DE19953478A1 - Genetically modified yeast lacking endogenous potassium transport activity, useful for identifying e.g. antiarrhythmic agents, includes a functional human potassium channel - Google Patents

Abstract

Genetically modified Saccharomyces cerevisiae (A) in which (i) the endogenous potassium-translocation systems (TRK1, TRK2 and TOK1) are specifically deleted and (ii) the human erg potassium ion channel (HERG) is stably integrated and expressed, is new. Independent claims are also included for the following: S. cerevisiae mutants with defects in potassium uptake as a result of mutation of the TRK1/2 and TOK1 genes, by introducing one or more selection markers (for auxotrophy or resistance); (1) stably integrating human potassium ion channel genes for expression in the genome of a S. cerevisiae host that lacks functional TRK1/2 and TOK1; and (2) identifying specific modulators of HERG.

Description

The invention relates to a Saccharomyces cerevisiae yeast host strain in which the Nucleic acid sequence for the human erg potassium ion channel (HERG) gene stable in the Yeast genome is integrated and expressed.

Another object of the invention is a Saccharomyces cerevisiae yeast host strain in which all identified potassium translocation systems have been specifically deleted, the method for the stable integration of human heterologous potassium ion channel proteins in this Saccharomyces cerevisiae yeast host strain and a method for the detection of specific modulators of the HERG potassium ion Channel, including:

• a) the treatment of the Saccharomyces cerevisiae yeast host strain by the Nucleic acid sequence for the human erg potassium ion channel (HERG) gene stable in the Yeast genome is integrated and expressed, but not that of the yeast Saccharomyces cerevisiae own TRK1, TRK2 or TOK1 potassium translocation systems expressed are, with test substances,
• b) growth determinations in the presence or after application of a test substance,
• c) measurements of the increase or decrease in the potassium transport of this strain in Presence or after application of a test substance.

field of use

Identification of modulators of human potassium ion channel genes or their Protein products that are involved in genetic diseases. The Yeast host strain is the basis of a pharmacological screening system more natural and / or chemical libraries with high efficiency for industrial use.  

State of the art

Cells of the yeast Saccharomyces cerevisiae express a double-affine potassium uptake system encoded by the genes TRK1 and TRK2, as described in:

• - Rodriguez-Navarro & Ramos (1984). J. Bacteriol. 159: 940-945
• - Ko & Gaber (1991). Mol. Cell. Biol. 11: 4266-4273

and an outbound voltage-gated potassium ion channel for potassium efflux encoded by the TOK1 gene, as described in:

• - Ketchum, K.A., Joiner, W.J., Sellers, A.J., Kaczmarek, L.K. and Goldstein, S.A.N. (1995). Nature 376: 690-695.

This protein is the only voltage-dependent potassium ion channel in the world Plasma membrane from S. cerevisiae.

S. cerevisiae yeast strains that contain mutations in the potassium transport proteins TRK1 and TRK2 show growth defects on media with low potassium ion concentrations. Compared to the S. cerevisiae wild strain, these mutant yeast strains require an addition of 10 mM potassium in the medium. Complementing this potassium uptake defect through the use of expression cloning strategies led to the isolation and cloning of various potassium transport proteins from plants as described in:

• - Anderson, J.A., Huprikar, S.S., Kochian, L.V., Lucas, W.J. and R.F. Gaber (1992). Proc. Natl. Acad. Science USA 89: 3736-3740;
• - Sentenac, H., Bonneaud, N., Minet, M., Lacroute, F., Salmon, J-M., Gaymard, F. and Grignon, C. (1992). Science 256: 663-665
• - Schachtman, D.P. and Schroeder, J.I. (1994). Nature 370: 655-658

as well as an inward rectifying potassium ion channel (gpIRK) from guinea pigs as described in:

• - Tang, W., Ruknudin, A., Yang, W-P., Shaw, S.-Y., Knickerbocker, A. and Kurtz, S. (1995). Mol. Biol. Cell 6: 1231-1240.

However, Saccharomyces cerevisiae yeast strains that contain mutations in the potassium transport proteins TRK1 and TRK2 still have additional effective mechanisms for potassium uptake, mediated by the potassium ion channel TOK1, as recently described in:

• - Fairman, C., Zhou, X. and Kung, C. (1999). J Membr. Biol. 168: 149-57.

