# Patent

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

## Abstract

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 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.

## Background
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.
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.
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.
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 K+ (Gaber, R. F. et al., 1988).
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).
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).
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).
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).
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.
The invention relates to a process for identifying inhibitors of a eukaryotic potassium channel, in which

    
    
        a) a mutated S. cerevisiae cell is used which does not express the three endogenous potassium channels TRK1, TRK2 and TOK1;
        b) a eukaryotic potassium channel is expressed heterologously in this mutated S. cerevisiae cell;
        c) the mutated S. cerevisiae cell is incubated together with a substance to be tested; and
        d) the effect of the substance to be tested on the eukaryotic potassium channel is determined.
    
    


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.
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.
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.
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.
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.
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.
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.
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 Mikroorganismen und 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.
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 Assession Number AF 006823] or ROMK2 [Genbank Accession Number U 12542] and where the potassium channel has the natural sequence or can be mutated.
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.
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 toxic substances.
The invention also relates to a process for identifying activators of a eukaryotic potassium channel, in which

    
    
        a) a mutated S. cerevisiae cell is used which does not express the three endogenous potassium channels TRK1, TRK2 and TOK1;
        b) a eukaryotic potassium channel is expressed heterologously in this mutated S. cerevisiae cell;
        c) the mutated S. cerevisiae cell is incubated together with a substance to be tested; and
        d) the effect of the substance to be tested on the eukaryotic potassium channel is determined.
    
    


The invention furthermore relates to a process for identifying activators of a eukaryotic potassium channel, in which

    
    
        a) a mutated S. cerevisiae cell is used which does not express the three endogenous potassium channels TRK1, TRK2 and TOK1;
        b) a eukaryotic potassium channel is expressed heterologously in this mutated S. cerevisiae cell;
        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; and
        d) the effect of the substance to be tested on the eukaryotic potassium channel is determined.
    
    


The invention also relates to a process for the preparation of a medicament, in which

    
    
        a) an Inhibitor of a eukaryotic potassium channel is identified,
        b) the Inhibitor is prepared or isolated by known chemical processes, and
        c) physiologically acceptable additives are added to the inhibitor.
    
    


The invention also relates to a process for the preparation of a medicament, in which

    
    
        a) an activator of a eukaryotic potassium channel is identified,
        b) the activator is prepared or isolated by known chemical processes, and
        c) physiologically acceptable additives are added to the activator.
    
    





FIGURES
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.
FIG. 2: Growth of strains YM168 (Δtrk1 Δtrk2) and YM182 (Δtrk1 Δtrk2 Δtok1) on DPM medium with defined KCl concentrations at pH 6.5.
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 RbCl at pH 6.5.
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.
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)
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)
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.
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.
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.
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.
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.
The values correspond to the average of in each case 4 measurements ±SD. The two diferent assays were carried out independently of each other on two different days.
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.
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.).
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 pages12 et seq. and 15 et seq.).
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.
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.
FIG. 14: Plasmid map of p423 GPD-ROMK2.



EXAMPLES
Materials and Strains
Media
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 acids, 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-FOA
All media are described in: (Fink, G. R. et al., 1991)
Amino Acid Dropout Mix:
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:















mM
g/l



















Adenine (100×)
30
5.53
heating (up to not more than 60° C.)


Leucine (60×)
100
13.12
heating


Lysine (100×)
100
18.26
—


Histidine (200×)
60
12.57
—


Tryptophan (100×)
40
8.17
—


Uracil (100×)
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):















(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

6
g


mix


Lysine

330
ml of 100× stock


Adenine
0.9 mM  
30
ml of 100× stock







→ bring to pH 6.5 (or an other pH) with HCl, autoclave










Glucose
2%

from 40% stock


KCI


from 1 M stock







essential amino acids (with the exception of Lys/Ade) from stocks


Agar









Buffer and Solutions:
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 citrate*2 H2O 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):












Name
Sequence (5′→3′)
RE


















TRK1-FL-BamHI-Fo
GCG′GATCCATGCATTTTAGAAGAACGATGAGTAG:
SEQ ID NO. 7
BamHI


 


TRK1-FL-PstI-Re
AGGTTCTGCTGCA′GTTGGTGT:
SEQ ID NO. 8
PstI


 


TRK1-FL-PstI-Fo
ACACCAACTGCA′GCAGAACCT:
SEQ ID NO. 9
PstI


 


TRK1-FL-XhoI-Re
CGC′TCGAGTTAGAGCGTTGTGCTGCTCCT:
SEQ ID NO. 10
XhoI


 


TRK1-Dia-Fo
CCTTACCATTAGCATCACTGAT:
SEQ ID NO. 11
—


 


TRK1-Dia-Re1
CTATTAACCATTTCTCCGCTG:
SEQ ID NO. 12
—


 


URA-Rev
GATTTATCTTCGTTTCCTGCAGGT:
SEQ ID NO. 13
—


 


TRK2-DEL-5-Fo-B
CAC′GTACGTCCAGCACAATTTCACAACAGCT:
SEQ ID NO. 14
Bs/WI


 


TRK2-DEL-5-Re
CAG′TCGACCTGGATGACGTCCTCTTAGCTG:
SEQ ID NO. 15
Sa/I


 


TRK2-DEL-3-Fo
CAGAT′ATCATGCTGCCAAGTGACAAACTG:
SEQ ID NO. 16
EcoRV


 


TRK2-DEL-3-Re
TCA′CTAGTTGTTGATGGCTTTGGTTGGT:
SEQ ID NO. 17
SpeI


 


TRK2-Dia-Fo
GCGAAGAATAGGATGAGATGTG:
SEQ ID NO. 18
—


 


TRK2-Dia-Re1
TTGTCGTGGGTCTTCTCTGG:
SEQ ID NO. 19
—


 


KAN-Rev
GCTACCTTTGCCATGTTTCAGAA:
SEQ ID NO. 20
—


 


TOK1-DEL-5-Fo
CAC′GTACGGCAAATTTATCGAGACTCTGCGA:
SEQ ID NO. 21
Bs/WI


 


TOK1-DEL-5-Re
AGG′TCGACCATATTGCCATATCCCAGCGT:
SEQ ID NO. 22
Sa/I


 


TOK1-DEL-3-Fo
TGGAT′ATCACCTGATACGCCC:
SEQ ID NO. 23:
EcoRV


 


TOK1-DEL-3-Re
CAA′CTAGTGCATACCAGTAGTATGAGACATGCTTG:
SEQ ID NO. 24
Spel


 


TOK1-Dia-Fo
CCTGAGTACTCAGTACCATCTTG:
SEQ ID NO. 25
—


 


TOK1-Dia-Re1
CTGTAGATGCTGGGCATG:
SEQ ID NO. 26
—


 


Kv1.5-GFP-Fo
TACG′TCGACATGGAGATCGCCCTGGTG:
SEQ ID NO. 27
Sa/I


 


Kv1.5-GFP-Re
TACG′TCGACATCTGTTTCCCGGCTGGTG:
SEQ ID NO. 28
Sa/I


 


HERG1-GFP-Fo
TACAT′CGATATGCCGGTGCGGAGGG:
SEQ ID NO. 29
C/aI


 


HERG1-GFP-Re
TACG′TCGACACTGCCCGGGTCCGA:
SEQ ID NO. 30
Sa/I









Vectors:










5 Bacterial vectors









Name
Size (bp)
Genes





pcDNA3
5446
CMV prom., T7 prom., polylinker, Sp6 prom., BGH poly


(Invitrogen)

(A), SV40 prom., SV 40 ori, NeomycinR, SV 40 poly




(A), ColE1 ori, AmpR


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, NeomycinR, SV 40 poly (A), ColE1 ori,




AmpR


pUG6
4009
loxP-TEF2 prom.-kanMX-loxP-TEF2 term., ori, AmpR


pCR ®-Blunt II-
3519
lac prom./op., M13 reverse prim. site, LacZ-α ORF,


TOPO

SP6 prom. prim. site, MCS, TOPO TM 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









Name
Size (bp)
Genes





pSH47
6786
CEN6/ARSH4, URA3, CYC1 term., CRE, GAL1 prom.,




Amp


p414 GAL1
5474
CEN6/ARSH4, TRP1, CYC1 term., GAL1 prom., AmpR


p416 GAL1
5584
CEN6/ARSH4, URA3, CYC1 term., GAL1 prom., AmpR


p416 ADH
6624
CEN6/ARSH4, URA3, CYC1 term., ADH prom., AmpR


p423 GPD3
6678
2μ, HIS3, CYC1 term., GPD3 prom., AmpR


p426 GAL1
6417
2μ, URA3, CYC1 term., GAL1 prom., AmpR


p426 GAL1-
7140
2μ, URA3, CYC1 term., yEGFP3, GAL1 prom., AmpR


yEGFP3


p426 GAL1-SP-
7227
2μ, URA3, CYC1 term., N-terminal 24 aa of Ste2,


yEGFP3

yEGFP3, GAL1 prom., AmpR









Strains:
Bacterial strains: DH5α; One ShotTM TOP10 (Invitrogen)
Yeast Strains:
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.














