between segments S4 and S5, the region," and sequences of segment S6 form part of the inner mouth and pore of ion channels, whereas part of the H5 region forms part of the outer mouth and outer half of the pore. See AHP 98133 P1 Heinemann, et al., J. Physiol. 88:173-180 (1994); Durrell, S.R. and R. Guy, Biophys. J. 62:238-250 (1992). Segment S4 contains several positively charged amino acids and is believed to be the voltage-sensing a helix. The amino-terminal domain of ion channels is involved in subunit assembly and channel inactivation. Li, etal., Science 257:1225-1 230 (1992); Hoshi, et Science 250:533-538 (1990). Rapidly inactivating A-type ion channels have an amino-terminal inactivation domain which is able to close the open channel from the inside at depolarized membrane potentials, as will be discussed more fully below. This type of inactivation is often referred to as "N-type" inactivation. Hoshi, et al., supra.

N-type inactivation operates in a ball-and-chain type mechanism. Hoshi, et al., supra; Zagotta, et al., Science 250:568-570 (1990). The amino terminus of the a-subunit is the "ball" which swings into the open pore, binds to a receptor site (the intracellular receptor region) and thereby plugs the ion channel pore. This mechanism has been confirmed by several researchers. Mutations within the aminoterminal ball or a deletion of this ball abolishes rapid N-type inactivation. Also, mutations within the S4-S5 region disrupt N-type inactivation. Isacoff, et al., Nature 353:86-90 (1991). The on-rate time constant for binding the inactivating domain to the receptor is voltage-dependent, such that depolarization of the membrane accelerates the binding. Conversely, the off-rate time constant is also voltage-dependent, but is significantly faster at negative than at more positive membrane potentials. S. Heinemann, supra; Ruppersberg, et al., Nature 353:657-660 (1991). Upon depolarization, the ball moves into the electric field of the membrane and obstructs the open channel pore. Upon repolarization of the membrane the off-rate is faster than the on-rate time constant. This causes the ball to swing away from the ion channel pore and to free the ion channel from inactivation.

Thus, the ratio between on- and off-rate at negative membrane potentials may also be an important determinant for the refactory period which A-type ion channels require for recovery from inactivation. S. Heinemann, supra.

As described above for a-subunits, the amino terminus of p-subunits also functions as a tethered inactivating ball which swings into the inner mouth of the ion channels and occludes the pore upon depolarization of the membrane. The amino terminus of p-subunits has been shown to be structurally and functionally similar to the inactivating ball domain of a-subunits. S. Heinemann, supra. A hallmark of the AHP 98133 P1 inactivating domain of both a and P-subunits is the presence of an amino terminal cysteine followed by a cluster of positively charged amino acids (lysines and arginines). Ruppersberg, et al., supra. The latter may be important for moving the inactivating ball into the electric field, the cysteine for interaction with the intracellular receptor region at or near the entrance of the ion channel pore. S. Heinemann, supra.

In one aspect, the invention provides methods for detecting a compound that inhibits binding of an intracellular receptor region of an a-subunit and an aminoterminal inactivation region of an ion channel protein, thereby keeping rapidly inactivating channels open longer. The methods comprise contacting the compound with the intracellular receptor region and the amino-terminal inactivation region, and determining the ability of the compound to interfere with the functional interaction or binding of these two regions. A decrease in binding in the presence of the compound compared to the binding in the absence of the compound indicates that the compound inhibits binding interaction between these two regulatory regions.

Although this method will work using any appropriately constructed in vitro or in vivo system which allows monitoring of these specific interactions, the invention is preferably practiced using a modified host cell which expresses these heterologous regulatory regions, such as the two-hybrid system described below. The method is generally applicable to voltage-gated ion channels which inactivate via an N-type inactivation mechanism, and particularly voltage-gated potassium and sodium ion channels.

In one embodiment, the method comprises adding a candidate compound to a modified host cell and comparing the exhibition of a selected phenotype in the presence and absence of the compound, wherein the modified host cell is adapted to exhibit a change in phenotype only in the presence of a molecule which inhibits the binding of the intracellular receptor region to the amino-terminal inactivation region.

Preferably, the modified host cell comprises an inverse selection (also known as "counter" or "rescue") "two-hybrid" system, such as the modified yeast two-hybrid screen described herein.

In another aspect, the invention provides modified host cells which are useful for screening candidate compounds for ion channel modulatory activity. In a preferred embodiment, the modified host cell comprises a first hybrid protein comprising a DNA-binding domain of a transcriptional activator in polypeptide linkage AHP 98133 P1 to either an intracellular receptor region of an a-subunit of a voltage-gated ion channel or (ii) an amino-terminal inactivation region of an ion channel protein, and a second hybrid protein comprising an activation domain of a transcriptional activator in polypeptide linkage to the intracellular receptor region if the DNA-binding domain is in polypeptide linkage to the amino-termina! inactivation region or to the amino-terminal inactivation region if the DNA-binding domain is in polypeptide linkage to the intracellular receptor region.

The intracellular receptor region of the a-subunit of a voltage-gated ion channel, for purposes of the present invention, are those regions of the a-subunit that bind to the amino-terminal inactivation region of an ion channel protein. By way of example, the amino acid sequences for the intracellular receptor regions of certain asubunits are set forth herein in Figure 1. These sequences can be easily identified in any a-subunit of a voltage-gated ion channel given the high degree of homology among these sequences. In the example of Figure 1, the intracellular receptor regions ("loops") of the human Kv1.2 human Kv1.3 human Kv1.4 human Kv1.5 human Kv1.6 ("hKvl.6") and human Kv3.4 ("hKv3.4") are shown in alignment with human Kvl.1 [As used herein and consistent with art-recognized usage, "Kv" refers to a voltage-gated potassium ion channel protein.] It is expected that intracellular receptor regions of currently unidentified a-subunits will contain a homology of at least 60%, preferably at least 75%, more preferably at least 85%, and most preferably at least 90 to based on the homologies present in the a-subunits of the hKvl and hKv3 channel proteins. Due to the high degree of conservation of sequences among all known asubunits of voltage-gated ion channels, additional members of the potassium channel family, as well as members of the sodium channel family, are expected to comprise intracellular receptor regions which are structurally and functionally equivalent to those of the hKv1.1 and hKvl.4 a-subunits exemplified in the Examples hereof.

Thus, the general features contained and described herein will be applicable to newly discovered ion channel proteins.

The amino-terminal inactivation region of an ion channel protein, for purposes of the present invention, are those regions of the ion channel protein that bind to the intracellular receptor region of an a-subunit. By way of example, the amino acid sequences for the amino-terminal inactivation regions of certain ion channel proteins are set forth herein in Figure 2. In the example of Figure 2, the amino-terminal AHP 98133 P1 inactivation regions of human Kvp1b ("Kvp1b"; also known as "Kvp1.2"), human Kvplc ("Kvplc"; also known as "Kvpl.3"), Kvp3 human Kv1.4 and human Kv3.4 are shown in alignment with human KvP1 As can be seen in this figure, the amino-terminal inactivation regions in each of these subunits can be readily identified by the presence of an amino terminal cysteine residue connected to a string of positively charged amino acids lysines and arginines). It is expected that amino-terminal inactivation regions of currently unidentified ion channel proteins will contain a homology of at least 60%, preferably of at least 75%, more preferably at least 85%, and most preferably at least 90 to based on the homologies present in the amino-terminal inactivation regions of the Kvpl, Kvp1.2, Kvp1.3, Kvp3, Kv1.4, and Kv3.4 channel proteins. Due to the characteristic chemical composition and structure of the globular domain on the amino-terminus of ion channel proteins, additional members of the potassium channel family, as well as members of the sodium channel family, are expected to comprise amino-terminal activation regions which are structurally and functionally equivalent to those of the hKvp1 and hKv1.4 subunits exemplified in the Examples hereof. Thus, the general features contained and described herein will be applicable to newly discovered ion channel proteins.

In preferred embodiments, the voltage-gated ion channel is a potassium or sodium channel, the intracellular receptor region is an S4-S5 cytoplasmic receptor domain of an a-subunit or a biologically active fragment thereof, and the aminoterminal inactivation region is the amino-terminal domain of an a- or p-subunit of a potassium or sodium channel protein, or a biologically active fragment thereof.

Preferably, the intracellular receptor region comprises the S4-S5 cytoplasmic receptor domain of a potassium channel protein selected from the group consisting of Kv1.1, Kv1.4, and Kv3.4, and the amino-terminal inactivation region comprises the aminoterminal inactivation domain of a potassium channel protein selected from the group consisting of Kvpl, Kvpl.2, Kvp1.3, Kvp3, Kv1.4, and Kv3.4. In particularly preferred embodiments, the intracellular receptor region has an amino acid sequence as set forth in SEQUENCE (SEQ) ID NO:1 (GenBank Accession No. L02750) and SEQ ID NO:2 (GenBank Accession No. M55514), as well as DNA sequences encoding these sequences, such as the sequences shown in SEQ ID NO:3 (GenBank Accession No.

L02750) and SEQ ID NO:4 (GenBank Accession No. M55514), and the aminoterminal inactivation region has an amino acid sequence as set forth in SEQ ID AHP 98133 P1 (GenBank Accession No. X83127) and SEQ ID NO:6 (GenBank Accession No.

L02751), as well as DNA sequences encoding these sequences, such as the sequences shown in SEQ ID NO:7 (GenBank Accession No. X83127) and SEQ ID NO:8 (GenBank Accession No. L02751). Also included are naturally occurring allelic sequences of SEQ ID NO:3, 4, 7 and 8, and equivalent degenerative codon sequences of the above.

The invention further provides methods for detecting a compound that inhibits binding of an intracellular receptor region of an a-subunit and an amino-terminal inactivation region of an ion channel protein utilizing an improved two-hybrid system, such as the yeast two-hybrid screen exemplified herein. The yeast two-hybrid screen is generally known in the art. See, e.g. Fields, et al., Nature 340:245-246 (1989), and as modified by Young, K.H. and B.A. Ozenberger in PCT WO 95/34646 (Dec. 21, 1995), the whole of which is incorporated by reference herein. The present invention provides an improved two-hybrid system by utilizing two vectors which have not heretofore been utilized in such a system. In particular, the present invention provides an improved two-hybrid system, wherein the improvement comprises a first vector containing nucleic acid sequences encoding a fusion protein of a DNA binding domain of a transcriptional activator and either an intracellular receptor region of an a-subunit of a voltage-gated ion channel or (ii) an amino-terminal inactivation region of an ion channel protein, and a second vector containing nucleic acid sequences encoding a fusion protein of an activation domain of a transcriptional activator and the intracellular receptor region if the first vector encodes a fusion protein comprising the amino-terminal inactivation region or to the amino-terminal inactivation region if the first vector encodes a fusion protein comprising the intracellular receptor region. As will be appreciated by those skilled in this art, the expression of the DNA binding fusion protein and the activation fusion protein can be interchanged, such that the intracellular receptor region is expressed as a fusion with either the transcription DNA binding domain or the activation domain of the transcriptional activator.

