Potassium channel mutants of the yeast Saccharomyces cerevisiae and their use for screening eukaryotic potassium channels

Abstract

processes for identifying inhibitors and activators of eukaryotic potassium channels, in which a mutated S. cerevisiae cell is used whose endogenous potassium channels TRK1, TRK2 and TOK1 are not expressed functionally, but which expresses heterologously a eukaryotic potassium channel to be studied. Other subject matters of the invention are mutated S. cerevisiae cells which do not express TRK1, TRK2 and TOK1, and the preparation and use of these mutated S. cerevisiae cells.

Description

[0001]The invention relates to processes for identifying inhibitors and activators of eukaryotic potassium channels, in which a mutated S. cerevisiae cell is used whose endogenous potassium channels TRK1, TRK2 and TOK1 are not expressed functionally, but which heterologously expresses a eukaryotic potassium channel to be studied. Other subject matters of the invention are mutated S. cerevisiae cells which do not express TRK1, TRK2 and TOK1, and the preparation and use of these mutated S. cerevisiae cells.

[0002]Each cell is enclosed by a plasma membrane with a thickness of approximately 6-8 nm. This membrane determines the cell's dimensions and separates the cell content from its environment. All biomembranes are composed of a connected bilayer of lipid molecules, which bilayer accommodates a variety of membrane proteins. While the lipid bilayer determines the basic structure of biomembranes, the proteins are responsible for most of their functions. Owing to its hydrophobic interior, the lipid bilayer acts as an impermeable barrier for most polar molecules. Only membrane proteins such as receptors, ion channels and transporters allow controlled ion flux and the transport of polar molecules (Alberts et al., 1995). Thus, proteins contribute to different ion concentrations in the cell's interior and its environment and govern the entry of nutrients and the exit of breakdown products. Most of the membrane proteins span the plasma membrane repeatedly, as do the ion channels, which thus belong to the group of the integral membrane proteins. These proteins have both hydrophobic regions, which span the lipid bilayer, and hydrophilic sections, which are exposed to the aqueous medium on either side of the membrane. Ion channels are found in all cells and, in nerve cells, are responsible for the generation of action potentials (Alberts et al., 1995). Ion channels can be differentiated on the basis of their different ion selectivity and with reference to their different opening and closing mechanisms.

[0003]Potassium channels are ubiquitous membrane proteins found both in excitable and in nonexcitable cells (for review see (Jan, L. Y. et al., 1997). Open potassium channels shift the membrane potential closer to the potassium equilibrium potential and thus away from the threshold potential for triggering an action potential. Thus, potassium channels strengthen the resting membrane potential, repolarizing the cell and in this way determine the length of the frequency of action potentials (Sanguinetti, M. C. et al., 1997; Wilde, A. A. et al., 1997; Wang, Q. et al., 1998). Owing to these functions, potassium channels also constitute the molecular cause for the generation of a number of pathological situations and are thus an interesting target for the development of therapeutical agents.

[0004]The yeast Saccharomyces cerevisiae (hereinbelow S. cerevisiae) has three potassium channels, namely TRK1, TRK2 and TOK1. The potassium channel TRK1 (YJL129c) belongs to the family of the "major facilitator" potassium permeases and, being a high-affinity potassium transporter, is responsible for the influx of potassium ions from the medium into the cell (Gaber, R. F. et al., 1988; Ko, C. H. et al., 1990; Ko, C. H. et al., 1991). The deletion mutant Δtrk1 is viable and highly polarized on at least 10 mM K⁺ (Gaber, R. F. et al., 1988; Madrid, R. et al., 1998). A Δtrk1 strain does not survive on 1 mM⁺ (Gaber, R. F. et al., 1988).

[0005]The potassium channel TRK2 (YKR050w) also belongs to the family of the "major facilitator" potassium permeases and, being a low-affinity potassium transporter, is responsible for the influx of potassium ions from the medium into the cell (Ko, C. H. et al., 1990; Ko, C. H. et al., 1991; Madrid, R. et al., 1998). The phenotype of the Δtrk2 deletion mutant is less pronounced than in the case of the Δtrk1 mutant. A Δtrk2 strain also survives on 1 mM K⁺ (Ko, C. H. et al., 1990; Madrid, R. et al., 1998).

[0006]The potassium channel TOK1 (also known as DVK1 or YORK) is responsible for the influx of potassium ions from the medium into the cell (Ketchum, K. A. et al., 1995; Fairman, C. et al., 1999). However, the direction of the ion fluxes is reversible, and, depending on the culture conditions, can therefore also take the opposite direction Fairman, C. et al., 1999).

[0007]The deletion mutant Δtrk1 Δtrk2 has already been described repeatedly (Ko, C. H. et al., 1990; Ko, C. H. et al., 1991; Madrid, R. et al., 1998; Fairman, C. et al., 1999).

[0008]In the past, this mutant was also used for identifying and describing K⁺ channels of higher eukaryotes by complementation of the phenotype. Described to date is the complementation by the inward rectifier channels KAT1 cDNA (Arabidopsis thaliana), HKT1 cDNA (Triticum aestivum), IRK1 (Mus musculus) and HKT1 K⁺/Na⁺ transporters (Triticum aestivum) (Tang, W. et al., 1995; Smith, F. W. et al., 1995; Goldstein, S. A. et al., 1996; Nakamura, R. L. et al., 1997). In addition, it has been described that the overexpression of TOK1 and its homologue ORK1 from Drosophila melanogaster in yeast cells can complement the growth deficiency of the Δtrk1 Δtrk2 mutant (Fairman, C. et al. 1999).

[0009]However, the study of a large number of eukaryotic potassium channels and the identification of substances which can modify the activity of the potassium channels is difficult since, for example, the human channels HERG1 or Kv1.5 cannot complement the lethal phenotype of Δtrk1 Δtrk2 on 5 mM KCl. Thus, no screening is possible.

[0010]The invention relates to a process for identifying inhibitors of a eukaryotic potassium channel, in which [0011]a) a mutated S. cerevisiae cell is used which does not express the three endogenous potassium channels TRK1, TRK2 and TOK1; [0012]b) a eukaryotic potassium channel is expressed heterologously in this mutated S. cerevisiae cell; [0013]c) the mutated S. cerevisiae cell is incubated together with a substance to be tested; [0014]and [0015]d) the effect of the substance to be tested on the eukaryotic potassium channel is determined.

[0016]In the mutated S. cerevisiae cell used in the method, the genes TRK1, TRK2 and TOK1 (SEQ ID NO. 1, SEQ ID NO. 2 and SEQ ID NO. 3) are switched off (Δtrk1, Δtrk2, Δtok1), preferably by knock-out, it being preferred for large portions of the genes to be deleted.

[0017]The eukaryotic potassium channel used in the process is the potassium channel to be studied, the channel for which inhibitors or activators are to be identified.

[0018]For example, the eukaryotic potassium channel is a human HERG1, a human Kv1.5, a human ROMK2 or gpIRK1 (guinea pig) channel. The eukaryotic potassium channel preferably has the natural sequence of the potassium channel in question, for example encoded by one of the sequences SEQ ID NO. 4, SEQ ID. NO. 5, SEQ ID NO. 7 (ROMK2) or SEQ ID NO. 6. However, the natural sequence of the potassium channel can also be modified, for example mutated.

[0019]Preferably, the nucleotide sequence encoding the eukaryotic potassium channel is integrated into a yeast expression plasmid, for example p423 GPD3 or a vector, for example of the pRS 42x or pRS 32x series, and the recombinant expression plasmid is introduced into the mutated S. cerevisiae cell.

[0020]The process is intended to identify substances which have an effect on the eukaryotic potassium channel. These substances inhibit the growth of the mutated S. cerevisiae cell. A substance to be studied which inhibits the heterologously expressed eukaryotic potassium channel causes the mutated S. cerevisiae cell--since it does not express endogenous potassium channels--to divide and multiply with greater difficulty or more slowly or, in a particular embodiment of the invention, to die.

[0021]The effect of the substance to be tested can be determined for example directly by measuring the optical density at 600 nm or with the aid of a growth reporter which is expressed constitutively in the mutated S. cerevisiae cell. The constitutively expressed growth reporter preferably encodes a protein which either shows fluorescence or luminescence itself or which participates in a reaction which gives a fluorescence or luminescence signal. The sequence encoding the growth reporter is preferably of a vector. Suitable growth reporters are, for example, the LacZ gene for β-galactosidase or acid phosphatase PH03, both of which are expressed under the control of a constitutive yeast promoter. The measurable fluorescence or luminescence allows conclusions regarding the cell count of the mutated S. cerevisiae cells. If no, or less, fluorescence or luminescence is measured, then the sample in question contains fewer mutated S. cerevisiae cells. If fewer mutated S. cerevisiae cells are present, then the substance to be tested has an inhibitor effect on the eukaryotic potassium channel.

[0022]The processes described can be automated with particular ease and carried out in parallel for a multiplicity of substances to be tested. In particular embodiments of the invention, two or more processes are carried out in a comparative fashion, where two or more mutated S. cerevisiae cells are analyzed in a comparative fashion. These mutated S. cerevisiae cells are preferably incubated together with the same amount of substance to be tested, but express the eukaryotic potassium channel in question to a different extent. In another particular embodiment of the invention, mutated S. cerevisiae cells which express the eukaryotic potassium channel in question to the same extent, but which are incubated together with different amounts of substance to be tested, are analyzed in a comparative fashion.

[0023]Subject matter of the invention is also a mutated S. cerevisiae cell in which the endogenous potassium channels TRK1, TRK2 and TOK1 are not expressed. A further embodiment relates to a mutated S. cerevisiae cell in which the genes TRK1, TRK2 and TOK1 are switched off; these genes have preferably been removed by knock-out in their entirety or in part, or have been mutated. A further embodiment relates to a mutated S. cerevisiae cell which is deposited at the Deutsche Sammlung von Mikroorganlsmen and Zellkulturen GmbH (Mascheroder Weg 1b. D-38124 Braunschweig) in compliance with the provisions of the Budapest Treaty on the International recognition of the deposit of microorganisms for the purposes of patent procedure; deposit number DSM 13197.

[0024]A particular embodiment of the invention relates to a mutated S. cerevisiae cell which heterologously expresses a eukaryotic potassium channel, the eukaryotic potassium channel preferably being a human potassium channel, for example a HERG1, Kv1.5 or gpIRK1 or a human Kv 4.3 [Genbank Accession Number AF 187963], TASK (Genbank Accession Number AF 006823] or ROMK2 [Genbank Accession Number U 12542] and where the potassium channel has the natural sequence or can be mutated.

[0025]The invention also relates to a process for the preparation of a mutated S. cerevisiae cell which does not express the potassium channels TRK, TRK2 and TOK1 the genes TRK1, TRK2 and TOK1 having been destroyed or deleted by knock-out.

[0026]The mutated S. cerevisiae cell can be used for example in processes for identifying substances which inhibit or activate the activity of the eukaryotic potassium channel, or it can be part of a test kit which can be used for example for determining to substances.

[0027]The invention also relates to a process for identifying activators of a eukaryotic potassium channel, in which [0028]a) a mutated S. cerevisiae cell is used which does not express the three endogenous potassium channels TRK1, TRK2 and TOK1; [0029]b) a eukaryotic potassium channel is expressed heterologously in this mutated S. cerevisiae cell; [0030]c) the mutated S. cerevisiae cell is incubated together with a substance to be tested; [0031]and [0032]d) the effect of the substance to be tested on the eukaryotic potassium channel is determined.

[0033]The invention furthermore relates to a process for identifying activators of a eukaryotic potassium channel, in which [0034]a) a mutated S. cerevisiae cell is used which does not express the three endogenous potassium channels TRK1, TRK2 and TOK1; [0035]b) a eukaryotic potassium channel is expressed heterologously in this mutated S. cerevisiae cell; [0036]c) the mutated S. cerevisiae cell is incubated together with a substance to be tested in the presence of an inhibitor of the eukaryotic potassium channel; [0037]and [0038]d) the effect of the substance to be tested on the eukaryotic potassium channel is determined.

[0039]The invention also relates to a process for the preparation of a medicament, in which [0040]a) an inhibitor of a eukaryotic potassium channel is identified, [0041]b) the inhibitor is prepared or isolated by known chemical processes, and [0042]c) physiologically acceptable additives are added to the inhibitor.

[0043]The invention also relates to a process for the preparation of a medicament, in which [0044]a) an activator of a eukaryotic potassium channel is identified, [0045]b) the activator is prepared or isolated by known chemical processes, and [0046]c) physiologically acceptable additives are added to the activator.

FIGURES

[0047]FIG. 1: Diagnostic PCR for verifying the triple knock-out. Explanation of the rows/lanes in the gel, see text, Example 2, triple knock-out.

[0048]FIG. 2: Growth of strains YM168 (Δtrk1 Δtrk2) and YM182 (Δtrk1 Δtrk2 Δtok1) on DPM medium with defined KCl concentrations at pH 6.5.

[0049]FIG. 3: Growth of strains YM189 and YM190 (in Δtrk1 Δtrk2), and YM194 and YM195 (in Δtrk1 Δtrk2 Δtok1) on DPM medium with 5 mM KCl+2 mM CsCl at pH 6.5.

[0050]FIG. 4: Growth of strains YM189 and YM191 (in Δtrk1 Δtrk2), and YM194 and YM196 (in Δtrk1 Δtrk2 Δtok1) on DPM medium with 5 mM KCl+2 mM CsCl at pH 6.5.

[0051]FIG. 5: Growth of strains YM194 and YM195 (in Δtrk1 Δtrk2 Δtok1) in DPM medium with 5 mM KCl+1 mM RbCl at pH 6.5. ("KON"=control)

[0052]FIG. 6: Growth of strains YM194 and YM196 (in Δtrk1 Δtrk2 Δtok1) in DPM medium with 5 mM KCl+1 mM CsCl at pH 6.5. ("KON"=control)

[0053]FIG. 7: Expression of the human potassium channel HERG1 in the triple mutant Δtrk1Δtrk2Δtok1 in DPM-HIS/-TRP 5 mM KCl medium in 96-well ELISA plates in the presence of 0.5 mM CsCl as activator.

[0054]The various inhibitors were employed at a final concentration of in each case 30 μM. To measure the cell density, a commercially available LacZ reporter system pYX232 by Ingenius (cat. No. MBV-032-10) was transformed into the yeast strains to be studied. Expression of the LacZ reporter gene was under the control of the constitutive Saccharomyces cerevisiae promotor TPI for the triose phosphate isomerase gene. The LacZ enzyme activity was measured via detecting the luminescence after 24 hours' growth (density of the starter culture: 0.01 OD₆₂₀) using a commercially available assay system by TROPIX. The values correspond to the average of in each case 4 measurements±SD. The two different assays were carried out independently of each other on two different days.

[0055]FIG. 8: Expression of the human potassium channel HERG1 in the triple mutant Δtrk1Δtrk2Δtok1 in DPM-HIS 5 mM KCl medium in 96-well ELISA plates in the presence of 0.5 mM CsCl as activator.

[0056]The various inhibitors were employed at a final concentration of in each case 30 μM. The cell density was measured after 38 hours' growth (density of the starter culture: 0.03 OD₆₂₀) via determination of the optical density at a wavelength of 620 nm. The values corresponded to the average of in each case 4 measurements±SD.

[0057]FIG. 9: Growth of the Saccharomyces cerevisiae wild-type strain in DPM-HIS/-TRP 5 mM KCl medium in 96-well ELISA plates in the presence of 0.5 mM CsCl. The various inhibitors were employed at a final concentration of in each case 30 μM. To measure the cell density, a commercially available LacZ reporter system pYX232 by Ingenius (cat. No. MBV-032-10) was transformed into the yeast strains to be studied. Expression of the LacZ reporter gene was under the control of the constitutive Saccharomyces cerevisiae promotor TPI for the triose phosphate isomerase gene. The LacZ enzyme activity was measured via detecting the luminescence after 24 hours' growth (density of the starter culture: 0.01 OD₆₂₀) using a commercially available assay system by TROPIX.

[0058]The values correspond to the average of in each case 4 measurements±SD. The two different assays were carried out independently of each other on two different days.

[0059]FIG. 10: Growth of the Saccharomyces cerevisiae wild-type strain in DPM medium in 96-well ELISA plates in the presence of 5 mM KCl or in the presence of 80 mM KCl. The inhibitors Ziprasidone and Pimozide were employed at a final concentration of in each case 30 μM. The cell density was measured after 24 hours' growth (density of the starter culture: 0.01 OD₆₂₀) via determination of the optical density at a wavelength of 620 nm. The values corresponded to the average of in each case 4 measurements±SD.

[0060]FIG. 11: Expression of the human potassium channel HERG1 in triple mutant Δtrk1Δtrk2Δtok1, and in the double mutant Δtrk1Δtrk2 on DPM-HIS medium in the presence of 5 mM KCl and 0.5 mM CsCl as activator.

1: Growth of the triple mutant Δtrk1Δtrk2Δtok1 upon expression of the blank vector p423GPD as negative control. 2: Growth of the triple mutant Δtrk1Δtrk2Δtok1 upon expression of p423GPD-TRK1 as positive control. 3: Growth of the triple mutant Δtrk1Δtrk2Δtok1 upon expression of p423GPD-HERG1. 4: Growth of the double mutant Δtrk1Δtrk2 upon expression of p423GPD-HERG1. The vectors and constructs used are explained in the patent application (see pages 12 et seq. and 15 et seq.).

[0061]FIG. 12: Expression of the human potassium channel Kv1.5 in triple mutant Δtrk1Δtrk2Δtok1, and in the double mutant Δtrk1Δtrk2 on DPM-HIS medium in the presence of 5 mM KCl and 2 mM RbCl as activator.

1: Growth of the triple mutant Δtrk1Δtrk2Δtok1 upon expression of the blank vector p423GPD as negative control. 2: Growth of the triple mutant Δtrk1Δtrk2Δtok1 upon expression of p423GPD-TRK1 as positive control. 3: Growth of the triple mutant Δtrk1Δtrk2Δtok1 upon expression of p423GPD-Kv1.5. 4: Expression of the double mutant Δtrk1Δtrk2 upon expression of p423GPD-Kv1.5. The vectors and constructs used are explained in the patent application (see pages 12 et seq. and 15 et seq.).

[0062]FIG. 13: Expression of the human potassium channel ROMK2 and of the yeast vector p423GPD as negative control in the triple mutant Δtrk1Δtrk2Δtok1 in DPM-HIS 5 mM KCl medium in 96-well ELISA plates.

[0063]The cell density was measured after 24 hours' growth (density of the starter culture: 0.01 OD₆₂₀) via determination of the optical density at a wavelength of 620 nm. The values corresponded to the average of in each case 4 measurements±SD.

[0064]FIG. 14: Plasmid map of p423 GPD-ROMK2.

EXAMPLES

Materials and Strains

Media

[0065]YPD (complete yeast medium): 1% Bacto yeast extract, 2% Bacto peptone, 2% Bacto agar, 2% glucose.SC (synthetic complete) Medium: 0.67% Bacto yeast nitrogen base, amino adds, 2% glucose.Sporulation medium: 1% potassium acetate, amino acids.5-FOA medium: 0.67% Bacto yeast nitrogen base, amino acids, Uracil (50 μg/ml), 2% sugar (galactose or glucose), 0.1% 5-FOAAll media are described in: (Fink, G. R. et al., 1991)

Amino Acid Dropout Mix:

[0066]L-alanine 2 g; L-arginine 2 g; L-asparagine*H₂O 2.27 g; L-aspartic acid 2 g; L-cysteine*HCl 2.6 g; L-glutamine 2 g; L-glutamic acid 2 g; glycine 2 g; myoinositol 2 g; L-isoleucine 2 g; L-methionine 2 g; PABA 0.2 g; L-phenylalanine 2 g; L-proline 2 g; L-serine 2 g; L-threonine 2 g; L-tyrosine 2 g; L-valine 2 g.