Original
Mating



Strain
Name
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:















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


YM 140
Δtok1 in YM 123
MATα
trk1::hisG-URA3-hisG, tok1::loxP-KanMX-loxP


YM 141
Δtok1 in YM 123
MATα
trk1::hisG-URA3-hisG, tok1::loxP-KanMX-loxP


YM 142
Δtok1 in YM 96
MATa/α
tok1::loxP-KanMX-loxP


YM 143
Δtok1 in YM 124
MATa
trk1::hisG-URA3-hisG, tok1::loxP-KanMX-loxP


YM 144
Δtok1 in YM 124
MATa
trk1::hisG-URA3-hisG, tok1::loxP-KanMX-loxP


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 123
MATα
trk1::hisG, tok1::loxP


YM 163
Δtok1 in YM 123
MATα
trk1::hisG, tok1::loxP


YM 164
Δtok1 in YM 124
MATa
trk1::hisG, tok1::loxP


YM 165
Δtok1 in YM 124
MATa
trk1::hisG, tok1::loxP


YM 166
YM 124 × YM 160
MATa
trk1::hisG-URA3-hisG, trk2::loxP-KanMX-loxP


YM 167
YM 124 × YM 160
MATa
trk1::hisG-URA3-hisG, trk2::loxP-KanMX-loxP


YM 168
YM 124 × YM 160
MATα
trk1::hisG-URA3-hisG, trk2::loxP-KanMX-loxP


YM 169
YM 124 × YM 160
MATα
trk1::hisG-URA3-hisG, trk2::loxP-KanMX-loxP


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:















A)










Systematic name
KCNA5



Synonyms
Kv1.5, (HK2, HPCN1)



Family
voltage-gated potassium channel,




shaker-related subfamily (member No. 5),




delayed rectifier



Chromosomal
12p13.32-p13.31



localization



Accession
NID g4504818



Protein
613 aa, 67 kD



Distribution in
heart, pancreatic islets and insulinoma



the tissue



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
19p13.3



localization



Accession
NID g4996893 g4775348



Protein
889 aa



Function
pacemaker



Distribution in
brain, heart



the tissue



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
7q35-q36



localization



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
brain, heart, lung, kidney, placenta,



the tissue
skeletal musculature



References
(Tang, W. et al., 1995)









Methods:



ROMK2 (see appendix “Sequence ROMK2”)



PCR:



Protocol for Powerscript polymerase (PAN Biotech):



Mix for lower reagent (hotstart protocol) (25 μl):



3 μl H2O; 2.5 μl 10× OptiPerform TM III buffer, pH 9.2;



10 μl 1.25 mM dNTPs (=200 uM);



1.5 μl forward primer (20 pmol/μl); 1.5 μl reverse primer



(20 pmol/μl); 1.5 μl 50 mM



MgCl2 (=1.25 mM); 5 μl 5× OptiZyme TM enhancer.



Mix for upper reagent (35 μl):



23 μl H2O; 3.5 μl 10× OptiPerform TM III buffer;



1.5 μl 50 mM MgCl2; 0.5 μl



PowerScript DNA polymerase; 7 μl 5× OptiZyme TM enhancer.



PCR program (hotstart):



1. 1 min at 94° C.



2. 1 min at 94° C.



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):
18.1 μl H2O; 4.2 μl 10× buffer II; 16.7 μl dNTPs; 2.5 μl forward primer; 2.5 μl reverse primer; 6 μl 25 mM MgCl2 (=1.5 mM).
Mix for Lower Reagent (50 μl):
42 μl, H2O; 5 μl 10× buffer II; 1 μl AmpliTaq polymerase; 2 μl template.
DNA Purification
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: 
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 pellel 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):
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.
Add the following in the exact sequence stated:
240 μl PEG (50%), mix suspension by gently pipetting
36 μl M LiAc, mix suspension by gently pipetting
10 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 pipetting
Incubate transformation reaction for 30 minutes at 30° C. in an overhead rotator at slow speed
Transformation 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 PRISMTMred. protokoll/AmpliTaq® FS 1⁄4 BIGDYE TERMINATOR










Reaction:



















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):
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 Columns, Princeton Separations):
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:
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):
Base unit, vacuum platform, porous vacuum slab, filter paper, nylon membrane, vinyl window, agarose gel, final frame, lid
Preheat BioRad vacuum pump for 10 minutes, apply vacuum (5 inches Hg)
Press gel gently along the edge
Place 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 STRATALINKER® UV CROSSLINKER 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):
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:
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):
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 A
Signal 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 (HyperfilmTM 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
All deletions were carried out by standard methods (Fink, G. R. et al., 1991; Wach, A. et al., 1994; Guldener, U. et al., 1996; Goldstein, A. L. et al., 1999).
Fragments of about 500 bp 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-Xhol-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 Δ trk1 acted as DNA template.
Example 2
Construction of the Single, Double and Triple Mutants
Example 2a
Single Knock-Out
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-(YM158-161) 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.
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
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).
Single colonies were streaked out as patches, replica-plated on 5-FOA, and colonies ere 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.
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
Overnight cultures in YPD were set up with single Δtrk1 Δtok1 single colonies (YM162 and YM164), and, next day, transformed with the BsiWI/Spel-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 1






Top row, left to right:
Bottom row, left to right:


















1.
marker
1.
marker


2.
YM 97 with TRK1
2.
YM 182 with TRK1



DiaFo/Re1

DiaFo/Re1


3.
YM 97 with TRK2
3.
YM 182 with TRK2



DiaFo/Re1

DiaFo/Re1


4.
YM 97 with TOK1
4.
YM 182 with TOK1



DiaFo/Re1

DiaFo/Re1


5.
YM 97 with TRK1
5.
YM 182 with TRK1



DiaFo/URARe

DiaFo/URARe


6.
YM 97 with TRK2
6.
YM 182 with TRK2



DiaFo/KANRe

DiaFo/KANRe


7.
YM 97 with TOK1
7.
YM 182 with TOK1



DiaFo/KANRe

DiaFo/KANRe


8.
free
8.
free


9.
YM 97 with TRK1
9.
YM 182 with TRK1



DiaFo/Re2

DiaFo/Re2


10.
YM 97 with TRK2
10.
YM 182 with TRK2



DiaFo/Re2

DiaFo/Re2


11.
YM 97 with TOK1
11.
YM 182 with TOK1



DiaFo/Re2

DiaFo/Re2









Example 3
Subcloning and Transformation of the Human Potassium Channels into the Double and Triple Mutants
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 Nhel/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 Δtrk1 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
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.
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.
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
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.
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
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 he potassium deficiency.
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 he 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
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.
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.
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
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.
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.
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
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. 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
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.
The substances employed as inhibitors of the human HERG1 K+ channel were terfenadine (α-(4-tert-butylphenyl)-4-(α-hydroxy-aphenylbenzyl)-1-piperidinebutanol; 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-piperidinecarboxylate; 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,α-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.
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.
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
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).
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.
These studies demonstrated (FIG. 10) that pimozide inhibits the activity of the essential endogenous potassium channels TRK1 and TRK2 in an unspecific fashion.
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.
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.
The results can be seen from FIG. 10.
Example 8
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.
FIGS. 11 and 12 demonstrate that the human potassium channels HERG1 and Kv1.5 do not complement the growth deficiency of the double mutantΔtrk1Δtrk2 (in each case 4th segment in FIGS. 11 and 12). The comparison with the negative control, i.e. the blank vector üp423GPD in the triple mutant Δtrk1Δtrk2Δtok1 (in each case 1st 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
The human potassium channel ROMK2 ((Shuck, M. E. et al., 1994; Bock, J. H. et al., 1997); Sequence SEQ ID NO.7 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.
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.
The results can be seen from FIG. 13.
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TABLE 1





Nucleotide sequence of TRK1















SEQ ID. NO. 1







ATGCATTTTAGAAGAACGATGAGTAGAGTGCCCACATTGGCATCTCTTGAAATACGATATAAAAAATCTTTCGGCC


 


ATAAATTTCGTGATTTTATTGCTCTATGTGGTCACTATTTTGCTCCAGTTAAAAAATATATCTTCCCCAGTTTTAT


 


CGCGGTTCACTACTTCTACACGATATCCCTGACATTAATAACTTCAATCCTGCTATATCCCATTAAGAATACCAGA


 