Briefly, using a two-hybrid system, a candidate compound is introduced into the system (a host cell), and a change in a reporter or marker protein product is assayed. Any compound which alters the level of expression of the reporter or marker, as monitored by a suitable assay, is a potential drug candidate and may be suitable for further, in-depth studies of therapeutic applications. The candidate AHP 98133 P1 compound may be of any form suitable for entry into the cytoplasm and/or nucleus of the modified host cell. Under appropriate conditions, the candidate compound may be allowed to freely diffuse into the cell, or the delivery of the compound may be facilitated by techniques and substances which enhance cell permeability, a wide variety of which are known in the art. Methods for increasing cell permeability include, without limitation, the use of organic solvents such as dimethylsulfoxide, hydrolytic enzymes (which degrade cell walls), yeast cell mutants erg-), liposomes, application of electrical current, and physical means such as compoundcoated teflon pellets.

The host organism ("modified host cell") may be any eukaryotic or prokaryotic cell, or multicellular organism. Many strains of yeast cells known to those skilled in the art may be available as host cells for practicing the present invention. Suitable host cells may also be mammalian cells, such as Chinese hamster ovary cells (CHO), the monkey COS-1 cell line, and the mammalian cell CV-1, or amphibian cells, such as a Xenopus egg cell. Bacterial cells may also be suitable hosts. For example, the various strains of E. coli HB101, MC1061) are well-known as host cells in the field of biotechnology. Various strains of B. subtilis, Pseudomonas, other bacilli and the like may also be employed in this method. Additionally, where desired, insect cells may be utilized as host cells in the method of the present invention. See, e.g.

Miller et al, Genetic Engineering, 8:277-298 (Plenum Press 1986) and references cited therein. In preferred embodiments, the modified host cell is a yeast or mammalian cell. More preferably, the modified host cell is a yeast cell selected from the group consisting of Saccharomyces cerevisiae, Schizosaccharomyces pombe, and Pichia pastoris. In a particularly preferred embodiment, the modified host cell is a yeast cell derived from a Saccharomyces organism having the genotype MATa, gal 4, his3, ade2-101, leu2-3, 112 trpl-901, ura3-52 cyh' LYS2::GALUAs-HIS3.

The transcriptional activation protein ("transcriptional activator") may vary widely as long as the DNA binding domains and the activation domains are known or can be deduced by available scientific methods. The transcriptional activator may be any protein having two components, a DNA binding component and an activation component, wherein the transcriptional activator contains an acidic a-helix for the activation of transcription. Preferably, the transcriptional activator is selected from the group consisting of Gal4, Gcn4, Hapi, Adrl, Swi5, Stel2, Mcml, Yap1, Acel, Pprl, Arg81, Lac9, QalF, VP16, LexA, non-mammalian nuclear receptors ecdysone) AHP 98133 P1 or mammalian nuclear receptors estrogen, androgens, glucocorticoids, mineralocorticoids, retinoic acid and progesterone). See Picard, et al., Gene 86:257-261 (1990). Preferably, the transcriptional activator is a yeast protein, and more preferably, the transcriptional yeast protein is Gal4, Gcn4 or Adrl. In general, any DNA binding protein which functions with an activation domain may be used. A DNA binding protein may be substituted for the DNA binding domain of a transcriptional activation protein if the recognition sequences operatively associated with the reporter gene are correspondingly engineered. Illustrative of non-yeast DNA binding proteins are mammalian steroid receptors and bacterial LexA. See Wilson, et al., Science 252:1296-1300 (1990).

The modified host cell may comprise a reporter gene whose transcription is dependent upon binding between the first and second hybrid proteins, thereby reconstituting a transcriptional activator. The reporter gene is generally selected in order that the binding of the domains of the transcriptional activation protein can be monitored by well-known and straightforward techniques. Preferably, the reporter gene is selected based on its cost, ease of measuring its activity, and low background the activity can be determined at relatively low levels of expression of the reporter gene because of a high signal to background ratio and/or minimal or no uninduced activity). Suitable reporter genes include, for example, genes which confer a selectable phenotype to cells in which the reporter gene is efficiently expressed, and/or encode a gene product enzyme) which is conveniently detected such as by in situ assay, or the like. Illustrative of reporter genes which may be used in the present invention are reporter genes selected from the following: genes which confer sensitivity to a chemical, such as CYH2 (cyclohexamide sensitivity) and CAN1 (canavine); genes which confer resistance to a chemical an antibiotic), such as neor and KAN; genes which complement auxotrophic mutations in a host organism, such as HIS3, URA3, LEU2, ARG, MET, ADE, LYS, and TRP, and the like; genes which encode toxic gene products, such as ricin; and LACZ, LAC1, firefly luciferase, bacterial luciferase, green fluorescent protein, CAT (chloramphenicol acetyl transferase), alkaline phosphatase, horseradish peroxidase, and the like.

In one embodiment, the present invention may be practiced using a conventional two-hybrid system which relies upon a positive association between two Gal4 fusion proteins, thereby reconstituting a functional Gal4 transcriptional activator which then induces transcription of a reporter gene operably linked to a Gal4 binding AHP 98133 P1 site. Transcription of the reporter gene generally produces a positive readout, typically manifested either as an enzyme activity p-galactosidase) that can be identified by a colorimetric enzyme assay, or as enhanced cell growth on a defined medium HIS3). Using conventional two-hybrid systems, a compound which is capable of inhibiting N-type inactivation of a voltage-gated ion channel is identified by its inhibitory affect on reporter gene expression reduced enzyme activity or cell growth).

In a preferred embodiment, the methods of the present invention are practiced using an "inverse" (also known as "counter selection" or "reverse") two-hybrid system. Using an inverse two-hybrid system, compounds which are capable of affecting N-type inactivation of a voltage-gated ion channel will generate a selectable and/or detectable readout complementation of an auxotrophic phenotype, expression of a detectable reporter molecule, and the like). Typically, an inverse two-hybrid system produces a positive readout under conditions wherein an agent blocks or otherwise inhibits the intermolecular binding of the interacting polypeptides an intracellular receptor region of an a-subunit and an amino-terminal inactivation region of an ion channel protein). A positive readout condition is generally identified as one or more of the following detectable conditions: an increased transcription rate of a reporter gene, an increased concentration or abundance of a polypeptide product encoded by a reporter gene, typically such as an enzyme which can be readily assayed in vivo, and/or a selectable or otherwise identifiable phenotypic change in the organism harboring the inverse two-hybrid system. Generally, a selectable or otherwise identifiable phenotypic change that characterizes a positive readout condition confers upon the organism either a selective growth advantage on a defined medium, drug resistance, a characteristic morphology or developmental stage, or a detectable enzymatic activity P-galactosidase, luciferase, alkaline phosphatase, and the like). In this manner, it is possible to efficiently identify agents (including but not limited to small molecules, polypeptides, peptides and oligonucleotides) which inhibit intermolecular binding between an intracellular receptor region of a voltage-gated ion channel and an aminoterminal inactivation region of an ion channel protein.

In another aspect, the invention provides a modified host cell comprising a first hybrid protein comprising an intracellular receptor region of an a-subunit of a voltage-gated ion channel in polypeptide linkage to a first peptide of a peptide binding AHP 98133 P1 pair, and a second hybrid protein comprising an amino-terminal inactivation region of an ion channel protein in polypeptide linkage to a second peptide of the peptide binding pair, wherein a functional or binding interaction between the two peptides causes activation of a signal transduction pathway in the modified host cell. In accordance with this aspect of the invention, one of the two peptides of the peptide binding pair is a cell compartment localization domain and the other peptide is an effector molecule. In this aspect of the invention, the activity of an endogenous effector molecule in the host cell is defective due, for example, to a mutation which prevents activation of the indicator molecule, or the effector molecule is expressed at a level that does not produce maximum activation of the indicator molecule. When the two hybrid proteins are expressed in an appropriate host cell, one hybrid protein is localized to the appropriate cell compartment via the cell compartment localization domain to the inner surface of the cell membrane via a myristylation tag).

Functional interaction between the intracellular receptor region and the aminoterminal inactivation region facilitates activation of an indicator molecule by the effector molecule, wherein activation of the indicator molecule generates a selectable or otherwise identifiable phenotypic change that characterizes a positive readout condition, as described above. In this manner, it is possible to efficiently identify agents which affect intermolecular binding between an intracellular receptor region of a voltage-gated ion channel and an amino-terminal inactivation region of an ion channel protein.

Preferably, the cell compartment localization domain is a plasma membrane localizing domain such as the sequence of v-Src that acts as a myristoylation signal, the sequence of H-Ras that acts as a signal for farnesylation and palmitoylation, or the sequence of K-Ras4B that acts a farnesylation signal. Expression of a fusion protein containing one of these domains results in farnesylation or myristoylation of the fusion protein and localization of the fusion protein or a complex containing the fusion protein to the plasma membrane. In addition, a domain such as a pleckstrin homology domain can be useful for localizing a fusion protein to the plasma membrane. For a description of plasma membrane localizing domains useful in the present invention, see, for example, Buss et al., Mol. Cell. Biol. (1988) 8:3960-3963; Karin et al., U.S. Pat. No. 5,776,689; Hancock et al., EMBO J. (1991) 10:4033-4039; and Shaw, BioEssays (1996) 18:35-46, each of which is incorporated by reference in its entirety herein. See also Broder, et al., Curr. Biol. (1998) 8(20):1121-1124, AHP 98133 P1 and Aronheim, et al., Mol. Cell. Biol. (1997) 17(6):3094-3102, both of which are incorporated by reference herein.