Stock Solutions for Marker Amino Acids:

TABLE-US-00001 [0067] mM g/l Adenine (100x) 30 5.53 heating (up to not more than 60° C.) Leucine (60x) 100 13.12 heating Lysine (100x) 100 18.26 -- Histidine (200x) 60 12.57 -- Tryptophan (100x) 40 8.17 -- Uracil (100x) 20 2.24 heating in 0.5% NaHCO₃ solution

Vitamin stock (50 ml): biotin 20 μg/l; calcium pantothenate 40 μg/l; thiamine 40 μg/l.Defined potassium medium (DPM): for 1.5 l (2× stock):

TABLE-US-00002 (NH₄)₂HPO₄ 8 mM 3.2 g (NH₄)₂SO₄ 29 mM 11.5 g MgSO₄ 2 mM 0.8 g (or 6 ml of 1 M stock) CaCl₂ 0.2 mM 90 μg (or 1.2 ml of 0.5 M stock) Vitamin stock 120 μl Amino acid dropout mix 6 g Lysine 330 ml of 100x stock Adenine 0.9 mM 30 ml of 100x stock → bring to pH 6.5 (or another pH) with HCl, autoclave Glucose 2% from 40% stock KCl from 1 M stock essential amino acids (with the exception of Lys/Ade) from stocks Agar

Buffer and Solutions:

[0068]TE buffer: Tris/HCl (pH 7.5) 10 mM; EDTA (pH 8.0) 1 mM;TAE buffer: Tris 40 mM; EDTA 1 mM; acetic acid 0.2 mM;SSC buffer (20×): NaCl3 M; sodium citate*2H₂O 0.3 M;Gel loading buffer. Bromphenol Blue 0.05% (w/v); sucrose 40% (w/v); EDTA, pH 8.0 0.1 M; SDS 0.5% (w/v);Hybridization buffer. SSC 5×; SDS 0.1% (w/v); dextran sulfate 5% (w/v); stop reagent 1:20;Buffer A (sterile): Tris-HCl 100 mM; NaCl, pH 9.5 300 mM;Depurination solution: HCl 0.25 M;Denaturation solution: NaCl 1.5 M; NaOH 0.5 M;Neutralization solution: NaCl 1.5 M; Tris, pH 8.0 0.5 M.Oligonucleotides (PCR primers):

TABLE-US-00003 Name Sequence (5'→ 3') RE TRK1-FL-BamHI-Fo SEQ ID NO. 7: BamHI GCG'GATCCATGCATTTTAGAAGAACGATGAGTAG TRK1-FL-PstI-Re SEQ ID NO. 8: PstI AGGTTCTGCTGCA'GTTGGTGT TRK1-FL-PstI-Fo SEQ ID NO. 9: PstI ACACCAACTGCA'GCAGAACCT TRK1-FL-XhoI-Re SEQ ID NO. 10: XhoI CGC'TCGAGTTAGAGCGTTGTGCTGCTCCT TRK1-Dia-Fo SEQ ID NO. 11: -- CCTTACCATTAGCATCACTGAT TRK1-Dia-Re1 SEQ ID NO. 12: -- CTATTAACCATTTCTCCGCTG URA-Rev SEQ ID NO. 13: -- GATTTATCTTCGTTTCCTGCAGGT TRK2-DEL-5-Fo-B SEQ ID NO. 14: BslWI CAC'GTACGTCCAGCACAATTTCACAACAGCT TRK2-DEL-5-Re SEQ ID NO. 15: SalI CAG'TCGACCTGGATGACGTCCTCTTAGCTG TRK2-DEL-3-Fo SEQ ID NO. 16: EcoRV CAGAT'ATCATGCTGCCAAGTGACAAACTG TRK2-DEL-3-Re SEQ ID NO. 17: SpeI TCA'CTAGTTGTTGATGGCTTTGGTTGGT TRK2-Dia-Fo SEQ ID NO. 18: -- GCGAAGAATAGGATGAGATGTG TRK2-Dia-Re1 SEQ ID NO. 19: -- TTGTCGTGGGTCTTCTCTGG KAN-Rev SEQ ID NO. 20: -- GCTACCTTTGCCATGTTTCAGAA TOK1-DEL-5-Fo SEQ ID NO. 21: BslWI CAC'GTACGGCAAATTTATCGAGACTCTGCGA TOK1-DEL-5-Re SEQ ID NO. 22: SalI AGG'TCGACCATATTGGCATATCCCAGCGT TOK1-DEL-3-Fo SEQ ID NO. 23: EcoRV TGGAT'ATCACCTGATACGCCC TOK1-DEL-3-Re SEQ ID NO. 24: SpeI CAA'CTAGTGCATACCAGTAGTATGAGACATGCTTG TOK1-Dia-Fo SEQ ID NO. 25: -- CCTGAGTACTCAGTACCATCTTG TOK1-Dia-Re1 SEQ ID NO. 26: -- CTGTAGATGCTGGGCATG Kv1.5-GFP-Fo SEQ ID NO. 27: SalI TACG'TCGACATGGAGATCGCCCTGGTG Kv1.5-GFP-Re SEQ ID NO. 28: SalI TACG'TCGACATCTGTTTCCCGGCTGGTG HERG1-GFP-Fo SEQ ID NO. 29: ClaI TACAT'CGATATGCCGGTGCGGAGGG HERG1-GFP-Re SEQ ID NO. 30: SalI TACG'TCGACACTGCCCGGGTCCGA

Vectors:

TABLE-US-00004 [0069]Name Size (bp) Genes Bacterial vectors pcDNA3 5446 CMV prom., T7 prom., polylinker, Sp6 prom., BGH poly (Invitrogen) (A), SV40 prom., SV 40 ori, Neomycin^R, SV 40 poly (A), ColE1 ori, Amp^R pcDNA3.1 (+/-) 5432 CMV prom., T7 prom./priming site, MCS, pcDNA3.1 (Invitrogen) reverse priming site, BGH poly (A), F1 ori, SV40 prom., SV 40 ori, Neomycin^R, SV 40 poly (A), ColE1 ori, Amp^R pUG6 4009 loxP-TEF2 prom.-kanMX-loxP-TEF2 term., ori, Amp^R pCR ® -Blunt II- 3519 lac prom./op., M13 reverse prim. site, LacZ-α ORF, TOPO SP6 prom. prim. site, MCS, TOPO ® cloning site, T7 prom. prim. site, M13 (-20) forward prim. site, M13 (-40) prim. site, fusion point, ccdB lethal gene ORF, kan gene, (kan prom., kanamycin resistance gene ORF), zeocin resistence ORF, pMB1 origin (pUC- derived) pCR ® II-TOPO 3900 LacZ-α gene, M13 reverse prim. site, SP6 prom., MCS, T7 prom., M13 (-20) forward prim. site, M13 (-40) forward prim. site, f1 origin, kanamycin resistance ORF, ampicillin resistence ORF, pMB1 origin (pUC- derived) Yeast vectors pSH47 6786 CEN6/ARSH4, URA3, CYC1 term., CRE, GAL1 prom., Amp p414 GAL1 5474 CEN6/ARSH4, TRP1, CYC1 term., GAL1 prom., Amp^R p416 GAL1 5584 CEN6/ARSH4, URA3, CYC1 term., GAL1 prom., Amp^R p416 ADH 6624 CEN6/ARSH4, URA3, CYC1 term., ADH prom., Amp^R p423 GPD3 6678 2μ, HIS3, CYC1 term., GPD3 prom., Amp^R p426 GAL1 6417 2μ, URA3, CYC1 term., GAL1 prom., Amp^R p426 GAL1- 7140 2μ, URA 3, CYC1 term., yEGFP3, GAL1 prom., Amp^R yEGFP3 p426 GAL1-SP- 7227 2μ, URA 3, CYC1 term., N-terminal 24 aa of Ste2, yEGFP3 yEGFP3, GAL1 prom., Amp^R

Strains:

[0070]Bacterial strains: DH5Δ; One Shot® TOP10 (Invitrogen)

Yeast Strains:

[0071]All yeast strains generated for this work are based on the diploid wild-type strain: W303 MATa/α ade2, his3-11-15, leu2-3-112, trp1-1, ura3-1, can1-100; ATCC No. 208352.

TABLE-US-00005 Original Strain Name Mating type Genes YM 96 w303 MATa/α ade2, his3-11-15, leu2-3-112, trp1-1, ura3-1, can1-100 YM 97 w303 MATa ade2, his3-11-15, leu2-3-112, trp1-1, ura3-1, can1-100 YM 98 w303 MATα ade2, his3-11-15, leu2-3-112, trp1-1, ura3-1, can1-100

The following yeast strains were generated:

TABLE-US-00006 Mating Genes (with the exception of ade2, his3-11-15, Strain Original Name type leu2-3-112, trp1-1, ura3-1, can1-100) YM 123 Δtrk1 in YM 96 MATα trk1::hisG-URA3-hisG YM 124 Δtrk1 in YM 96 MATa trk1::hisG-URA3-hisG YM 139 Δtok1 in YM 96 MATa/α tok1::loxP-KanMX-loxP YM140 Δtok1 in YM MATα trk1::hisG-URA3-hisG, tok1::loxP-KanMX-loxP 123 YM 141 Δtok1 in YM MATα trk1::hisG-URA3-hisG, tok1::loxP-KanMX-loxP 123 YM 142 Δtok1 in YM 96 MATa/α tok1::loxP-KanMX-loxP YM 143 Δtok1 in YM MATa trk1::hisG-URA3-hisG, tok1::loxP-KanMX-loxP 124 YM144 Δtok1 in YM MATa trk1::hisG-URA3-hisG, tok1::loxP-KanMX-loxP 124 Gal1-yEGFP3 tok1::loxP, trk2::loxP-KanMX-loxP in YM 97 YM 186 Kv1.5-pRS426- MATa pRS426-GAL1 with N24 Ste2-Kv1.5-GFP3, YM 154 Δtok1 in YM 96 MATα tok1::loxP-KanMX-loxP YM 155 Δtok1 in YM 96 MATa tok1::loxP-KanMX-loxP YM 156 Δtok1 in YM 96 MATa tok1::loxP-KanMX-loxP YM 157 Δtok1 in YM 96 MATα tok1::loxP-KanMX-loxP YM 158 Δtrk2 in YM 96 MATα trk2::loxP-KanMX-loxP YM 159 Δtrk2 in YM 96 MATa trk2::loxP-KanMX-loxP YM 160 Δtrk2 in YM 96 MATa trk2::loxP-KanMX-loxP YM 161 Δtrk2 in YM 96 MATα trk2::loxP-KanMX-loxP YM 162 Δtok1 in YM MATα trk1::hisG, tok1::loxP 123 YM 163 Δtok1 in YM MATα trk1::hisG, tok1::loxP 123 YM 164 Δtok1 in YM MATa trk1::hisG, tok1::loxP 124 YM 165 Δtok1 in YM MATa trk1::hisG, tok1::loxP 124 YM 166 YM 124 × YM MATa trk1::hisG-URA3-hisG, trk2::loxP-KanMX-loxP 160 YM 167 YM 124 × YM MATa trk1::hisG-URA3-hisG, trk2::loxP-KanMX-loxP 160 YM 168 YM 124 × YM MATα trk1::hisG-URA3-hisG, trk2::loxP-KanMX-loxP 160 YM 169 YM 124 × YM MATα trk1::hisG-URA3-hisG, trk2::loxP-KanMX-loxP 160 YM 182 Δtrk2 in YM 165 MATa trk1::hisG, tok1::loxP, trk2::loxP-KanMX-loxP YM 183 YM 166 MATa trk1::hisG, tok1::loxP, trk2::loxP-KanMX-loxP YM 184 YM 168 MATa trk1::hisG, tok1::loxP, trk2::loxP-KanMX-loxP YM 185 Kv1.5-pRS426- MATa pRS426-GAL1 with Kv1.5-GFP3, trk1::hisG, Gal1-yEGFP3 tok1::loxP, trk2::loxP-KanMX-loxP in YM 97 YM 186 Kv1.5-pRS426- MATa pRS426-GAL1 with N24 Ste2-Kv1.5-GFP3, Gal1-SP- trk1::hisG, tok1::loxP, trk2::loxP-KanMX-loxP yEGFP3 in YM 97 YM 187 Kv1.5-pRS426- MATa pRS426-GAL1 with Kv1.5-GFP3, trk1::hisG, Gal1-yEGFP3 tok1::loxP, trk2::loxP-KanMX-loxP in YM 182 YM 188 Kv1.5-pRS426- MATa pRS426-GAL1 with N24 Ste2-Kv1.5-GFP3, Gal1-SP- trk1::hisG, tok1::loxP, trk2::loxP-KanMX-loxP yEGFP3 in YM 182 YM 189 p423-GPD3 in MATa p423-GPD3, trk1::hisG-URA3-hisG, trk2::loxP- YM 168 KanMX-loxP YM 190 Kv1.5-p423- MATa p423-GPD3 with Kv1.5, trk1::hisG-URA3-hisG, GPD3 in YM trk2::loxP-KanMX-loxP 168 YM 191 HERG-p423- MATa p423-GPD3 with HERG, trk1::hisG-URA3-hisG, GPD3 in YM trk2::loxP-KanMX-loxP 168 YM 192 HCN2-p423- MATa p423-GPD3 with HCN2, trk1::hisG-URA3-hisG, GPD3 in YM trk2::loxP-KanMX-loxP 168 YM 193 IRK1-p423- MATa p423-GPD3 with IRK1, trk1::hisG-URA3-hisG, GPD3 in YM trk2::loxP-KanMX-loxP 168 YM 194 p423-GPD3 in MATa p423-GPD3, trk1::hisG, tok1::loxP, trk2::loxP- YM 182 KanMX-loxP YM 195 Kv1.5-p423- MATa p423-GPD3 with Kv1.5, trk1::hisG, tok1::loxP, GPD3 in YM trk2::loxP-KanMX-loxP 182 YM 196 HERG-p423- MATa p423-GPD3 with HERG, trk1::hisG, tok1::loxP, GPD3 in YM trk2::loxP-KanMX-loxP 182 YM 197 HCN2-p423- MATa p423-GPD3 with HCN2, trk1::hisG, tok1:loxP, GPD3 in YM trk2::loxP-KanMX-loxP 182 YM 198 IRK1-p423- MATa p423-GPD3 with IRK1, trk1::hisG, tok1::loxP, GPD3 in YM trk2::loxP-KanMX-loxP 182 YM 199 TRK1-p423- MATa p423-GPD3 with TRK1, trk1::hisG, tok1::loxP, GPD3 in YM trk2::loxP-KanMX-loxP 182

Cloned Potassium Channels:

TABLE-US-00007 [0072]A) Systematic name KCNA5 Synonyms Kv1.5, (HK2, HPCN1) Family voltage-gated potassium channel, shaker-related subfamily (member No. 5), delayed rectifier Chromosomal localization 12p13.32-p13.31 Accession NID g4504818 Protein 613 aa, 67 kD Distribution in the tissue heart, pancreatic islets and insulinoma Homologs mKcna5 (Mus musculus), 70% with hHCN4 References (Roberds, S. L. et al., 1991; Curran, M. E. et al., 1992; Snyders, D. J. et al., 1993) B) Systematic name HCN2 Synonyms BCNG2 (brain cyclic nucleotide gated channel), HAC1 Family hyperpolarization-activated and cyclic nucleotide gate potassium channel, belongs to the superfamily of the voltage-gated potassium channels Chromosomal localization 19p13.3 Accession NID g4996893 g4775348 Protein 889 aa Function pacemaker Distribution in the tissue brain, heart Homologs mHcn2 (Mus musculus) References (Ludwig, A. et al., 1999) C) Systematic name KCNH2 Synonyms HERG1 (longer splice variant) Family voltage-gated potassium channel, eag related subfamily, member No. 2 Chromosomal localization 7q35-q36 Accession NID g4557728 g4156210 Properties channel activation by K⁺channel regulator 1 accelerated References (Taglialatela, M. et al., 1998; Itoh, T. et al., 1998) D) Systematic name KCNJ2 (guinea pig) Synonyms Kir2.1, IRK1 Family inwardly rectifying potassium channel Occurrence in the tissue brain, heart, lung, kidney, placenta, skeletal musculature References (Tang, W. et al., 1995)

Methods:

[0073]ROMK2 (see appendix "Sequence ROMK2")

PCR:

[0074]Protocol for Powerscript polymerase (PAN Biotech):

Mix for Lower Reagent (Hotstart Protocol) (25 μl):

[0075]3 μl H₂O; 2.5 μl 10× OptiPerform® III buffer, pH 9.2; 10 μl 1.25 mM dNTPs (=200 μM);1.5 μl forward primer (20 μmol/μl); 1.5 μl reverse primer (20 pmol/μl); 1.5 μl 50 mM MgCl₂ (=1.25 mM); 5 μl 5× OptiZyme® enhancer.

Mix for Upper Reagent (35 μl):

[0076]23 μl H₂O; 3.5 μl 10× OptiPerform® III buffer, 1.5 μl 50 mM MgCl₂; 0.5 μl PowerScript DNA polymerase; 7 μl 5× OptiZyme® enhancer.

PCR Program (Hotstart):

1. 1 min at 94° C.

2. 1 min at 94° C.

[0077]3. 1.5 minutes at 50-55° C. (depending on primer)4. 4 minutes at 69-72° C. (depending on polymerase)5. Repeat 27× from 2.

6. 4° C. ∞

7. End.

Protocol for Amplitaq Polymerase (Perkin Elmer):

Mix for Upper Reagent (Hotstart Protocol) (50 μl):

[0078]18.1 μl H₂O; 4.2 μl 10× buffer II; 16.7 μl dNTPs; 2.5 μl forward primer, 2.5 μlreverse primer, 6 μl 25 mM MgCl₂ (=1.5 mM).

Mix for Lower Reagent (50 μl):

[0079]42 μl H₂O; 5 μl 10× buffer II; 1 μl AmpliTaq polymerase; 2 μl template.

DNA Purification

[0080]Purification of PCR reactions: The purification of PCR amplification products was carried out using the High Pure PCR Product Purification Kit (Roche)Phenol extraction: Make up sample volume to 200 μl with TE buffer. Add 200 μl of phenol/chloroform/isoamyl alcohol (25:24:1), mix and spin for 1 minute at maximum speed. Transfer top phase into new Eppendorf tube, add 200 μl of chloroform/isoamyl alcohol, mix, spin for 1 minute. Remove top phase, then precipitate with ethanol.Ethanol precipitation: To a sample volume of approx. 200 μl pipette 5 μl 5 M NaCl and 20 μl 3 M NaAc (pH 5.7). Add 2.5 volumes of 100% ethanol, mix, store for at least 30 minutes or longer at -20° C., spin for 10 minutes at 4° C., wash the pellet in 170 μl of 70% cold ethanol, spin for 3 minutes, and dry pellet at 37° C. and resuspend in 30 μl of H₂O.Isolation of plasmid DNA from E. coli: The isolation of plasmid DNA from E. coli overnight cultures was carried out using the QIAprep Spin Miniprep Kit Protocol (Qiagen)DNA Preparation from Saccharomyces cerevisiae:

[0081]Incubate the yeast cells overnight at 30° C. in 10 ml of YPD, in the morning: spin for 10 minutes at 3000 rpm, and resuspend pellet in 500 μl of 1 M sorbitol, 0.1 M EDTA (pH 7.5), and transfer into an Eppendorf tube. Add 50 μl of Zymolase (5 mg/ml, in sorbitol/EDTA), incubate for 1 hour at 37° C. and spin for 1 minute. Resuspend the pellet in 500 μl 50 mM Tris, 20 mM EDTA (pH 7.4). Add 50 μl 10% SDS, mix thoroughly and incubate for 30 minutes at 65° C., add 200 μl 5 M KAc, place on ice for 1 hour and spin for 10 minutes. Transfer the supernatant (approx. 650 μl) into a new Eppendorf tube, add 1 volume of isopropanol, mix gently and leave to stand for 5 minutes. Either spin down briefly or extract precipitated DNA with a glass hook and dry the pellet in the air. Resuspend the pellet or the DNA in 150 μl of TE buffer and dissolve for 10 minutes at 65° C.

DNA cloning techniques: All DNA cloning techniques were carried out following standard protocols.

Yeast Transformation (Lithium Acetate Method):

[0082]Incubate the yeast strain to be transformed overnight at 30° C. on the shaker in 5 ml of suitable medium; in the morning dilute the overnight culture with suitable medium (OD₆₀₀=0.4-0.5) and incubate for a further 2 hours on the shaker at 30° C. (OD₆₀₀=0.4-0.8). Spin for 3 minutes at 2500 rpm, wash pellet with 25 ml of sterile H₂O, spin for 3 minutes at 2500 rpm; resuspend pellet in 1 ml of LITE (100 mM LiAc, TE pH 7.5) and transfer suspension into an Eppendorf tube. Incubate for 5 minutes at RT, spin for 15 sec (Quickspin); wash pellet with 1 ml of 100 mM LiAc, quick-spin; depending on the cell density, resuspend pellet in 200-400 μl of 100 mM LiAc and divide into 50 μl aliquots.

[0083]Add the following in the exact sequence stated:

240 μl PEG (50%), mix suspension by gently pipetting36 μl 1 M LiAc, mix suspension by gently pipetting10 μl ss-sperm DNA (stored at -20° C.; prior to use, heat for 10 minutes at 80-90° C., then transfer to ice)2-3 μg plasmid DNA (or 8-10 μl of Miniprep in the case of knock-out transformation), mix suspension by gently pipettingIncubate transformation reaction for 30 minutes at 30° C. in an overhead rotator at slow speedTransformation reaction for 15 minutes at 42° C.Quick-spin, resuspend pellet in 200 μl of TE buffer (in the case of knock-out: resuspend pellet in 300 μl of YPD and incubate in an overhead rotator for 4 hours at 30° C.Plate 100 μl per agar plate (in the case of knock-out of all of the reaction) and incubate for 3-4 days at 30° C.Sequencing: ABI PRISM®red. protokoll/AmpliTaq®FS 1/4 BigDyeTerminator

Reaction:

TABLE-US-00008 [0084] Premix 2 μl DNA template ss DNA 50 ng ds DNA 250 ng PCR products (0.2-5 kB) 10-50 ng Primer 3-10 pmol H₂O to final volume 10 μl

Thermocycler Protocol (25 Cycles):

[0085]1. 15 seconds at 96° C.2. 15 seconds at 96° C.3. 10 seconds at 55° C.4. 4 minutes at 60° C.5. return to 2., 24×

6. 4° C. ∞

7. End.

Purification Reaction (Centri Sep Spin Colums, Princeton Separations):

[0086]Pre-soak column with 750 μl of H₂O for 30 minutes; drain liquid; spin for 2 minutes at 3000 rpm; make up reaction to 20 μl with H₂O and apply to column; spin for 2 minutes at 3000 rpm.Sample application: in sequencing tubes, 4 μl of Centri Sep eluate+20 μl of TSR (template suppression reagent); denature for 2 minutes at 90° C.

Southern Blot:

[0087]Digest DNA probe with suitable restriction enzymes, separate by gel electrophoresis and extract from the gel. Digest genomic DNA overnight with suitable restriction enzymes and separate by gel electrophoresis (1% agarose gel)

Pretreatment of the gel: Remove loading wells from the agarose gel. Depurinate the agarose gel for 15 minutes in 0.25 M HCl, then wash 2× in distilled water, denature the agarose gel for 30 minutes in 0.5 M NaOH; transfer using the Vacuum Blotter Model 785 (BioRad): into the center of the vinyl sheet, cut a window (window seal), trim the edges of the nylon membrane and the filter paper in each case 0.5 cm smaller than the gel, moisten the edge of the nylon membrane with distilled water in each case 0.5 cm wider than the window in the vinyl sheet, then moisten nylon membrane and filter paper with transfer solution

Construction of the Apparatus (Bottom to Top):

[0088]Base unit, vacuum platform, porous vacuum slab, fitter paper, nylon membrane, vinyl window, agarose gel, final frame, lidPreheat BioRad vacuum pump for 10 minutes, apply vacuum (5 inches Hg)Press gel gently along the edgePlace transfer solution (approx. 1 l 10×SSC) into upper reservoir, transfer time: 90 minutes; switch off vacuum, remove nylon membrane and rinse for 5 minutes in 2×SSC, then leave to dry in the air between filter paper. DNA immobilization: place nylon membrane on UV-permeable cling-film and apply probe at the edge as positive control; place into the UV stratalinker and start crosslinking (1200000 J→0); membrane may be stored in cling-film or between Whatman filter paper at room temperature or 4° C.