TACATTGATACATTGTTTTTAGCAGCGGGCGCAGTTACACAAGGTGGCTTAAATACTGTGGATATCAACAATCTAA


 


GCTTATACCAACAAATTGTTCTGTATATCGTATGCTGCATATCAACACCAATTGCAGTTCATAGTTGCTTGGCATT


 


TGTACGGCTTTACTGGTTTGAGCGCTACTTCGATGGTATTAGAGACTCTTCTAGACGAATTTTAAAGATGAGAAGA


 


ACGAAAACAATCTTAGAAAGGGAACTAACAGCAAGAACCATGACCAAGAATAGAACAGGTACCCAAAGAACGTCTT


 


ATCCTAGGAAACAAGCTAAAACAGATGATTTCCAAGAAAAATTGTTCAGCGGAGAAATGGTTAATAGAGATGAGCA


 


GGACTCAGTTCACAGCGACCAGAATTCTCATGACATTAGTAGGGACAGCAGCAATAATAATACGAATCACAATGGT


 


AGCAGTGGCAGTTTAGATGATTTCGTTAAGGAAGACGAAACGGATGACAATGGAGAATATCAGGAGAACAACTCCT


 


ACTCGACGGTAGGTAGTTCGTCTAACACAGTTGCAGACGAAAGTTTAAATCAGAAGCCCAAGCCAAGCAGTCTTCG


 


GTTTGATGAGCCACACAGCAAACAAAGACCCGCAAGAGTTCCCTCAGAGAAATTTGCAAAAAGAAGGGGTTCAAGA


 


GATATTAGCCCAGCCGATATGTATCGATCCATTATGATGCTACAAGGTAAGCATGAAGCAACTGCTGAAGATGAAG


 


GTCCCCCTTTAGTCATCGGGTCCCCTGCGGATGGCACAAGATATAAAAGTAATGTCAATAAGCTAAAGAAGGCCAC


 


CGGCATAAATGGTAACAAAATCAAGATTCGAGATAAGGGAAATGAAAGTAACACTGATCAAAATTCCGTGTCAAGT


 


GAAGCAAACAGTACGGCGAGCGTTTCGGACGAAAGCTCGTTACACCAAAATTTTGGTAACAAAGTACCTTCATTAA


 


GAACAAATACTCATAGATCAAATTCGGGCCCGATAGCCATTACTGATAACGCAGAAACAGACAAAAAGCATGGGCC


 


ATCAATTCAATTCGATATAACTAAACCTCCTAGAAAAATTTCAAAAAGAGTTTCAACCTTCGATGATTTGAACCCA


 


AAATCTTCCGTTCTTTATCGAAAAAAAGCATCGAAGAAGTACCTCATGAAAACATTTTCCTAAGCGCGGCGAATAC


 


GGCAACAAATTAAGAGAAGGCTTTCTACTGGTTCAATTGAGAAAAACAGCAGTAACAATGTTTCAGATAGAAAACC


 


TATTACTGATATGGATGATGATGATGATGACGATGACAACGACGGCGATAACAACGAAGAATACTTTGCTGACAAC


 


GAAAGCGGCGATGAAGATGAACGAGTACAGCAGTCTGAACCACATTCTGATTCAGAACTCAAATCGCACCAACAAC


 


AGCAAGAAAAACACCAACTGCAGCAGAACCTGCACCGCATGTATAAAACCAAATCATTTGATGATAATCGTTCAAG


 


AGCAGTTCCTATGGAACGTTCCAGGACCATCGATATGGCAGAGGCTAAGGATCTAAATGAGCTCGCAAGGACGCCT


 


GATTTTCAAAAAATGGTCTATCAAAATTGGAAAGCCCATCATAGAAAAAAACCGAACTTTAGGAAGAGGGGATGGA


 


ATAACAAGATATTTGAACATGGTCCCTATGCATCTGACAGCGATCGCAATTATCCTGATAATAGTAATACTGGAAA


 


CAGTATTCTTCATTACGCAGAGTCTATTTTACATCATGATGGCTCTCATAAAAATGGAAGCGAAGAAGCCTCTTCC


 


GACTCTAATGAGAATATCTATTCCACGAATGGAGGAAGCGACCACAATGGTCTTAACAACTATCCTACTTACAACG


 


ACGATGAAGAAGGCTATTATGGTTTACATTTCGATACCGATTATGACCTAGATCCTCGTCATGATTTATCTAAAGG


 


CAGTGGTAAACGTATCTATCATGGCAACCAACTATTGGACGTAAACTCAAACTTCCTTGGATTAACAAGAGCCCAG


 


AAAGATGAATTAGGTGGTGTCGAGTACAGAGCAATCAAACTTTTATGCACCATATTGGTTGTCTACTACGTTGGAT


 


GGCATATTGTTGCTTTTGTTATGTTAGTACCTTGGATTATTTTGAAAAAGCATTATAGTGAAGTTGTTAGAGATGA


 


TGGTGTTTCACCTACATGGTGGGGATTTTGGACAGCAATGAGTGCATTTAATGATTTAGGTTTGACATTAACTCCA


 


AATTCAATGATGTCGTTTAACAAAGCTGTATACCCATTGATCGTTATGATTTGGTTTATCATTATCGGAAATACAG


 


GGTTTCCCATCCTTCTTAGATGCATCATTTGGATAATGTTTAAAATTTCTCCTGATTTATCACAGATGAGAGAAAG


 


TTTAGGTTTTCTCTTAGACCATCCACGTCGTTGTTTCACCTTGCTATTTCCTAAGGCAGCTACATGGTGGCTACTT


 


TTAACGCTTGCAGGATTGAATATAACTGATTGGATTTTATTTATTATTCTAGATTTTGGCTCAACAGTTGTGAAAT


 


CATTATCGAAAGGCTATAGAGTCCTTGTCGGCCTGTTTCAATCTGTTAGCACAAGAACTGCTGGATTCAGCGTTGT


 


CGATTTAAGTCAACTGCATCCTTCTATCCAAGTCTCCTATATGCTAATGATGTATGTCTCCGTATTACCATTGGCC


 


ATCTCTATTCGACGGACAAATGTTTACGAGGAGCAATCTTTAGGACTATATGGAGATATGGGGGGAGAACCAGAAG


 


ATACGGATACTGAAGACGATGGTAACGATGAAGATGACGACGAGGAAAACGAGAGTCACGAAGGTCAAAGTAGTCA


 


AAGAAGTAGTTGAACAACAACAACAATAACAACAGGAAAAAGAAAAAGAAAAAGAAAAACTGAAAATCCAAATGAA


 


ATATCTACAAAATCCTTTATCGGTGCCCATTTAAGGAAACAGCTTTCATTTGACTTGTGGTTTCTATTTTTAGGGT


 


TATTTATCATTTGCATTTGTGAAGGGGACAAGATAAAGGACGTACAAGAACCAAACTTTAATATATTTGCAATTCT


 


TTTTGAAATTGTTAGCGCTTACGGTACAGTTGGGCTATCGCTAGGTTATCCGGACACCAACCAATCGTTTTCAAGA


 


CAGTTTACTACATTATCTAAGTTGGTGATCATAGCTATGCTGATCAGAGGCAAGAATAGAGGTCTACCATACTCAC


 


TGGATCGTGCAATTATCTTGCCTAGTGATAGACTTGAACATATTGACCACCTTGAGGGCATGAAATTGAAGAGACA


 


GGCTAGAACCAATACAGAAGACCCAATGACGGAACATTTCAAGAGAAGTTTCACTGATGTGAAACATCGTTGGGGA


 


GCTCTTAAGCGTAAGACCACACATTCCCGAAATCCTAAAAGGAGCAGCACAACGCTCTAA
















TABLE 2





Nucleotide sequence of TRK2















SEQ ID. NO. 2







ATGCCAACAGCTAAGAGGACGTCATCCAGGGCTTCGTTGGCACTGCCCTTCCAGTTACGGTTGGTGCACAAGAAAT


 


CATGGGGCCATCGGCTAAGAGACTTCATTTCCGGGTTCTTAAAATCATGCAGACCCATTGCTAAATACGTTTTCCC


 


CAACTTCATCGTGGTGCACTATATCTACCTGATCACGCTGTCGATTATCGGGTCCATTCTGTTATATCCGTGCAAG


 


AACACGGCGTTCATCGATGTGCTATTTCTGGCTGCTGGAGCGTCTACACAGGGCGGGCTGGCCACCAAGAGCACTA


 


ACGATTTCAACCTGTACCAGCAGATAGTGGTGTACGTCATTACATTGCTGTCCACGCCTATACTTATTCATGGGTT


 