The effector molecule may be any peptide or polypeptide that can be expressed as a fusion protein and, when so expressed, can activate an indicator molecule. For example, the effector molecule may be an active fragment of an effector protein such as a guanine nucleotide exchange factor provided the active fragment comprises a sufficient portion of the effector protein so as to confer the effector function. In an exemplified embodiment, the effector molecule is human Sos which is known to activate Ras. See Chardin et al., Science (1993) 260:1338-1343, which is incorporated by reference herein. Activation of Ras by hSos does not require the full length Sos protein, but requires, at a minimum, an active fragment that maintains guanine nucleotide exchange activity and converts Ras-GDP to Ras-GTP. See Aronheim et al., Cell. (1994) 78:949-961; Quilliam et al., Proc. Natl.

Acad. Sci. USA (1994) 91:8512-8516; Lai et al., Mol. Cell. Biol. (1993) 13:1345-1352; and Boguski and McCormick, Nature (1993) 366:643-654. Such active fragments of an effector protein are considered to be within the meaning of the term "effector molecule" as used herein.

A wide variety of indicator molecules which cause activation of a signaling pathway, and which may be used in the practice of the present invention, are well known and readily available in the art, including, without limitation, G protein-linked receptors and their ligands receptors for epinephrine, serotonin, and glucagon, and the like, and their respective ligands); ion-channel receptors and ligands acetylcholine receptor and ligand); receptors associated with cytosolic protein tyrosine kinases and their ligands; receptors with intrinsic enzymatic activity and their ligands receptor serine/threonine kinases and receptor tyrosine kinases, and their respective ligands); and yeast G protein coupled receptors and their ligands yeast phereomone receptors such as STE2 and STE3, whose ligands are alpha-factor or a-factor). Preferably, the indicator molecule is a protein selected from the group consisting of a mitogen-activated protein (MAP) kinase, a MAP kinase related protein, a RAS protein, a RAS related protein, a Janus kinase (JAK), a JAK related protein, a c-Jun N terminal kinase (JNK), a JNK related protein, insulin receptor substrate 1 (IRS-1), and an IRS-1 related protein. The term "related," as applied in this context, refers to a protein or polypeptide having similar biological AHP 98133 P1 activity to one of the above-referenced proteins. In particularly preferred embodiments, the first or second peptide of the peptide binding pair is a MAP kinase or a Ras protein. For a description of indicator molecules useful in the present invention, see, for example, Gustin, et al., Microbiol. Mol. Biol. Rev. (1998) 62(4):1264-1300 [MAP kinase]; Force, T. and J.V. Bonventre, Hypertension (1998) 31(1):152-161 [MAP kinase]; Carter-Su, C. and L.S. Smit, Hormone Res. (1998) 53:61-83 [JAK]; Avruch, Mol. Cell. Biochem. (1998) 182(1-2):31-48 [IRS-1]; Whitmarsh, A.J. and R.J. Davis, J. Mol. Med. (1996) 74(10):589-607 [JNK and MAP kinase]; Karin et al., U.S. Pat. No. 5,776,689; and Aronheim, supra, each of which is incorporated by reference in its entirety herein.

The invention includes polynucleotides, expression vectors, and host cells transfected or transformed with expression vectors containing nucleotide sequences which encode an intracellular receptor region of an a-subunit of a voltage-gated ion channel and an amino-terminal inactivation region of an ion channel protein, or biologically active fragments thereof.

The modified host cells of the present invention comprise hybrid proteins containing polypeptides intracellular receptor regions and amino-terminal inactivation regions) or fragments thereof having amino acid sequence lengths that are at least 25%(more preferably at least 50%, and most preferably at least 75%) of the length of a disclosed polypeptide a polypeptide having an amino acid sequence as set forth in SEQ ID NO:1, SEQ ID NO:2, SEQ ID NO:5, or SEQ ID NO:6), and have at least 60% sequence identity (preferably at least 75% identity; more preferably at least 85%, and most preferably at least 90% to 95% identity) with that disclosed polypeptide, where sequence identity is determined by comparing the amino acid sequences of the polypeptides when aligned so as to maximize overlap and identity while minimizing sequence gaps. Also included in the present invention are polypeptides and fragments thereof that contain a segment comprising 6 or more (preferably 8 or more, more preferably 10 or more, and most preferably 12 or more) contiguous amino acids that shares at least 60% sequence identity (preferably at least 75% identity, more preferably at least 85% identity; and most preferably at least to 95% identity) with any such segment of any of the disclosed polypeptides.

In particular, sequence identity may be determined using WU-BLAST (Washington University BLAST) version 2.0 software, which builds upon WU-BLAST version 1.4, which in turn is based on the public domain NCBI-BLAST version 1.4 AHP 98133 P1 (Altschul and Gish, Local alignment statistics, Doolittle ed., Methods in Enzymology 266:460-480 (1996); Altschul et al., Basic local alignment search tool, Journal of Molecular Biology 215:403-410 (1990); Gish and States, Identification of protein coding regions by database similarity search, Nature Genetics 3:266-272 (1993); Karlin and Altschul, Applications and statistics for multiple high-scoring segments in molecular sequences, Proc. Natl. Acad. Sci. USA 90:5873-5877 (1993); all of which are incorporated by reference herein). WU-BLAST version 2.0 executable programs for several UNIX platforms can be downloaded from ftp://blast.wustl.edu/blast/executables. The complete suite of search programs (BLASTP, BLASTN, BLASTX, TBLASTN, and TBLASTX) is provided at that site, in addition to several support programs. WU-BLAST 2.0 is copyrighted and may not be sold or redistributed in any form or manner without the express written consent of the author; but the posted executables may otherwise be freely used for commercial, nonprofit, or academic purposes. In all search programs in the suite BLASTP, BLASTN, BLASTX, TBLASTN and TBLASTX the gapped alignment routines are integral to the database search itself, and thus yield much better sensitivity and selectivity while producing the more easily interpreted output. Gapping can optionally be turned off in all of these programs, if desired. The default penalty for a gap of length one is Q=9 for proteins and BLASTP, and Q=10 for BLASTN, but may be changed to any integer value including zero, one through eight, nine, ten, eleven, twelve through twenty, twenty-one through fifty, fifty-one through one hundred, etc.

The default per-residue penalty for extending a gap is R=2 for proteins and BLASTP, and R=10 for BLASTN, but may be changed to any integer value including zero, one, two, three, four, five, six, seven, eight, nine, ten, eleven, twelve through twenty, twenty-one through fifty, fifty-one through one hundred, etc. Any combination of values for Q and R can be used in order to align sequences so as to maximize overlap and identity while minimizing sequence gaps. The default amino acid comparison matrix is BLOSUM62, but other amino acid comparison matrices such as PAM can be utilized.

AHP 98133 P1 The present invention also includes polynucleotides that hybridize under reduced stringency conditions, more preferably stringent conditions, and most preferably highly stringent conditions, to polynucleotides described herein the polynucleotides as depicted in SEQ ID NO:3, SEQ ID NO:4, SEQ ID NO:7, or SEQ ID NO:8). Examples of stringency conditions are shown in the table below: highly stringent conditions are those that are at least as stringent as, for example, conditions A-F; stringent conditions are at least as stringent as, for example, conditions G-L; and reduced stringency conditions are at least as stringent as, for example, conditions M-

R.

Stringency Polynucleotide Hybrid Hybridization Temperature Wash Condition Hybrid Length and Buffert Temperature and Buff er A DNA:DNA 50 65 0 C; 1xSSC -or- 4200; 1SSC, 50% tormamide 0.3xSSC B DNA:DNA <50 TB*; 1XSSC TB*; 1XSSC C DNA:RNA t 50 67*C; 1xSSC -or- 45*C; 67'C; 1 xSSC, 50% formamide 0.3xSSC DDNA:RNA <50 TD*; 1XSSC TD*; 1xSSC E RNA:RNA 50 70*C; 1xSSC -or- 50'C; 7000; 1 SSC, 50% formamide 0.3xSSC F RNA:RNA <50 TF*; 1XSSC TF*; 1XSSC G DNA:DNA 50 65*C; 4xSSC -or- 42'C; 65'C; 1xSSC 4xSSC, 50% formamide H DNA:DNA <50 TH*; 4xSSC TH*; 4xSSC IDNA:RNA 2! 50 6700; 4xSSC -or- 4500; 67*C; 1xSSC 4xSSC, 50% formamide J DNA:RNA <50 Tj*; 4xSSC Tj*; 4xSSC K RNA:RNA 2! 50 70'C; 4xSSC -or- 670C; 1xSSC 4xSSC, formamide L RNA:RNA <50 TL*; 2xSSC TL*; 2xSSC M DNA:DNA 2: 50 50 0 C; 4xSSC -or- 4000; 50'C; 2xSSC 50% formamide N DNA:DNA <50 TN*; 6xSSC TN*; 6xSSC 0 DNA:RNA 50 55 0 C; 4xSSC -or- 4200; 55*C; 2xSSC 6xSSC, 50% formamide P DNA:RNA <50 1Tp*; 6xSSC Tp*; 6xSSC o RNA:RNA 50 6000; 4xSSC -or- 4500; 60'C; 2xSSC 50% formamide R RNA:RNA <50 TR*; 4xSSC TR*; 4xSSC AHP 98133 P1 The hybrid length is that anticipated for the hybridized region(s) of the hybridizing polynucleotides. When hybridizing a polynucleotide to a target polynucleotide of unknown sequence, the hybrid length is assumed to be that of the hybridizing polynucleotide. When polynucleotides of known sequence are hybridized, the hybrid length can be determined by aligning the sequences of the polynucleotides and identifying the region or regions of optimal sequence complementarity.

t: SSPE (1xSSPE is 0.15M NaCI, 10mM NaH 2

PO

4 and 1.25mM EDTA, pH 7.4) can be substituted for SSC (1xSSC is 0.15M NaCI and 15mM sodium citrate) in the hybridization and wash buffers; washes are performed for 15 minutes after hybridization is complete.

*TB TR: The hybridization temperature for hybrids anticipated to be less than 50 base pairs in length should be 5-100C less than the melting temperature (Tm) of the hybrid, where Tm is determined according to the following equations. For hybrids less than 18 base pairs in length, Tm(oC) of A T bases) of G C bases). For hybrids between 18 and 49 base pairs in length, Tm(oC) 81.5 16.6(logio[Na']) 0.41(%G+C) (600/N), where N is the number of bases in the hybrid, and is the concentration of sodium ions in the hybridization buffer for 1xSSC 0.165 M).