Gene Images Random Prime Labelling Module (Amersham):

[0089]Labeling of the DNA probe: Denature DNA probe for 5 minutes at 96° C. (heat shock), then place on ice. 10 μl reaction mix (nucleotide mix (5×), fluorescein-11-dUTP, dATP, dCTP, dGTP and dTTP in Tris-HCl, pH 7.8, 2-mercaptoethanol and MgCl₂); 5 μl of primer (Random Nonamers); 1 μl of enzyme solution (Klenow fragment, 5 units/ml); 22 μl of denatured DNA probe; 12 μl of H₂O. Incubate for 2 hours at 37° C. and add 2 μl of 0.5 M EDTA (=20 mM), store aliquots at -20° C. Verification of the labeling efficiency: dilute 5× nucleotide mix with TE buffer 1/5, 1/10, 1/25, 1/50, 1/100, 1/250 and 1/500; to a nylon membrane strip, apply 5 μl of DNA probe together with 5 μl of 1/5 dilution, allow to absorb briefly and wash for 15 minutes at 60° C. in prewarmed 2×SSC; apply to a reference membrane strip the remaining solutions without the 1/5 dilution and observe both membrane strips under UV light→determination of the sample intensity.

Hybridization: Prehybridize nylon membrane (blot) with warmed hybridization buffer (0.3 ml/cm²) for 2 hours at 60° C. in a rotating oven; drain buffer and retain 10 ml thereof, denature DNA probe (20 μl); (5 minutes at 96° C., then cool on ice); place probe with the 10 ml of buffer onto blot and hybridize overnight at 60° C. in the rotating oven.

Wash Steps:

[0090]15 minutes on platform shaker in warmed 1×SSC, 0.1% (w/v) SDS; 15 minutes on a platform shaker in warmed 0.5×SSC, 0.1% (w/v) SDS

Gene Images CDP-Star Detection Module (Amersham):

[0091]Stop and antibody reaction: On a shaker, incubate the blot at room temperature for 1 hour in a 1/10 dilution of stop reagent in buffer A; dilute antibody solution (alkaline phosphatase coupled to antifluorescein, 5000×) with 0.5% (w/v) BSA/buffer A, together with the blot seal into foil and incubate for 1 hour at room temperature on a shaker; remove unbound antibody solution by washing three times for 10 minutes in 0.3% Tween 10 in buffer ASignal generation and detection: Drain wash buffer, place blot on cling-film; apply 5 ml of detection reagent, allow to react for 2-5 minutes and again drain (the alkaline phosphatase causes the generation of light); wrap in cling-film and, in a dark room in red light, apply the film (Hyperfilm® MP, Amersham), expose for 0.5 2 hours in a film cassette (BioMax, Kodak), develop and scan; the blot can be stored in cling-film at 4° C.

Example 1

Construction of the Specific Deletion Cassettes

[0092]All deletions were carried out by standard methods (Fink, G. R. et al., 1991; Wall, A. et al., 1994; Guldener, U. et al., 1996; Goldstein, A. L et al., 1999).

[0093]Fragments of about 500 by each, each of which represents the region at the beginning and the end of the gene, was amplified by PCR with the primers TRK1-FL-BamHI-Fo, TRK1-FL-Pstl-Re. TRK1-FL-Pstl-Fo and TRK1-FL-XhoI-Re for TRK1 or TRK2-DEL-5-Fo-B. TRK2-DEL-5-Re, TRK2-DEL-3-Fo and TRK2-DEL-3-Re for TRK2 and TOK1-DEL-5-Fo, TOK1-DEL-5-Re, TOK1-DEL-3-Fo and TOK1-DEL-3-Re for TOK1 (see Chapter 2.3). The amplified termini later allow correct integration into the yeast genome. The yeast strain w303 a/α or w303 a/α Δtrk1 acted as DNA template.

Example 2

Construction of the Single, Double and Triple Mutants

Example 2a

Single Knock-Out

[0094]The constructed deletion cassettes for TRK1, TRK2 and TOK1 were each transformed into the diploid yeast strain YM 96 (MATa/MATα). Integration of the deletion cassettes to the genome was verified by growing the trk1 mutants (YM123/124) on (-)URA/Glc and the trk2- (YM 158-161) and tok1 mutants (YM154-157) on YPD/geniticin, since the URA3 marker in the TRK1 deletion cassette allows growth on (-)URA medium and the KAN marker in the TRK2 or TRK1 deletion cassette allows growth on geneticin (Fink, G. R. et al., 1991). The positive colonies were transferred to a sporulation plate by replica plating, whereupon MATa/MATα diploid cells sporulate after 18-24 hours without vegetative growth. After they were treated with Zymolase and regrown on YPD, tetrads of some colonies were then divided into 4 individual spores with the aid of a dissecting microscope.

[0095]The mating type of the spore colonies was determined by pairing with matching tester strains (Fink, G. R. et al., 1991). Selection for the presence of the deletion cassette was done by replica-plating on -URA medium (for trk1) and on geneticin-containing medium for trk2 and tok1. After obtaining the genomic DNA of the transformants by yeast DNA preparation, the result was verified by diagnostic PCR and Southern blot.

Example 2b

Double Knock-Out

[0096]The TOK1 deletion cassette was transformed into the haploid Δtrk1 yeast strains YM123 and Y124 and selected for integration of the TOK1 deletion cassette by growth on YPD/geneticin. The result was verified by diagnostic PCR and Southern blot. Glycerol cultures were made with the (+)URA3, (+)KAN (Δtrk1 Δtok1) strains (YM140, YM141, YM143 and YM144).

[0097]Single colonies were streaked out as patches, replica-plated on 5-FOA, and colonies were selected which had eliminated the URA3 marker and a hisG repeat from the TRK1 deletion cassette (Fink, G. R. et al., 1991). Accordingly, no colonies which lacked the URA3 gene (in TRK1) for uracil synthesis grew on (-)URA/Glc, while all colonies survived on YPD/gen owing to the resistance gene in the TOK1 deletion cassette. To remove the Kan marker from the genome, the (-)URA3 mutants were transformed with plasmid pSH47, on which the genes for Cre recombinase and uracil synthesis (URA3) are located. Positive transformants grew on (-)URA/Glc and it was then possible to induce Cre recombinase by incubation in (-)URA/Gal liquid medium. In this process, the Kan marker together with one loxP repeat is eliminated, and one loxP remains.

[0098]After the overnight culture was brought to OD₆₀₀=5, the dilutions 1:10 000 and 1:50 000 were plated onto (-)URA/Gal. Patches of single colonies, replica-plated on YPD/gen, showed no growth (this means that the Kan marker had been eliminated successfully). To remove plasmid pSH47, the cells were subsequently reselected twice 5-FOA. Glycerol cultures were made with the (-)URA(-)KAN (Δtrk1 Δtok1) strains (YM162, YM163 and YM164).

Example 2c

Triple Knock-Out

[0099]Overnight cultures in YPD were set up with single Δtrk1 Δtok1 single colonies (YM162 and YM164), and, next day, transformed with the Bs/WI/SpeI-digested TRK2 deletion cassette and plated onto YPD/KCl/geneticin. After a yeast DNA preparation, the triple knock-out was verified by diagnostic PCR and Southern blot.

TABLE-US-00009 TABLE 1 Top row, left to right: Bottom row, left to right: 1. marker 1. marker 2. YM 97 with 2. YM 182 with TRK1 DiaFo/Re1 TRK1 DiaFo/Re1 3. YM 97 with 3. YM 182 with TRK2 DiaFo/Re1 TRK2 DiaFo/Re1 4. YM 97 with 4. YM 182 with TOK1 DiaFo/Re1 TOK1 DiaFo/Re1 5. YM 97 with 5. YM 182 with TRK1 DiaFo/URARe TRK1 DiaFo/URARe 6. YM 97 with 6. YM 182 with TRK2 DiaFo/KANRe TRK2 DaiFo/KANRe 7. YM 97 with 7. YM 182 with TOK1 DiaFo/KANRe TOK1 DiaFo/KANRe 8. free 8. free 9. YM 97 with 9. YM 182 with TRK1 DiaFo/Re2 TRK1 DiaFo/Re2 10. YM 97 with 10. YM 182 with TRK2 DiaFo/Re2 TRK2 DiaFo/Re2 11. YM 97 with 11. YM 182 with TOK1 DiaFo/Re2 TOK1 DiaFo/Re2

Example 3

Subcloning and Transformation of the Human Potassium Channels into the Double and Triple Mutants

[0100]The human genes HERG, HCN2, Kv1.5 and, as positive controls, TRK1 and IRK1 (guinea pig) were excised from the plasmids harboring them (HERG between BamHI in pcDNA; HCN2 between NcoI/XhoI in pTLN; Kv1.5 between NheI/EcoRI in pcDNA3.1(-); IRK1 between BamHI/EcoRI in pSGEM) by cleavage with restriction enzymes, separated by gel electrophoresis and extracted from the gel. The individual human potassium channels were ligated into the yeast vector p423-GPD3 (Mumberg, D. et al., 1995; Ronicke, V. et al., 1997) and transformed into E. coli. Control digestion of the plasmid preparations and sequencing permitted the identification of the clones which had integrated the human gene. The plasmids were subsequently transformed into the Δtrk2 Δtrk2 double knock-out (YM 168) and into the Δtrk1 Δtrk2 Δtok1 triple knock-out (YM 182) and plated onto (-)HIS/80 mM KCl.

Example 4

Characterization of the Knock-Out Strains

Example 4a

Growth of the Double and Triple Mutants on Culture Plates at Various K⁺ Concentrations and pH Values

[0101]To compare the different potassium requirements of the various knockouts, yeast strains YM 182, YM 168 and YM 97 (WT) were incubated on DPM plates with different K⁺ concentrations and different pH values. To this end, patches of the glycerol cultures were first streaked onto 100 mM KCl/pH 6.5. After 2 days' growth, 50 mM, 30 mM and 5 mM KCl were replica-plated.

[0102]This experiment showed that both strain YM168 (Δtrk1 Δtrk2) and strain YM182 (Δtrk1 Δtrk2 Δtok1) are viable on 50 mM and 30 mM KCl. Additionally, it emerged that strain YM182 grew better in the presence of 30 mM KCl than strain YM168. None of the two strains was viable in the presence of 5 mM KCl, in contrast to the wild-type strain YM97.

[0103]To test for pH dependency, the three strains were additionally replica-plated on 100 mM and 5 mM KCl/pH 5.0 and on 100 mM and 5 mM KCl/pH 4.0. This experiment demonstrated that neither YM168 nor YM182 are viable at pH 4.0 in the presence of 100 mM KCl and 5 mM KCl. At pH 5.0 and 100 mM KCl, the growth deficiency of YM168 is more pronounced than in the case of strain YM182. Expression of TRK1 of vector pRS416GAL1 fully compensates for the growth deficiency of strains YM168 (Δtrk1 Δtrk2) and YM182 (Δtrk1 Δtrk2 Δtok1).

Example 4b

Growth of Double and Triple Mutants in Liquid Medium at Various K⁺ Concentrations

[0104]To characterize strains YM168 (Δtrk1 Δtrk2) and YM182 (Δtrk1 Δtrk2 Δtok1), on which all further experiments are based, the growth behavior of the yeast strains in liquid culture was studied. First, overnight cultures were set up in DPM/80 mM KCl, and, next morning, the cultures were brought to an OD=0.05 with DPM/5 mM KCl and with DPM/15 mM KCl. The optical density at 600 nm was determined after defined intervals with the aid of a photometer.

[0105]These studies demonstrate that the growth deficiency of strain YM182 is less pronounced at 5 mM KCl and at 15 mM KCl than in the case of strain YM168.

Example 5

Characterization of the Human Potassium Channels in Double and Triple Knock-Outs

Example 5a

Complementation Capacity for K⁺ Deficiency on Culture Plates

[0106]Each of the strains YM168 (Δtrk1 Δtrk2) and YM182 (Δtrk1 Δtrk2 Δtok1) was transformed with the human potassium channels Kv1.5 ((Fedida, D. et al, 1998); YM190 and YM195) and HERG1 ((Fedida, D. et al., 1998); YM191 and YM196) in p423-GPD3, respectively, as yeast expression vector. gpIRK1 ((Tang, W. et al., 1995); YM193 and YM198) acted as positive control in p423-GPD3 as yeast expression vector (Mumberg, D. et al., 1995; Ronicke, V. et al., 1997). The blank vector p423-GPD3 (YM189 and YM194) acted as negative control. The transformed yeast strains were plated onto (-)HIS/80 mM KCl medium. After this, patches of single colonies were replica-plated onto DPM/5 mM KCl (pH 6.5) to check the capacity of complementing the potassium deficiency.

[0107]These experiments demonstrated that the positive control gpIRK1 (YM193 and YM198) in p423-GPD3 fully complemented growth deficiency of double and triple knock-outs. The blank vector p423-GPD3 (YM189 and YM194) as negative control is not capable of complementing the growth deficiency. While the human potassium channel Kv1.5 complements the growth deficiency of triple knock-out, it does so significantly less effectively than the positive control gpIRK1. It was also observed that the human potassium channel Kv 1.5 does not complement the double knock-out Δtrk1 Δtrk2. Under the given experimental conditions, the HERG1 channel does not complement the growth deficiency of double and triple knock-outs.

Example 5b

Growth on Culture Plates in the Presence of Activators

[0108]To demonstrate the effect of activators on the various potassium channels, the strains stated above were incubated in media containing the following specific activators. Kv1.5: Rb⁺ extends the hyperpolarization phase. This means that the inwardly directed K⁺ flux is more prolonged and increases the possibility of complementing the growth deficiency.

HERG: Cs⁺ extends the hyperpolarization phase. This means that the inwardly directed K⁺ flux is more prolonged and increases the possibility of complementing the growth deficiency. This channel is inhibited by Cs⁺.IRK1: Cs⁺ blocks this channel.

[0109]The experiments with p423-GPD3-Kv1.5 demonstrated that the human Kv1.5 channel is capable of fully complementing the growth deficiency of the Δtrk1 Δtrk2 Δtok1 mutant in the presence of 2 mM RbCl (FIG. 3). Complementation of the growth deficiency of the Δtrk1 Δtrk2 mutant is markedly less effective (FIG. 3). This tallies with the results shown in Example 6a.

[0110]The experiments with p423-GPD3-HERG demonstrated that the human HERG1 channel is capable of fully complementing the growth deficiency of the Δtrk1 Δtrk2 tok1 mutant in the presence of 2 mM CsCl (FIG. 4). Complementation of the growth deficiency of the Δtrk1 Δtrk2 mutant is markedly less effective (FIG. 4). This tallies with the results shown in Example 6a.

Example 5c

Complementation by the Kv1.5 Channel in the Δtrk1 Δtrk2 Δtok1 Mutant in the Presence of RbCl in Liquid Medium

[0111]The yeast strains YM 194 and YM 195 were tested in DPM/-HIS/5 mM KCl with 1 mM RbCl for the different growth behavior in liquid medium. To this end, 10 ml of overnight culture were set up in DPM/-HIS/80 mM KCl and, next morning, brought to an OD₆₀₀ of 0.05 with the relevant media (final volume: 20 ml). The optical density at 600 nm was determined at defined intervals with the aid of a photometer.

[0112]These experiments demonstrate unambiguously that the expression of Kv1.5 of vector p423-GPD3 in a yeast strain which is deleted for TRK1, TRK2 and TOK1 is capable of complementing the growth deficiency caused thereby.

[0113]In further experiments, it was demonstrated that the complementation of the growth deficiency by Kv1.5 and also by gpIRK1 is inhibited in the presence of 2 mM CsCl.

Example 5d

Complementation by the HERG1 Channel in the Δtrk1 Δtrk2 Δtok1 Mutant in the Presence of CsCl in Liquid Medium

[0114]The yeast strains YM 194 and YM 196 were tested in DPM/-HIS/5 mM KCl with 1 mM CsCl for their different growth behavior in liquid medium. To this end, 10 ml of overnight culture were set up in DPM/-HIS/80 mM KCl and, next morning, brought to an OD₆₀₀ of 0.05 with the relevant media (final volume: 20 ml). The optical density at 600 nm was determined at defined intervals with the aid of a photometer.

[0115]These experiments demonstrate unambiguously that the expression of HERG1 of vector p423-GPD3 in a yeast strain which is deleted for TRK1, TRK2 and TOK1 is capable of complementing the growth deficiency caused thereby.

Example 6

[0116]All growth assays in the triple mutant Δtrk1Δtrk2Δtok1 were carried out in growth medium DPM (defined potassium medium) at the pH and the potassium concentration stated in each case.

[0117]The substances employed as inhibitors of the human HERG1 K⁺ channel were terfenadine (α-(4-tert-butylphenyl)-4-(α-hydroxy-αphenylbenzyl)-1-p- iperidinebutanol; HMR), pimozide (1-(4,4-bis(P-fluorophenyl)butyl)-4-(2-oxo-1-benzimidazolinyl)-piperidine- ; Sigma, Cat. No. P100), ziprasidone (5-(2-[4-(1,2-benzisothiazol-3-yl)piperazino]-ethyl)-6-chloro-1,3-dihydro- -2H-indol-2-one; HMR), loratidine (ethyl 4-(8-chloro-5,6-dihydro-11H-benzo[5,6]cyclohepta[1,2-b]pyridin-11-ylidene- )-1-piperidinecarboxytate; HMR) and sertindole (1-(2-[4-[5-chloro-1-(4-fluorophenyl)-1H-indol-3-yl]-1-piperidinyl]ethyl)- -2-imidazolidinone; HMR) (Richelson, E. 1996; Richelson, E. 1999; Delpon, E. et al., 1999; Kobayashi, T. et al., 2000; Drici, M. D. et al., 2000). Diphenyhydramine (Sigma, Cat. No. D3630) and fexofenadine (4-[hydroxy-4-[4-(hydroxydiphenylmethyl)-1-piperidinyl]butyl]-α,.al- pha.-dimethyl benzeneacetic acid hydrochloride; HMR) (Taglialatela, M. et al., 1999; DuBuske, L M. 1999), substances which should not have inhibitory effect on potassium channels, were also employed.

[0118]All substances, dissolved in DMSO, were employed in a final concentration of 30 μM. As a control, cells were measured with the same final concentration of 0.5% DMSO without substance, without DMSO addition or without substance.

[0119]As described in FIGS. 1 and 2, the human HERG1 channel is capable of complementing the growth deficiency of the triple mutant Δtrk1Δtrk2Δtok1 on medium which only contains 5 mM KCl. It was possible to demonstrate (FIG. 7, FIG. 8) that, in the presence of the substances terfenadine, pimozide, ziprasidone, sertindole and loratadine, the human HERG1 channel can no longer complement the growth deficiency of the triple mutant Δtrk1Δtrk2Δtok1 on medium which only contains 5 mM KCl.

Example 7

[0120]Incubation with the substances terfenadine, pimozide, diphenhydramine, ziprasidone, loratidine, fexofenadine and sertindole of the wild-type strain which expresses all three endogenous potassium channel proteins of yeast demonstrated that terfenadine, loratidine and sertindole are specific inhibitors of the human HERG1 channel (FIG. 9).

[0121]According to the present results, pimozide and ziprasidone must be considered as rather unspecific inhibitors. This means that these substances possibly inhibit not only the human HERG1 channel, but also the endogenous potassium channels of the yeast Saccharomyces cerevisiae. However, the present results could not exclude that the inhibitory effect found for these substances can possibly also be attributed to an inhibition of other proteins which are essential for the growth of yeast cells. To study this possibility, the action of these substances was also tested in a growth medium containing 80 mM KCl.

[0122]These studies demonstrated (FIG. 10) that pimozide inhibits the activity of the essential endogenous potassium channels TRK1 and TRK2 in an unspecific fashion.

[0123]The absence of an inhibitory effect of higher potassium concentrations allows the conclusion that pimozide has no generally toxic effect on yeast cells. In contrast, it was demonstrated that ziprasidone inhibits the growth of the yeast cells even at higher potassium concentrations and therefore has a toxic effect on Saccharomyces cerevisiae. The identification of the target protein in the yeast which might be responsible for this effect is as yet outstanding.

[0124]In conclusion, these experiments demonstrate that the above-described system makes it possible in practice to identify, in the yeast Saccharomyces cerevisiae, substances which specifically inhibit the human potassium channels.

[0125]The results can be seen from FIG. 10.

Example 8

[0126]The human potassium channels HERG1 and Kv1.5 do not complement the growth deficiency of the double mutant Δtrk1Δtrk2 (FIG. 11 and FIG. 12).

Results: FIGS. 11 and 12.

[0127]FIGS. 11 and 12 demonstrate that the human potassium channels HERG1 and Kv1.5 do not complement the growth deficiency of the double mutant Δtrk1Δtrk2 (in each case 4th segment in FIGS. 11 and 12). The comparison with the negative control, i.e. the blank vector up423GPD in the triple mutant Δtrk1Δtrk2Δtok1 (in each case let segment of FIGS. 11 and 12), shows no improved growth. The negative control p423GPD in the double mutant Δtrk1Δtrk2 is not shown, but does not differ from the negative control p423GPD in the triple mutant Δtrk1Δtrk2Δtok1. In contrast, the human potassium channels HERG1 and Kv1.5 complement the growth deficiency of the triple mutant Δtrk1Δtrk2Δtok1 (in each case 3rd segment of FIGS. 11 and 12).

Example 9

[0128]The human potassium channel ROMK2 ((Shuck, M. E. et al., 1994; Bock, J. H. et al., 1997); Sequence SEQ ID NO. 31 hROMK2) was subcloned into the yeast vector p423GPD and transformed into the triple mutant Δtrk1Δtrk2Δtok1. The studies demonstrated that this human potassium channel too is capable of complementing the growth deficiency of the triple mutant Δtrk1Δtrk2Δtok1.

[0129]The capability of this human potassium channel to complement the growth deficiency of the double mutant Δtrk1Δtrk2 has not been studied as yet. No substances are known as yet which specifically inhibit the ROMK2 channel.

[0130]The results can be seen from FIG. 13.