TTTGGCCTTTGTCAGGCTGTATTGGTTTGAAAGGTACTTCGACAACATTAGGGATATCTCCAACAGAATTTTAAAA


 


CTAAGAAGGACCATGACGTTGCAACAAAGGGAACTATCGGGCAGCAGTGGCAATGCCGCTCGAAGTAGGAGTTTCA


 


AGGACAACCTGTTCCGTGGGAAGTTTGTTTCCAGAGAAGACCCACGACAATCCGCTTCAGATGTGCCGATGGACTC


 


TCCTGACACGTCCGCATTGTCCTCAATCTCACCGTTGAATGTTTCCTCCTCTAAGGAGGAATCCAGTGACACGCAA


 


AGCTCGCCTCCAAACTTCTCAAGTAAGCGCCAACCCTCAGACGTTGACCCAAGAGACATTTACAAATCGATAATGA


 


TGCTACAAAAACAACAAGAGAAGAGCAACGCAAACTCCACGGATTCTTTTTCGAGCGAGACCAATGGACCCGCTTT


 


CATTGTGCAGGAACGTCATGAGAGAAGAGCCCCCCACTGCTCACTGAAACGCCATTCTGTCCTGCCATCTTCTCAG


 


GAATTGAACAAGCTAGCCCAGACGAAAAGTTTCCAGAAATTGCTTGGCTTGCGGAGAGATGAAGGTGACCATGACT


 


ACTTTGACGGTGCTCCTCACAAATATATGGTCACCAAGAAGAAAAAAATATCTAGAACGCAATCATGTAACATCCC


 


AACGTATACTGCTTCACCGAGTCCTAAAACCTCAGGCCAAGTAGTTGAAAATCATAGAAACTTGGCCAAGTCGGCG


 


CCTTCATCTTTTGTTGATGAGGAGATGAGCTTTTCACCGCAAGAGTCTTTGAATTTACAGTTCCAAGCGCACCCGC


 


CCAAACCAAAACGACGTGAAGGTGATATAGGCCACCCCTTCACCAGAACAATGAGCACCAACTATCTATCGTGGCA


 


GCCAACCTTTGGCAGAAACTCCGTCTTCATTGGACTCACAAAGCAACAAAAGGAGGAACTCGGCGGTGTCGAATAT


 


CGTGCTTTGAGATTGCTGTGCTGCATTCTCATGGTATACTACATCGGATTCAACATTTTGGCGTTTGTGACCATCG


 


TTCCATGGGCCTGTACGAGGCACCACTACTCAGAGATTATTAGACGAAATGGAGTTTCTCCAACCTGGTGGGGGTT


 


TTTCACTGCAATGAGTGCATTCAGCAACTTGGGTCTGTCTTTGACCGCTGATTCAATGGTTTCCTTTGATACTGCG


 


CCGTATCCGCTGATTTTCATGATGTTCTTCATCATCATAGGCAATACAGGCTTCCCAATTATGTTACGATTTATCA


 


TTTGGATCATGTTCAAGACCTCGAGAGACCTATCTCAGTTTAAGGAAAGTCTTGGGTTTCTCTTGGATCATCCGCG


 


CAGGTGTTTTACGTTGCTGTTCCCCAGCGGCCCCACATGGTGGCTGTTTACAACTTTAGTCGTCTTAAACGCTACG


 


GATTGGATTCTTTTCATAATTCTGGATTTCAACTCCGCTGTAGTAAGGCAGGTTGCTAAAGGTTATCGAGCTCTCA


 


TGGGCCTCTTCCAGTCTGTATGCACAAGAACTGCTGGATTCAACGTTGTTGACTTAAGTAAATTACACCCGTCCAT


 


TCAGGTGTCTTATATGCTAATGATGTACGTTTCGGTCCTGCCGCTGGCGATTTCCATTAGAAGAACGAATGTTTAT


 


GAGGAGCAATCGTTGGGACTATACGATAGTGGACAAGATGACGAAAATATCACCCACGAAGACGATATAAAGGAAA


 


CAGACCATGATGGCGAATCCGAAGAGCGAGACACTGTATCTACAAAGTCCAAGCCGAAGAAACAGTCCCCAAAATC


 


GTTTGTTGGTGCTCATTTGAGGAGGCAACTCTCTTTTGATTTATGGTACCTATTCCTTGGATTATTTATAATATGC


 


ATATGCGAGGGCAGAAAAATCGAAGACGTTAATAAACCTGATTTCAATGTCTTTGCTATATTGTTTGAAGTTGTTA


 


GCGCTTATGGTACAGTGGGTTTGTCATTGGGTTACCCAAACACCAACACATCACTATCTGCCCAGTTCACCGTATT


 


ATCGAAGCTAGTCATAATTGCCATGCTAATAAGAGGAAGAAATAGAGGTTTACCATACACTTTGGATCGTGCCATC


 


ATGCTGCCAAGTGACAAACTGGAACAAATTGATCGTTTACAAGATATGAAAGCTAAGGGTAAGTTGTTAGCCAAAG


 


TTGGTGAGGATCCAATGACTACTTACGTCAAAAAGAGATCCCACAAACTGAAAAAAATAGCAACAAAGTTTTGGGG


 


GAAGCATTA
















TABLE 3





Nucleotide sequence of TOK1















SEQ ID NO. 3







ATGACAAGGTTCATGAACAGCTTTGCCAAACAAACGCTGGGATATGGCAATATGGCGACAGTGGAGCAAGAGAGCT


 


CAGCTCAGGCTGTTGATTCTCATTCAAACAACACACCGAAGCAAGCTAAGGGTGTTCTTGCAGAGGAACTAAAGGA


 


TGCATTGCGGTTCCGGGACGAAAGAGTTAGTATTATTAATGCAGAGCCTTCTTCAACACTGTTCGTCTTTTGGTTT


 


GTGGTTTCATGCTATTTCCCTGTGATTACTGCCTGCTTGGGTCCCGTAGCTAACACTATCTCGATAGCCTGTGTAG


 


TTGAAAAATGGAGATCCTTAAAGAACAACTCCGTGGTGACAAATCCACGAAGCAATGACACCGATGTTTTGATGAA


 


TCAAGTAAAGACAGTTTTTGATCCTCCTGGTATTTTTGCCGTTAATATCATCTCTTTGGTACTGGGTTTTACGTCA


 


AATATTATACTAATGCTACATTTCAGTAAGAAGTTGACGTATCTTAAATCTCAGTTAATAAATATAACAGGATGGA


 


CAATAGCTGGAGGGATGCTTTTGGTGGACGTGATTGTATGCTCCTTGAATGACATGCCCAGCATCTACAGTAAGAC


 


TATCGGATTTTGGTTTGCCTGTATCAGTTCTGGTCTATATTTGGTATGCACCATTATTTTAACAATACATTTTATT


 


GGATATAAATTAGGAAAATATCCTCCAACGTTCAACCTTTTGCCCAATGAAAGAAGTATCATGGCATACACTGTAC


 


TATTGTCTTTATGGTTGATTTGGGGTGCGGGTATGTTTAGCGGTTTATTGCACATCACTTACGGAAATGCATTATA


 


TTTCTGCACGGTATCATTATTAACCGTGGGACTAGGTGACATCCTGCCCAAGTCGGTTGGCGCCAAAATCATGGTT


 


TTAATCTTTTCGCTATCTGGTGTTGTCTTGATGGGTTTAATAGTGTTTATGACAAGATCCATCATTCAAAAGTCCT


 


CTGGCCCAATTTTCTTTTTCCACAGAGTTGAAAAAGGCAGGTCCAAATCGTGGAAACATTATATGGATAGTAGTAA


 


AAATTTATCTGAAAGGGAAGCGTTCGACTTAATGAAGTGTATCCGACAAACGGCCTCAAGGAAGCAGCATTGGTTT


 


TCTTTGTCGGTGACTATTGCAATTTTCATGGCTTTTTGGTTATTGGGAGCTCTTGTATTCAAATTCGCAGAAAATT


 


GGTCGTACTTCAATTGTATTTACTTTTGTTTCTTGTGCTTATTAACCATTGGATACGGAGACTATGCTCCAAGGAC


 


TGGTGCAGGCCGTGCTTTTTTTGTGATTTGGGCGTTGGGAGCCGTGCCATTAATGGGGGCTATCCTATCTACAGTC


 


GGTGATCTGTTGTTTGACATTTCCACTTCTCTGGATATTAAGATCGGTGAATCATTCAATAATAAAGTCAAGTCCA


 


TCGTTTTTAATGGGCGTCAAAGAGCACTTTCCTTTATGGTGAACACTGGAGAAATTTTCGAAGAATCTGACACAGC


 