Thus, Saccharomyces cerevisiae strains that only contain mutations in the TRK1 and TRK2 genes for potassium transport are not suitable for biotechnological purposes (heterologous expression of potassium ion channel proteins). A large number of the mutants defective in potassium transport have also been generated by simple mutations in haploid laboratory strains. Saccharomyces cerevisiae wild-type strains are diploid organisms with a double set of chromosomes. The modified alleles in haploid strains are therefore not genetically stable. The disadvantages of using such mutant yeast host strains are in particular:

• - no defined genetic background,
• - is the phenotypic selection for expressed heterologous potassium ion channel genes unsure there
• - The expression of heterologous potassium ion channel genes the growth of the mutants Yeast host strains only minimally and under certain conditions, and
• - thus the screening of pharmacologically active substances for heterologous expressed potassium ion channels is difficult.

Ion channels are transmembrane proteins that selectively mediate the flow of certain ions through membranes. Among the ion channels known to date, potassium ion channels represent the most numerous and heterogeneous group. They are responsible for maintaining the resting potential in both excitable and non-excitable cells. The outward currents mediated by potassium ion channels are the basis of both the shape and frequency as well as the repolarization of action potentials in excitable tissue. Potassium ion channels are ubiquitous membrane proteins with an amazing variety of electrical properties. There are potassium ion channels that inactivate quickly (A-type channels) and those that transmit inward currents (K _IR channels). The variety of functions of the potassium ion channels is just as much reflected in the diversity of their structure. "Classic" voltage-dependent potassium ion channels of the so-called Kv family consist of four subunits, with six transmembrane regions and a pore-forming domain being postulated for each subunit, K _IR ion channel subunits of the inwardly rectifying potassium ion channels consist of only two transmembrane and one pore region. Only recently was the S. cerevisiae TOK1 potassium ion channel found, a group of potassium ion channels, for which a particularly different topological structure was suggested by duplicated pore regions, as described in:

• - Ketchum, K.A., Joiner, W.J., Sehers, A.J., Kaczmarek, L.K. and Goldstein, S.A.N. (1995). Nature 376: 690-695.

In recent years, a number of papers have shown an increasing number of human diseases as a result of mutations in genes coding for potassium ion channels. So z. B. a point mutation in Kv1.1 episodic ataxia, as described in:

• - Adelmann, J.P., Bond, C.T., Pessia, M. and Mylie, J. (1995). Neuron 15: 1449-1454

LQT syndrome is a disease that affects ventricular repolarization. A delayed repolarization of ventricular myocytes causes QT prolongation Intervals in the electrocardiogram (EKG). The LQT syndrome is mostly called autosomal dominant or recessive disease inherited. When the ventricular arrhythmias are used Fibrillation degenerate (cardiac fibrillation), sudden death can result.

In this context, the role of the human erg (human eag related gene, HERG) potassium ion channel in the heart is of particular interest. Repolarizing potassium currents, called "delayed rectifiers", are active in ventricular myocytes and are composed of several components. These multiple components are differentiated by the speed of their activation (fast = rapid = I _Kr ; slow = slow = I _Ks ) and their different pharmacology. The fast component I _Kr is blocked by class III anti-arrhythmic agents such as D-Solatol, Dofetilid and Clofilium. The currents mediated by the HERG potassium ion channel correspond to the fast I _Kr component of the "delayed rectifier" measured in isolated heart muscle cells, as described in:

• - Lees-Miller, J.P., Kondo, C., and Wang, L. (1997). Circ. Res. 81: 719-723

HERG-mediated potassium ion currents contribute to the shortening of the action potential with a faster heartbeat rate. Mutations in HERG cause an inherited form of polymorphic ventricular arrhythmia (torsades des pointes), also known as LQT2 syndrome, as described in:

• - Sanguinetti, M.C., Jiang, C., Curran, M.E., and Keating, M.T. (1995) Cell 81: 299-307
• - Curran, M.E., Splaski, L, Timothy, K.W., Vincent, G.M., Green, E.D. and keating, M.T. (1995). Cell 80: 795-803  
• - Keating, M.T. and Sanguinetti, M.C. (1996). Science 272: 681-685

Another syndrome (LQT 1) is caused by a defect in the Kv-LQT-1 gene, as described in:

• - Wang, Q., M.E. Curran, I. Splawski, T.C. Burn, J.M. Millholland, T.J. VanRaay, J. Shen, K.W. Timothy, G.M. Vincent, T. de Jager, P.J. Schwartz, J.A. Toubin, A.J. Moss, D.L. Atkinson, G.M. Landes, T.D. Connors & M.T. Keating (1996). Nat Genet 12: 17-23

The loss of functional outward-rectifying K _ATP channels in the pancreatic β-cells causes persistent hypersecretion of insulin, the hyperglycemia, as described in:

• - Kane, C., Shepherd, R.M., Squires, P.E., Johnson, P.R.V., James, R.F.L., Milla, P.J., Aynsley-Green, A., Lindley, K.J., Dunne, M.J. (1996). Nature Medicine 2: 1344-1347.