Additional examples of stringency conditions for polynucleotide hybridization are provided in Sambrook, E.F. Fritsch, and T. Maniatis, 1989, Molecular Cloning: A Laboratory Manual, Cold Spring Harbor Laboratory Press, Cold Spring Harbor, NY, chapters 9 and 11, and Current Protocols in Molecular Biology, 1995, F.M. Ausubel et al., eds., John Wiley Sons, Inc., sections 2.10 and 6.3-6.4, incorporated herein by reference.

Preferably, each such hybridizing polynucleotide has a length that is at least preferably at least 50%, and most preferably at least 75%) of the length of the polynucleotide of the present invention to which it hybridizes, and has at least sequence identity (more preferably, at least 75% identity; most preferably at least 90% or 95% identity) with the polynucleotide of the present invention to which it hybridizes, where sequence identity is determined by comparing the sequences of the hybridizing polynucleotides when aligned so as to maximize overlap and identity while minimizing sequence gaps.

A further aspect of the invention includes vectors comprising a DNA sequence as described above in operative association with an expression control sequence.

AHP 98133 P1 Regulatory sequences for such vectors are known to those skilled in the art and may be selected depending upon the host cells. Such selection is routine and does not form part of the present invention. These vectors may be employed in a novel process of the present invention in which a cell line transformed with a DNA sequence encoding an intracellular receptor region and an amino-termina! inactivation region, or biologically active fragments thereof, in operative association with an expression control sequence, is cultured under suitable conditions for growth.

The resulting modified host cells are placed in a growth medium, which optionally contains agar, with the test sample applied to the surface of the growth medium. The growth medium is preferably a conventional liquid medium of growth reagents and water, such as yeast synthetic medium (YSM available from B10101 (also see Rose et al., Methods in Yeast Genetics, 1990). As discussed above, this process may employ a number of known cells both prokaryotic and eukaryotic as host cells for expression of the polypeptide.

In order to illustrate the invention, description of preferred embodiments are presented below. One embodiment comprises an inverse two-hybrid system in heterologous yeast cells comprising the interacting components of the human Kvl.1 channel and Kv31 which are expressed as fusion proteins to the DNA binding domain or the activation domain, respectively, of the yeast Gal4 transcription activation protein. The expression plasmids were transfected into a host strain which contains an operatively linked CYH2 counter-selection reporter gene. Functional interaction of the Kvl.1 and KvP13 domains reconstitutes the function of the yeast Gal4 protein, drives expression of the downstream reporter gene and results in attenuation of yeast cell growth on selective media. Disruption, or blocking, of the interaction between Kvl.1 and KvP1 fusion proteins prevents the functional reconstitution of the yeast Gal4 protein, the reporter gene is not activated, and permissive yeast cell growth on selective media (rescue) is observed. A second embodiment comprises a nontranscription based two-hybrid system in heterologous yeast cells comprising the interacting components of the human Kvl.1 channel and KvP1 which are expressed as fusion proteins to the C-terminally truncated human SOS (hSOS) or the human myristoylation (hMyr) signal, respectively. The expression plasmids are transfected into a host strain which expresses a temperature sensitive cdc25 gene product.

Functional interaction of the Kvl.1 and KvP1 domains results in recruitment of the complex to the plasma membrane, allowing growth at the nonpermissive temperature.

AHP 98133 P1 Disruption of the interaction between Kvl.1 and Kv31 fusion proteins prevents the SOS-mediated rescue of the temperature sensitive cdc25 allele, thereby inhibiting yeast cell growth at the nonpermissive temperature.

A variety of alternative embodiments and variations will be apparent to those of skill in the art, including alternative host cells mammalian, bacterial, fungal, insect, and the like), alternative reporter genes, variations of the basic inverse two-hybrid method, variations of the peptide binding pair, alternative indicator molecules, and others. Moreover, in addition to high-throughput screening for potential modulators (both inhibitors and agonists) of N-type ion channel inactivation, the present invention is susceptible to a number of additional uses, such as to further define interaction domains of ion channel proteins, to characterize analogs, and to evaluate candidate compounds generated in structure-activity-relationship programs or using combinatorial chemistry. These variations, modifications, and additional applications constitute part of the present invention.

EXAMPLES:

EXAMPLE 1: CONSTRUCTION OF RECOMBINANT PLASMIDS Molecular Reagents: Generation of recombinant plasmids employed standard molecular techniques. Oligonucleotides are prepared on an ABI automated synthesizer (Perkin Elmer Cetus, Norwalk, CT). Polymerase chain reactions (PCR) employ standard techniques (Finney Current Protocols in Molecular Biology, Wiley Sons, NY, 1993). In general, PCR products are cloned into pCRII (Invitrogen, Carlsbad, CA 92008) as an interim step for PCR product sequence confirmation and propagation for further cloning. The pCRII recombinant plasmids are transfected into OneShot competent E. colicells (Invitrogen, Carlsbad, CA 92008), and other recombinant plasmids were transfected into DH5a competent E.

colicells (GIBCO Life Technologies, Rockville, Maryland 20849), following manufacturer's instructions. Recombinant plasmid stocks are prepared using Qiagen Mini preps (Qiagen, Valencia, CA). DNA sequencing is performed using di-deoxy terminator reactions (Perkin Elmer Cetus, Norwalk, CT) and an ABI 373 automated sequencer (Applied Biosystems, Foster City, CA).

AHP 98133 P1 A. Kvl.1 Kv1 channel molecular reagents: Kv 1.1 alpha S4-5 loop: The cDNA encoding the intracellular loop between the S4 and S5 transmembrane domains of the Kv1.1 potassium channel alpha subunit is generated as oligonucleotides. A 59 base sense oligonucleotide is generated with a 5' precut Ncol site, a stop codon and a 3' precut BamH! site which has the following sequence: 5'-C ATG GAG CAG ATC CTG GGC CAG ACC CTC AAA GCT AGT ATG AGA GAG CTA GGG CTG TAA G-3' [SEQ. ID NO:9] and a 59 base reverse complement oligonucleotide containing a 5' precut BamHI site, a stop codon, and a 3' precut Ncol site which has the following sequence: 5'-GATCC TTA CAG CCC TAG CTC TCT CAT ACT AGC TTT GAG GGT CTG GCC CAG GAT CTG CTC-3' [SEQ.

ID The oligonucleotides are phosphorylated and annealed by standard techniques and cloned into Ncol-BamHI sites of pAS1 (2 micron plasmid) (Durfee, T., et al., Gene Devel. (1993) 7:555-569) to generated S45-pAS1 and cloned into the Ncol-BamHI sites of pUN30AS (CEN plasmid) to generate S45-pUN30AS. See Young, et al., Nature Biotechnology (1998) 16:946-950, which is incorporated by reference in its entirety herein. Both plasmids express the S4-5 loop as a 3' fusion to the yeast Gal4 DNA binding domain protein. Confirmation and orientation of the insert is confirmed by di-deoxy terminator sequence analysis using an ABI 373 automated sequencer (Perkin Elmer Cetus, Norwalk, CT).

Kvp1 full length: The cDNA encoding the full length P1 cytoplasmic protein is generated by PCR using a 55 base sense oligonucleotide containing 5' EcoRI and Ndel restriction sites having the following sequence: 5'-C CGA ATT CGA CAT ATG AAA ATG CAA GTC TCC ATA GCC TGC ACA GAG CAC AAT TTG-3' [SEQ. ID NO:11] and a 42 base antisense oligonucleotide containing a stop codon, EcoRI and BamHI restriction sites having the following sequence: 5'-ACG GAT CCC CGA ATT CCA TTA TGA TCT ATA GTC CTT CTT GCT-3' [SEQ. ID NO:12] and human KvP1 as template (GenBank Accession No. U33428). The 1205 bp PCR product encodes bp 28-1233 of human KvP1. The cDNA encoding KvP1 is cloned into the EcoRI site of pUN100ACT (Young and Ozenberger, PCT WO 95/34646; and Young, et al.

(1998), supra), as a 3' fusion to the yeast Gal4 activation domain cDNA to generate recombinant plasmid Kv1 -pUN1OOACT.

AHP 98133 P1 Kv1 1-100 amino acids: The cDNA encoding amino acids 1-100 of the KvP1 subunit which contains the ball peptide inactivation domain is generated by PCR. The 5' oligonucleotide described for the cloning of the full length p1 protein is used in conjunction with a 45 base antisense oligonucleotide containing a stop codon and EcoRI and BamHI restriction sites having the fo!!owing sequence: 5'-ACG GAT CCC CGA ATT CCA TTA ATC TGA AAT TTG ACC TCC AAA TGT-3' [SEQ. ID NO:13] and human KvP1 as template (GenBank Accession U33428). The cDNA encoding P1 1-100 is obtained as an EcoRI fragment and cloned in the EcoRI site of pACT2 (Clontech, Palo Alto, CA 94303) to generate P1 1-100-pACT2, as well as cloned into the EcoRI site of pUN100ACT to generate P1 1-100-pUN100ACT. Both recombinant plasmids will generate KvP1 1-100 as a 3' fusion to the yeast Gal4 activation domain.

B. Kv1.4 molecular channel reagents: Kv1.4 channel Molecular Reagents: The cDNA encoding the intracellular loop between the S4 and S5 transmembrane regions of the human Kv1.4 subunit (GenBank accession M55514) was generated using a 59 base sense oligonucleotide containing a 5' precut Ncol site, and a 3' stop codon and precut BamHI site having the following sequence: C ATG GAG CAG ATC CTG GGC CAC ACC CTG AGA GCC AGC ATG CGG GAA CTG GGC CTT TAA G-3' (SEQ. ID NO:14] and a 59 base reverse complement oligonucleotide having the following sequence: GA TCC TTA AAG GCC CAG TTC CGG CAT GCT GGC TCT GAG GGT GTG GCC CAG GAT CTG CTC [SEQ. ID The oligonucleotides were phosphorylated and annealed by standard techniques (Manniatus et al., 1982, supra) and directionally cloned into the Ncol- BamHI sites of pAS1 to generate S4-S5 loop#105-pAS1. The amino terminal region of the human Kv1.4 channel containing the inactivation ball peptide region was generated by PCR using a 60 base sense oligonucleotide containing EcoRI, BamHI, Ndel sites, and a start codon having the following sequence: 5'-CGA ATT CAT ATG CGG ATC CGT AGA ATG GAG GTT GCA ATG GTG AGT GCG GAG AGC TCA GGG-3' [SEQ. ID NO:16] and a 41 base antisense oligonucleotide containing a stop codon followed by EcoRI and Sail restriction sites having the following sequence: GGT CGA CGA ATT CGT TAC CTT GCA GGA TCG GAG CTC TCG TG-3' [SEQ.