TGATGGTGATCTGGAAGAAAATACAACGAGCTCACAATCCAGTCAAATTTCTGAATTCAACGATAATAATTCAGAA


 


GAGAATGATTCTGGAGTGACATCCCCTCCTGCAAGCCTGCAAGAATCATTTTCTTCATTATCAAAAGCATCTAGCC


 


CAGAGGGAATACTTCCTCTAGAATATGTTTCTTCTGCTGAATATGCACTACAGGACTCGGGGACCTGTAATTTAAG


 


GAACTTGCAAGAGCTACTTAAAGCCGTCAAAAAACTACATCGGATATGTCTGGCGGATAAAGATTACACACTTAGT


 


TTTTCCGACTGGTCGTACATTCATAAACTACATTTGAGGAACATTACAGATATTGAGGAGTACACACGCGGACCCG


 


AATTTTGGATATCACCTGATACGCCCCTCAAGTTCCCGTTAAATGAACCTCATTTTGCTTTTATGATGCTTTTCAA


 


GAACATAGAAGAATTAGTTGGTAATCTAGTAGAAGACGAAGAGCTTTATAAAGTTATAAGCAAAAGAAAATTTTTG


 


GGTGAGCATAGAAAGACACTTTGA
















TABLE 4





Nucleotide sequence of HERG1















SEQ ID NO.4







ATGCCGGTGCGGAGGGGCCACGTCGCGCCGCAGAACACCTTCCTGGACACCATCATCCGCAAGTTTGAGGGCCAGA


 


GCCGTAAGTTCATCATCGCCAACGCTCGGGTGGAGAACTGCGCCGTCATCTACTGCAACGACGGCTTCTGCGAGCT


 


GTGCGGCTACTCGCGGGCCGAGGTGATGCAGCGACCCTGCACCTGCGACTTCCTGCACGGGCCGCGCACGCAGCGC


 


CGCGCTGCCGCGCAGATCGCGCAGGCACTGCTGGGCGCCGAGGAGCGCAAAGTGGAAATCGCCTTCTACCGGAAAG


 


ATGGGAGCTGCTTCCTATGTCTGGTGGATGTGGTGCCCGTGAAGAACGAGGATGGGGCTGTCATCATGTTCATCCT


 


CAATTTCGAGGTGGTGATGGAGAAGGACATGGTGGGGTCCCCGGCTCATGACACCAACCACCGGGGCCCCCCCACC


 


AGCTGGCTGGCCCCAGGCCGCGCCAAGACCTTCCGCCTGAAGCTGCCCGCGCTGCTGGCGCTGACGGCCCGGGAGT


 


CGTCGGTGCGGTCGGGCGGCGCGGGCGGCGCGGGCGCCCCGGGGGCCGTGGTGGTGGACGTGGACCTGACGCCCGC


 


GGCACCCAGCAGCGAGTCGCTGGCCCTGGACGAAGTGACAGCCATGGACAACCACGTGGCAGGGCTCGGGCCCGCG


 


GAGGAGCGGCGTGCGCTGGTGGGTCCCGGCTCTCCGCCCCGCAGCGCGCCCGGCCAGCTCCCATCGCCCCGGGCGC


 


ACAGCCTCAACCCCGACGCCTCGGGCTCCAGCTGCAGCCTGGCCCGGACGCGCTCCCGAGAAAGCTGCGCCAGCGT


 


GCGCCGCGCCTCGTCGGCCGACGACATCGAGGCCATGCGCGCCGGGGTGCTGCCCCCGCCACCGCGCCACGCCAGC


 


ACCGGGGCCATGCACCCACTGCGCAGCGGCTTGCTCAACTCCACCTCGGACTCCGACCTCGTGCGCTACCGCACCA


 


TTAGCAAGATTCCCCAAATCACCCTCAACTTTGTGGACCTCAAGGGCGACCCCTTCTTGGCTTCGCCCACCAGTGA


 


CCGTGAGATCATAGCACCTAAGATAAAGGAGCGAACCCACAATGTCACTGAGAAGGTCACCCAGGTCCTGTCCCTG


 


GGCGCCGACGTGCTGCCTGAGTACAAGCTGCAGGCACCGCGCATCCACCGCTGGACCATCCTGCATTACAGCCCCT


 


TCAAGGCCGTGTGGGACTGGCTCATCCTGCTGCTGGTCATCTACACGGCTGTCTTCACACCCTACTCGGCTGCCTT


 


CCTGCTGAAGGAGACGGAAGAAGGCCCGCCTGCTACCGAGTGTGGCTACGCCTGCCAGCCGCTGGCTGTGGTGGAC


 


CTCATCGTGGACATCATGTTCATTGTGGACATCCTCATCAACTTCCGCACCACCTACGTCAATGCCAACGAGGAGG


 


TGGTCAGCCACCCCGGCCGCATCGCCGTCCACTACTTCAAGGGCTGGTTCCTCATCGACATGGTGGCCGCCATCCC


 


CTTCGACCTGCTCATCTTCGGCTCTGGCTCTGAGGAGCTGATCGGGCTGCTGAAGACTGCGCGGCTGCTGCGGCTG


 


GTGCGCGTGGCGCGGAAGCTGGATCGCTACTCAGAGTACGGCGCGGCCGTGCTGTTCTTGCTCATGTGCACCTTTG


 


CGCTCATCGCGCACTGGCTAGCCTGCATCTGGTACGCCATCGGCAACATGGAGCAGCCACACATGGACTCACGCAT


 


CGGCTGGCTGCACAACCTGGGCGACCAGATAGGCAAACCCTACAACAGCAGCGGCCTGGGCGGCCCCTCCATCAAG


 


GACAAGTATGTGACGGCGCTCTACTTCACCTTCAGCAGCCTCACCAGTGTGGGCTTCGGCAACGTCTCTCCCAACA


 


CCAACTCAGAGAAGATCTTCTCCATCTGCGTCATGCTCATTGGCTCCCTCATGTATGCTAGCATCTTCGGCAACGT


 


GTCGGCCATCATCCAGCGGCTGTACTCGGGCACAGCCCGCTACCACACACAGATGCTGCGGGTGCGGGAGTTCATC


 


CGCTTCCACCAGATCCCCAATCCCCTGCGCCAGCGCCTCGAGGAGTACTTCCAGCACGCCTGGTCCTACACCAACG


 


GCATCGACATGAACGCGGTGCTGAAGGGCTTCCCTGAGTGCCTGCAGGCTGACATCTGCCTGCACCTGAACCGCTC


 


ACTGCTGCAGCACTGCAAACCCTTCCGAGGGGCCACCAAGGGCTGCCTTCGGGCCCTGGCCATGAAGTTCAAGACC


 


ACACATGCACCGCCAGGGGACACACTGGTGCATGCTGGGGACCTGCTCACCGCCCTGTACTTCATCTCCCGGGGCT


 


CCATCGAGATCCTGCGGGGCGACGTCGTCGTGGCCATCCTGGGGAAGAATGACATCTTTGGGGAGCCTCTGAACCT


 


GTATGCAAGGCCTGGCAAGTCGAACGGGGATGTGCGGGCCCTCACCTACTGTGACCTACACAAGATCCATCGGGAC


 


GACCTGCTGGAGGTGCTGGACATGTACCCTGAGTTCTCCGACCACTTCTGGTCCAGCCTGGAGATCACCTTCAACC


 


TGCGAGATACCAACATGATCCCGGGCTCCCCCGGCAGTACGGAGTTAGAGGGTGGCTTCAGTCGGCAACGCAAGCG


 


CAAGTTGTCCTTCCGCAGGCGCACGGACAAGGACACGGAGCAGCCAGGGGAGGTGTCGGCCTTGGGGCCGGGCCGG


 


GCGGGGGCAGGGCCGAGTAGCCGGGGCCGGCCGGGGGGGCCGTGGGGGGAGAGCCCGTCCAGTGGCCCCTCCAGCC


 


CTGAGAGCAGTGAGGATGAGGGCCCAGGCCGCAGCTCCAGCCCCCTCCGCCTGGTGCCCTTCTCCAGCCCCAGGCC


 


CCCCGGAGAGCCGCCGGGTGGGGAGCCCCTGATGGAGGACTGCGAGAAGAGCAGCGACACTTGCAACCCCCTGTCA


 


GGCGCCTTCTCAGGAGTGTCCAACATTTTCAGCTTCTGGGGGGACAGTCGGGGCCGCCAGTACCAGGAGCTCCCTC


 


GATGCCCCGCCCCCACCCCCAGCCTCCTCAACATCCCCCTCTCCAGCCCGGGTCGGCGGCCCCGGGGCGACGTGGA


 


GAGCAGGCTGGATGCCCTCCAGCGCCAGCTCAACAGGCTGGAGACCCGGCTGAGTGCAGACATGGCCACTGTCCTG


 