REFERENCES

[0131]Curran, M. E., Landes, G. M., and Keating. M. T. Molecular cloning, characterization, and genomic localization of a human potassium channel gene. Genomics 12: 729-737. (1992) [0132]Dascal, N., Schreibmayer, W., Lim, N. F., Wang, W., Chavkin, C., DiMagno, L, Labarca, C., Kieffer, B. L., Gaveriaux-Ruff, C., and Trollinger, D. Atrial G protein-activated K+ channel: expression cloning and molecular properties. Proc. Natl. Acad. Sci. U.S.A. 90: 10235-10239. (1993) [0133]Fairman, C., Zhou, X., and Kung, C. Potassium uptake through the TOK1 K+ channel in the budding yeast. J. Membr. Biol. 168: 149-157. (1999) [0134]Fedida, D., Chen, F. S., and Zhang, X. The 1997 Stevenson Award Lecture. Cardiac K+ channel gating: cloned delayed rectifier mechanisms and drug modulation. Can. J. Physiol. Pharmacol. 76: 77-89. (1998) [0135]Fink, G. R. and Guthrie, C. Guide to Yeast Genetics and Molecular Biology. Guthrie, C. and Fink, G. R. (194). 1991. Academic Press, Inc. Methods in Enzymology. Ref Type: Book, Whole [0136]Gaber, R. F., Styles, C. A., and Fink, G. R. TRK1 encodes a plasma membrane protein required for high-affinity potassium transport in Saccharomyces cerevisiae. Mol. Cell Biol. 8: 2848-2859. (1988) [0137]Goldstein, A. L. and McCusker, J. H. Three new dominant drug resistance cassettes for gene disruption in Saccharomyces cerevisiae [In Process Citation] Yeast. 15: 1541-1553. (1999) [0138]Goldstein, S. A., Price, L. A., Rosenthal, D. N., and Pausch, M. H. ORK1, a potassium-selective leak channel with two pore domains cloned from Drosophila melanogaster by expression in Saccharomyces cerevisiae [published erratum appears in Proc Natl Acad Sci USA 1999 Jan. 5; 96(1):318] Proc. Natl. Acad. Sci. U.S.A. 93: 13256-13261. (1996) [0139]Guldener, U., Heck, S., Fielder, T., Beinhauer, J., and Hegemann, J. H. A new efficient gene disruption cassette for repeated use in budding yeast. Nucleic. Acids. Res. 24: 2519-2524. (1996) [0140]Ikeda, K., Kobayashi, K., Kobayashi, T., Ichikawa, T., Kumanishi, T., Kishida, H., Yano, R., and Manabe, T. Functional coupling of the nociceptin/orphanin FQ receptor with the G-protein-activated K+(GIRK) channel. Brain Res. Mol. Brain Res. 45: 117-126. (1997) [0141]Itoh, T., Tanaka, T., Nagai, R., Kamiya, T., Sawayama, T., Nakayama, T., Tomoike, H., Sakurada, H., Yazaki, Y., and Nakamura, Y. Genomic organization and mutational analysis of HERG, a gene responsible for familial long QT syndrome. Hum. Genet. 102: 435-439. (1998) [0142]Jan, L. Y. and Jan, Y. N. Cloned potassium channels from eukaryotes and prokaryotes. Annu. Rev. Neurosci. 20:91-123: 91-123. (1997) [0143]Jelacic, T. M., Sims, S. M., and Clapham, D. E. Functional expression and characterization of G-protein-gated inwardly rectifying K+ channels containing GIRK3. J. Membr. Biol. 169: 123-129. (1999) [0144]Ketchum, K. A., Joiner, W. J., Sellers, A. J., Kaczmarek, L. K., and Goldstein, S. A. A new family of outwardly rectifying potassium channel proteins with two pore domains in tandem. Nature 376: 690-695. (1995) [0145]Ko, C. H. Buckley, A. M. and Gaber, R. F. TRK2 is required for low affinity K+ transport in Saccharomyces cerevisiae. Genetics 125: 305-312. (1990) [0146]Ko, C. H. and Gaber, R. F. TRK1 and TRK2 encode structurally related K+ transporters in Saccharomyces cerevisiae. Mol. Cell Biol. 11: 4266-4273. (1991) [0147]Kubo, Y., Reuveny, E., Slesinger, P. A., Jan, Y. N., and Jan, L. Y. Primary structure and functional expression of a rat G-protein-coupled muscarinic potassium channel [see comments] Nature 364: 802-806. (1993) [0148]Ludwig, A., Zong, X., Stieber, J., Hullin, R., Hofmann, F., and Biel, M. Two pacemaker channels from human heart with profoundly different activation kinetics. EMBO J. 18: 2323-2329. (1999) [0149]Madrid, R., Gomez, M. J., Ramos, J., and Rodriguez-Navarro, A. Ectopic potassium uptake in trk1 trk2 mutants of Saccharomyces cerevisiae correlates with a highly hyperpolarized membrane potential. J. Biol. Chem. 273: 14838-14844. (1998) [0150]Main, M. J., Brown, J., Brown, S., Fraser, N. J., and Foord, S. M. The CGRP receptor can couple via pertussis toxin sensitive and insensitive G proteins. FEBS Lett. 441: 6-10. (1998) [0151]Mumberg, D., Muller, R., and Funk, M. Yeast vectors for the controlled expression of heterologous proteins in different genetic backgrounds. Gene 156: 119-122. (1995) [0152]Myers, A. M., Pape, L. K., and Tzagoloff, A. Mitochondrial protein synthesis is required for maintenance of intact mitochondrial genomes in Saccharomyces cerevisiae. EMBO J. 4: 2087-2092. (1985) [0153]Nakamura, R. L, Anderson, J. A., and Gaber, R. F. Determination of key structural requirements of a K+ channel pore. J. Biol. Chem. 272: 1011-1018. (1997) [0154]Roberds, S. L. and Tamkun, M. M. Cloning and tissue-specific expression of five voltage-gated potassium channel cDNAs expressed in rat heart. Proc. Natl. Acad. Sci. U.S.A. 88: 1798-1802. (1991) [0155]Ronicke, V., Graulich, W., Mumberg, D., Muller, R., and Funk, M. Use of conditional promoters for expression of heterologous proteins in Saccharomyces cerevisiae. Methods Enzymol. 283:313-22: 313-322. (1997) [0156]Sanguinetti, M. C. and Zou, A. Molecular physiology of cardiac delayed rectifier K+ channels. Heart Vessels Suppl 12: 170-172. (1997) [0157]Schreibmayer, W., Dessauer, C. W., Vorobiov, D., Gilman, A. G., Lester, H. A., Davidson. N., and Dascal, N. Inhibition of an inwardly rectifying K+ channel by G-protein alpha- subunits. Nature 380: 624-627. (1996) [0158]Smith, F. W., Ealing, P. M., Hawkesford, M. J., and Clarkson, D. T. Plant members of a family of sulfate transporters reveal functional subtypes. Proc. Natl. Acad. Sci. U.S.A. 92: 9373-9377. (1995) [0159]Snyders, D. J., Tamkun, M. M., and Bennett, P. B. A rapidly activating and slowly inactivating potassium channel cloned from human heart. Functional analysis after stable mammalian cell culture expression. J. Gen. Physiol. 101: 513-543. (1993) [0160]Taglialatela, M., Castaldo, P., Pannaccione, A., Giorgio, G., and Annunziato, L. Human ether-a-gogo related gene (HERG) K+ channels as pharmacological targets: present and future implications. Biochem. Pharmacol. 55:1741-1746. (1998) [0161]Tang, W., Ruknudin, A., Yang, W. P., Shaw, S. Y., Knickerbocker, A., and Kurtz. S. Functional expression of a vertebrate inwardly rectifying K+ channel in yeast. Mol. Biol. Cell 6: 1231-1240. (1995) [0162]Wach, A. Brachat. A. Pohlmann. R. and Philippsen. P. New heterologous modules for classical or PCR-based gene disruptions in Saccharomyces cerevisiae. Yeast 10: 1793-1808. (1994) [0163]Wang, Q., Chen, Q., and Towbin, J. A. Genetics, molecular mechanisms and management of long QT syndrome. Ann. Med. 30: 58-65. (1998) [0164]Wilde. A. A. and Veldkamp, M. W. Ion channels, the QT interval, and arrhythmias. Pacing. Clin. Electrophysiol. 20: 2048-2051. (1997) [0165]Wischmeyer, E., Doring, F., Spauschus, A., Thomzig, A., Veh, R., and Karschin, A. Subunit interactions in the assembly of neuronal Kir3.0 inwardly rectifying K+ channels. Mol. Cell Neurosci. 9: 194-206. (1997) [0166]Yamada, M., Inanobe, A., and Kurachi, Y. G protein regulation of potassium ion channels. Pharmacol. Rev. 50: 723-760. (1998) [0167]Bock, J. H., Shuck, M. E., Benjamin, C. W., Chee, M., Bienkowski, M. J., and Slightom, J. L Nucleotide sequence analysis of the human KCNJ1 potassium channel locus Gene 188: 9-16. (1997) [0168]Delpon, E., Valenzuela, C., and Tamargo, J. Blockade of cardiac potassium and other channels by antihistamines Drug Saf 21 Suppl 1:11-8; discussion 81-7: 11-18. (1999) [0169]Drici, M. D. and Barhanin, J. Cardiac K+ channels and drug-acquired long QT syndrome Therapie 55: 185-193. (2000) [0170]DuBuske, L. M. Second-generation antihistamines: the risk of ventricular arrhythmias Clin. Ther. 21: 281-295. (1999) [0171]Itoh, T., Tanaka, T., Nagai, R., Kikuchi, K., Ogawa, S., Okada, S., Yamagata, S., Yano, K., Yazaki, Y., and Nakamura, Y. Genomic organization and mutational analysis of KVLQT1, a gene responsible for familial long QT syndrome Hum. Genet. 103: 290-294. (1998) [0172]Kobayashi, T., Ikeda, K., and Kumanishi, T. Inhibition by various antipsychotic drugs of the G-protein-activated inwardly rectifying K(+) (GIRK) channels expressed in Xenopus oocytes Br. J. Pharmacol. 129: 1716-1722. (2000) [0173]Richelson, E. Preclinical pharmacology of neuroleptics: focus on new generation compounds J. Clin. Psychiatry 57 Suppl 11: 4-11: 4-11. (1996) [0174]Richelson, E. Receptor pharmacology of neuroleptics: relation to clinical effects [see comments]J. Clin. Psychiatry 60 Suppl 10:5.14: 5-14. (1999) [0175]Shuck, M. E., Bock, J. H., Benjamin, C. W., Tsai, T. D., Lee, K. S., Slightom, J. L, and Bienkowski, M. J. Cloning and characterization of multiple forms of the human kidney ROM-K potassium channel J. Biol. Chem. 269: 24261-24270. (1994) [0176]Taglialatela, M., Castaldo, P., Pannaccione, A., Giorgio, G., Genovese, A., Marone, G., and Annunziato, L. Cardiac ion channels and antihistamines: possible mechanisms of cardiotoxicity Clin. Exp. Allergy 29 Suppl 3:182-9: 182-189. (1999)

TABLE-US-00010 [0176]TABLE 1 SEQ ID. NO. 1 Nucleotide sequence of TRK1 ATGCATTTTAGAAGAACGATGAGTAGAGTGCCCACATTGGCATCTCTTGAAATACGATATAAAAAATCTTTCGG- CC ATAAATTTCGTGATTTTATTGCTCTATGTGGTCACTATTTTGCTCCAGTTAAAAAATATATCTTCCCCAGTTTT- AT CGCGGTTCACTACTTCTACACGATATCCCTGACATTAATAACTTCAATCCTGCTATATCCCATTAAGAATACCA- GA TACATTGATACATTGTTTTTAGCAGCGGGCGCAGTTACACAAGGTGGCTTAAATACTGTGGATATCAACAATCT- AA GCTTATACCAACAAATTGTTCTGTATATCGTATGCTGCATATCAACACCAATTGCAGTTCATAGTTGCTTGGCA- TT TGTACGGCTTTACTGGTTTGAGCGCTACTTCGATGGTATTAGAGACTCTTCTAGACGAAATTTTAAGATGAGAA- GA ACGAAAACAATCTTAGAAAGGGAACTAACAGCAAGAACCATGACCAAGAATAGAACAGGTACCCAAAGAACGTC- TT ATCCTAGGAAACAAGCTAAAACAGATGATTTCCAAGAAAAATTGTTCAGCGGAGAAATGGTTAATAGAGATGAG- CA GGACTCAGTTCACAGCGACCAGAATTCTCATGACATTAGTAGGGACAGCAGCAATAATAATACGAATCACAATG- GT AGCAGTGGCAGTTTAGATGATTTCGTTAAGGAAGACGAAACGGATGACAATGGAGAATATCAGGAGAACAACTC- CT ACTCGACGGTAGGTAGTTCGTCTAACACAGTTGCAGACGAAAGTTTAAATCAGAAGCCCAAGCCAAGCAGTCTT- CG GTTTGATGAGCCACACAGCAAACAAAGACCCGCAAGAGTTCCCTCAGAGAAATTTGCAAAAAGAAGGGGTTCAA- GA GATATTAGCCCAGCCGATATGTATCGATCCATTATGATGCTACAAGGTAAGCATGAAGCAACTGCTGAAGATGA- AG GTCCCCCTTTAGTCATCGGGTCCCCTGCGGATGGCACAAGATATAAAAGTAATGTCAATAAGCTAAAGAAGGCC- AC CGGCATAAATGGTAACAAAATCAAGATTCGAGATAAGGGAAATGAAAGTAACACTGATCAAAATTCCGTGTCAA- GT GAAGCAAACAGTACGGCGAGCGTTTCGGACGAAAGCTCGTTACACACAAATTTTGGTAACAAAGTACCTTCATT- AA GAACAAATACTCATAGATCAAATTCGGGCCCGATAGCCATTACTGATAACGCAGAAACAGACAAAAAGCATGGG- CC ATCAATTCAATTCGATATAACTAAACCTCCTAGAAAAATTTCAAAAAGAGTTTCAACCTTCGATGATTTGAACC- CA AAATCTTCCGTTCTTTATCGAAAAAAAGCATCGAAGAAGTACCTCATGAAACATTTTCCTAAAGCGCGGCGAAT- AC GGCAACAAATTAAGAGAAGGCTTTCTACTGGTTCAATTGAGAAAAACAGCAGTAACAATGTTTCAGATAGAAAA- CC TATTACTGATATGGATGATGATGATGATGACGATGACAACGACGGCGATAACAACGAAGAATACTTTGCTGACA- AC GAAAGCGGCGATGAAGATGAACGAGTACAGCAGTCTGAACCACATTCTGATTCAGAACTCAAATCGCACCAACA- AC AGCAAGAAAAACACCAACTGCAGCAGAACCTGCACCGCATGTATAAAACCAAATCATTTGATGATAATCGTTCA- AG AGCAGTTCCTATGGAACGTTCCAGGACCATCGATATGGCAGAGGCTAAGGATCTAAATGAGCTCGCAAGGACGC- CT GATTTTCAAAAAATGGTCTATCAAAATTGGAAAGCCCATCATAGAAAAAAACCGAACTTTAGGAAGAGGGGATG- GA ATAACAAGATATTTGAACATGGTCCCTATGCATCTGACAGCGATCGCAATTATCCTGATAATAGTAATACTGGA- AA CAGTATTCTTCATTACGCAGAGTCTATTTTACATCATGATGGCTCTCATAAAAATGGAAGCGAAGAAGCCTCTT- CC GACTCTAATGAGAATATCTATTCCACGAATGGAGGAAGCGACCACAATGGTCTTAACAACTATCCTACTTACAA- CG ACGATGAAGAAGGCTATTATGGTTTACATTTCGATACCGATTATGACCTAGATCCTCGTCATGATTTATCTAAA- GG CAGTGGTAAAACGTATCTATCATGGCAACCAACTATTGGACG$$AACTCAAACTTCCTTGGATTAACAAGAGCC- CAG AAAGATGAATTAGGTGGTGTCGAGTACAGAGCAATCAAACTTTTATGCACCATATTGGTTGTCTACTACGTTGG- AT GGCATATTGTTGCTTTTGTTATGTTAGTACCTTGGATTATTTTGAAAAAGCATTATAGTGAAGTTGTTAGAGAT- GA TGGTGTTTCACCTACATGGTGGGGATTTTGGACAGCAATGAGTGCATTTAATGATTTAGGTTTGACATTAACTC- CA AATTCAATGATGTCGTTTAACAAAGCTGTATACCCATTGATCGTTATGATTTGGTTTATCATTATCGGAAATAC- AG GGTTTCCCATCCTTCTTAGATGCATCATTTGGATAATGTTTAAAATTTCTCCTGATTTATCACAGATGAGAGAA- AG TTTAGGTTTTCTCTTAGACCATCCACGTCGTTGTTTCACCTTGCTATTTCCTAAGGCAGCTACATGGTGGCTAC- TT TTAACGCTTGCAGGATTGAATATAACTGATTGGATTTTATTTATTATTCTAGATTTTGGCTCAACAGTTGTGAA- AT CATTATCGAAAGGCTATAGAGTCCTTGTCGGCCTGTTTCAATCTGTTAGCACAAGAACTGCTGGATTCAGCGTT- GT CGATTTAAGTCAACTGCATCCTTCTATCCAAGTCTCCTATATGCTAATGATGTATGTCTCCGTATTACCATTGG- CC ATCTCTATTCGACGGACAAATGTTTACGAGGAGCAATCTTTAGGACTATATGGAGATATGGGGGGAGAACCAGA- AG ATACGGATACTGAAGACGATGGTAACGATGAAGATGACGACGAGGAAAACGAGAGTCACGAAGGTCAAAGTAGT- CA AAGAAGTAGTTCGAACAACAACAACAATAACAACAGGAAAAAGAAAAAGAAAAAGAAAACTGAAAATCCAAATG- AA ATATCTACAAAATCCTTTATCGGTGCCCATTTAAGGAAACAGCTTTCATTTGACTTGTGGTTTCTATTTTTAGG- GT TATTTATCATTTGCATTTGTGAAGGGGACAAGATAAAGGACGTACAAGAACCAAACTTTAATATATTTGCAATT- CT TTTTGAAATTGTTAGCGCTTACGGTACAGTTGGGCTATCGCTAGGTTATCCGGACACCAACCAATCGTTTTCAA- GA CAGTTTACTACATTATCTAAGTTGGTGATCATAGCTATGCTGATCAGAGGCAAGAATAGAGGTCTACCATACTC- AC TGGATCGTGCAATTATCTTGCCTAGTGATAGACTTGAACATATTGACCACCTTGAGGGCATGAAATTGAAGAGA- CA GGCTAGAACCAATACAGAAGACCCAATGACGGAACATTTCAAGAGAAGTTTCACTGATGTGAAACATCGTTGGG- GA GCTCTTAAGCGTAAGACCACACATTCCCGAAATCCTAAAAGGAGCAGCACAACGCTCTAA

TABLE-US-00011 TABLE 2 SEQ ID. NO. 2 Nucleotide sequence of TRK2 ATGCCAACAGCTAAGAGGACGTCATCCAGGGCTTCGTTGGCACTGCCCTTCCAGTTACGGTTGGTGCACAAGAA- AT CATGGGGCCATCGGCTAAGAGACTTCATTTCCGGGTTCTTAAAATCATGCAGACCCATTGCTAAATACGTTTTC- CC CAACTTCATCGTGGTGCACTATATCTACCTGATCACGCTGTCGATTATCGGGTCCATTCTGTTATATCCGTGCA- AG AACACFFCFTTCATCGATGTGCTATTTCTGGCTGCTGGAGCGTCTACACAGGGCGGGCTGGCCACCAAGAGCAC- TA ACGATTTCAACCTGTACCAGCAGATAGTGGTGTACGTCATTACATTGCTGTCCACGCCTATACTTATTCATGGG- TT TTTGGCCTTTGTCAGGCTGTATTGGTTTGAAAGGTACTTCGACAACATTAGGGATATCTCCAAACAGAATTTTA- AA CTAAGAAGGACCATGACGTTGCAACAAAGGGAACTATCGGGCAGCAGTGGCAATGCCGCTCGAAGTAGGAGTTT- CA AGGACAACCTGTTCCGTGGGAAGTTTGTTTCCAGAGAAGACCCACGACAATCCGCTTCAGATGTGCCGATGGAC- TC TCCTGACACGTCCGCATTGTCCTCAATCTCACCGTTGAATGTTTCCTCCTCTAAGGAGGAATCCAGTGACACGC- AA AGCTCGCCTCCAAACTTCTCAAGTAAGCGCCAACCCTCAGACGTTGACCCAAGAGACATTTACAAATCGATAAT- GA TGCTACAAAAACAACAAGAGAAGAGCAACGCAAACTCCACGGATTCTTTTTCGAGCGAGACCAATGGACCCGCT- TT CATTGTGCAGGAACGTCATGAGAGAAGAGCCCCCCACTGCTCACTGAAACGCCATTCTGTCCTGCCATCTTCTC- AG GAATTGAACAAGCTAGCCCAGACGAAAAGTTTCCAGAAATTGCTTGGCTTGCGGAGAGATGAAGGTGACCATGA- CT ACTTTGACGGTGCTCCTCACAAATATATGGTCACCAAGAAGAAAAAAATATCTAGAACGCAATCATGTAACATC- CC AACGTATACTGCTTCACCGAGTCCTAAAACCTCAGGCCAAGTAGTTGAAAATCATAGAAACTTGGCCAAGTCGG- CG CCTTCATCTTTTGTTGATGAGGAGATGAGCTTTTCACCGCAAGAGTCTTTGAATTTACAGTTCCAAGCGCACCC- GC CCAAACCAAAACGACGTGAAGGTGATATAGGCCACCCCTTCACCAGAACAATGAGCACCAACTATCTATCGTGG- CA GCCAACCTTTGGCAGAAACTCCGTCTTCATTGGACTCACAAAGCAACAAAAGGAGGAACTCGGCGGTGTCGAAT- AT CGTGCTTTGAGATTGCTGTGCTGCATTCTCATGGTATACTACATCGGATTCAACATTTTGGCGTTTGTGACCAT- CG TTCCATGGGCCTGTACGAGGCACCACTACTCAGAGATTATTAGACGAAATGGAGTTTCTCCAACCTGGTGGGGG- TT TTTCACTGCAATGAGTGCATTCAGCAACTTGGGTCTGTCTTTGACCGCTGATTCAATGGTTTCCTTTGATACTG- CG CCGTATCCGCTGATTTTCATGATGTTCTTCATCATCATAGGCAATACAGGCTTCCCAATTATGTTACGATTTAT- CA TTTGGATCATGTTCAAGACCTCGAGAGACCTATCTCAGTTTAAGGAAAGTCTTGGGTTTCTCTTGGATCATCCG- CG CAGGTGTTTTACGTTGCTGTTCCCCAGCGGCCCCACATGGTGGCTGTTTACAACTTTAGTCGTCTTAAACGCTA- CG GATTGGATTCTTTTCATAATTCTGGATTTCAACTCCGCTGTAGTAAGGCAGGTTGCTAAAGGTTATCGAGCTCT- CA TGGGCCTCTTCCAGTCTGTATGCACAAGAACTGCTGGATTCAACGTTGTTGACTTAAGTAAATTACACCCGTCC- AT TCAGGTGTCTTATATGCTAATGATGTACGTTTCGGTCCTGCCGCTGGCGATTTCCATTAGAAGAACGAATGTTT- AT GAGGAGCAATCGTTGGGACTATACGATAGTGGACAAGATGACGAAAATATCACCCACGAAGACGATATAAAGGA- AA CAGACCATGATGGCGAATCCGAAGAGCGAGACACTGTATCTACAAAGTCCAAGCCGAAGAAACAGTCCCCAAAA- TC GTTTGTTGGTGCTCATTTGAGGAGGCAACTCTCTTTTGATTTATGGTACCTATTCCTTGGATTATTTATAATAT- GC ATATGCGAGGGCAGAAAAATCGAAGACGTTAATAAACCTGATTTCAATGTCTTTGCTATATTGTTTGAAGTTGT- TA GCGCTTATGGTACAGTGGGTTTGTCATTGGGTTACCCAAACACCAACACATCACTATCTGCCCAGTTCACCGTA- TT ATCGAAGCTAGTCATAATTGCCATGCTAATAAGAGGAAGAAATAGAGGTTTACCATACACTTTGGATCGTGCCA- TC ATGCTGCCAAGTGACAAACTGGAACAAATTGATCGTTTACAAGATATGAAAGCTAAGGGTAAGTTGTTAGCCAA- AG TTGGTGAGGATCCAATGACTACTTACGTCAAAAAGAGATCCCACAAACTGAAAAAAATAGCAACAAAGTTTTGG- GG GAAGCATTA