ID NO:17] and using a human the Kv1.4 clone (GenBank accession M55514) H3MV226str.seq as template. A 950 bp PCR product was generated encoding AHP 98133 P1 approximately the first 307 amino acids of the human Kv1.4 alpha subunit. This domain was obtained as a BamHI-Sall fragment from pCRII and subcloned into the BamHI-Xhol site of pACT2 to generate Nfull#49-pACT2.

Kv1.4 EAQ mutation in S4-5 loop region: A mutated version of the human IKv1.4 S4-5 loop containing a Giu substitution for Gin at amino acid position 395 (Isacoff, et al., Nature 353:86-90 (1991)) was generated using a 59 base sense oligonucleotide containing a 5' precut Ncol site, and a stop codon, and a 3' precut BamHI site having the following sequence: 5'-C ATG GAG CAG ATC CTG GGC CAC ACC CTC AGA GCC AGC ATG CGG CAA CTG GGC CTT TAA G-3' [SEQ. ID NO:18] and a 59 base reverse complement oligonucleotide containing a 5' precut BamHI site, a 3' stop codon, and precut Ncol site having the following sequence: GA TCC TTA AAG GCC CAG TTG CCG CAT GCT GGC TCT GAG GGT GTG GCC CAG GAT CTG CTC-3' [SEQ. ID NO:19].

The oligonucleotides were phosphorylated and annealed by standard techniques (Manniatus et al., 1982, supra) and directionally cloned into the Ncol- BamHI sites of pAS1 to generate EAQS45-pAS1.

EXAMPLE 2: CONSTRUCTION OF YEAST STRAINS A. Generation of Kvl.1 KvSl yeast strains: All strains are generated by transforming expression plasmids into CY770 (Ozenberger and Young, 1995, supra) using the lithium chloride method and grown on synthetic drop-out media to maintain plasmids (Rose, et al., Methods in Yeast Genetics, Cold Spring Harbor Laboratory Press, Cold Spring Harbor, NY, 1990). 3-amino-1,2,4-triazole is used to counteract background expression of the GALUAs-HIS3 reporter. All strains will contain a UASGAL-CYH2 reporter plasmids with the following combination of plasmids: S45-pUN30AS and p1-pUN100ACT (strain YKY4/Kv1.1) S45-pUN30AS and pUN100ACT (strain YKY15/Kv1.1) and 1 -pUN100ACT (strain YKY16/Kv1.1) SNF1-pAS1 and SNF4-pACT (strain CY856) S45-pUN30AS and SNF4-pACT (strain YKY2/Kv1.1) SNF1-pAS and p1-pUN100ACT (strain YKY5/Kvl.1) and p1 1-100-pUN100ACT (strain YKY17/Kv1.1) AHP 98133 P1 S45-pUN30AS and pUN100ACT (strain YKY15/Kv1.1) B. Generation of Kv1.4 yeast strains: All strains were generated by transforming expression plasmids into CY770 (Ozenberger and Young, 1995, supra) using the lithium chloride method and grown on synthetic drop-out media to maintain p!asmids (Rose et supra). 3-amino 1,2,4triazole was used to counteract background expression of the GALuAS -HIS3 reporter.

All strains contained a ura marked third plasmid, pCUP (Ozenberger and Young, 1995, supra) and the following combination of plasmids: S45#105-pAS and Nfull#49-pACT2 (strain YKY15a/Kv1.4) S45#105-pAS and SNF4-pACT (strain YKY17a/Kv1.4) SNF1-pAS1 and Nfull#49-pACT2 (strain YKY19/Kv1.4) SNF1-pAS1 and SNF4-pACT (strain CY856) EAQS45-pAS1 and Nfull#49-pACT2 (strain YKY/Kv1.4) EXAMPLE 3: YEAST TWO-HYBRID SCREEN A. Kvl.1 11 yeast two-hybrid screen: The interacting components of the human Kvl.1 channel and KvP1 are expressed as fusion proteins to the DNA binding domain or the activation domain, respectively, of the yeast Gal4 transcription activation protein. The expression plasmids are transfected into a host strain which contains a counter selection reporter gene. Functional interaction of the Kvl.1 and KvP1 domains will reconstitute the function of the yeast Gal4 protein, drive expression of the downstream reporter gene and result in attenuation of yeast cell growth on selective media. Disruption, or blocking, of the interaction between Kvl.1 and KvP1 fusion proteins, will prevent the functional reconstitution of the yeast Gal4 protein, the reporter gene will not be activated, and permissive yeast cell growth on selective media (rescue) will be observed.

B. Bioassay conditions: The N-terminal 307 amino acids of the human Kv1.4 (hKv1.4) a-subunit and the S4-S5 cytoplasmic loop of human Kv1.4 (amino acids 382-398; GenBank M05514) were expressed as fusion partners. These two channel domains interact strongly in the yeast two-hybrid system.

The human Kv1.1 potassium channel yeast strain (YKY4c/Kv1.1; 1 x 10 cells/ml) is embedded in selective media [SC-ura-leu-trp] containing 11 pg/ml AHP 98133 P1 cycloheximide. The negative control strain (CY856) is plated identically but at 2 x 104 cells/ml. Candidate compounds are applied (18 pg in 100% DMSO) to the agar surface using six 96-well microplate arrays per assay plate for a total of 576 compounds per assay plate. (Walsh, Proceeding: Intl. Symp. Lab. Auto. Robotics, Oct 19-22, 1997, Boston MA). Assay plates are incubated at 300 C for 48 hours.

Evidence of yeast growth at the point of compound application is scored visually and 'positive' compounds are retested to confirm activity. All compounds are also tested against a negative control strain expressing an unrelated but interacting protein pair, plated under identical conditions. Compounds that are selective for the Kv1.1/Kvp1 strain are scored as active and analyzed in electrophysiological assays, as described below.

Compounds active in yeast assays were examined for their ability to disrupt N-type Kv channel inactivation in CHO cells transfected with hKvl.1 alone, hKvl.1 plus hKvpl, or hKv1.4, and in Xenopus oocytes injected with the corresponding mRNA's. Several compounds eliminate N-type inactivation of hKv1.4 channels with a potency of IC50 2.5 18 pM. The compounds, however, have no effect on the inactivation kinetics of rKv4.2 channels (which inactivate by a combined N- and Cterminal mechanism), indicating that they selectively block N-type inactivation (Figure In addition to the disinactivating effect of these compounds, a parallel dose-dependent increase in peak current occurs simultaneously with the loss of inactivation. This increase in peak current is probably at least partially due to the removal of inactivation, which uncovers the "true" peak current of the channels in the cell, although other mechanisms such as a stabilization of the open state of the channel may also be occurring. At concentrations that eliminated N-type inactivation (up to 30 pM in CHO and 100 pM in oocytes) these compounds exhibited no effect on hKvl.1 channels expressed in the absence of hKvp1 (Figure At 100 pM, however, Compound blocked approximately 37% of the hKv1.1 current in CHO cells, thus high concentrations of some of these compounds are able to block Kv1.1 channels.

Further analysis of Compound using radioligand binding assays (NovaScreen, 7170 Standard Drive, Hanover, MD 21076-1334) indicates that at concentrations up to 10 pM this compound has little or no affinity for neurotransmitter receptors, uptake sites, or other ion channels (including Na+ or Ca2+ channels).

AHP 98133 P1 The mechanism of action of these Kv channel modulators (referred to herein as "disinactivators") was further explored by altering the voltage protocol so that single voltage steps to 50 mV are applied once every 20 sec (Figure This protocol significantly alters the IC50 for Compound from 2.5 pM to 13.2 pM, thus indicating a use-dependence to the disinactivation. It is known that N-termina! inactivating channel complexes are sensitive to the redox state of the intracellular medium.

When glutathione is applied to maintain channels in their reduced (and inactivating) state, however, Compound still disinactivates hKv1.4 in CHO cells (Figure 4).

Glutathione is able to block the disinactivation of the oxidizing agent H 2 0 2 thus the "disinactivators" do not appear to disinactivate channels by oxidizing their N-terminals.

The activities of Kv channel modulators were examined in several in vitro and in vivo seizure models. Field potential recordings are performed in the CA1 region of rat hippocampal slices treated with 10 pM bicuculline to induce multiple population spikes (Albus, H. and R. Williamson, Epilepsia (1998) 39(2):124-139). Bath application of Compound hippocampal slices dramatically reduces bicucculine-induced hyperexcitability. These effects are similar to, though somewhat more rapid in onset and more potent than, the known anticonvulsant phenytoin, and suggest that the disinactivators are likely to have anticonvulsant activity in vivo. To test this hypothesis directly, the disinactivators were examined for their ability to protect mice from pentylenetetrazol-induced seizures. Mice were pretreated with Compound or Compound (30-300 mg/kg valproic acid (156 mg/kg i.p.) or saline. Thirty minutes later, pentylenetetrazol (PTZ; 85 mg/kg, is administered and the mice were monitored for development of spontaneous, recurrent seizures (a seizure was defined as loss of righting reflex; see Frey, and I. Bartels, Epilepsy Res. (1997) 27:151-164). Compound and Compound protect mice against PTZ-induced seizures, with estimated ED50s of 84 and 36 mg/kg respectively for valproic acid 141 mg/kg These compounds were also examined for their ability to block shock-induced seizures using the maximal electroshock paradigm. Interestingly, none of the disinactivators described above were active in this model, indicating that their efficacy against PTZ-induced seizures is not due to global suppression of neuronal excitability and that the disinactivators likely exert their effects on specific CNS pathways. The hippocampal slice and PTZ data demonstrate AHP 98133 P1 that inhibitors of N-type Kv channel inactivation ("disinactivators") inhibit seizure activity in vitro and in vivo.

As is evident from these examples, a functional interaction between the N-terminal inactivation region and the cytoplasmic S4-S5 linker can be reconstituted in the yeast two-hybrid system, and sma!! molecule inhibitors of this interaction ("disinactivators") eliminate N-type inactivation in cells coexpressing the two regions.