CAGCTGCTACAGAGGCAGATGACGCTGGTCCCGCCCGCCTACAGTGCTGTGACCACCCCGGGGCCTGGCCCCACTT


 


CCACATCCCCGCTGTTGCCCGTCAGCCCCCTCCCCACCCTCACCTTGGACTCGCTTTCTCAGGTTTCCCAGTTCAT


 


GGCGTGTGAGGAGCTGCCCCCGGGGGCCCCAGAGCTTCCCCAAGAAGGCCCCACACGACGCCTCTCCCTACCGGGC


 


CAGCTGGGGGCCCTCACCTCCCAGCCCCTGCACAGACACGGCTCGGACCCGGGCAGTTA
















TABLE 5





Nucleotide sequence of Kv1-5















SEQ ID NO. 5







ATGGAGATCGCCCTGGTGCCCCTGGAGAACGGCGGTGCCATGACCGTCAGAGGAGGCGATGAGGCCCGGGCAGGCT


 


GCGGCCAGGCCACAGGGGGAGAGCTCCAGTGTCCCCCGACGGCTGGGCTCAGCGATGGGCCCAAGGAGCCGGCGCC


 


AAAGGGGCGCGCGCAGAGAGACGCGGACTCGGGAGTGCGGCCCTTGCCTCCGCTGCCGGACCCGGGAGTGCGGCCC


 


TTGCCTCCGCTGCCAGAGGAGCTGCCACGGCCTCGACGGCCGCCTCCCGAGGACGAGGAGGAAGAAGGCGATCCCG


 


GCCTGGGCACGGTGGAGGACCAGGCTCTGGGCACGGCGTCCCTGCACCACCAGCGCGTCCAAATCAACATCTCCGG


 


GCTGCGCTTTGAGACGCAGCTGGGCACCCTGGCGCAGTTCCCCAACACACTCCTGGGGGACCCCGCCAAGCGCCTG


 


CCGTACTTCGACCCCCTGAGGAACGAGTACTTCTTCGACCGCAACCGGCCCAGCTTCGACGGTATCCTCTACTACT


 


ACCAGTCCGGGGGCCGCCTGCGAGGGGTCAACGTCTCCCTGGACGTGTTCGCGGACGAGATACGCTTCTACCAGCT


 


GGGGGACGAGGCCATGGAGCGCTTCCGCGAGGATGAGGGCTTCATTAAAGAAGAGGAGAAGCCCCTGCCCCGCAAC


 


GAGTTCCAGCGCCAGGTGTGGCTTATCTTCGAGTATCCGGAGAGCTCTGGGTCCGCGCGGGCCATCGCCATCGTCT


 


CGGTCTTGGTTATCCTCATCTCCATCATCACCTTCTGCTTGGAGACCCTGCCTGAGTTCAGGGATGAACGTGAGCT


 


GCTCCGCCACCCTCCGGCGCCCCACCAGCCTCCCGCGCCCGCCCCTGGGGCCAACGGCAGCGGGGTCATGGCCCCC


 


GCCTCTGGCCCTACGGTGGCACCGCTCCTGCCCAGGACCCTGGCCGACCCCTTCTTCATCGTGGAGACCACGTGCG


 


TGATCTGGTTCACCTTCGAGCTGCTCGTGCGCTTCTTCGCCTGCCCCAGCAAGGCAGGGTTCTCCCGGAACATCAT


 


GAACATCATCGATGTGGTGGCCATCTTCCCCTACTTCATCACCCTGGGCACCGAACTGGCAGAGCAGCAGCCAGGG


 


GGCGGAGGAGGCGCCCAGAATGGGCAGCAGGCCATGTCCCTGGCCATCCTCCGAGTCATCCGCCTGGTCCGGGTGT


 


TCCGCATCTTCAAGCTCTCCCGCCACTCCAAGGGGCTGCAGATCCTGGGCAAGACCTTGCAGGCCTCCATGAGGGA


 


GCTGGGGCTGCTCATCTTCTTCCTCTTCATCGGGGTCATCCTCTTCTCCAGTGCCGTCTACTTCGCAGAGGCTGAC


 


AACCAGGGAACCCATTTCTCTAGCATCCCTGACGCCTTCTGGTGGGCAGTGGTCACCATGACCACTGTGGGCTACG


 


GGGACATGAGGCCCATCACTGTTGGGGGCAAGATCGTGGGCTCGCTGTGTGCCATCGCCGGGGTCCTCACCATTGC


 


CCTGCCTGTGCCCGTCATCGTCTCCAACTTCAACTACTTCTACCACCGGGAAACGGATCACGAGGAGCCGGCAGTC


 


CTTAAGGAAGAGCAGGGCACTCAGAGCCAGGGGCCGGGGCTGGACAGAGGAGTCCAGCGGAAGGTCAGCGGGAGCA


 


GGGGATCCTTCTGCAAGGCTGGGGGGACCCTGGAGAATGCAGACAGTGCCCGAAGGGGCAGCTGCCCCCTAGAGAA


 


GTGTAACGTCAAGGCCAAGAGCAACGTGGACTTGCGGAGGTCCCTTTATGCCCTCTGCCTGGACACCAGCCGGGAA


 


ACAGATTTGTGA
















TABLE 6





Nucleotide sequence of IRK1















SEQ ID NO. 6







ATGGGCAGTGTGCGAACCAACCGCTATAGCATTGTCTCTTCGGAAGAGGACGGCATGAAGTTGGCCACCATGGCAG


 


TTGCCAATGGCTTTGGGAATGGGAAAAGTAAAGTCCACACTCGGCAACAGTGTAGGAGCCGCTTTGTGAAGAAAGA


 


TGGCCACTGTAATGTTCAGTTCATCAACGTTGGGGAAAAGGGACAACGGTACCTTGCTGACATTTTTACTACGTGT


 


GTGGACATTCGCTGGCGGTGGATGCTGGTTATCTTTTGCCTAGCTTTTGTTCTCTCGTGGCTGTTTTTTGGCTGTG


 


TGTTTTGGCTGATAGCTTTGCTCCATGGAGATCTGGATGCATCTAAGGAGAGCAAAGCCTGTGTGTCTGAGGTCAA


 


CAGCTTCACAGCTGCCTTTCTTTTCTCCATTGAGACCCAGACAACCATCGGCTATGGGTTCCGATGTGTCACGGAT


 


GAATGCCCGATTGCGGTGTTCATGGTTGTGTTCCAGTCAATTGTGGGCTGCATTATTGATGCTTTTATCATTGGTG


 


CCGTCATGGCAAAGATGGCAAAGCCAAAGAAAAGAAATGAGACTCTTGTCTTCAGTCACAATGCTGTGATTGCCAT


 


GAGAGATGGCAAGCTGTGTTTGATGTGGCGAGTAGGCAACCTTCGGAAAAGCCACTTGGTAGAAGCTCATGTTCGA


 


GCCCAGCTCCTCAAATCCAGAATTACTTCTGAAGGGGAATACATCCCCTTGGATCAAATAGACATCAATGTTGGCT


 


TTGACAGTGGAATTGACCGTATATTTCTGGTATCCCCAATCACTATTGTCCATGAAATAGATGAAGATAGTCCTTT


 


ATATGATTTGAGCAAGCAGGACATTGATAATGCAGACTTTGAAATTGTTGTGATACTAGAAGGCATGGTGGAAGCC


 


ACTGCCATGACAACACAGTGTCGTAGTTCTTATTTGGCCAACGAGATCCTTTGGGGCCACCGCTATGAGCCAGTGC


 


TCTTTGAGGAGAAGCACTACTATAAAGTGGACTATTCGAGGTTTCATAAGACTTACGAAGTACCCAACACTCCCCT


 


TTGTAGTGCCAGAGACTTAGCAGAAAAGAAATATATTCTCTCAAATGCTAACTCATTTTGCTATGAAAATGAAGTT


 


GCCCTTACAAGCAAAGAGGAAGATGACAGTGAAAATGGGGTTCCAGAAAGCACCAGTACAGACACACCTCCTGACA


 


TCGACCTTCACAACCAGGCAAGTGTACCTCTAGAGCCCAGACCCTTACGGCGAGAATCGGAGATATGA
















TABLE 7





Nucleotides 4307-5425 of SEQ ID NO. 31: human ROMK2 (Genbank accession number U12542)















AATGTTCAACATCTTCGGAAATGGGTCGTCACTCGCTTTTTTGGGCATTCTCGGCAAAGAGCAAGGCTAGTCTCCA


 


AAGATGGAAGGTGCAACATAGAATTTGGCAATGTGGAGGCACAGTCAAGGTTTATATTCTTTGTGGACATCTGGAC


 