In this glucose homeostasis defect, "therapeutic" potassium channel openers (KCOs), synthetic pharmacological molecules, were successfully used for treatment, as described in:

• - Lawson, K. (1996). Clinical Science 91: 651-663
• - Ashcroft, F.M. (1996). Nature Medicine 2: 1301-1302

Currents through voltage-dependent potassium ion channels are modulated and / or blocked by several substances that are either organic salts or animal toxins (protein derivatives), as described in:

• - Mosczydlowski, E., Lucchesi, K. and Ravindran, A. (1988) J. Membrane Biol. 105: 95-111
• - Rudy, B. (1988). Neuroscience 25: 729-749
• - Pongs, O. (1992). Physiological Rev. 72: 69-87
• - Defarias, F.P., Stevens, S.P. and Leonard, R.D. (1995). Recept. and Channels 3: 273-281
• - Gomez-Lagungs, F., Olamendi-Portugal, T., Zamudio, F.Z. and Oossani, L.D. (1996). J. Membrane Biol. 152: 49-56

Potassium ion channel modulators are therefore valuable pharmacological agents with potential therapeutic applications. Highly selective blockers (inhibitors) are only known for a relatively limited number of voltage-dependent potassium ion channels; potassium channel openers (activators) are unknown. Additional substances are for possible therapeutic use in acquired or inherited diseases by mutations in potassium ion channels such. B. necessary in HERG. New substances are usually tested in mammalian cell lines that express foreign potassium ion channel genes. However, this method is complicated by the presence of endogenous channels in the cell lines used, and understandably only of limited suitability for biotechnological purposes (homologous and heterologous expression of potassium ion channels). The disadvantages of using animal cell lines are in particular:

• - the cultivation of animal cell lines is much more complicated, financially complex and prone to contamination.
• - Homolog and / or heterologously expressed potassium ion channels influence this Growth of animal cell lines not
• - The presence of endogenous potassium ion channels complicates the evaluation of experimental data.

An alternative approach to using animal cell lines is to develop Yeast strains, the potassium ion channels from mammals, especially humans, express functionally.

Traditionally, most pharmacologically active substances are Screening of chemical or natural banks discovered. "Screening" - Programs related to the sensitivity with which the active material is identified the number of samples that can be tested as well as different types molecular targets and their recognizable cellular functions optimized.

Based on the application of yeast genetics and molecular biology, various test systems that meet these criteria have been developed over the past decade. So z. B. Yeast test systems with G protein-coupled receptors, as described in:

• - King, K., Dohlmann, H.G., Thorner, J., Caron, M.G., and Lefkowitz, R.J. (1990).
• - Science 250: 121-123

cytoplasmic receptors as described in:

• - Metzger, D., White, J.H., and Chambon, P. (1988). Nature 334: 31-36
• - Schena, M., Yamamoto, K.R. (1988). Science 251: 965-967

a human ion channel (VDAC) as described in:

• - Blachly-Dyson, E., Brygida Zambronicz, E., Hong Yu, W., Adams, V., McCabe, E.R.B., Adelman, J., Colombini, M., and Forte, M. (1993). J. Bio. Chem. 268: 1835-1841

immunosuppressive agents, as described in:

• - Foor, F., Parent, S.A., Morin, N., Dahl, A.M., Ramadan, N., Cherbet, G., Bostian, K.A. and Nielsen J.B. (1992). Nature 360: 682-684

Sterol biosynthesis enzymes as described in:

• - Basson, M.E., Thorsness, M. Finer-Moore, J., Stroud, R.M., and Rine, J. (1988). Mol. Cell. Biol. 8: 3797-3808

Oncogenes, as described in:

• - Broach, J.R. (1991). Trends Genet. 7: 28-33,

and proteins for "multiple drug resistance" as described in:

• - Raymond, M., Gros, P., Whiteway, M., and Thomas, D.Y. (1992). Science 256: 232-234

has been developed. The high homology of essential cellular processes in yeast and cells of higher eukaryotes opens up far-reaching possibilities of using yeast as an expression system for the investigation of heterologously expressed potassium ion channel proteins. These recombinant potassium ion channel proteins can be used for the following studies:

• - discovery of substances effective on mammalian cells (screening tests),
• - Use as biotherapeutics and as
• - Substrates for targeted drug development.