TABLE-US-00012 TABLE 3 SEQ ID NO. 3 Nucleotide sequence of TOK1 ATGACAAGGTTCATGAACAGCTTTGCCAAACAAACGCTGGGATATGGCAATATGGCGACAGTGGAGCAAGAGAG- CT CAGCTCAGGCTGTTGATTCTCATTCAAACAACACACCGAAGCAAGCTAAGGGTGTTCTTGCAGAGGAACTAAAG- GA TGCATTGCGGTTCCGGGACGAAAGAGTTAGTATTATTAATGCAGAGCCTTCTTCAACACTGTTCGTCTTTTGGT- TT GTGGTTTCATGCTATTTCCCTGTGATTACTGCCTGCTTGGGTCCCGTAGCTAACACTATCTCGATAGCCTGTGT- AG TTGAAAAATGGAGATCCTTAAAGAACAACTCCGTGGTGACAAATCCACGAAGCAATGACACCGATGTTTTGATG- AA TCAAGTAAAGACAGTTTTTGATCCTCCTGGTATTTTTGCCGTTAATATCATCTCTTTGGTACTGGGTTTTACGT- CA AATATTATACTAATGCTACATTTCAGTAAGAAGTTGACGTATCTTAAATCTCAGTTAATAAATATAACAGGATG- GA CAATAGCTGGAGGGATGCTTTTGGTGGACGTGATTGTATGCTCCTTGAATGACATGCCCAGCATCTACAGTAAG- AC TATCGGATTTTGGTTTGCCTGTATCAGTTCTGGTCTATATTTGGTATGCACCATTATTTTAACAATACATTTTA- TT GGATATAAATTAGGAAAATATCCTCCAACGTTCAACCTTTTGCCCAATGAAAGAAGTATCATGGCATACACTGT- AC TATTGTCTTTATGGTTGATTTGGGGTGCGGGTATGTTTAGCGGTTTATTGCACATCACTTACGGAAATGCATTA- TA TTTCTGCACGGTATCATTATTAACCGTGGGACTAGGTGACATCCTGCCCAAGTCGGTTGGCGCCAAAATCATGG- TT TTAATCTTTTCGCTATCTGGTGTTGTCTTGATGGGTTTAATAGTGTTTATGACAAGATCCATCATTCAAAAGTC- CT CTGGCCCAATTTTCTTTTTCCACAGAGTTGAAAAAGGCAGGTCCAAATCGTGGAAACATTATATGGATAGTAGT- AA AAATTTATCTGAAAGGGAAGCGTTCGACTTAATGAAGTGTATCCGACAAACGGCCTCAAGGAAGCAGCATTGGT- TT TCTTTGTCGGTGACTATTGCAATTTTCATGGCTTTTTGGTTATTGGGAGCTCTTGTATTCAAATTCGCAGAAAA- TT GGTCGTACTTCAATTGTATTTACTTTTGTTTCTTGTGCTTATTAACCATTGGATACGGAGACTATGCTCCAAGG- AC TGGTGCAGGCCGTGCTTTTTTTGTGATTTGGGCGTTGGGAGCCGTGCCATTAATGGGGGCTATCCTATCTACAG- TC GGTGATCTGTTGTTTGACATTTCCACTTCTCTGGATATTAAGATCGGTGAATCATTCAATAATAAAGTCAAGTC- CA TCGTTTTTAATGGGCGTCAAAGAGCACTTTCCTTTATGGTGAACACTGGAGAAATTTTCGAAGAATCTGACACA- GC TGATGGTGATCTGGAAGAAAATACAACGAGCTCACAATCCAGTCAAATTTCTGAATTCAACGATAATAATTCAG- AA GAGAATGATTCTGGAGTGACATCCCCTCCTGCAAGCCTGCAAGAATCATTTTCTTCATTATCAAAAGCATCTAG- CC CAGAGGGAATACTTCCTCTAGAATATGTTTCTTCTGCTGAATATGCACTACAGGACTCGGGGACCTGTAATTTA- AG GAACTTGCAAGAGCTACTTAAAGCCGTCAAAAAACTACATCGGATATGTCTGGCGGATAAAGATTACACACTTA- GT TTTTCCGACTGGTCGTACATTCATAAACTACATTTGAGGAACATTACAGATATTGAGGAGTACACACGCGGACC- CG AATTTTGGATATCACCTGATACGCCCCTCAAGTTCCCGTTAAATGAACCTCATTTTGCTTTTATGATGCTTTTC- AA GAACATAGAAGAATTAGTTGGTAATCTAGTAGAAGACGAAGAGCTTTATAAAGTTATAAGCAAAAGAAAATTTT- TG GGTGAGCATAGAAAAGACACTTTGA

TABLE-US-00013 TABLE 4 SEQ ID NO. 4 Nucleotide sequence of HERG1 ATGCCGGTGCGGAGGGGCCACGTCGCGCCGCAGAACACCTTCCTGGACACCATCATCCGCAAGTTTGAGGGCCA- GA GCCGTAAGTTCATCATCGCCAACGCTCGGGTGGAGAACTGCGCCGTCATCTACTGCAACGACGGCTTCTGCGAG- CT GTGCGGCTACTCGCGGGCCGAGGTGATGCAGCGACCCTGCACCTGCGACTTCCTGCACGGGCCGCGCACGCAGC- GC CGCGCTGCCGCGCAGATCGCGCAGGCACTGCTGGGCGCCGAGGAGCGCAAAGTGGAAATCGCCTTCTACCGGAA- AG ATGGGAGCTGCTTCCTATGTCTGGTGGATGTGGTGCCCGTGAAGAACGAGGATGGGGCTGTCATCATGTTCATC- CT CAATTTCGAGGTGGTGATGGAGAAGGACATGGTGGGGTCCCCGGCTCATGACACCAACCACCGGGGCCCCCCCA- CC AGCTGGCTGGCCCCAGGCCGCGCCAAGACCTTCCGCCTGAAGCTGCCCGCGCTGCTGGCGCTGACGGCCCGGGA- GT CGTCGGTGCGGTCGGGCGGCGCGGGCGGCGCGGGCGCCCCGGGGGCCGTGGTGGTGGACGTGGACCTGACGCCC- GC GGCACCCAGCAGCGAGTCGCTGGCCCTGGACGAAGTGACAGCCATGGACAACCACGTGGCAGGGCTCGGGCCCG- CG GAGGAGCGGCGTGCGCTGGTGGGTCCCGGCTGTCCGCCCCGCAGCGCGCCCGGCCAGCTCCCATCGCCCCGGGC- GC ACAGCCTCAACCCCGACGCCTCGGGCTCCAGCTGCAGCCTGGCCCGGACGCGCTCCCGAGAAAGCTGCGCCAGC- GT GCGCCGCGCCTCGTCGGCCGACGACATCGAGGCCATGCGCGCCGGGGTGCTGCCCCCGCCACCGCGCCACGCCA- GC ACCGGGGCCATGCACCCACTGCGCAGCGGCTTGCTCAACTCCACCTCGGACTCCGACCTCGTGCGCTACCGCAC- CA TTAGCAAGATTCCCCAAATCACCCTCAACTTTGTGGACCTCAAGGGCGACCCCTTCTTGGCTTCGCCCACCAGT- GA CCGTGAGATCATAGCACCTAAGATAAAGGAGCGAACCCACAATGTCACTGAGAAGGTCACCCAGGTCCTGTCCC- TG GGCGCCGACGTGCTGCCTGAGTACAAGCTGCAGGCACCGCGCATCCACCGCTGGACCATCCTGCATTACAGCCC- CT TCAAGGCCGTGTGGGACTGGCTCATCCTGCTGCTGGTCATCTACACGGCTGTCTTCACACCCTACTCGGCTGCC- TT CCTGCTGAAGGAGACGGAAGAAGGCCCGCCTGCTACCGAGTGTGGCTACGCCTGCCAGCCGCTGGCTGTGGTGG- AC CTCATCGTGGACATCATGTTCATTGTGGACATCCTCATCAACTTCCGCACCACCTACGTCAATGCCAACGAGGA- GG TGGTCAGCCACCCCGGCCGCATCGCCGTCCACTACTTCAAGGGCTGGTTCCTCATCGACATGGTGGCCGCCATC- CC CTTCGACCTGCTCATCTTCGGCTCTGGCTCTGAGGAGCTGATCGGGCTGCTGAAGACTGCGCGGCTGCTGCGGC- TG GTGCGCGTGGCGCGGAAGCTGGATCGCTACTCAGAGTACGGCGCGGCCGTGCTGTTCTTGCTCATGTGCACCTT- TG CGCTCATCGCGCACTGGCTAGCCTGCATCTGGTACGCCATCGGCAACATGGAGCAGCCACACATGGACTCACGA- CT CGGCTGGCTGCACAACCTGGGCGACCAGATAGGCAAACCCTACAACAGCAGCGGCCTGGGCGGCCCCTCCATCA- AG GACAAGTATGTGACGGCGCTCTACTTCACCTTCAGCAGCCTCACCAGTGTGGGCTTCGGCAACGTCTCTCCCAA- CA CCAACTCAGAGAAGATCTTCTCCATCTGCGTCATGCTCATTGGCTCCCTCATGTATGCTAGCATCTTCGGCAAC- GT GTCGGCCATCATCCAGCGGCTGTACTCGGGCACAGCCCGCTACCACACACAGATGCTGCGGGTGCGGGAGTTCA- TC CGCTTCCACCAGATCCCCAATCCCCTGCGCCAGCGCCTCGAGGAGTACTTCCAGCACGCCTGGTCCTACACCAA- CG GCATCGACATGAACGCGGTGCTGAAGGGCTTCCCTGAGTGCCTGCAGGCTGACATCTGCCTGCACCTGAACCGC- TC ACTGCTGCAGCACTGCAAACCCTTCCGAGGGGCCACCAAGGGCTGCCTTCGGGCCCTGGCCATGAAGTTCAAGA- CC ACACATGCACCGCCAGGGGACACACTGGTGCATGCTGGGGACCTGCTCACCGCCCTGTACTTCATCTCCCGGGG- CT CCATCGAGATCCTGCGGGGCGACGTCGTCGTGGCCATCCTGGGGAAGAATGACATCTTTGGGGAGCCTCTGAAC- CT GTATGCAAGGCCTGGCAAGTCGAACGGGGATGTGCGGGCCCTCACCTACTGTGACCTACACAAGATCCATCGGG- AC GACCTGCTGGAGGTGCTGGAACTGTACCCTGAGTTCTCCGACCACTTCTGGTCCAGCCTGGAGATCACCTTCAA- CC TGCGAGATACCAACATGATCCCGGGCTCCCCCGGCAGTACGGAGTTAGAGGGTGGCTTCAGTCGGCAACGCAAG- CG CAAGTTGTCCTTCCGCAGGCGCACGGACAAGGACACGGAGCAGCCAGGGGAGGTGTCGGCCTTGGGGCCGGGCC- GG GCGGGGGCAGGGCCGAGTAGCCGGGGCCGGCCGGGGGGGCCGTGGGGGGAGAGCCCGTCCAGTGGCCCCTCCAG- CC CTGAGAGCAGTGAGGATGAGGGCCCAGGCCGCAGCTCCAGCCCCCTCCGCCTGGTGCCCTTCTCCAGCCCCAGG- CC CCCCGGAGACCGCCGGGTGGGGAGCCCCTGATGGAGGACTGCGAGAAAGAGCAGCGACACTTGCAACCCCCTGT- CA GGCGCCTTCTCAGGAGTGTCCAACATTTTCAGCTTCTGGGGGGACAGTCGGGGCCGCCAGTACCAGGAGCTCCC- TC GATGCCCCGCCCCCACCCCCAGCCTCCTCAACATCCCCCTCTCCAGCCCGGGTCGGCGGCCCCGGGGCGACGTG- GA GAGCAGGCTGGATGCCCTCCAGCGCCAGCTCAACAGGCTGGAGACCCGGCTGAGTGCAGACATGGCCACTGTCC- TG CAGCTGCTACAGAGGCAGATGACGCTGGTCCCGCCCGCCTACAGTGCTGTGACCACCCCGGGGCCTGGCCCCAC- TT CCACATCCCCGCTGTTGCCCGTCAGCCCCCTCCCCACCCTCACCTTGGACTCGCTTTCTCAGGTTTCCCAGTTC- AT GGCGTGTGAGGAGCTGCCCCCGGGGGCCCCAGAGCTTCCCCAAGAAGGCCCCACACGACGCCTCTCCCTACCGG- GC CAGCTGGGGGCCCTCACCTCCCAGCCCCTGCACACACGGCTCGGACCCCGGCAGTTA

TABLE-US-00014 TABLE 5 SEQ ID NO. 5 Nucleotide sequence of K.sub.v1-5 ATGGAGATCGCCCTGGTGCCCCTGGAGAACGGCGGTGCCATGACCGTCAGAGGAGGCGATGAGGCCCGGGCAGG- CT GCGGCCAGGCCACAGGGGGAGAGCTCCAGTGTCCCCCGACGGCTGGGCTCAGCGATGGGCCCAAGGAGCCGGCG- CC AAAGGGGCGCGCGCAGAGAGACGCGGACTCGGGAGTGCGGCCCTTGCCTCCGCTGCCGGACCCGGGAGTGCGGC- CC TTGCCTCCGCTGCCAGAGGAGCTGCCACGGCCTCGACGGCCGCCTCCCGAGGACGAGGAGGAAGAAGGCGATCC- CG GCCTGGGCACGGTGGAGGACCAGGCTCTGGGCACGGCGTCCCTGCACCACCAGCGCGTCCACATCAACATCTCC- GG GCTGCGCTTTGAGACGCAGCTGGGCACCCTGGCGCAGTTCCCCAACACACTCCTGGGGGACCCCGCCAAGCGCC- TG CCGTACTTCGACCCCCTGAGGAACGAGTACTTCTTCGACCGCAACCGGCCCAGCTTCGACGGTATCCTCTACTA- CT ACCAGTCCGGGGGCCGCCTGCGAGGGGTCAACGTCTCCCTGGACGTGTTCGCGGACGAGATACGCTTCTACCAG- CT GGGGGACGAGGCCATGGAGCGCTTCCGCGAGGATGAGGGCTTCATTAAAGAAGAGGAGAAGCCCCTGCCCCGCA- AC GAGTTCCAGCGCCAGGTGTGGCTTATCTTCGAGTATCCGGAGAGAGCTCTGGGTCCGCGGGCCATCGCCATCGT- CT CGGTCTTGGTTATCCTCATCTCCATCATCACCTTCTGCTTGGAGACCCTGCCTGAGTTCAGGGATGAACGTGAG- CT GCTCCGCCACCCTCCGGCGCCCCACCAGCCTCCCGCGCCCGCCCCTGGGGCCAACGGCAGCGGGGTCATGGCCC- CC GCCTCTGGCCCTACGGTGGCACCGCTCCTGCCCAGGACCCTGGCCGACCCCTTCTTCATCGTGGAGACCACGTG- CG TGATCTGGTTCACCTTCGAGCTGCTCGTGCGCTTCTTCGCCTGCCCCAGCAAGGCAGGGTTCTCCCGGAACATC- AT GAACATCATCGATGTGGTGGCCATCTTCCCCTACTTCATCACCCTGGGCACCGAACTGGCAGAGCAGCAGCCAG- GG GGCGGAGGAGGCGGCCAGAATGGGCAGCAGGCCATGTCCCTGGCCATCCTCCGAGTCATCCGCCTGGTCCGGGT- GT TCCGCATCTTCAAGCTCTCCCGCCACTCCAAGGGGCTGCAGATCCTGGGCAAGACCTTGCAGGCCTCCATGAGG- GA GCTGGGGCTGCTCATCTTCTTCCTCTTCATCGGGGTCATCCTCTTCTCCAGTGCCGTCTACTTCGCAGAGGCTG- AC AACCAGGGAACCCATTTCTCTAGCATCCCTGACGCCTTCTGGTGGGCAGTGGTCACCATGACCACTGTGGGCTA- CG GGGACATGAGGCCCATCACTGTTGGGGGCAAGATCGTGGGCTCGCTGTGTGCCATCGCCCGGGTCCTCACCATT- GC CCTGCCTGTGCCCGTCATCGTCTCCAACTTCAACTACTTCTACCACCGGGAAACGGATCACGAGGAGCCGGCAG- TC CTTAAGGAAGAGCAGGGCACTCAGAGCCAGGGGCCGGGGCTGGACAGAGGAGTCCAGCGGAAGGTCAGCGGGAG- CA GGGGATCCTTCTGCAAGGCTGGGGGGACCCTGGAGAATGCAGACAGTGCCCGAAGGGGCAGCTGCCCCCTAGAG- AA GTGTAACGTCAAGGCCAAGAGCAACGTGGACTTGCGGAGGTCCCTTTATGCCCTCTGCCTGGACACCAGCCGGG- AA ACAGATTTGTGA

TABLE-US-00015 TABLE 6 SEQ ID NO. 6: Nucleotide sequence of IRK1 ATGGGCAGTGTGCGAACCAACCGCTATAGCATTGTCTCTTCGGAAGAGGACGGCATGAAGTTGGCCACCATGGC- AG TTGCCAATGGCTTTGGGAATGGGAAAAGTAAAGTCCACACTCGGCAACAGTGTAGGAGCCGCTTTGTGAAGAAA- GA TGGCCACTGTAATGTTCAGTTCATCAACGTTGGGGAAAAGGGACAACGGTACCTTGCTGACATTTTTACTACGT- GT GTGGACATTCGCTGGCGGTGGATGCTGGTTATCTTTTGCCTAGCTTTTGTTCTCTCGTGGCTGTTTTTTGGCTG- TG TGTTTTGGCTGATAGCTTTGCTCCATGGAGATCTGGATGCATCTAAGGAGAGCAAAGCCTGTGTGTCTGAGGTC- AA CAGCTTCACAGCTGCCTTTCTTTTCTCCATTGAGACCCAGACAACCATCGGCTATGGGTTCCGATGTGTCACGG- AT GAATGCCCGATTGCGGTGTTCATGGTTGTGTTCCAGTCAATTGTGGGCTGCATTATTGATGCTTTTATCATTGG- TG CCGTCATGGCAAAGATGGCAAAGCCAAAGAAAAGAAATGAGACTCTTGTCTTCAGTCACAATGCTGTGATTGCC- AT GAGAGATGGCAAGCTGTGTTTGATGTGGCGAGTAGGCAACCTTCGGAAAAGCCACTTGGTAGAAGCTCATGTTC- GA GCCCAGCTCCTCAAATCCAGAAATTACTTCTGAAGGGGAATACATCCCCTGGATCAAATAGACATCAATGTTGG- CT TTGACAGTGGAATTGACCGTATATTTCTGGTATCCCCAATCACTATTGTCCATGAAATAGATGAAGATAGTCCT- TT ATATGATTTGAGCAAGCAGGACATTGATAATGCAGACTTTGAAATTGTTGTGATACTAGAAGGCATGGTGGAAG- CC ACTGCCATGACAACACAGTGTCGTAGTTCTTATTTGGCCAACGAGATCCTTTGGGGCCACCGCTATGAGCCAGT- GC TCTTTGAGGAGAAGCACTACTATAAAGTGGACTATTCGAGGTTTCATAAGACTTACGAAGTACCCAACACTCCC- CT TTGTAGTGCCAGAGACTTAGCAGAAAAGAAATATATTCTCTCAAATGCTAACTCATTTTGCTATGAAAATGAAG- TT GCCCTTACAAGCAAAGAGGAAGATGACAGTGAAAATGGGGTTCCAGAAAGCACCAGTACAGACACACCTCCTGA- CA TCGACCTTCACAACCAGGCAAGTGTACCTCTAGAGCCCAGACCCTTACGGCGAGAATCGGAGATATGA