AACGGTACTTGACCTCAAGTGGAGATACAAAATGACCATTTTCATCACAGCCTTCTTGGGGAGTTGGTTTTTCTTT


 


GGTCTCCTGTGGTATGCAGTAGCGTACATTCACAAAGACCTCCCGGAATTCCATCCTTCTGCCAATCACACTCCCT


 


GTGTGGAGAATATTAATGGCTTGACCTCAGCTTTTCTGTTTTCTCTGGAGACTCAAGTGACCATTGGATATGGATT


 


CAGGTGTGTGACAGAACAGTGTGCCACTGCCATTTTTCTGCTTATCTTTCAGTCTATACTTGGAGTTATAATCAAT


 


TCTTTCATGTGTGGGGCCATCTTAGCCAAGATCTCCAGGCCCAAAAAACGTGCCAAGACCATTACGTTCAGCAAGA


 


ACGCAGTGATCAGCAAACGGGGAGGGAAGCTTTGCCTCCTAATCCGAGTGGCTAATCTCAGGAAGAGCCTTCTTAT


 


TGGCAGTCACATTTATGGAAAGCTTCTGAAGACCACAGTCACTCCTGAAGGAGAGACCATTATTTTGGACCAGATC


 


AATATCAACTTTGTAGTTGACGCTGGGAATGAAAATTTATTCTTCATCTCCCCATTGACAATTTACCATGTCATTG


 


ATCACAACAGCCCTTTCTTCCACATGGCAGCGGAGACCCTTCTCCAGCAGGACTTTGAATTAGTGGTGTTTTTAGA


 


TGGCACAGTGGAGTCCACCAGTGCTACCTGCCAAGTCCGGACATCCTATGTCCCAGAGGAGGTGCTTTGGGGCTAC


 


CGTTTTGCTCCCATAGTATCCAAGACAAAGGAAGGGAAATACCGAGTGGATTTCCATAACTTTAGCAAGACAGTGG


 


AAGTGGAGACCCCTCACTGTGCCATGTGCCTTTATAATGAGAAAGATGTTAGAGCCAGGATGAAGAGAGGCTATGA


 


CAACCCCAACTTCATCTTGTCAGAAGTCAATGAAACAGATGACACCAAAATGTAA

















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




Mean
St.



(30 μM)
Exp. 1
Exp. 2
Exp. 3
Exp. 4
growth
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


Serlindole
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 KCI
StD

80 mM KCI
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 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
3181
4178
4864
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
3561
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
3569
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 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 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
0.30362738


Pimo (30 μM)
0.904
0.823
0.305
0.614
0.6615
0.26723086
3.673
3.505
3.46
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 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 13





Nucleotide sequence of p423 GPD-hROMK2


(Accession No. U 12542)















SEQ ID NO. 31







gacgaaagggcctcgtgatacgcctatttttataggttaatgtcatgataataatggtttcttagatgatccaatatcaaagg


 


aaatgatagcattgaaggatgagactaatccaattgaggagtggcagcatatagaacagctaaagggtagtgctgaag


 


gaagcatacgataccccgcatggaatgggataatatcacaggaggtactagactacctttcatcctacataaatagacg


 


catataagtacgcatttaagcataaacacgcactatgccgttcttctcatgtatatatatatacaggcaacacgcagatata


 


ggtgcgacgtgaacagtgagctgtatgtgcgcagctcgcgttgcattttcggaagcgctcgttttcggaaacgctttgaagt


 


tcctattccgaagttcctattctctagaaagtataggaacttcagagcgcttttgaaaaccaaaagcgctctgaagacgca


 


ctttcaaaaaaccaaaaacgcaccggactgtaacgagctactaaaatattgcgaataccgcttccacaaacattgctca


 


aaagtatctctttgctatatatctctgtgctatatccctatataacctacccatccacctttcgctccttgaacttgcatctaaact


 


cgacctctacattttttatgtttatctctagtattactctttagacaaaaaaattgtagtaagaactattcatagagtgaatcgaa


 


aacaatacgaaaatgtaaacatttcctatacgtagtatatagagacaaaatagaagaaaccgttcataattttctgaccaa


 


tgaagaatcatcaacgctatcactttctgttcacaaagtatgcgcaatccacatcggtatagaatataatcggggatgccttt


 


atcttgaaaaaatgcacccgcagcttcgctagtaatcagtaaacgcgggaagtggagtcaggctttttttatggaagaga


 


aaatagacaccaaagtagccttcttctaaccttaacggacctacagtgcaaaaagttatcaagagactgcattatagagc


 


gcacaaaggagaaaaaaagtaatctaagatgctttgttagaaaaatagcgctctcgggatgcatttttgtagaacaaaa


 


aagaagtatagattctttgttggtaaaatagcgctctcgcgttgcatttctgttctgtaaaaatgcagctcagattctttgtttgaa


 


aaattagcgctctcgcgttgcatttttgttttacaaaaatgaagcacagattcttcgttggtaaaatagcgctttcgcgttgcatt


 


tctgttctgtaaaaatgcagctcagattctttgtttgaaaaattagcgctctcgcgttgcatttttgttctacaaaatgaagcaca


 


gatgcttcgttcaggtggcacttttcggggaaatgtgcgcggaacccctatttgtttatttttctaaatacattcaaatatgtatc


 


cgctcatgagacaataaccctgataaatgcttcaataatattgaaaaaggaagagtatgagtattcaacatttccgtgtcg


 


cccttattcccttttttgcggcattttgccttcctgtttttgctcacccagaaacgctggtgaaagtaaaagatgctgaagatca


 


gttgggtgcacgagtgggttacatcgaactggatctcaacagcggtaagatccttgagagttttcgccccgaagaacgttt


 


tccaatgatgagcacttttaaagttctgctatgtggcgcggtattatcccgtattgacgccgggcaagagcaactcggtcgc


 


cgcatacactattctcagaatgacttggttgagtactcaccagtcacagaaaagcatcttacggatggcatgacagtaag


 


agaattatgcagtgctgccataaccatgagtgataacactgcggccaacttacttctgacaacgatcggaggaccgaag


 


gagctaaccgcttttttgcacaacatgggggatcatgtaactcgccttgatcgttgggaaccggagctgaatgaagccata


 


ccaaacgacgagcgtgacaccacgatgcctgtagcaatggcaacaacgttgcgcaaactattaactggcgaactactt


 


actctagcttcccggcaacaattaatagactggatggaggcggataaagttgcaggaccacttctgcgctcggcccttcc


 


ggctggctggtttattgctgataaatctggagccggtgagcgtgggtctcgcggtatcattgcagcactggggccagatgg


 


taagccctcccgtatcgtagttatctacacgacggggagtcaggcaactatggatgaacgaaatagacagatcgctgag


 


ataggtgcctcactgattaagcattggtaactgtcagaccaagtttactcatatatactttagattgatttaaaacttcatttttaa


 


tttaaaaggatctaggtgaagatcctttttgataatctcatgaccaaaatcccttaacgtgagttttcgttccactgagcgtca


 


gaccccgtagaaaagatcaaaggatcttcttgagatcctttttttctgcgcgtaatctgctgcttgcaaacaaaaaaaccac


 


cgctaccagcggtggtttgtttgccggatcaagagctaccaactctttttccgaaggtaactggcttcagcagagcgcaga


 


taccaaatactgtccttctagtgtagccgtagttaggccaccacttcaagaactctgtagcaccgcctacatacctcgctct


 


gctaatcctgttaccagtggctgctgccagtggcgataagtcgtgtcttaccgggttggactcaagacgatagttaccggat


 


aaggcgcagcggtcgggctgaacggggggttcgtgcacacagcccagcttggagcgaacgacctacaccgaactg


 


agatacctacagcgtgagctatgagaaagcgccacgcttcccgaagggagaaaggcggacaggtatccggtaagcg


 


gcagggtcggaacaggagagcgcacgagggagcttccagggggaaacgcctggtatctttatagtcctgtcgggtttc


 


gccacctctgacttgagcgtcgatttttgtgatgctcgtcaggggggcggagcctatggaaaaacgccagcaacgcggc


 


ctttttacggttcctggccttttgctggccttttgctcacatgttctttcctgcgttatcccctgattctgtggataaccgtattaccgc


 


ctttgagtgagctgataccgctcgccgcagccgaacgaccgagcgcagcgagtcagtgagcgaggaagcggaaga


 


gcgcccaatacgcaaaccgcctctccccgcgcgttggccgattcattaatgcagctggcacgacaggtttcccgactgg


 


aaagcgggcagtgagcgcaacgcaattaatgtgagttacctcactcattaggcaccccaggctttacactttatgcttccg