Task / goal

The object and the aim of the present invention was to achieve the above-mentioned. Disadvantages too avoid and a genetically defined (isogenic) Saccharomyces cerevisiae To develop a yeast host strain in which all the yeast genes described for transport and channel proteins are specifically deleted and in which the human potassium ion channel erg (HERG) is stably integrated and expressed in the yeast genome.

In biotechnology, there is a need for well-growing, genetically defined yeast strains for the heterologous expression of potassium ion-transporting potassium ion channel genes involved in human diseases for pharmacological purposes. The biotechnological usability of stable potassium ion channel genes expressing good growing yeast strains consists in the functional and pharmacological analysis of the heterologous potassium ion channel target proteins:
Firstly, yeasts can be used in growth-based test series for screening many different substance libraries on a very small scale and with high efficiency ("screening" method in microtiter dishes) and
secondly, can be used as a recombinant system for the analysis and confirmation of therapeutically active substances on target proteins of human origin and
third, the use as biotherapeutics and as substrates for targeted drug development.

This problem is solved by a genetically modified isogenic Saccharomyces cerevisiae yeast host strain with deletions in the TRK1, TRK2 and TOK1 genes which are responsible for Potassium intake is necessary, obtainable by introducing one or more selective marker (auxotrophy and / or resistance), subsequent crossing for preservation isogenic strains and selection of those strains which are more stable after transformation Integration of the plasmid p679pma1HERG into the yeast genome and cultivation in culture media grow with a limited potassium concentration of 10 mg / l or less.

To obtain the trk1trk2tok1 triple mutant Saccharomyces cerevisiae Yeast host strain can have the genes for both potassium transport proteins (TRK1, TRK2) and the gene for the potassium ion channel (TOK1) by introducing one or more selective Markers (auxotrophy and / or resistance) are deleted and / or disrupted.

The selectable biosynthetic marker genes (auxotrophy needs and / or Resistance) can be introduced into the loci of wild-type Potassium transporter genes are introduced. Suitable selectable markers are e.g. B. the Auxotrophy markers URA3 and LEU2 or genes that show resistance e.g. B. against G418 (Aminoglycoside phosphotransferase gene). Such modified alleles can then can be transformed into Saccharomyces cerevisiae, where they undergo homologous recombination replace the wild type loci. The strains with modified alleles can be selected by the biosynthetic marker or markers are determined.

The selectable ones introduced into the loci of the potassium translocation systems Biosynthetic markers also provide an easy way to transfer them Mutations in genetically different lines (crossing). A strain that has a mutation in contains one of the potassium translocation genes (e.g. TRK1 or TRK2 or TOK1) can with a strain of the opposite mating type that mutates in another Potassium translocation gene (e.g. TRK2 or TRK1 or TOK1) carries, crossed to diploids  become. Subsequent sporulation to haploids (tetrad analysis) can cause isogenic Progeny then selected for the presence of the biosynthetic markers become. The trk1trk2tok1 triple mutant Saccharomyces cerevisiae phenotype can by Growth tests on selective culture media with potassium concentrations of 10 mM or can be determined less.

A yeast strain according to the invention can be determined by a test in which is analyzed whether the expressed gene to be tested trk1trk2tok1 triple mutants Saccharomyces cerevisiae phenotype complemented. To do this, the stem to be tested by growth tests on selective culture media with potassium concentrations of 10 mM or less incubated.