TABLE-US-00016 TABLE 7 SEQ ID NO. 7: human ROMK2 (Genbank accession number U12542) ATGTTCAAACATCTTCGGAAATGGGTCGTCACTCGCTTTTTTGGGCATTCTCGGCAAAGAGCAAGGCTAGTCTC- CA AAGATGGAAGGTGCAACATAGAATTTGGCAATGTGGAGGCACAGTCAAGGTTTATATTCTTTGTGGACATCTGG- AC AACGGTACTTGACCTCAAGTGGAGATACAAAATGACCATTTTCATCACAGCCTTCTTGGGGAGTTGGTTTTTCT- TT GGTCTCCTGTGGTATGCAGTAGCGTACATTCACAAAGACCTCCCGGAATTCCATCCTTCTGCCAATCACACTCC- CT GTGTGGAGAATATTAATGGCTTGACCTCAGCTTTTCTGTTTTCTCTGGAGACTCAAGTGACCATTGGATATGGA- TT CAGGTGTGTGACAGAACAGTGTGCCACTGCCATTTTTCTGCTTATCTTTCAGTCTATACTTGGAGTTATAATCA- AT TCTTTCATGTGTGGGGCCATCTTAGCCAAGATCTCCAGGCCCAAAAAACGTGCCAAGACCATTACGTTCAGCAA- GA ACGCAGTGATCAGCAAACGGGAGGGAAGCTTTGCCTCCTAATCCGAGTGGCTAATCTCAGGAAGAGCCTTCTTA- T TGGCAGTCACATTTATGGAAAGCTTCTGAAGACCACAGTCACTCCTGAAGGAGAGACCATTATTTTGGACCAGA- TC AATATCAACTTTGTAGTTGACGCTGGGAATGAAAATTTATTCTTCATCTCCCCATTGACAATTTACCATGTCAT- TG ATCACAACAGCCCTTTCTTCCACATGGCAGCGGAGACCCTTCTCCAGCAGGACTTTGAATTAGTGGTGTTTTTA- GA TGGCACAGTGGAGTCCACCAGTGCTACCTGCCAAGTCCGGACATCCTATGTCCCAGAGGAGGTGCTTTGGGGCT- AC CGTTTTGCTCCCATAGTATCCAAGACAAAGAAGGAAATACCGAGTGGATTTCCATAACTTTAGCAAGACAGTGG AAGTGGAGACCCCTCACTGTGCCATGTGCCTTTATAATGAGAAAGATGTTAGAGCCAGGATGAAGAGAGGCTAT- GA CAACCCCAACTTCATCTTGTCAGAAGTCAATGAAACAGATGACACCAAAATGTAA

TABLE-US-00017 TABLE 8 HERG in Δtrk1Δtrk2Δtok1 in DPM -HIS medium with 0.5 mM CsCl as activator after 38 hours growth. Starting culture 0.03 OD. Detection OD620 nm. Inhibitors (30 μM) Exp. 1 Exp. 2 Exp. 3 Exp. 4 Mean growth St. Dev. Terfenadine 0.006 0.006 0.005 0.007 0.006 0.0 Pimozide 0.004 0.004 0.005 0.005 0.0045 0.0 Diphenhydramine 0.095 0.151 0.17 0.186 0.1505 0.04 Ziprasidone 0.006 0.01 0.012 0.015 0.01075 0.0 Fexofenadine 0.082 0.144 0.159 0.156 0.13525 0.0 Sertindole 0.007 0.004 0.007 0.005 0.00575 0.0 Loratadine 0.024 0.016 0.062 0.014 0.029 0.0 DMSO control 0.162 0.163 0.136 0.146 0.15175 0.0 Wild-type cells in DPM medium with 5 mM or 80 mM KCl after 24 hours growth. Starting culture 0.01 OD. Detection at OD 620 nm. 5 mM KCl StD 80 mM KCl StD DMSO 2.791 3.437 3.959 3.875 3.5155 0.5 3.814 3.959 3.319 3.959 3.7 0.3 Pimo (30 μM) 0.904 0.823 0.305 0.614 0.6615 0.27 3.673 3.505 3.46 3.441 3.5 0.1 Zipra (30 μM) 0.943 0.877 0.675 0.701 0.799 0.1 0.836 0.681 0.717 0.606 0.7 0.1 control 2.953 2.902 3.781 3.353 3.24725 0.4 3.228 3.264 3.781 3.947 3.6 0.4

TABLE-US-00018 TABLE 9 Inhibitors (30 μM) Exp. 1 Exp. 2 Exp. 3 Exp. 4 Mean growth St. Dev. LacZ in wild-type cells In DPM -HIS/-TRP medium with 0.5 mM CsCl as activator after 24 hours growth; detection with the TROPIX kit. ASSAY 1 Terfenadine 4497 4481 5058 5381 4854.25 441.936176 Pimozide 357.4 747.9 804.6 585.4 623.825 200.443648 Diphenhydramine 2806 3161 4178 4884 3757.25 937.881789 Ziprasidone 55.32 70.29 70.3 77.18 68.2725 9.22481933 Fexofenadine 3326 2938 3377 4659 3575 748.783458 Sertindole 4165 2099 4588 3069 3480.25 1121.45304 Loratadine 4905 5141 1857 3266 3792.25 1536.17173 DMSO control 3172 4129 4984 5077 4340.5 888.190858 LacZ in wild-type cells In DPM -HIS/-TRP medium with 0.5 mM CsCl as activator after 24 hours growth; detection with TROPIX-kit. ASSAY 2 Terfenadine (0.5 Cs) 3439 3795 3698 3388 3580 197.394698 Pimozide (0.5 Cs) 905 2176 496.5 573.4 1037.725 779.2749 Diphenhydramine (0.5 Cs) 3468 2980 3062 3581 3267.75 289.383684 Ziprasidone (0.5 Cs) 62.52 44.3 49.71 51.87 52.1 7.64158361 Fexofenadine (0.5 Cs) 3533 3502 3661 3589 3566.25 68.8446318 Sertindole (0.5 Cs) 3992 3076 3972 2782 3455.5 619.738386 Loratadine (0.5 Cs) 3553 1965 3590 2478 2896.5 807.211042 DMSO control (0.5 Cs) 3520 3218 3460 3087 3321.25 203.540946

TABLE-US-00019 TABLE 10 HERG in Δtrk1Δtrk2Δtok1 in DPM -HIS medium with 0.5 mM CsCl as activator after 38 hours growth. Starting culture 0.03 OD. Detection at OD 620 nm. Inhibitors (30 μM) Exp. 1 Exp. 2 Exp. 3 Exp. 4 Mean growth St. Dev. Terfenadine 0.006 0.006 0.005 0.007 0.006 0.0008165 Pimozide 0.004 0.004 0.005 0.005 0.0045 0.00057735 Diphenhydramine 0.095 0.151 0.17 0.186 0.1505 0.03966947 Ziprasidone 0.006 0.01 0.012 0.015 0.01075 0.00377492 Fexofenadine 0.082 0.144 0.159 0.156 0.13525 0.0360867 Sertindole 0.007 0.004 0.007 0.005 0.00575 0.0015 Loratadine 0.024 0.016 0.062 0.014 0.029 0.02242023 DMSO control 0.162 0.163 0.136 0.146 0.15175 0.01307351

TABLE-US-00020 TABLE 11 Wild-type cells in DPM medium with 5 mM or 80 mM KCl after 24 hours growth. Starting culture 0.01 OD. Detection at OD 620 nm. 5 mM KCl StD 80 mM KCl StD DMSO 2.791 3.437 3.959 3.875 3.5155 0.53447638 3.814 3.959 3.319 3.959 3.76275 0.30382738 Pimo (30 μM) 0.904 0.823 0.305 0.614 0.6615 0.26723086 3.673 3.505 3.48 3.441 3.51975 0.10563262 Zipra (30 μM) 0.943 0.877 0.675 0.701 0.799 0.13140269 0.836 0.681 0.717 0.606 0.71 0.09588535 control 2.953 2.902 3.781 3.353 3.24725 0.40900397 3.228 3.264 3.781 3.947 3.555 0.36347856

TABLE-US-00021 TABLE 12 p423GPD (YM194) and p423GPD-ROMK2 (YM256) In Δtrk1Δtrk2Δtok1 in DPM-HIS 5 mM KCl, pH 6.5 after 24 hours growth; starting OD 0.01. Averages 194 SD 256 SD DMSO 0.023 0.0036 0.19 0.013 (0.5%) Cells 0.028 0.0012 0.23 0.011 2 mM RbCl 0.048 0.0052 0.44 0.033 Signal to noise ratio S/N DMSO 8.2 (0.5%) Cells 8.3 2 mM RbCl 8.46

TABLE-US-00022 TABLE 13 SEQ ID NO. 31 - Nucleotide sequence of p423 GPD-hROMK2 (Accession No. U 12542) gacgaaagggcctcgtgatacgcctatttttataggttaatgtcatgataataatggtttcttagatgatccaa- tatcaaagg aaatgatagcattgaaggatgagactaatccaattgaggagtggcagcatatagaacagctaaagggtagtgct- gaag gaagcatacgataccccgcatggaatgggataatatcacaggaggtactagactacctttcatcctacataaat- agacg catataagtacgcatttaagcataaacacgcactatgccgttcttctcatgtatatatatacaggcaacacgca- gatata ggtgcgacgtgaacagtgagctgtatgtgcgcagctcgcgttgcattttcggaagcgctcgttttcggaaacgc- tttgaagt tcctattccgaagttcctattctctagaaagtataggaacttcagagcgcttttgaaaaccaaaagcgctctga- agacgca ctttcaaaaaaccaaaaacgcaccggactgtaacgagctactaaaatattgcgaataccgcttccacaaacatt- gctca aaagtatctctttgctatatatctctgtgctatatccctatataacctacccatccacctttcgctccttgaac- ttgcatctaaact cgacctctacattttttatgtttatctctagtattactctttagacaaaaaaattgtagtaagaactattcata- gagtgaatcgaa aacaatacgaaaatgtaaacatttcctatacgtagtatatagagacaaaatagaagaaaccgttcataattttc- tgaccaa tgaagaatcatcaacgctatcactttctgttcacaaagtatgcgcaatccacatcggtatagaatataatcggg- gatgccttt atcttgaaaaaatgcacccgcagcttcgctagtaatcagtaaacgcgggaagtggagtcaggctttttttatgg- aagaga aaatagacaccaaagtagccttcttctaaccttaacggacctacagtgcaaaaagttatcaagagactgcatta- tagagc gcacaaaggagaaaaaaagtaatctaagatgctttgttagaaaaatagcgctctcgggatgcatttttgtagaa- caaaa aagaagtatagattctttgttggtaaaatagcgctctcgcgttgcatttctgttctgtaaaaatgcagctcaga- ttctttgtttgaa aaattagcgctctcgcgttgcatttttgttttacaaaaatgaagcacagattcttcgttggtaaaatagcgctt- tcgcgttgcatt tctgttctgtaaaaatgcagctcagattctttgtttgaaaaattagcgctctcgcgttgcatttttgttctaca- aaatgaagcaca gatgcttcgttcaggtggcacttttcggggaaatgtgcgcggaacccctatttgtttatttttctaaatacatt- caaatatgtatc cgctcatgagacaataaccctgataaatgcttcaataatattgaaaaaggaagagtatgagtattcaacatttc- cgtgtcg cccttattcccttttttgcggcattttgccttcctgtttttgctcacccagaaacgctggtgaaagtaaaagat- gctgaagatca gttgggtgcacgagtgggttacatcgaactggatctcaacagcggtaagatccttgagagttttcgccccgaag- aacgttt tccaatgatgagcacttttaaagttctgctatgtggcgcggtattatcccgtattgacgccgggcaagagcaac- tcggtcgc cgcatacactattctcagaatgacttggttgagtactcaccagtcacagaaaagcatcttacggatggcatgac- agtaag agaattatgcagtgctgccataaccatgagtgataacactgcggccaacttacttctgacaacgatcggaggac- cgaag gagctaaccgcttttttgcacaacatgggggatcatgtaactcgccttgatcgttgggaaccggagctgaatga- agccata ccaaacgacgagcgtgacaccacgatgcctgtagcaatggcaacaacgttgcgcaaactattaactggcgaact- actt actctagcttcccggcaacaattaatagactggatggaggcggataaagttgcaggaccacttctgcgctcggc- ccttcc ggctggctggtttattgctgataaatctggagccggtgagcgtgggtctcgcggtatcattgcagcactggggc- cagatgg taagccctcccgtatcgtagttatctacacgacggggagtcaggcaactatggatgaacgaaatagacagatcg- ctgag ataggtgcctcactgattaagcattggtaactgtcagaccaagtttactcatatatactttagattgatttaaa- acttcatttttaa tttaaaaggatctaggtgaagatcctttttgataatctcatgaccaaaatcccttaacgtgagttttcgttcca- ctgagcgtca gaccccgtagaaaagatcaaaggatcttcttgagatcctttttttctgcgcgtaatctgctgcttgcaaacaaa- aaaaccac cgctaccagcggtggtttgtttgccggatcaagagctaccaactctttttccgaaggtaactggcttcagcaga- gcgcaga taccaaatactgtccttctagtgtagccgtagttaggccaccacttcaagaactctgtagcaccgcctacatac- ctcgctct gctaatcctgttaccagtggctgctgccagtggcgataagtcgtgtcttaccgggttggactcaagacgatagt- taccggat aaggcgcagcggtcgggctgaacggggggttcgtgcacacagcccagcttggagcgaacgacctacaccgaact- g agatacctacagcgtgagctatgagaaagcgccacgcttcccgaagggagaaaggcggacaggtatccggtaag- cg gcagggtcggaacaggagagcgcacgagggagcttccagggggaaacgcctggtatctttatagtcctgtcggg- tttc gccacctctgacttgagcgtcgatttttgtgatgctcgtcaggggggcggagcctatggaaaaacgccagcaac- gcggc ctttttacggttcctggccttttgctggccttttgctcacatgttctttcctgcgttatcccctgattctgtgg- ataaccgtattaccgc ctttgagtgagctgataccgctcgccgcagccgaacgaccgagcgcagcgagtcagtgagcgaggaagcggaag- a gcgcccaatacgcaaaccgcctctccccgcgcgttggccgattcattaatgcagctggcacgacaggtttcccg- actgg aaagcgggcagtgagcgcaacgcaattaatgtgagttacctcactcattaggcaccccaggctttacactttat- gcttccg gctcctatgttgtgtggaattgtgagcggataacaatttcacacaggaaacagctatgaccatgattacgccaa- gcgcgc aattaaccctcactaaagggaacaaaagctggagctcagtttatcattatcaatactgccatttcaaagaatac- gtaaata attaatagtagtgattttcctaactttatttagtcaaaaaattagccttttaattctgctgtaacccgtacatg- cccaaaataggg ggcgggttacacagaatatataacatcgtaggtgtctgggtgaaccgtttattcctggcatccactaaatataa- tggagcc cgctttttaagctggcatccagaaaaaaaaagaatcccagcaccaaaatattgttttcttcaccaaccatcagt- tcataggt ccattctcttagcgcaactacagagaacaggggcacaaacaggcaaaaaacgggcacaacctcaatggagtgat- gc aacctgcctggagtaaatgatgacacaaggcaattgacccacgcatgtatctatctcattttcttacaccttct- attaccttctg ctctctctgatttggaaaaagctgaaaaaaaaggttgaaaccagttccctgaaattattcccctacttgactaa- taagtatat aaagacggtaggtattgattgtaattctgtaaatctatttcttaaacttcttaaattctacttttatagttagt- cttttttttagttttaaa acaccagaacttagtttcgacggattctagaactagtggatcccccgggctgcagccatgttcaaacatcttcg- gaaatg ggtcgtcactcgttttttgggcattctcggcaaagagcaaggctagtctccaaagatggaaggtgcaacataga- atttgg caatgtggaggcacagtcaaggtttatattctttgtggacatctggacaacggtacttgacctcaagtggagat- acaaaat gaccattttcatcacagccttcttggggagttggtttttcttggtctcctgtggtatgcagtagcgtacattca- caaagacctcc cggaattccatccttctgccaatcacactccctgtgtggagaatattaatggcttgacctcagcttttctgttt- tctctggagact caagtgaccattggatatggattcaggtgtgtgacagaacagtgtgccactgccatttttctgcttatctttca- gtctatacttg gagttataatcaattctttcatgtgtggggccatcttagccaagatctccaggcccaaaaaacgtgccaagacc- attacgtt cagcaagaacgcagtgatcagcaaacggggagggaagcttgcctcctaatccgagtggctaatctcaggaagag- cc ttcttattggcagtcacatttatggaaagcttctgaagaccacagtcactcctgaaggagagaccattattttg- gaccagatc aatatcaactttgtagttgacgctgggaatgaaaatttattcttcatctccccattgacaatttaccatgtcat- tgatcacaaca gccctttcttccacatggcagcggagacccttctccagcaggactttgaattagtggtgtttttagatggcaca- gtggagtcc accagtgctacctgccaagtccggacatcctatgtcccagaggaggtgctttggggctaccgttttgctcccat- agtatcca agacaaaggaagggaaataccgagtggatttccataactttagcaagacagtggaagtggagacccctcactgt- gcc atgtgcctttataatgagaaagatgttagagccaggatgaagagaggctatgacaaccccaacttcatcttgtc- agaagt caatgaaacagatgacaccaaaatgtaacagtcgacctcgagtcatgtaattagttatgtcacgcttacattca- cgccctc cccccacatccgctctaaccgaaaaggaaggagttagacaacctgaagtctaggtccctatttatttttttata- gttatgttag tattaagaacgttatttatatttaaatttttcttttttttctgtacagacgcgtgtacgcatgtaacattatac- tgaaaaccttgcttg agaaggttttgggacgctcgaaggctttaatttgcggccggtacccaattcgccctatagtgagtcgtattacg- cgcgctca ctggccgtcgttttacaacgtcgtgactgggaaaaccctggcgttacccaacttaatcgccttgcagcacatcc- ccctttcg ccagctggcgtaatagcgaagaggcccgcaccgatcgcccttcccaacagttgcgcagcctgaatggcgaatgg- cgc gacgcgccctgtagcggcgcattaagcgcggcgggtgtggtggttacgcgcagcgtgaccgctacacttgccag- cgcc ctagcgcccgctcctttcgctttcttcccttcctttctcgccacgttcgccggctttccccgtcaagctctaaa- tcgggggctcc ctttagggttccgatttagtgctttacggcacctcgaccccaaaaaacttgattagggtgatggttcacgtagt- gggccatcg ccctgatagacggtttttcgccctttgacgttggagtccacgttctttaatagtggactcttgttccaaactgg- aacaacactc aaccctatctcggtctattcttttgatttataagggattttgccgatttcggcctattggttaaaaaatgagct- gatttaacaaaa atttaacgcgaattttaacaaaatattaacgtttacaatttcctgatgcggtattttctccttacgcatctgtg- cggtatttcacac cgcatagatccgtcgagttcaagagaaaaaaaaagaaaaagcaaaaagaaaaaaggaaagcgcgcctcgttcag aatgacacgtatagaatgatgcattaccttgtcatcttcagtatcatactgttcgtatacatacttactgacat- tcataggtata catatatacacatgtatatatatcgtatgctgcagctttaaataatcggtgtcactacataagaacacctttgg- tggagggaa catcgttggtaccattgggcgaggtggcttctcttatggcaaccgcaagagccttgaacgcactctcactacgg-

tgatgat cattcttgcctcgcagacaatcaacgtggagggtaattctgctagcctctgcaaagctttcaagaaaatgcggg- atcatct cgcaagagagatctcctactttctccctttgcaaaccaagttcgacaactgcgtacggcctgttcgaaagatct- accaccg ctctggaaagtgcctcatccaaaggcgcaaatcctgatccaaacctttttactccacgcgccagtagggcctct- ttaaaag cttgaccgagagcaatcccgcagtcttcagtggtgtgatggtcgtctatgtgtaagtcaccaatgcactcaacg- attagcg accagccggaatgcttggccagagcatgtatcatatggtccagaaaccctatacctgtggacgttaatcacttg- cgatt gtgtggcctgttctgctactgcttctgcctctttttctgggaagatcgagtgctctatcgctaggggaccaccc- tttaaagagat cgcaatctgaatcttggtttcatttgtaatacgctttactagggctttctgctctgtcatctttgccttcgttt- atcttgcctgctcatttt ttagtatattcttcgaagaaatcacattactttatataatgtataattcattatgtgataatgccaatcgctaa- gaaaaaaaaa gagtcatccgctaggggaaaaaaaaaaatgaaaatcattaccgaggcataaaaaaatatagagtgtactagagg- ag gccaagagtaatagaaaaagaaaattgcgggaaaggactgtgttatgacttccctgactaatgccgtgttcaaa- cgata cctggcagtgactcctagcgctcaccaagctcttaaaacgggaatttatggtgcactctcagtacaatctgctc- tgatgccg catagttaagccagccccgacacccgccaacacccgctgacgcgccctgacgggcttgtctgctcccggcatcc- gctta cagacaagctgtgaccgtctccgggagctgcatgtgtcagaggttttcaccgtcatcaccgaaacgcgcga