 


gctcctatgttgtgtggaattgtgagcggataacaatttcacacaggaaacagctatgaccatgattacgccaagcgcgc


 


aattaaccctcactaaagggaacaaaagctggagctcagtttatcattatcaatactgccatttcaaagaatacgtaaata


 


attaatagtagtgattttcctaactttatttagtcaaaaaattagccttttaattctgctgtaacccgtacatgcccaaaataggg


 


ggcgggttacacagaatatataacatcgtaggtgtctgggtgaacagtttattcctggcatccactaaatataatggagcc


 


cgctttttaagctggcatccagaaaaaaaaagaatcccagcaccaaaatattgttttcttcaccaaccatcagttcataggt


 


ccattctcttagcgcaactacagagaacaggggcacaaacaggcaaaaaacgggcacaacctcaatggagtgatgc


 


aacctgcctggagtaaatgatgacacaaggcaattgacccacgcatgtatctatctcattttcttacaccttctattaccttctg


 


ctctctctgatttggaaaaagctgaaaaaaaaggttgaaaccagttccctgaaattattcccctacttgactaataagtatat


 


aaagacggtaggtattgattgtaattctgtaaatctatttcttaaacttcttaaattctacttttatagttagtcttttttttagttttaaa


 


acaccagaacttagtttcgacggattctagaactagtggatcccccgggctgcagccatgttcaaacatcttcggaaatg


 


ggtcgtcactcgcttttttgggcattctcggcaaagagcaaggctagtctccaaagatggaaggtgcaacatagaatttgg


 


caatgtggaggcacagtcaaggtttatattctttgtggacatctggacaacggtacttgacctcaagtggagatacaaaat


 


gaccattttcatcacagccttcttggggagttggtttttctttggtctcctgtggtatgcagtagcgtacattcacaaagacctcc


 


cggaattccatccttctgccaatcacactccctgtgtggagaatattaatggcttgacctcagcttttctgttttctctggagact


 


caagtgaccattggatatggattcaggtgtgtgacagaacagtgtgccactgccatttttctgcttatctttcagtctatacttg


 


gagttataatcaattctttcatgtgtggggccatcttagccaagatctccaggcccaaaaaacgtgccaagaccattacgtt


 


cagcaagaacgcagtgatcagcaaacggggagggaagctttgcctcctaatccgagtggctaatctcaggaagagcc


 


ttcttattggcagtcacatttatggaaagcttctgaagaccacagtcactcctgaaggagagaccattattttggaccagatc


 


aatatcaactttgtagttgacgctgggaatgaaaatttattcttcatctccccattgacaatttaccatgtcattgatcacaaca


 


gccctttcttccacatggcagcggagacccttctccagcaggactttgaattagtggtgtttttagatggcacagtggagtcc


 


accagtgctacctgccaagtccggacatcctatgtcccagaggaggtgctttggggctaccgttttgctcccatagtatcca


 


agacaaaggaagggaaataccgagtggatttccataactttagcaagacagtggaagtggagacccctcactgtgcc


 


atgtgcctttataatgagaaagatgttagagccaggatgaagagaggctatgacaaccccaacttcatcttgtcagaagt


 


caatgaaacagatgacaccaaaatgtaacagtcgacctcgagtcatgtaattagttatgtcacgcttacattcacgccctc


 


cccccacatccgctctaaccgaaaaggaaggagttagacaacctgaagtctaggtccctatttatttttttatagttatgttag


 


tattaagaacgttatttatatttcaaatttttcttttttttctgtacagacgcgtgtacgcatgtaacattatactgaaaaccttgcttg


 


agaaggttttgggacgctcgaaggctttaatttgcggccggtacccaattcgccctatagtgagtcgtattacgcgcgctca


 


ctggccgtcgttttacaacgtcgtgactgggaaaaccctggcgttacccaacttaatcgccttgcagcacatccccctttcg


 


ccagctggcgtaatagcgaagaggcccgcaccgatcgcccttcccaacagttgcgcagcctgaatggcgaatggcgc


 


gacgcgccctgtagcggcgcattaagcgcggcgggtgtggtggttacgcgcagcgtgaccgctacacttgccagcgcc


 


ctagcgcccgctcctttcgctttcttcccttcctttctcgccacgttcgccggctttccccgtcaagctctaaatcgggggctcc


 


ctttagggttccgatttagtgctttacggcacctcgaccccaaaaaacttgattagggtgatggttcacgtagtgggccatcg


 


ccctgatagacggtttttcgccctttgacgttggagtccacgttctttaatagtggactcttgttccaaactggaacaacactc


 


aaccctatctcggtctattcttttgatttataagggattttgccgatttcggcctattggttaaaaaatgagctgatttaacaaaa


 


atttaacgcgaattttaacaaaatattaacgtttacaatttcctgatgcggtattttctccttacgcatctgtgcggtatttcacac


 


cgcatagatccgtcgagttcaagagaaaaaaaaagaaaaagcaaaaagaaaaaaggaaagcgcgcctcgttcag


 


aatgacacgtatagaatgatgcattaccttgtcatcttcagtatcatactgttcgtatacatacttactgacattcataggtata


 


catatatacacatgtatatatatcgtatgctgcagctttaaataatcggtgtcactacataagaacacctttggtggagggaa


 


catcgttggtaccattgggcgaggtggcttctcttatggcaaccgcaagagccttgaacgcactctcactacggtgatgat


 


cattcttgcctcgcagacaatcaacgtggagggtaattctgctagcctctgcaaagctttcaagaaaatgcgggatcatct


 


cgcaagagagatctcctactttctccctttgcaaaccaagttcgacaactgcgtacggcctgttcgaaagatctaccaccg


 


ctctggaaagtgcctcatccaaaggcgcaaatcctgatccaaacctttttactccacgcgccagtagggcctctttaaaag


 


cttgaccgagagcaatcccgcagtcttcagtggtgtgatggtcgtctatgtgtaagtcaccaatgcactcaacgattagcg


 


accagccggaatgcttggccagagcatgtatcatatggtccagaaaccctatacctgtgtggacgttaatcacttgcgatt


 


gtgtggcctgttctgctactgcttctgcctctttttctgggaagatcgagtgctctatcgctaggggaccaccctttaaagagat


 


cgcaatctgaatcttggtttcatttgtaatacgctttactagggctttctgctctgtcatctttgccttcgtttatcttgcctgctcatttt


 


ttagtatattcttcgaagaaatcacattactttatataatgtataattcattatgtgataatgccaatcgctaagaaaaaaaaa


 


gagtcatccgctaggggaaaaaaaaaaatgaaaatcattaccgaggcataaaaaaatatagagtgtactagaggag


 


gccaagagtaatagaaaaagaaaattgcgggaaaggactgtgttatgacttccctgactaatgccgtgttcaaacgata


 


cctggcagtgactcctagcgctcaccaagctcttaaaacgggaatttatggtgcactctcagtacaatctgctctgatgccg


 


catagttaagccagccccgacacccgccaacacccgctgacgcgccctgacgggcttgtctgctcccggcatccgctta


 


cagacaagctgtgaccgtctccgggagctgcatgtgtcagaggttttcaccgtcatcaccgaaacgcgcga

## Claims

1. 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 and which is not complemented by an expressed HERG1;
b) treating said mutated cell with a mutated Kv1.5 human potassium channel wherein said human potassium channel is expressed heterologously in this mutated S. cerevisiae cell;
c) incubating the S. cerevisiae cell expressing the human potassium channel together with a substance to be tested; and
d) 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 if the human potassium channel.

2. The process as claimed in claim 1, wherein the genes TRK1, TRK2 and TOK1 are switched off in the mutated S. cerevisiae cell (Δtrk1, Δtrk2, Δtok1).

3. The process as claimed in claim 2, wherein the human potassium channel is present in a yeast expression plasmid.

4. The process as claimed in claim 2, wherein the mutated S. cerevisiae cell expresses constitutively a growth reporter.

5. The process as claimed in claim 4, wherein the substance to be tested, which has an effect on the human potassium channel, inhibits the growth of the mutated S. cerevisiae cell.

6. The process as claimed in claim 4, 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.

7. The process as claimed in claim 6, wherein the cell count is determined via fluorescence or luminescence of the constitutively expressed growth reporter.

8. 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 and which is not complemented by an expressed HERG1;
b) reacting said mutated cell with a mutant Kv1.5 human potassium channel wherein said human potassium channel is expressed heterologously in this mutated S. cerevisiae cell;
c) incubating the S. cerevisiae cell expressing the human potassium channel together with a substance to be tested; and
d) 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.

9. 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 and which is not complemented by an expressed HERG1;
b) reacting said mutated cell with a mutant Kv1.5 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; and
d) 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.