The growth defect of the trk1trk2tok1 triple mutant Saccharomyces cerevisiae Yeast host strain can be used to isolate and enrich potassium ion channel genes Gene libraries (e.g. human cDNA libraries) are used when the genes expressed the mutations in the triple mutant yeast host strain complement and a wild type phenotype with regard to the required potassium concentration results. It can be used to identify potassium ion channels that are physiological described, but have not yet been isolated. Alternatively, different ones Potassium ion channels are introduced into the triple mutant to test whether the Growth defect on potassium limited media is complemented. Heterologous proteins are obtained in that the Saccharomyces cerevisiae Yeast host strain can be transformed with a recombinant DNA which contains the gene of expressing potassium ion channel. The Saccharomyces cerevisiae wild type phenotype can be selected by selecting those strains which are based on transformation, selection and Cultivation can be determined under conditions of 10 mg / l potassium in the culture medium.

Each of these applications results in a yeast strain that is heterologous to a foreign one Potassium ion channel expressed and for screening modulators of the concerned Potassium ion channel can be used. A yeast strain that is heterologous to a stranger Potassium ion channel expressed can be used in simple screening procedures various test substance libraries (chemical and natural substance libraries) be used. These simple procedures in which changes in growth or increased and / or reduced potassium uptake by agar plate tests and / or in Liquid culture can be observed, therefore, specifically effective substances detect that modulate the potassium ion channel function. For screening on  The screening method can activate such a potassium ion channel function Changes such as metabolic activity, increased growth rate or increased Include potassium ion intake. To screen for inhibitors of a Potassium ion channel function, the screening process can make such changes as reduced growth rate or reduced potassium ion intake. The Test substances used in the process for the detection of specific modulators, can e.g. B. be synthetic or natural products. Include natural products vegetable, animal or microbial extracts.

Another object of the invention is a method for the detection of specific modulators of the expressed HERG potassium ion channel, including:

• a) Treatment of the Saccharomyces cerevisiae yeast host strain, which the Nucleic acid sequence for the human erg potassium ion channel (HERG), but not that of Expressed with yeast's own potassium translocation systems TRK1, TRK2 and TOK1 Test substances,
• b) growth determinations in the presence or after application of a test substance,
• c) measurements of the increase or decrease in the potassium transport of those strains in Presence or after application of a test substance.

This procedure is used to identify specific HERG ion channel modulators suitable.  

The following examples further illustrate the invention.

Examples General methods Recombinant DNA technology

Standard methods were used to enrich and manipulate DNA, such as see Sambrook, J. et al., In: Molecular cloning: A laboratory manual. Cold Spring Harbor Laboratory Press, Cold Spring Harbor, New York (1989). The used Molecular biological reagents were used according to the manufacturer's instructions.

Yeast transformation

Saccharomyces cerevisiae strains were made according to the lithium acetate method, as described by Rothstein, R. (1991) in Methods in Enzymology 194: 281-302, transformed.

Yeast genetic methods

Saccharomyces cerevisiae strains were obtained according to the method described in Sherman, F. et al. (Methods in Yeast Genetics: A Laboratory Manual, Cold Spring Harbor Laboratory, Cold Spring Harbor, New York, 1981) described crossed and diploid strains by method Micromanipulation isolated.

example 1 Isolation of the isogenic Saccharomyces cerevisiae yeast host strain with defective Potassium transport 1.1 Introduction of the biosynthetic markers

Principle: To obtain the specific gene disruptions, the technique of flanking homology-PCR ("polymerase chain reaction") was used, in which the coding region (open reading frame) of a gene was determined by the nucleic acid sequence for the aminoglycoside phosphotransferase (lox-kanMX-lox; kan ^R resistance marker), which mediates resistance to the antibiotic geneticin (G418, kanamycin sulfate), as described in Güldener, U., Heck, S., Fiedler, T., Beinhauer, J. and Hegemann, J. (1996 ). Nucleic Acid Research 24: 2519-2524.

A Saccharomyces cerevisiae yeast strain JRY379, described in Reid, JD, Lukas, W., Shafaatian, R., Bertl, A., Scheuermann-Kettner, C., Guy, HR and North, RA (1996). Receptors and Channels 4: 51-61, was transformed in the first step with the plasmid pGAL-HO, described in Rothstein (1991), to obtain the opposite mating type in the resulting strain HLY38 (see also FIG. 3).