Sequence CWU 1

3113708DNASaccharomyces cerevisiae 1atgcatttta gaagaacgat gagtagagtg cccacattgg catctcttga aatacgatat 60aaaaaatctt tcggccataa atttcgtgat tttattgctc tatgtggtca ctattttgct 120ccagttaaaa aatatatctt ccccagtttt atcgcggttc actacttcta cacgatatcc 180ctgacattaa taacttcaat cctgctatat cccattaaga ataccagata cattgataca 240ttgtttttag cagcgggcgc agttacacaa ggtggcttaa atactgtgga tatcaacaat 300ctaagcttat accaacaaat tgttctgtat atcgtatgct gcatatcaac accaattgca 360gttcatagtt gcttggcatt tgtacggctt tactggtttg agcgctactt cgatggtatt 420agagactctt ctagacgaaa ttttaagatg agaagaacga aaacaatctt agaaagggaa 480ctaacagcaa gaaccatgac caagaataga acaggtaccc aaagaacgtc ttatcctagg 540aaacaagcta aaacagatga tttccaagaa aaattgttca gcggagaaat ggttaataga 600gatgagcagg actcagttca cagcgaccag aattctcatg acattagtag ggacagcagc 660aataataata cgaatcacaa tggtagcagt ggcagtttag atgatttcgt taaggaagac 720gaaacggatg acaatggaga atatcaggag aacaactcct actcgacggt aggtagttcg 780tctaacacag ttgcagacga aagtttaaat cagaagccca agccaagcag tcttcggttt 840gatgagccac acagcaaaca aagacccgca agagttccct cagagaaatt tgcaaaaaga 900aggggttcaa gagatattag cccagccgat atgtatcgat ccattatgat gctacaaggt 960aagcatgaag caactgctga agatgaaggt ccccctttag tcatcgggtc ccctgcggat 1020ggcacaagat ataaaagtaa tgtcaataag ctaaagaagg ccaccggcat aaatggtaac 1080aaaatcaaga ttcgagataa gggaaatgaa agtaacactg atcaaaattc cgtgtcaagt 1140gaagcaaaca gtacggcgag cgtttcggac gaaagctcgt tacacacaaa ttttggtaac 1200aaagtacctt cattaagaac aaatactcat agatcaaatt cgggcccgat agccattact 1260gataacgcag aaacagacaa aaagcatggg ccatcaattc aattcgatat aactaaacct 1320cctagaaaaa tttcaaaaag agtttcaacc ttcgatgatt tgaacccaaa atcttccgtt 1380ctttatcgaa aaaaagcatc gaagaagtac ctcatgaaac attttcctaa agcgcggcga 1440atacggcaac aaattaagag aaggctttct actggttcaa ttgagaaaaa cagcagtaac 1500aatgtttcag atagaaaacc tattactgat atggatgatg atgatgatga cgatgacaac 1560gacggcgata acaacgaaga atactttgct gacaacgaaa gcggcgatga agatgaacga 1620gtacagcagt ctgaaccaca ttctgattca gaactcaaat cgcaccaaca acagcaagaa 1680aaacaccaac tgcagcagaa cctgcaccgc atgtataaaa ccaaatcatt tgatgataat 1740cgttcaagag cagttcctat ggaacgttcc aggaccatcg atatggcaga ggctaaggat 1800ctaaatgagc tcgcaaggac gcctgatttt caaaaaatgg tctatcaaaa ttggaaagcc 1860catcatagaa aaaaaccgaa ctttaggaag aggggatgga ataacaagat atttgaacat 1920ggtccctatg catctgacag cgatcgcaat tatcctgata atagtaatac tggaaacagt 1980attcttcatt acgcagagtc tattttacat catgatggct ctcataaaaa tggaagcgaa 2040gaagcctctt ccgactctaa tgagaatatc tattccacga atggaggaag cgaccacaat 2100ggtcttaaca actatcctac ttacaacgac gatgaagaag gctattatgg tttacatttc 2160gataccgatt atgacctaga tcctcgtcat gatttatcta aaggcagtgg taaaacgtat 2220ctatcatggc aaccaactat tggacgtaac tcaaacttcc ttggattaac aagagcccag 2280aaagatgaat taggtggtgt cgagtacaga gcaatcaaac ttttatgcac catattggtt 2340gtctactacg ttggatggca tattgttgct tttgttatgt tagtaccttg gattattttg 2400aaaaagcatt atagtgaagt tgttagagat gatggtgttt cacctacatg gtggggattt 2460tggacagcaa tgagtgcatt taatgattta ggtttgacat taactccaaa ttcaatgatg 2520tcgtttaaca aagctgtata cccattgatc gttatgattt ggtttatcat tatcggaaat 2580acagggtttc ccatccttct tagatgcatc atttggataa tgtttaaaat ttctcctgat 2640ttatcacaga tgagagaaag tttaggtttt ctcttagacc atccacgtcg ttgtttcacc 2700ttgctatttc ctaaggcagc tacatggtgg ctacttttaa cgcttgcagg attgaatata 2760actgattgga ttttatttat tattctagat tttggctcaa cagttgtgaa atcattatcg 2820aaaggctata gagtccttgt cggcctgttt caatctgtta gcacaagaac tgctggattc 2880agcgttgtcg atttaagtca actgcatcct tctatccaag tctcctatat gctaatgatg 2940tatgtctccg tattaccatt ggccatctct attcgacgga caaatgttta cgaggagcaa 3000tctttaggac tatatggaga tatgggggga gaaccagaag atacggatac tgaagacgat 3060ggtaacgatg aagatgacga cgaggaaaac gagagtcacg aaggtcaaag tagtcaaaga 3120agtagttcga acaacaacaa caataacaac aggaaaaaga aaaagaaaaa gaaaactgaa 3180aatccaaatg aaatatctac aaaatccttt atcggtgccc atttaaggaa acagctttca 3240tttgacttgt ggtttctatt tttagggtta tttatcattt gcatttgtga aggggacaag 3300ataaaggacg tacaagaacc aaactttaat atatttgcaa ttctttttga aattgttagc 3360gcttacggta cagttgggct atcgctaggt tatccggaca ccaaccaatc gttttcaaga 3420cagtttacta cattatctaa gttggtgatc atagctatgc tgatcagagg caagaataga 3480ggtctaccat actcactgga tcgtgcaatt atcttgccta gtgatagact tgaacatatt 3540gaccaccttg agggcatgaa attgaagaga caggctagaa ccaatacaga agacccaatg 3600acggaacatt tcaagagaag tttcactgat gtgaaacatc gttggggagc tcttaagcgt 3660aagaccacac attcccgaaa tcctaaaagg agcagcacaa cgctctaa 370822669DNASaccharomyces cerevisiae 2atgccaacag ctaagaggac gtcatccagg gcttcgttgg cactgccctt ccagttacgg 60ttggtgcaca agaaatcatg gggccatcgg ctaagagact tcatttccgg gttcttaaaa 120tcatgcagac ccattgctaa atacgttttc cccaacttca tcgtggtgca ctatatctac 180ctgatcacgc tgtcgattat cgggtccatt ctgttatatc cgtgcaagaa cacggcgttc 240atcgatgtgc tatttctggc tgctggagcg tctacacagg gcgggctggc caccaagagc 300actaacgatt tcaacctgta ccagcagata gtggtgtacg tcattacatt gctgtccacg 360cctatactta ttcatgggtt tttggccttt gtcaggctgt attggtttga aaggtacttc 420gacaacatta gggatatctc caaacagaat tttaaactaa gaaggaccat gacgttgcaa 480caaagggaac tatcgggcag cagtggcaat gccgctcgaa gtaggagttt caaggacaac 540ctgttccgtg ggaagtttgt ttccagagaa gacccacgac aatccgcttc agatgtgccg 600atggactctc ctgacacgtc cgcattgtcc tcaatctcac cgttgaatgt ttcctcctct 660aaggaggaat ccagtgacac gcaaagctcg cctccaaact tctcaagtaa gcgccaaccc 720tcagacgttg acccaagaga catttacaaa tcgataatga tgctacaaaa acaacaagag 780aagagcaacg caaactccac ggattctttt tcgagcgaga ccaatggacc cgctttcatt 840gtgcaggaac gtcatgagag aagagccccc cactgctcac tgaaacgcca ttctgtcctg 900ccatcttctc aggaattgaa caagctagcc cagacgaaaa gtttccagaa attgcttggc 960ttgcggagag atgaaggtga ccatgactac tttgacggtg ctcctcacaa atatatggtc 1020accaagaaga aaaaaatatc tagaacgcaa tcatgtaaca tcccaacgta tactgcttca 1080ccgagtccta aaacctcagg ccaagtagtt gaaaatcata gaaacttggc caagtcggcg 1140ccttcatctt ttgttgatga ggagatgagc ttttcaccgc aagagtcttt gaatttacag 1200ttccaagcgc acccgcccaa accaaaacga cgtgaaggtg atataggcca ccccttcacc 1260agaacaatga gcaccaacta tctatcgtgg cagccaacct ttggcagaaa ctccgtcttc 1320attggactca caaagcaaca aaaggaggaa ctcggcggtg tcgaatatcg tgctttgaga 1380ttgctgtgct gcattctcat ggtatactac atcggattca acattttggc gtttgtgacc 1440atcgttccat gggcctgtac gaggcaccac tactcagaga ttattagacg aaatggagtt 1500tctccaacct ggtgggggtt tttcactgca atgagtgcat tcagcaactt gggtctgtct 1560ttgaccgctg attcaatggt ttcctttgat actgcgccgt atccgctgat tttcatgatg 1620ttcttcatca tcataggcaa tacaggcttc ccaattatgt tacgatttat catttggatc 1680atgttcaaga cctcgagaga cctatctcag tttaaggaaa gtcttgggtt tctcttggat 1740catccgcgca ggtgttttac gttgctgttc cccagcggcc ccacatggtg gctgtttaca 1800actttagtcg tcttaaacgc tacggattgg attcttttca taattctgga tttcaactcc 1860gctgtagtaa ggcaggttgc taaaggttat cgagctctca tgggcctctt ccagtctgta 1920tgcacaagaa ctgctggatt caacgttgtt gacttaagta aattacaccc gtccattcag 1980gtgtcttata tgctaatgat gtacgtttcg gtcctgccgc tggcgatttc cattagaaga 2040acgaatgttt atgaggagca atcgttggga ctatacgata gtggacaaga tgacgaaaat 2100atcacccacg aagacgatat aaaggaaaca gaccatgatg gcgaatccga agagcgagac 2160actgtatcta caaagtccaa gccgaagaaa cagtccccaa aatcgtttgt tggtgctcat 2220ttgaggaggc aactctcttt tgatttatgg tacctattcc ttggattatt tataatatgc 2280atatgcgagg gcagaaaaat cgaagacgtt aataaacctg atttcaatgt ctttgctata 2340ttgtttgaag ttgttagcgc ttatggtaca gtgggtttgt cattgggtta cccaaacacc 2400aacacatcac tatctgccca gttcaccgta ttatcgaagc tagtcataat tgccatgcta 2460ataagaggaa gaaatagagg tttaccatac actttggatc gtgccatcat gctgccaagt 2520gacaaactgg aacaaattga tcgtttacaa gatatgaaag ctaagggtaa gttgttagcc 2580aaagttggtg aggatccaat gactacttac gtcaaaaaga gatcccacaa actgaaaaaa 2640atagcaacaa agttttgggg gaagcatta 266932076DNASaccharomyces cerevisiae 3atgacaaggt tcatgaacag ctttgccaaa caaacgctgg gatatggcaa tatggcgaca 60gtggagcaag agagctcagc tcaggctgtt gattctcatt caaacaacac accgaagcaa 120gctaagggtg ttcttgcaga ggaactaaag gatgcattgc ggttccggga cgaaagagtt 180agtattatta atgcagagcc ttcttcaaca ctgttcgtct tttggtttgt ggtttcatgc 240tatttccctg tgattactgc ctgcttgggt cccgtagcta acactatctc gatagcctgt 300gtagttgaaa aatggagatc cttaaagaac aactccgtgg tgacaaatcc acgaagcaat 360gacaccgatg ttttgatgaa tcaagtaaag acagtttttg atcctcctgg tatttttgcc 420gttaatatca tctctttggt actgggtttt acgtcaaata ttatactaat gctacatttc 480agtaagaagt tgacgtatct taaatctcag ttaataaata taacaggatg gacaatagct 540ggagggatgc ttttggtgga cgtgattgta tgctccttga atgacatgcc cagcatctac 600agtaagacta tcggattttg gtttgcctgt atcagttctg gtctatattt ggtatgcacc 660attattttaa caatacattt tattggatat aaattaggaa aatatcctcc aacgttcaac 720cttttgccca atgaaagaag tatcatggca tacactgtac tattgtcttt atggttgatt 780tggggtgcgg gtatgtttag cggtttattg cacatcactt acggaaatgc attatatttc 840tgcacggtat cattattaac cgtgggacta ggtgacatcc tgcccaagtc ggttggcgcc 900aaaatcatgg ttttaatctt ttcgctatct ggtgttgtct tgatgggttt aatagtgttt 960atgacaagat ccatcattca aaagtcctct ggcccaattt tctttttcca cagagttgaa 1020aaaggcaggt ccaaatcgtg gaaacattat atggatagta gtaaaaattt atctgaaagg 1080gaagcgttcg acttaatgaa gtgtatccga caaacggcct caaggaagca gcattggttt 1140tctttgtcgg tgactattgc aattttcatg gctttttggt tattgggagc tcttgtattc 1200aaattcgcag aaaattggtc gtacttcaat tgtatttact tttgtttctt gtgcttatta 1260accattggat acggagacta tgctccaagg actggtgcag gccgtgcttt ttttgtgatt 1320tgggcgttgg gagccgtgcc attaatgggg gctatcctat ctacagtcgg tgatctgttg 1380tttgacattt ccacttctct ggatattaag atcggtgaat cattcaataa taaagtcaag 1440tccatcgttt ttaatgggcg tcaaagagca ctttccttta tggtgaacac tggagaaatt 1500ttcgaagaat ctgacacagc tgatggtgat ctggaagaaa atacaacgag ctcacaatcc 1560agtcaaattt ctgaattcaa cgataataat tcagaagaga atgattctgg agtgacatcc 1620cctcctgcaa gcctgcaaga atcattttct tcattatcaa aagcatctag cccagaggga 1680atacttcctc tagaatatgt ttcttctgct gaatatgcac tacaggactc ggggacctgt 1740aatttaagga acttgcaaga gctacttaaa gccgtcaaaa aactacatcg gatatgtctg 1800gcggataaag attacacact tagtttttcc gactggtcgt acattcataa actacatttg 1860aggaacatta cagatattga ggagtacaca cgcggacccg aattttggat atcacctgat 1920acgcccctca agttcccgtt aaatgaacct cattttgctt ttatgatgct tttcaagaac 1980atagaagaat tagttggtaa tctagtagaa gacgaagagc tttataaagt tataagcaaa 2040agaaaatttt tgggtgagca tagaaagaca ctttga 207643479DNAHomo sapiens 4atgccggtgc ggaggggcca cgtcgcgccg cagaacacct tcctggacac catcatccgc 60aagtttgagg gccagagccg taagttcatc atcgccaacg ctcgggtgga gaactgcgcc 120gtcatctact gcaacgacgg cttctgcgag ctgtgcggct actcgcgggc cgaggtgatg 180cagcgaccct gcacctgcga cttcctgcac gggccgcgca cgcagcgccg cgctgccgcg 240cagatcgcgc aggcactgct gggcgccgag gagcgcaaag tggaaatcgc cttctaccgg 300aaagatggga gctgcttcct atgtctggtg gatgtggtgc ccgtgaagaa cgaggatggg 360gctgtcatca tgttcatcct caatttcgag gtggtgatgg agaaggacat ggtggggtcc 420ccggctcatg acaccaacca ccggggcccc cccaccagct ggctggcccc aggccgcgcc 480aagaccttcc gcctgaagct gcccgcgctg ctggcgctga cggcccggga gtcgtcggtg 540cggtcgggcg gcgcgggcgg cgcgggcgcc ccgggggccg tggtggtgga cgtggacctg 600acgcccgcgg cacccagcag cgagtcgctg gccctggacg aagtgacagc catggacaac 660cacgtggcag ggctcgggcc cgcggaggag cggcgtgcgc tggtgggtcc cggctctccg 720ccccgcagcg cgcccggcca gctcccatcg ccccgggcgc acagcctcaa ccccgacgcc 780tcgggctcca gctgcagcct ggcccggacg cgctcccgag aaagctgcgc cagcgtgcgc 840cgcgcctcgt cggccgacga catcgaggcc atgcgcgccg gggtgctgcc cccgccaccg 900cgccacgcca gcaccggggc catgcaccca ctgcgcagcg gcttgctcaa ctccacctcg 960gactccgacc tcgtgcgcta ccgcaccatt agcaagattc cccaaatcac cctcaacttt 1020gtggacctca agggcgaccc cttcttggct tcgcccacca gtgaccgtga gatcatagca 1080cctaagataa aggagcgaac ccacaatgtc actgagaagg tcacccaggt cctgtccctg 1140ggcgccgacg tgctgcctga gtacaagctg caggcaccgc gcatccaccg ctggaccatc 1200ctgcattaca gccccttcaa ggccgtgtgg gactggctca tcctgctgct ggtcatctac 1260acggctgtct tcacacccta ctcggctgcc ttcctgctga aggagacgga agaaggcccg 1320cctgctaccg agtgtggcta cgcctgccag ccgctggctg tggtggacct catcgtggac 1380atcatgttca ttgtggacat cctcatcaac ttccgcacca cctacgtcaa tgccaacgag 1440gaggtggtca gccaccccgg ccgcatcgcc gtccactact tcaagggctg gttcctcatc 1500gacatggtgg ccgccatccc cttcgacctg ctcatcttcg gctctggctc tgaggagctg 1560atcgggctgc tgaagactgc gcggctgctg cggctggtgc gcgtggcgcg gaagctggat 1620cgctactcag agtacggcgc ggccgtgctg ttcttgctca tgtgcacctt tgcgctcatc 1680gcgcactggc tagcctgcat ctggtacgcc atcggcaaca tggagcagcc acacatggac 1740tcacgcatcg gctggctgca caacctgggc gaccagatag gcaaacccta caacagcagc 1800ggcctgggcg gcccctccat caaggacaag tatgtgacgg cgctctactt caccttcagc 1860agcctcacca gtgtgggctt cggcaacgtc tctcccaaca ccaactcaga gaagatcttc 1920tccatctgcg tcatgctcat tggctccctc atgtatgcta gcatcttcgg caacgtgtcg 1980gccatcatcc agcggctgta ctcgggcaca gcccgctacc acacacagat gctgcgggtg 2040cgggagttca tccgcttcca ccagatcccc aatcccctgc gccagcgcct cgaggagtac 2100ttccagcacg cctggtccta caccaacggc atcgacatga acgcggtgct gaagggcttc 2160cctgagtgcc tgcaggctga catctgcctg cacctgaacc gctcactgct gcagcactgc 2220aaacccttcc gaggggccac caagggctgc cttcgggccc tggccatgaa gttcaagacc 2280acacatgcac cgccagggga cacactggtg catgctgggg acctgctcac cgccctgtac 2340ttcatctccc ggggctccat cgagatcctg cggggcgacg tcgtcgtggc catcctgggg 2400aagaatgaca tctttgggga gcctctgaac ctgtatgcaa ggcctggcaa gtcgaacggg 2460gatgtgcggg ccctcaccta ctgtgaccta cacaagatcc atcgggacga cctgctggag 2520gtgctggaca tgtaccctga gttctccgac cacttctggt ccagcctgga gatcaccttc 2580aacctgcgag ataccaacat gatcccgggc tcccccggca gtacggagtt agagggtggc 2640ttcagtcggc aacgcaagcg caagttgtcc ttccgcaggc gcacggacaa ggacacggag 2700cagccagggg aggtgtcggc cttggggccg ggccgggcgg gggcagggcc gagtagccgg 2760ggccggccgg gggggccgtg gggggagagc ccgtccagtg gcccctccag ccctgagagc 2820agtgaggatg agggcccagg ccgcagctcc agccccctcc gcctggtgcc cttctccagc 2880cccaggcccc ccggagagcc gccgggtggg gagcccctga tggaggactg cgagaagagc 2940agcgacactt gcaaccccct gtcaggcgcc ttctcaggag tgtccaacat tttcagcttc 3000tggggggaca gtcggggccg ccagtaccag gagctccctc gatgccccgc ccccaccccc 3060agcctcctca acatccccct ctccagcccg ggtcggcggc cccggggcga cgtggagagc 3120aggctggatg ccctccagcg ccagctcaac aggctggaga cccggctgag tgcagacatg 3180gccactgtcc tgcagctgct acagaggcag atgacgctgg tcccgcccgc ctacagtgct 3240gtgaccaccc cggggcctgg ccccacttcc acatccccgc tgttgcccgt cagccccctc 3300cccaccctca ccttggactc gctttctcag gtttcccagt tcatggcgtg tgaggagctg 3360cccccggggg ccccagagct tccccaagaa ggccccacac gacgcctctc cctaccgggc 3420cagctggggg ccctcacctc ccagcccctg cacagacacg gctcggaccc gggcagtta 347951836DNAHomo sapiens 5atggagatcg ccctggtgcc cctggagaac ggcggtgcca tgaccgtcag aggaggcgat 60gaggcccggg caggctgcgg ccaggccaca gggggagagc tccagtgtcc cccgacggct 120gggctcagcg atgggcccaa ggagccggcg ccaaaggggc gcgcgcagag agacgcggac 180tcgggagtgc ggcccttgcc tccgctgccg gacccgggag tgcggccctt gcctccgctg 240ccagaggagc tgccacggcc tcgacggccg cctcccgagg acgaggagga agaaggcgat 300cccggcctgg gcacggtgga ggaccaggct ctgggcacgg cgtccctgca ccaccagcgc 360gtccacatca acatctccgg gctgcgcttt gagacgcagc tgggcaccct ggcgcagttc 420cccaacacac tcctggggga ccccgccaag cgcctgccgt acttcgaccc cctgaggaac 480gagtacttct tcgaccgcaa ccggcccagc ttcgacggta tcctctacta ctaccagtcc 540gggggccgcc tgcgaggggt caacgtctcc ctggacgtgt tcgcggacga gatacgcttc 600taccagctgg gggacgaggc catggagcgc ttccgcgagg atgagggctt cattaaagaa 660gaggagaagc ccctgccccg caacgagttc cagcgccagg tgtggcttat cttcgagtat 720ccggagagct ctgggtccgc gcgggccatc gccatcgtct cggtcttggt tatcctcatc 780tccatcatca ccttctgctt ggagaccctg cctgagttca gggatgaacg tgagctgctc 840cgccaccctc cggcgcccca ccagcctccc gcgcccgccc ctggggccaa cggcagcggg 900gtcatggccc ccgcctctgg ccctacggtg gcaccgctcc tgcccaggac cctggccgac 960cccttcttca tcgtggagac cacgtgcgtg atctggttca ccttcgagct gctcgtgcgc 1020ttcttcgcct gccccagcaa ggcagggttc tcccggaaca tcatgaacat catcgatgtg 1080gtggccatct tcccctactt catcaccctg ggcaccgaac tggcagagca gcagccaggg 1140ggcggaggag gcggccagaa tgggcagcag gccatgtccc tggccatcct ccgagtcatc 1200cgcctggtcc gggtgttccg catcttcaag ctctcccgcc actccaaggg gctgcagatc 1260ctgggcaaga ccttgcaggc ctccatgagg gagctggggc tgctcatctt cttcctcttc 1320atcggggtca tcctcttctc cagtgccgtc tacttcgcag aggctgacaa ccagggaacc 1380catttctcta gcatccctga cgccttctgg tgggcagtgg tcaccatgac cactgtgggc 1440tacggggaca tgaggcccat cactgttggg ggcaagatcg tgggctcgct gtgtgccatc 1500gccggggtcc tcaccattgc cctgcctgtg cccgtcatcg tctccaactt caactacttc 1560taccaccggg aaacggatca cgaggagccg gcagtcctta aggaagagca gggcactcag 1620agccaggggc cggggctgga cagaggagtc cagcggaagg tcagcgggag caggggatcc 1680ttctgcaagg ctggggggac cctggagaat gcagacagtg cccgaagggg cagctgcccc 1740ctagagaagt gtaacgtcaa ggccaagagc aacgtggact tgcggaggtc cctttatgcc 1800ctctgcctgg acaccagccg ggaaacagat ttgtga 183661284DNACavia guianae 6atgggcagtg tgcgaaccaa ccgctatagc attgtctctt cggaagagga cggcatgaag 60ttggccacca tggcagttgc caatggcttt gggaatggga aaagtaaagt ccacactcgg 120caacagtgta ggagccgctt tgtgaagaaa gatggccact gtaatgttca gttcatcaac 180gttggggaaa agggacaacg gtaccttgct gacattttta ctacgtgtgt ggacattcgc 240tggcggtgga tgctggttat cttttgccta gcttttgttc tctcgtggct gttttttggc 300tgtgtgtttt ggctgatagc tttgctccat ggagatctgg atgcatctaa ggagagcaaa 360gcctgtgtgt ctgaggtcaa cagcttcaca gctgcctttc ttttctccat tgagacccag 420acaaccatcg gctatgggtt ccgatgtgtc acggatgaat gcccgattgc ggtgttcatg 480gttgtgttcc agtcaattgt gggctgcatt attgatgctt ttatcattgg tgccgtcatg 540gcaaagatgg caaagccaaa gaaaagaaat gagactcttg tcttcagtca caatgctgtg 600attgccatga gagatggcaa gctgtgtttg atgtggcgag taggcaacct tcggaaaagc 660cacttggtag aagctcatgt tcgagcccag ctcctcaaat ccagaattac ttctgaaggg 720gaatacatcc ccttggatca aatagacatc aatgttggct ttgacagtgg aattgaccgt 780atatttctgg tatccccaat cactattgtc catgaaatag atgaagatag tcctttatat 840gatttgagca agcaggacat tgataatgca gactttgaaa ttgttgtgat actagaaggc 900atggtggaag ccactgccat gacaacacag tgtcgtagtt cttatttggc caacgagatc 960ctttggggcc accgctatga gccagtgctc tttgaggaga agcactacta taaagtggac 1020tattcgaggt ttcataagac ttacgaagta