Moreover, these disinactivators have potent activity in predictive preclinical anticonvulsant models, suggesting that they may have therapeutic utility in treating specific types of human epilepsy. These disinactivators represent an entirely new pharmacological class of ion channel modulators that can be used to probe the role of N-type channel inactivation in native cells and tissues and may lead to improved mechanism-based therapeutics for neurodegenerative diseases and epilepsy.

EXAMPLE 4: SIGNAL TRANSDUCTION PATHWAY SCREEN This example demonstrates the non-transcription (or transcription independent) based two-hybrid system for identifying modulators of N-type ion channel inactivation using a signal transduction pathway screen. In this nontranscription based system, the modified host cell contains a first hybrid protein comprising an intracellular receptor region of an a-subunit of a voltage-gated ion channel in polypeptide linkage to a first peptide of a peptide binding pair, and a second hybrid protein comprising an amino-terminal inactivation region of an ion channel protein in polypeptide linkage to a second peptide of the peptide binding pair, wherein binding interaction between the two peptides causes activation of a signal transduction pathway in the modified host cell. Activation of the signal transduction pathway does not occur in the presence of a molecule which inhibits binding of the intracellular receptor region and the amino-terminal inactivation region of an ion channel protein.

A. Kvl.1 31 yeast two-hybrid screen: The standard methods used to generate recombinant reagents are as described in Example 1 above.

Kvl.1 alpha S4-5 loop: The cDNA encoding the intracellular loop between S4 and transmembrane domains of the Kv1.1 potassium channel alpha subunit are generated as oligonucleotides. A 59 base sense oligonucleotide is generated with a precut EcoRI site, a stop codon and a 3' precut Xhol site which has the following AHP 98133 P1 sequence: 5'-AA TTC CAG ATC CTG GGC CAG ACC CTC AAA GCT AGT ATG AGA GAG CTA GGG CTG TAA GCC-3' [SEQ. ID NO:20] and a 59 base reverse compliment oligonucleotide containing a 5' precut Xhol site, a stop codon, and a 3' precut EcoRI site which has the following sequence: 5'-T CGA GGC TTA CAG CCC TAG CTC TCT CAT ACT AGC TTT GAG GGT CTG GCC CAG GAT CTG G-3' [SEQ. ID NO:21].

The oligonucleotides are phosphorylated and annealed by standard techniques and cloned into the EcoRI Xhol site of pMyr vector (Strategene, La Jolla CA 92037) to generate In a similar manner, complimentary oligonucleotides are generated encoding precut BamHI, S4-S5 loop, stop codon and precut Sal I sites for cloning into the BamHI-Sall sites of pSOS vector (Stratagene, La Jolla, CA 92037).

Kvbl full length: The cDNA encoding the full length 31 cytoplasmic protein is generated by PCR using a 39 base sense oligonucleotide containing a 5' BamHI site with the following sequence: 5'-AGT AGG ATC CCC ATG CCA GTC TCC ATA GCC TGC ACA GAG-3' [SEQ. ID NO:22] and a 39 base antisense oligonucleotide containing a stop codon and a Sal I site having the following sequence: 5'-GGG ACG TCG ACG CCA TTA TGA TCT ATA GTC CTT CTT GCT-3' [SEQ. ID NO:23] and Kvp1 as template (Genbank Accession No. U33428). The 1205 basepair product encodes bp 28-1233 of human Kv1l. The cDNA is cloned into the BamHI Sal I site of pSOS vector (Strategene, La Jolla CA) as a 3' fusion to hSOS and generated recombinant plasmid Kvpl-pSOS.

In a similar manner, an sense oligonucleotide is generated containing a EcoRI site and an antisense oligonucleotide containing a Sal I site are used to generate a Kvp1 PCR product which is directionally cloned into the EcoRI Sal I site of pMyr to generated Kv1p-pMyr.

B. Bioassay Conditions: The plasmids generated for the non-transcription based two-hybrid system are used to generate appropriate experimental and control yeast strains. All strains are generated by transforming the above described expression plasmids into yeast strain (Strategene, La Jolla CA) using a lithium chloride method and grown on synthetic drop-out media to maintain plasmids, as described in Example 2 above.

Interaction is tested by plating yeast strains harboring plasmid combinations on AHP 98133 P1 selective media (SD glucose vs SD galactose) and assayed for growth at 250 C and 370 C, as described in Cytotrap Vector Kit, Strategene catalog no. 217438; Karin et al., U.S. Pat. No. 5,776,689; and Aronheim et al., Mol. Cell. Biol. (1997) 17:3094- 3102. Strains that contain a functional interaction fusion protein pair demonstrate growth at 250 C on both glucose and gaiactose containing media, and at 370 C on galactose containing media. Yeast strains containing only one fusion protein or a non-interacting peptide pair demonstrate growth on glucose or galactose containing plates at 250 C, but fail to grow on either media at 370 C.

Strains: S45-pMyr and P1 -pSOS (strain YKY/1NtKv1.1) and pSOS (strain YKY/2NtKv1.1) pMyr and p1 pSOS (strain YKY/3NtKv1.1) P1-pMyr and S45-pSOS (strain YKY/4NtKv1.1) 31-pMyr and pSOS (strain YKY/5NtKv1.1) pMyr and S45-pSOS (strain YKY/6NtKv1.1) The above-described system is also useful for identifying compounds capable of disruption of the S4-S5 loop and 31 protein "disinactivators"), wherein disruption of interaction is identified by lack of cell growth at 370 C, or by growth using a pathway responsive inverse selection reporter.

The foregoing descriptions detail presently embodiments of the present invention. Numerous modifications and variations in practice thereof are expected to occur to those skilled in the art upon consideration of these descriptions. Those modifications and variations are believed to be encompassed within the claims appended hereto.

EDITORIAL NOTE APPLICATION NUMBER 52753/02 The following Sequence Listing pages 1 to 7 are part of the description. The claims pages follow on pages 35 to 42.

SEQUENCE LISTING GENERAL INFORMATION: APPLICANT: YOUNG, KATHLEEN H.

RHODES, KENNETH J.

(ii) TITLE OF INVENTION: METHODS FOR IDENTIFYING MODULATORS OF N-TYPE ION CHANNEL INACTIVATION (iii) NUMBER OF SEQUENCES: 23 (iv) CORRESPONDENCE ADDRESS: ADDRESSEE: AMERICAN HOME PRODUCTS CORPORATION STREET: FIVE GIRALDA FARMS ATTN. KAY E. BRADY CITY: MADISON STATE: NEW JERSEY COUNTRY: US ZIP: 07940 COMPUTER READABLE FORM: MEDIUM TYPE: CD-R COMPUTER: IBM PC compatible OPERATING SYSTEM: WINDOWS NT SOFTWARE: PatentIn Release Version #1.30 (vi) CURRENT APPLICATION DATA: APPLICATION NUMBER: US FILING DATE:

CLASSIFICATION:

(viii) ATTORNEY/AGENT INFORMATION: NAME: GAVIN T. BOGLE REGISTRATION NUMBER: REFERENCE/DOCKET NUMBER: AHP 98133 P1 (ix) TELECOMMUNICATION INFORMATION: TELEPHONE: (617) 665-8079 TELEFAX: (617) 876-5851 INFORMATION FOR SEQ ID NO:1: SEQUENCE CHARACTERISTICS: LENGTH: 16 amino acids TYPE: amino acid STRANDEDNESS: not relevant TOPOLOGY: not relevant (ii) MOLECULE TYPE: peptide (vi) ORIGINAL SOURCE: ORGANISM: Homo sapiens (xi) SEQUENCE DESCRIPTION: SEQ ID NO:1: Gln Ile Leu Gly Gln Thr Leu Lys Ala Ser Met Arg Glu Leu Gly Leu 1 5 10 INFORMATION FOR SEQ ID NO:2: SEQUENCE CHARACTERISTICS: LENGTH: 16 amino acids TYPE: amino acid STRANDEDNESS: not relevant TOPOLOGY: not relevant (ii) MOLECULE TYPE: peptide (vi) ORIGINAL SOURCE: ORGANISM: Homo sapiens (xi) SEQUENCE DESCRIPTION: SEQ ID NO:2: Gin Ile Leu Gly His Thr Leu Arg Ala Ser Met Arg Glu Leu Gly Leu 1 5 10 INFORMATION FOR SEQ ID NO:3: SEQUENCE CHARACTERISTICS: LENGTH: 48 base pairs TYPE: nucleic acid STRANDEDNESS: single TOPOLOGY: linear (ii) MOLECULE TYPE: DNA (genomic) (xi) SEQUENCE DESCRIPTION: SEQ ID NO:3: GATCCTGGGC CAGACCCTCA AAGCTAGTAT GAGAGAGCTA GGGCTGCT 48 INFORMATION FOR SEQ ID NO:4: SEQUENCE CHARACTERISTICS: LENGTH: 48 base pairs TYPE: nucleic acid STRANDEDNESS: single TOPOLOGY: linear (ii) MOLECULE TYPE: DNA (genomic) (xi) SEQUENCE DESCRIPTION: SEQ ID NO:4: GATCCTGGGC CACACCCTCA GAGCCAGCAT GCGGGAACTG GGCCTTCT 48 INFORMATION FOR SEQ ID SEQUENCE CHARACTERISTICS: LENGTH: 30 amino acids TYPE: amino acid STRANDEDNESS: not relevant TOPOLOGY: not relevant (ii) MOLECULE TYPE: peptide (vi) ORIGINAL SOURCE: ORGANISM: Homo sapiens (xi) SEQUENCE DESCRIPTION: SEQ ID Met Gin Val Ser Ile Ala Cys Thr Glu His Asn Leu Lys Ser Arg Asn 1 5 10 Gly Glu Asp Arg Leu Leu Ser Lys Gin Ser Ser Thr Ala Pro 25 INFORMATION FOR SEQ ID NO:6: SEQUENCE CHARACTERISTICS: LENGTH: 30 amino acids TYPE: amino acid STRANDEDNESS: not relevant TOPOLOGY: not relevant (ii) MOLECULE TYPE: peptide (vi) ORIGINAL SOURCE: ORGANISM: Homo sapiens (xi) SEQUENCE DESCRIPTION: SEQ ID NO:6: Met Glu Val Ala Met Val Ser Ala Glu Ser Ser Gly Cys Asn Ser His 1 5 10 Met Pro Tyr Gly Tyr Ala Ala Gin Ala Arg Ala Arg Glu Arg 25 INFORMATION FOR SEQ ID NO:7: SEQUENCE CHARACTERISTICS: LENGTH: 90 base pairs TYPE: nucleic acid STRANDEDNESS: single TOPOLOGY: linear (ii) MOLECULE TYPE: DNA (genomic) (xi) SEQUENCE DESCRIPTION: SEQ ID NO:7: ATGCAAGTCT CCATAGCCTG CACAGAGCAC AATTTGAAGA GTCGGAATGG TGAGGACCGA CTTCTGAGCA AGCAGAGCTC CACCGCCCCC INFORMATION FOR SEQ ID NO:8: SEQUENCE CHARACTERISTICS: LENGTH: 90 base pairs TYPE: nucleic acid STRANDEDNESS: single TOPOLOGY: linear (ii) MOLECULE TYPE: DNA (genomic) (xi) SEQUENCE DESCRIPTION: SEQ ID NO:8: ATGGAGGTTG CAATGGTGAG TGCGGAGAGC TCAGGGTGCA ACAGTCACAT GCCTTATGGT TATGCTGCCC AGGCCCGGGC CCGGGAGCGG INFORMATION FOR SEQ ID NO:9: SEQUENCE CHARACTERISTICS: LENGTH: 59 base pairs TYPE: nucleic acid STRANDEDNESS: single TOPOLOGY: linear (ii) MOLECULE TYPE: DNA (genomic) (xi) SEQUENCE DESCRIPTION: SEQ ID NO:9: CATGGAGCAG ATCCTGGGCC AGACCCTCAA AGCTAGTATG AGAGAGCTAG GGCTGTAAG 59 INFORMATION FOR SEQ ID SEQUENCE CHARACTERISTICS: LENGTH: 59 base pairs TYPE: nucleic acid STRANDEDNESS: single TOPOLOGY: linear (ii) MOLECULE TYPE: DNA (genomic) (xi) SEQUENCE DESCRIPTION: SEQ ID GATCCTTACA GCCCTAGCTC TCTCATACTA GCTTTGAGGG TCTGGCCCAG GATCTGCTC 59 INFORMATION FOR SEQ ID NO:11: SEQUENCE CHARACTERISTICS: LENGTH: 55 base pairs TYPE: nucleic acid STRANDEDNESS: single TOPOLOGY: linear (ii) MOLECULE TYPE: DNA (genomic) (xi) SEQUENCE DESCRIPTION: SEQ ID NO:11: CCGAATTCGA CATATGAAAA TGCAAGTCTC CATAGCCTGC ACAGAGCACA ATTTG INFORMATION FOR SEQ ID NO:12: SEQUENCE CHARACTERISTICS: LENGTH: 42 base pairs TYPE: nucleic acid STRANDEDNESS: single TOPOLOGY: linear (ii) MOLECULE TYPE: DNA (genomic) (xi) SEQUENCE DESCRIPTION: SEQ ID NO:12: ACGGATCCCC GAATTCCATT ATGATCTATA GTCCTTCTTG CT 42 INFORMATION FOR SEQ ID NO:13: SEQUENCE CHARACTERISTICS: LENGTH: 45 base pairs TYPE: nucleic acid STRANDEDNESS: single TOPOLOGY: linear (ii) MOLECULE TYPE: DNA (genomic) (xi) SEQUENCE DESCRIPTION: SEQ ID NO:13: ACGGATCCCC GAATTCCATT AATCTGAAAT TTGACCTCCA AATGT INFORMATION FOR SEQ ID NO:14: SEQUENCE CHARACTERISTICS: LENGTH: 59 base pairs TYPE: nucleic acid STRANDEDNESS: single TOPOLOGY: linear (ii) MOLECULE TYPE: DNA (genomic) (xi) SEQUENCE DESCRIPTION: SEQ ID NO:14: CATGGAGCAG ATCCTGGGCC ACACCCTGAG AGCCAGCATG CGGGAACTGG GCCTTTAAG 59 INFORMATION FOR SEQ ID SEQUENCE CHARACTERISTICS: LENGTH: 59 base pairs TYPE: nucleic acid STRANDEDNESS: single TOPOLOGY: linear (ii) MOLECULE TYPE: DNA (genomic) (xi) SEQUENCE DESCRIPTION: SEQ ID GATCCTTAAA GGCCCAGTTC CGGCATGCTG GCTCTGAGGG TGTGGCCCAG GATCTGCTC 59 INFORMATION FOR SEQ ID NO:16: SEQUENCE CHARACTERISTICS: LENGTH: 60 base pairs TYPE: nucleic acid STRANDEDNESS: single TOPOLOGY: linear (ii) MOLECULE TYPE: DNA (genomic) (xi) SEQUENCE DESCRIPTION: SEQ ID NO:16: CGAATTCATA TGCGGATCCG TAGAATGGAG GTTGCAATGG TGAGTGCGGA GAGCTCAGGG INFORMATION FOR SEQ ID NO:17: SEQUENCE CHARACTERISTICS: LENGTH: 41 base pairs TYPE: nucleic acid STRANDEDNESS: single TOPOLOGY: linear (ii) MOLECULE TYPE: DNA (genomic) (xi) SEQUENCE DESCRIPTION: SEQ ID NO:17: GGTCGACGAA TTCGTTACCT TGCAGGATCG GAGCTCTCGT G 41 INFORMATION FOR SEQ ID NO:18: SEQUENCE CHARACTERISTICS: LENGTH: 59 base pairs TYPE: nucleic acid STRANDEDNESS: single TOPOLOGY: linear (ii) MOLECULE TYPE: DNA (genomic) (xi) SEQUENCE DESCRIPTION: SEQ ID NO:18: CATGGAGCAG ATCCTGGGCC ACACCCTCAG AGCCAGCATG CGGCAACTGG GCCTTTAAG 59 INFORMATION FOR SEQ ID NO:19: SEQUENCE CHARACTERISTICS: LENGTH: 59 base pairs TYPE: nucleic acid STRANDEDNESS: single TOPOLOGY: linear (ii) MOLECULE TYPE: DNA (genomic) (xi) SEQUENCE DESCRIPTION: SEQ ID NO:19: GATCCTTAAA GGCCCAGTTG CCGCATGCTG GCTCTGAGGG TGTGGCCCAG GATCTGCTC 59 INFORMATION FOR SEQ ID SEQUENCE CHARACTERISTICS: LENGTH: 59 base pairs TYPE: nucleic acid STRANDEDNESS: single TOPOLOGY: linear (ii) MOLECULE TYPE: DNA (genomic) (xi) SEQUENCE DESCRIPTION: SEQ ID AATTCCAGAT CCTGGGCCAG ACCCTCAAAG CTAGTATGAG AGAGCTAGGG CTGTAAGCC 59 INFORMATION FOR SEQ ID NO:21: SEQUENCE CHARACTERISTICS: LENGTH: 59 base pairs TYPE: nucleic acid STRANDEDNESS: single TOPOLOGY: linear (ii) MOLECULE TYPE: DNA (genomic) (xi) SEQUENCE DESCRIPTION: SEQ ID NO:21: TCGAGGCTTA CAGCCCTAGC TCTCTCATAC TAGCTTTGAG GGTCTGGCCC AGGATCTGG 59 INFORMATION FOR SEQ ID NO:22: SEQUENCE CHARACTERISTICS: LENGTH: 39 base pairs TYPE: nucleic acid STRANDEDNESS: single TOPOLOGY: linear (ii) MOLECULE TYPE: DNA (genomic) (xi) SEQUENCE DESCRIPTION: SEQ ID NO:22: AGTAGGATCC CCATGCCAGT CTCCATAGCC TGCACAGAG 39 INFORMATION FOR SEQ ID NO:23: SEQUENCE CHARACTERISTICS: LENGTH: 39 base pairs TYPE: nucleic acid STRANDEDNESS: single TOPOLOGY: linear (ii) MOLECULE TYPE: DNA (genomic) (xi) SEQUENCE DESCRIPTION: SEQ ID NO:23: GGGACGTCGA CGCCATTATG ATCTATAGTC CTTCTTGCT 39

Claims (40)

1. A method of evaluating a compound for the ability to inhibit binding of an intracellular receptor region of an a-subunit of a voltage-gated ion channel, or a biologically active fragment thereof and an amino-terminal inactivation region of an ion channel protein, comprising: contacting the compound with said intracellular receptor region or a biologically active fragment thereof and said amino-terminal inactivation region; and determining the ability of said compound to interfere with the binding of said intracellular receptor region with said amino-terminal inactivation region, wherein a decrease in said binding in the presence of said compound compared to said binding in the absence of said compound indicates that said compound inhibits binding of said intracellular receptor region to said amino-terminal inactivation region.

2. The method of claim 1, wherein the voltage-gated ion channel is a potassium channel or a sodium channel.

3. The method of claim 1, wherein the voltage-gated channel protein is a potassium channel protein selected from the group consisting of Kvl.1, Kv1.2, Kv1.3, Kv1.4, Kv1.5, Kv1.6, and Kv3.4.

4. The method of claim 1, wherein the amino-terminal inactivation region comprises an amino-terminal domain of a potassium channel protein or a sodium channel protein, or a biologically active fragment thereof. The method of claim 4, wherein the potassium channel protein is selected from the group consisting of Kv3l, Kv1.2, Kv3l1.3, KvP3, Kv1.4, and Kv3.4.

6. The method of screening a candidate compound for the ability to inhibit binding of an S4-S5 intracellular receptor region of an a-subunit of a voltage-gated ion channel or a biologically active fragment thereof, to an amino-terminal inactivation region of an ion channel protein, comprising: adding said candidate compound to a modified host cell comprising a reporter gene; and X.-ilesVlRN672369\872 3 6 9 _$PeO 270405.doc monitoring expression of said reporter gene, wherein a decrease in expression is an indication that said candidate compound inhibits binding of the S4-S5 intracellular receptor region of the a-subunit or a biologically active fragment thereof to the amino-terminal inactivation region of the ion channel protein.

7. The method of claim 6, wherein the voltage-gated ion channel is a potassium channel or a sodium channel.

8. The method of claim 6, wherein the voltage-gated ion channel is a potassium channel protein selected from the group consisting of Kv1.1, Kv1.2, Kv1.3, Kv1.4, Kv1.5, Kv1.6, and Kv3.4.