cccaacactc ccctttgtag tgccagagac 1080ttagcagaaa agaaatatat tctctcaaat gctaactcat tttgctatga aaatgaagtt 1140gcccttacaa gcaaagagga agatgacagt gaaaatgggg ttccagaaag caccagtaca 1200gacacacctc ctgacatcga ccttcacaac caggcaagtg tacctctaga gcccagaccc 1260ttacggcgag aatcggagat atga 1284734DNAArtificial SequencePrimer 7gcggatccat gcattttaga agaacgatga gtag 34821DNAArtificial SequencePrimer 8aggttctgct gcagttggtg t 21921DNAArtificial SequencePrimer 9acaccaactg cagcagaacc t 211029DNAArtificial SequencePrimer 10cgctcgagtt agagcgttgt gctgctcct 291122DNAArtificial SequencePrimer 11ccttaccatt agcatcactg at 221221DNAArtificial SequencePrimer 12ctattaacca tttctccgct g 211324DNAArtificial SequencePrimer 13gatttatctt cgtttcctgc aggt 241431DNAArtificial SequencePrimer 14cacgtacgtc cagcacaatt tcacaacagc t 311530DNAArtificial SequencePrimer 15cagtcgacct ggatgacgtc ctcttagctg 301629DNAArtificial SequencePrimer 16cagatatcat gctgccaagt gacaaactg 291728DNAArtificial SequencePrimer 17tcactagttg ttgatggctt tggttggt 281822DNAArtificial SequencePrimer 18gcgaagaata ggatgagatg tg 221920DNAArtificial SequencePrimer 19ttgtcgtggg tcttctctgg 202023DNAArtificial SequencePrimer 20gctacctttg ccatgtttca gaa 232131DNAArtificial SequencePrimer 21cacgtacggc aaatttatcg agactctgcg a 312229DNAArtificial SequencePrimer 22aggtcgacca tattgccata tcccagcgt 292321DNAArtificial SequencePrimer 23tggatatcac ctgatacgcc c 212435DNAArtificial SequencePrimer 24caactagtgc ataccagtag tatgagacat gcttg 352523DNAArtificial SequencePrimer 25cctgagtact cagtaccatc ttg 232618DNAArtificial SequencePrimer 26ctgtagatgc tgggcatg 182727DNAArtificial SequencePrimer 27tacgtcgaca tggagatcgc cctggtg 272828DNAArtificial SequencePrimer 28tacgtcgaca tctgtttccc ggctggtg 282925DNAArtificial SequencePrimer 29tacatcgata tgccggtgcg gaggg 253024DNAArtificial SequencePrimer 30tacgtcgaca ctgcccgggt ccga 24317772DNAHomo sapiens 31gacgaaaggg cctcgtgata cgcctatttt tataggttaa tgtcatgata ataatggttt 60cttagatgat ccaatatcaa aggaaatgat agcattgaag gatgagacta atccaattga 120ggagtggcag catatagaac agctaaaggg tagtgctgaa ggaagcatac gataccccgc 180atggaatggg ataatatcac aggaggtact agactacctt tcatcctaca taaatagacg 240catataagta cgcatttaag cataaacacg cactatgccg ttcttctcat gtatatatat 300atacaggcaa cacgcagata taggtgcgac gtgaacagtg agctgtatgt gcgcagctcg 360cgttgcattt tcggaagcgc tcgttttcgg aaacgctttg aagttcctat tccgaagttc 420ctattctcta gaaagtatag gaacttcaga gcgcttttga aaaccaaaag cgctctgaag 480acgcactttc aaaaaaccaa aaacgcaccg gactgtaacg agctactaaa atattgcgaa 540taccgcttcc acaaacattg ctcaaaagta tctctttgct atatatctct gtgctatatc 600cctatataac ctacccatcc acctttcgct ccttgaactt gcatctaaac tcgacctcta 660cattttttat gtttatctct agtattactc tttagacaaa aaaattgtag taagaactat 720tcatagagtg aatcgaaaac aatacgaaaa tgtaaacatt tcctatacgt agtatataga 780gacaaaatag aagaaaccgt tcataatttt ctgaccaatg aagaatcatc aacgctatca 840ctttctgttc acaaagtatg cgcaatccac atcggtatag aatataatcg gggatgcctt 900tatcttgaaa aaatgcaccc gcagcttcgc tagtaatcag taaacgcggg aagtggagtc 960aggctttttt tatggaagag aaaatagaca ccaaagtagc cttcttctaa ccttaacgga 1020cctacagtgc aaaaagttat caagagactg cattatagag cgcacaaagg agaaaaaaag 1080taatctaaga tgctttgtta gaaaaatagc gctctcggga tgcatttttg tagaacaaaa 1140aagaagtata gattctttgt tggtaaaata gcgctctcgc gttgcatttc tgttctgtaa 1200aaatgcagct cagattcttt gtttgaaaaa ttagcgctct cgcgttgcat ttttgtttta 1260caaaaatgaa gcacagattc ttcgttggta aaatagcgct ttcgcgttgc atttctgttc 1320tgtaaaaatg cagctcagat tctttgtttg aaaaattagc gctctcgcgt tgcatttttg 1380ttctacaaaa tgaagcacag atgcttcgtt caggtggcac ttttcgggga aatgtgcgcg 1440gaacccctat ttgtttattt ttctaaatac attcaaatat gtatccgctc atgagacaat 1500aaccctgata aatgcttcaa taatattgaa aaaggaagag tatgagtatt caacatttcc 1560gtgtcgccct tattcccttt tttgcggcat tttgccttcc tgtttttgct cacccagaaa 1620cgctggtgaa agtaaaagat gctgaagatc agttgggtgc acgagtgggt tacatcgaac 1680tggatctcaa cagcggtaag atccttgaga gttttcgccc cgaagaacgt tttccaatga 1740tgagcacttt taaagttctg ctatgtggcg cggtattatc ccgtattgac gccgggcaag 1800agcaactcgg tcgccgcata cactattctc agaatgactt ggttgagtac tcaccagtca 1860cagaaaagca tcttacggat ggcatgacag taagagaatt atgcagtgct gccataacca 1920tgagtgataa cactgcggcc aacttacttc tgacaacgat cggaggaccg aaggagctaa 1980ccgctttttt gcacaacatg ggggatcatg taactcgcct tgatcgttgg gaaccggagc 2040tgaatgaagc cataccaaac gacgagcgtg acaccacgat gcctgtagca atggcaacaa 2100cgttgcgcaa actattaact ggcgaactac ttactctagc ttcccggcaa caattaatag 2160actggatgga ggcggataaa gttgcaggac cacttctgcg ctcggccctt ccggctggct 2220ggtttattgc tgataaatct ggagccggtg agcgtgggtc tcgcggtatc attgcagcac 2280tggggccaga tggtaagccc tcccgtatcg tagttatcta cacgacgggg agtcaggcaa 2340ctatggatga acgaaataga cagatcgctg agataggtgc ctcactgatt aagcattggt 2400aactgtcaga ccaagtttac tcatatatac tttagattga tttaaaactt catttttaat 2460ttaaaaggat ctaggtgaag atcctttttg ataatctcat gaccaaaatc ccttaacgtg 2520agttttcgtt ccactgagcg tcagaccccg tagaaaagat caaaggatct tcttgagatc 2580ctttttttct gcgcgtaatc tgctgcttgc aaacaaaaaa accaccgcta ccagcggtgg 2640tttgtttgcc ggatcaagag ctaccaactc tttttccgaa ggtaactggc ttcagcagag 2700cgcagatacc aaatactgtc cttctagtgt agccgtagtt aggccaccac ttcaagaact 2760ctgtagcacc gcctacatac ctcgctctgc taatcctgtt accagtggct gctgccagtg 2820gcgataagtc gtgtcttacc gggttggact caagacgata gttaccggat aaggcgcagc 2880ggtcgggctg aacggggggt tcgtgcacac agcccagctt ggagcgaacg acctacaccg 2940aactgagata cctacagcgt gagctatgag aaagcgccac gcttcccgaa gggagaaagg 3000cggacaggta tccggtaagc ggcagggtcg gaacaggaga gcgcacgagg gagcttccag 3060ggggaaacgc ctggtatctt tatagtcctg tcgggtttcg ccacctctga cttgagcgtc 3120gatttttgtg atgctcgtca ggggggcgga gcctatggaa aaacgccagc aacgcggcct 3180ttttacggtt cctggccttt tgctggcctt ttgctcacat gttctttcct gcgttatccc 3240ctgattctgt ggataaccgt attaccgcct ttgagtgagc tgataccgct cgccgcagcc 3300gaacgaccga gcgcagcgag tcagtgagcg aggaagcgga agagcgccca atacgcaaac 3360cgcctctccc cgcgcgttgg ccgattcatt aatgcagctg gcacgacagg tttcccgact 3420ggaaagcggg cagtgagcgc aacgcaatta atgtgagtta cctcactcat taggcacccc 3480aggctttaca ctttatgctt ccggctccta tgttgtgtgg aattgtgagc ggataacaat 3540ttcacacagg aaacagctat gaccatgatt acgccaagcg cgcaattaac cctcactaaa 3600gggaacaaaa gctggagctc agtttatcat tatcaatact gccatttcaa agaatacgta 3660aataattaat agtagtgatt ttcctaactt tatttagtca aaaaattagc cttttaattc 3720tgctgtaacc cgtacatgcc caaaataggg ggcgggttac acagaatata taacatcgta 3780ggtgtctggg tgaacagttt attcctggca tccactaaat ataatggagc ccgcttttta 3840agctggcatc cagaaaaaaa aagaatccca gcaccaaaat attgttttct tcaccaacca 3900tcagttcata ggtccattct cttagcgcaa ctacagagaa caggggcaca aacaggcaaa 3960aaacgggcac aacctcaatg gagtgatgca acctgcctgg agtaaatgat gacacaaggc 4020aattgaccca cgcatgtatc tatctcattt tcttacacct tctattacct tctgctctct 4080ctgatttgga aaaagctgaa aaaaaaggtt gaaaccagtt ccctgaaatt attcccctac 4140ttgactaata agtatataaa gacggtaggt attgattgta attctgtaaa tctatttctt 4200aaacttctta aattctactt ttatagttag tctttttttt agttttaaaa caccagaact 4260tagtttcgac ggattctaga actagtggat cccccgggct gcagccatgt tcaaacatct 4320tcggaaatgg gtcgtcactc gcttttttgg gcattctcgg caaagagcaa ggctagtctc 4380caaagatgga aggtgcaaca tagaatttgg caatgtggag gcacagtcaa ggtttatatt 4440ctttgtggac atctggacaa cggtacttga cctcaagtgg agatacaaaa tgaccatttt 4500catcacagcc ttcttgggga gttggttttt ctttggtctc ctgtggtatg cagtagcgta 4560cattcacaaa gacctcccgg aattccatcc ttctgccaat cacactccct gtgtggagaa 4620tattaatggc ttgacctcag cttttctgtt ttctctggag actcaagtga ccattggata 4680tggattcagg tgtgtgacag aacagtgtgc cactgccatt tttctgctta tctttcagtc 4740tatacttgga gttataatca attctttcat gtgtggggcc atcttagcca agatctccag 4800gcccaaaaaa cgtgccaaga ccattacgtt cagcaagaac gcagtgatca gcaaacgggg 4860agggaagctt tgcctcctaa tccgagtggc taatctcagg aagagccttc ttattggcag 4920tcacatttat ggaaagcttc tgaagaccac agtcactcct gaaggagaga ccattatttt 4980ggaccagatc aatatcaact ttgtagttga cgctgggaat gaaaatttat tcttcatctc 5040cccattgaca atttaccatg tcattgatca caacagccct ttcttccaca tggcagcgga 5100gacccttctc cagcaggact ttgaattagt ggtgttttta gatggcacag tggagtccac 5160cagtgctacc tgccaagtcc ggacatccta tgtcccagag gaggtgcttt ggggctaccg 5220ttttgctccc atagtatcca agacaaagga agggaaatac cgagtggatt tccataactt 5280tagcaagaca gtggaagtgg agacccctca ctgtgccatg tgcctttata atgagaaaga 5340tgttagagcc aggatgaaga gaggctatga caaccccaac ttcatcttgt cagaagtcaa 5400tgaaacagat gacaccaaaa tgtaacagtc gacctcgagt catgtaatta gttatgtcac 5460gcttacattc acgccctccc cccacatccg ctctaaccga aaaggaagga gttagacaac 5520ctgaagtcta ggtccctatt tattttttta tagttatgtt agtattaaga acgttattta 5580tatttcaaat ttttcttttt tttctgtaca gacgcgtgta cgcatgtaac attatactga 5640aaaccttgct tgagaaggtt ttgggacgct cgaaggcttt aatttgcggc cggtacccaa 5700ttcgccctat agtgagtcgt attacgcgcg ctcactggcc gtcgttttac aacgtcgtga 5760ctgggaaaac cctggcgtta cccaacttaa tcgccttgca gcacatcccc ctttcgccag 5820ctggcgtaat agcgaagagg cccgcaccga tcgcccttcc caacagttgc gcagcctgaa 5880tggcgaatgg cgcgacgcgc cctgtagcgg cgcattaagc gcggcgggtg tggtggttac 5940gcgcagcgtg accgctacac ttgccagcgc cctagcgccc gctcctttcg ctttcttccc 6000ttcctttctc gccacgttcg ccggctttcc ccgtcaagct ctaaatcggg ggctcccttt 6060agggttccga tttagtgctt tacggcacct cgaccccaaa aaacttgatt agggtgatgg 6120ttcacgtagt gggccatcgc cctgatagac ggtttttcgc cctttgacgt tggagtccac 6180gttctttaat agtggactct tgttccaaac tggaacaaca ctcaacccta tctcggtcta 6240ttcttttgat ttataaggga ttttgccgat ttcggcctat tggttaaaaa atgagctgat 6300ttaacaaaaa tttaacgcga attttaacaa aatattaacg tttacaattt cctgatgcgg 6360tattttctcc ttacgcatct gtgcggtatt tcacaccgca tagatccgtc gagttcaaga 6420gaaaaaaaaa gaaaaagcaa aaagaaaaaa ggaaagcgcg cctcgttcag aatgacacgt 6480atagaatgat gcattacctt gtcatcttca gtatcatact gttcgtatac atacttactg 6540acattcatag gtatacatat atacacatgt atatatatcg tatgctgcag ctttaaataa 6600tcggtgtcac tacataagaa cacctttggt ggagggaaca tcgttggtac cattgggcga 6660ggtggcttct cttatggcaa ccgcaagagc cttgaacgca ctctcactac ggtgatgatc 6720attcttgcct cgcagacaat caacgtggag ggtaattctg ctagcctctg caaagctttc 6780aagaaaatgc gggatcatct cgcaagagag atctcctact ttctcccttt gcaaaccaag 6840ttcgacaact gcgtacggcc tgttcgaaag atctaccacc gctctggaaa gtgcctcatc 6900caaaggcgca aatcctgatc caaacctttt tactccacgc gccagtaggg cctctttaaa 6960agcttgaccg agagcaatcc cgcagtcttc agtggtgtga tggtcgtcta tgtgtaagtc 7020accaatgcac tcaacgatta gcgaccagcc ggaatgcttg gccagagcat gtatcatatg 7080gtccagaaac cctatacctg tgtggacgtt aatcacttgc gattgtgtgg cctgttctgc 7140tactgcttct gcctcttttt ctgggaagat cgagtgctct atcgctaggg gaccaccctt 7200taaagagatc gcaatctgaa tcttggtttc atttgtaata cgctttacta gggctttctg 7260ctctgtcatc tttgccttcg tttatcttgc ctgctcattt tttagtatat tcttcgaaga 7320aatcacatta ctttatataa tgtataattc attatgtgat aatgccaatc gctaagaaaa 7380aaaaagagtc atccgctagg ggaaaaaaaa aaatgaaaat cattaccgag gcataaaaaa 7440atatagagtg tactagagga ggccaagagt aatagaaaaa gaaaattgcg ggaaaggact 7500gtgttatgac ttccctgact aatgccgtgt tcaaacgata cctggcagtg actcctagcg 7560ctcaccaagc tcttaaaacg ggaatttatg gtgcactctc agtacaatct gctctgatgc 7620cgcatagtta agccagcccc gacacccgcc aacacccgct gacgcgccct gacgggcttg 7680tctgctcccg gcatccgctt acagacaagc tgtgaccgtc tccgggagct gcatgtgtca 7740gaggttttca ccgtcatcac cgaaacgcgc ga 7772

Claims

1-24. (canceled)

25. A process for identifying inhibitors of a human potassium channel,a) providing a mutated S. cerevisiae cell which does not express the three endogenous potassium channels TRK1, TRK2 and TOK1;b) treating said mutated cell with a human potassium channel wherein said human potassium channel is expressed heterologously in this mutated S. cerevisiae cell;c) incubating the mutated S. cerevisiae cell expressing the human potassium channel together with a substance to be tested; andd) determining the effect of the substance to be tested on the human potassium channel, wherein a decrease in the transport of potassium across the human potassium channel indicates that the substance is an inhibitor of the human potassium channel.

26. The process as claimed in claim 25, wherein the genes TRK1, TRK2 and TOK1 are switched off in the mutated S. cerevisiae cell (Δtrk1, Δtrk2, Δtok1).

27. The process as claimed in claim 25, wherein the human potassium channel is a HERG1, Kv1.5 or IRK1.

28. The process as claimed in claim 25, wherein the human potassium channel is mutated.

29. The process as claimed in claim 25, wherein the human potassium channel is present in a yeast expression plasmid.

30. The process as claimed in claim 25, wherein the mutated S. cerevisiae cell expresses constitutively a growth reporter.

31. The process as claimed in claim 25, wherein the substance to be tested, which has an effect on the human potassium channel, inhibits the growth of the mutated S. cerevisiae cell.

32. The process as claimed in claim 31, wherein the effect of the substance to be tested on the human potassium channel is determined by measuring the cell count of the mutated S. cerevisiae cells.

33. The process as claimed in claim 30, wherein the cell count is determined via fluorescence or luminescence of the constitutively expressed growth reporter.

34. A mutated S. cerevisiae cell in which TRK1, TRK2 and TOK1 are not expressed, and which expresses heterologously a human potassium channel.

35. The mutated S. cerevisiae cell as claimed in claim 34, wherein the human potassium channel is HERG1, Kv1.5 or IRK1.

36. The mutated S. cerevisiae cell as claimed in claim 34, wherein the human potassium channel is mutated.

37. A process of identifying activators of a human potassium channel,a) providing a mutated S. cerevisiae cell which does not express the three endogenous potassium channels TRK1, TRK2 and TOK1;b) reacting said mutated cell with a human potassium channel, wherein said human potassium channel is expressed heterologously in this mutated S. cerevisiae cell;c) incubating the mutated S. cerevisiae cell expressing the human potassium channel together with a substance to be tested; andd) determining the effect of the substance to be tested on the human potassium channel, wherein an increase in the transport of potassium across the human potassium channel indicates that the substance is an activator of the human potassium channel.

38. A process of identifying activators of a human potassium channel,a) providing a mutated S. cerevisiae cell which does not express the three endogenous potassium channels TRK1, TRK2 and TOK1;b) reacting said mutated cell with a human potassium channel wherein said human potassium channel is expressed heterologously in this mutated S. cerevisiae cell;c) incubating the mutated S. cerevisiae cell expressing the human potassium channel together with a substance to be tested in the presence of an inhibitor of the human potassium channel; andd) determining the effect of the substance to be tested on the human potassium channel, wherein an increase in the transport of potassium across the human potassium channel indicates that the substance is an activator of the human potassium channel.

39. A test kit comprising the mutated S. cerevisiae cell as claimed in claim 34.

Images