9. The method of claim 6, wherein the amino-terminal inactivation region is an amino-terminal domain of a potassium channel protein selected from the group consisting of Kv31, Kv1.2, Kvp1.3, Kvp3, Kv1.4, and Kv3.4. A modified host cell comprising: a first hybrid protein comprising a DNA-binding domain of a transcriptional activator in polypeptide linkage to either an intracellular receptor region of an a-subunit of a voltage-gated ion channel or a biologically active fragment thereof or (ii) an amino-terminal inactivation region of an ion channel protein; and a second hybrid protein comprising an activation domain of a transcriptional activator in polypeptide linkage to said intracellular receptor region if said DNA-binding domain is in polypeptide linkage to said amino-terminal inactivation region or to said amino-terminal inactivation region if said DNA-binding domain is in polypeptide linkage to said intracellular receptor region.

11. The modified host cell of claim 10, wherein the voltage-gated ion channel is a potassium channel or a sodium channel.

12. The modified host cell of claim 10, wherein the intracellular receptor region is an S4-S5 cytoplasmic receptor domain of a potassium channel protein selected from the group consisting of Kv1.1, Kv1.2, Kv1.3, Kv1.4, Kv1.5, Kv1.6, and Kv3.4. X \Fles\IRN67236g\672369_spec 270405 doc

13. The modified host cell of claim 10, wherein the intracellular receptor region of an a-subunit comprises an amino acid sequence selected from the group consisting of: an amino acid sequence as set forth in SEQ ID NO:1, or a biologically active fragment thereof; an amino acid sequence as set forth in SEQ ID NO:2, or a biologically active fragment thereof; and an amino acid sequence which is at least 90 to 95% identical to the amino acid sequence of SEQ ID NO:1 or SEQ ID NO:2.

14. The modified host cell of claim 10, wherein the amino-terminal inactivation region is an amino-terminal domain of a potassium channel protein or a sodium channel protein.

15. The modified host cell of claim 10, wherein the amino-terminal inactivation region is an amino-terminal domain of a potassium channel protein selected from the group consisting of Kvpl, Kv31.2, Kvp1.3, Kvp3, Kv1.4, and Kv3.4.

16. The modified host cell of claim 10, wherein the amino-terminal inactivation region comprises an amino acid sequence selected from the group consisting of: an amino acid sequence as set forth in SEQ ID NO:5, or a biologically active fragment thereof; an amino acid sequence as set forth in SEQ ID NO:6, or a biologically active fragment thereof; and an amino acid sequence which is at least 90 to 95% identical to the amino acid sequence of SEQ ID NO:5 or SEQ ID NO:6.

17. The modified host cell of claim 10, wherein said host cell is selected from the group consisting of a yeast cell, a mammalian cell, an amphibian cell, and a bacterial cell.

18. The modified host cell of claim 17, wherein said yeast cell is selected from the group consisting of Saccharomyces cerevisiae, Schizosaccharomyces pombe, and Pichia pastoris. X \Fles'IRN67236e9672369_peCi 270405 doc

19. The modified host cell of claim 10, wherein the transcriptional activator is selected from the group consisting of Gal4, Gcn4, Hap1, Adrl, Swi5, Stel2, Mcml, Yap1, Acel, Pprl, Arg81, Lac9, QalF, VP16, LexA, and a mammalian nuclear receptor. The modified host cell of claim 10, wherein the transcriptional activator is Gal4.

21. The modified host cell of claim 10, further comprising a reporter gene whose transcription is dependent upon the first hybrid protein and the second hybrid protein being bound to each other, thereby reconstituting a transcriptional activator.

22. The modified host cell of claim 21, wherein the reporter gene is selected from the group consisting of: genes conferring sensitivity to a chemical; genes conferring resistance to a chemical; genes complementing auxotrophies; and LACZ, Luciferase gene, green fluorescent protein gene, URA, CAT, LAC1, and

23. The modified host cell of claim 21, wherein the reporter gene is a HIS gene or a CYH2 gene.

24. The modified host cell of claim 21, wherein: the first hybrid protein comprises a Gal4 DNA-binding domain in polypeptide linkage to an S4-S5 cytoplasmic receptor domain of an a-subunit of a Kv1.1 channel protein, or a biologically active fragment thereof; the second hybrid protein comprises a Gal4 activation domain in polypeptide linkage to the amino-terminal inactivation region of a KvP1 cytoplasmic protein, or a biologically active fragment thereof; and the reporter gene comprises CYH2. The modified host cell of claim 21, wherein: X TdesIRN87236M723698_spea 270405doC the first hybrid protein comprises a Gal4 DNA-binding domain in polypeptide linkage to an S4-S5 cytoplasmic receptor domain of an a-subunit of a Kv1.4 channel protein, or a biologically active fragment thereof; the second hybrid protein comprises a Gal4 activation domain in polypeptide linkage to the amino-terminal inactivation region of an a-subunit of an Kv1.4 channel protein, or a biologically active fragment hereof; and the reporter gene comprises CYH2.

26. The modified host cell of claim 21, wherein the modified host cell is a yeast cell derived from a Saccharomyces organism having the genotype MATa, gal80, gal 4, his3, ade2-101, leu2-3, 112 trpl-901, ura3-52 cyh' LYS2::GALUAs-HIS3.

27. A method for identifying compounds which inhibit N-type inactivation of a voltage-gated ion channel, comprising: administering a compound to the modified host cell of claim 21 and incubating the modified host cell for a suitable period; determining whether the administration of the compound inhibits expression of the reporter gene; and identifying a compound which inhibits expression of the reporter gene as an inhibitor of N-type inactivation of said voltage-gated ion channel.

28. A modified host cell comprising: a first hybrid protein comprising an S4-S5 intracellular receptor region of an a- subunit of a voltage-gated ion channel or a biologically active fragment thereof in polypeptide linkage to a first peptide of a peptide binding pair; and a second hybrid protein comprising an amino-terminal inactivation region of an ion channel protein in polypeptide linkage to a second peptide of the peptide binding pair; wherein binding interaction between the first peptide and the second peptide in the modified host cell causes activation of a signal transduction pathway in said modified host cell. WAFies%67236QX672389_sped 270405 doC

29. The modified host cell of claim 28, wherein the voltage-gated ion channel is a potassium channel or a sodium channel. The modified host cell of claim 28, wherein the intracellular receptor region is an S4-S5 cytoplasmic receptor domain or a potassium channel protein selected from the group consisting of Kv1.1, Kv1.2, Kv1.3, Kv1.4, Kv1.5, Kv1.6, and Kv3.4.

31. The modified host cell of claim 28, wherein the amino-terminal inactivation region is an amino-terminal domain of a potassium channel protein selected from the group consisting of Kvp1, Kv31.2, Kv1p.3, Kv33, Kv1.4, and Kv3.4.

32. The modified host cell of claim 28, wherein said host cell is selected from the group consisting of a yeast cell, a mammalian cell, an amphibian cell, and a bacterial cell.

33. The modified host cell of claim 32, wherein said yeast cell is selected from the group consisting of Saccharomyces cerevisiae, Schizosaccharomyces pombe, and Pichia pastoris.

34. The modified host cell of claim 28, wherein said first peptide of the peptide binding pair is either an effector molecule or a cell compartment localization domain, and wherein said second peptide of the peptide binding pair is a cell compartment localization domain if said first peptide is an effector molecule or (ii) an effector molecule if said first peptide is a cell compartment localization domain. The modified host cell of claim 34, wherein said effector molecule is a guanine nucleotide exchange factor and said cell compartment localization domain is a plasma membrane localization domain.

36. The modified host cell of claim 35, wherein said guanine nucleotide exchange factor is an SOS and said plasma membrane localization domain is a myristoylation signal. X Files\lRN6723691872369 sped 270405 doc

37. The modified host cell of claim 36, wherein said effector molecule activates an indicator molecule selected from the group consisting of a MAP kinase, a RAS protein, a JAK protein, a JNK protein, and IRS-1 protein.

38. A polynucleotide encoding a DNA-binding domain or an activation domain or a transcriptional activator and comprising a nucleotide sequence selected from the group consisting of: the nucleotide sequence of SEQ ID NO:3; the nucleotide sequence of SEQ ID NO:4; a nucleotide sequence which is at least 90% identical to the nucleic acid of of and which encodes a peptide that is capable of binding to an amino-terminal inactivation region of an ion channel protein; and a nucleotide sequence which is degenerate as a result of the genetic code to a nucleic acid defined in or and which encodes a peptide that is capable of binding to an amino-terminal inactivation region of an ion channel protein.

39. A polynucleotide encoding a DNA-binding domain or an activation domain of a transcriptional activator and comprising a nucleotide sequence selected from the group consisting of: the nucleotide sequence of SEQ ID NO:7; the nucleotide sequence of SEQ ID NO:8; a nucleic acid molecule which is at least 80% identical to the nucleic acid of of and which encodes a peptide that is capable of binding to an intracellular receptor region of an a-subunit of a voltage-gated ion channel; and a nucleic acid molecule which is degenerate as a result of the genetic code to a nucleic acid defined in or and which encodes a peptide that is capable of binding to an intracellular receptor region of an a- subunit of a voltage-gated ion channel. An expression vector comprising the polynucleotide of claim 38.

41. An expression vector comprising the polynucleotide of claim 39.

42. A host cell transfected or transformed with the expression vector of claim X:1FilesJRN672369g672369 sped 270405.doc

43. A host cell transfected or transformed with the expression vector of claim 41.

44. A method according to any one of claims 1 to 9 or 27 substantially as hereinbefore described, with reference to any of the Figures, Table, Sequences and/or Examples. A modified host cell according to any one of claims 10 to 26 or 28 to 37 substantially as hereinbefore described, with reference to any of the Figures, Table, Sequences and/or Examples.

46. A polynucleotide according to claims 38 or 39 substantially as hereinbefore described, with reference to any of the Figures, Table, Sequences and/or Examples.

47. An expression vector according to claims 40 or 41 substantially as hereinbefore described, with reference to any of the Figures, Table, Sequences and/or Examples.

48. A host cell according to claims 42 or 43 substantially as hereinbefore described, with reference to any of the Figures, Table, Sequences and/or Examples. DATED: 29 April 2005 PHILLIPS ORMONDE FITZPATRICK Attorneys for: WYETH W;AFesN6?238g72369_spea 270405 doc