By "polymerase chain reaction" (PCR) technique, the structural gene of the aminoglycoside phosphotransferase in the plasmid pUG6 (kan ^R has been described in Güldener, U., Heck, S. Fiedler, T., Hauer leg, J. and Hegemann, J (1996). Nucleic Acid Research 24: 2519-2524) with oligonucleotide primers, each with 20-22 homologous bases to the sequence of the aminoglycoside phosphotransferase gene (kan ^R ) in the plasmid pUG6, and then 40 flanking bases to the 5 'upstream of the START codon and 3' downstream of the STOP codon regions of the genes to be disrupted TRK1, TRK2 and TOK1 amplified. Using the oligonucleotide pairs (see also FIG. 2)

(SEQ. ID. NO. 1) / (SEQ. ID. NO. 2),

TRK1-sense: 5 'GAA GGA GGG TAT TCT ATT GGC TCT CAA GGA AGT CAT TCC TGC TGA AGC TTC GTA CGC TGC 3'
TRK1-antisense 5 'ACG TAG GCA AAT GTA TAT CAC AAT GTT ACT AAT CAA GGT TGC ATA GGC CAC TAG TGG ATC TG 3'

(SEQ. ID. NO. 3) / (SEQ. ID. NO. 4),

TRK2-sense: 5 'CCC CTC GGC CGT GCG GCT GAA AAA GAG AAA TGA TAT TGG AGC TGA AGC TTC GTA CGC TGC 3'
TRK2-antisense: 5 'AAT TAC GTT GGC TCT TAT GTA GGT AAA GAG GGG TAA ACT TGC ATA GGC CAC TAG TGG ATC TG 3''

and (SEQ. ID. NO. 5) / (SEQ. ID. NO. 6)

TOK1-sense: 5 'GGT TCC GGG ACG AAA GAG IITA GTA TTA IFFA ATG CCA GCT GAA GCT TCG TAC GC 3'
TOK1-antisense: 5 'AGC TCT TCG TCT TCT ACT AGA TTA CCA ACT AAG CAT AGG CCA CTA GTG GAT CTG 3'

and the plasmid pUG6 as template DNA, the corresponding disruption constructs trk1 d51 :: lox-kanMX lox, trk2Δ50 :: lox-kanMX-lox and tok1Δ50 :: lox-kanMX-lox were generated. In Fig. 1 is a schematic representation of the genomic fragments containing the Saccharomyces cerevisiae wild-type TRK1, TRK2 and TOKI loci and the corresponding mutant alleles trk1 :: kanMX-Δ51lox, trk2Δ50 :: lox :: kanMX and tok1Δ1 lox shown kanMX (For clarification, the kan ^R gene is not drawn to scale, the size of this gene is 1.0 kb). The regions encoding TRK1, TRK2 and TOK1 are identified by black bars. The mutant alleles were transformed into Saccharomyces cerevisiae strains to replace the corresponding wild-type loci. The oligonucleotides TRK1-sense (SEQ. ID. NO. 1), TRK1-antisense (SEQ. ID. NO. 2), TRK2-sense (SEQ. ID. NO. 3), TRK2-antisense used to construct the mutated alleles (SEQ. ID. NO. 4), TOK1-sense (SEQ. ID. NO. 5) and TOK1-antisense (SEQ. ID. NO. 6) are shown as solid arrows. These three deletion cassettes contain the aminoglycoside phosphotransferase structural gene, flanked on both sides by the 34 base pairs long loxP "direct repeats" and flanked by approx. 40 homologous base pairs to the regions 5 'upstream of the START codon and 3' downstream of the STOP codon the genes TRK1, TRK2 and TOK1 to be disrupted ( FIG. 1).

1.2 Selection of mutants for G418 resistance

The strain JRY379, described in Reid, J.D., Lukas, W., Shafaatian, R., Bertl, A., Scheuermann-Kettner, C., Guy, H.R. and North, R.A. (1996). Receptors and Channels 4: 51- 61, was transformed separately with each of the PCR generated cassettes and transformed yeast colonies on culture media with the antibiotic G418 (200 µg / ml, Kanamycin sulfate) selected as described by Güldener, U., Heck, 5th, Fiedler, T., Beinhauer, J. and Hegemann, J. (1996). Nucleic Acid Research 24: 2519-2524.

The yeast strains HLY39 and HLY40 (see also FIG. 3) have replaced the complete coding regions of the genes TRK1, TRK2 with the lox-kanMX-lox module (aminoglycoside-phosphotransferase gene; kan ^R ) (see also FIGS. 5A, 5B for TRK1 and 6A, 6B for TRK2).

In the yeast strain HLY41 (see also FIG. 3), the TOK1 gene is not completely replaced by the lox-kanMX-lox module (aminoglycoside phosphotransferase gene; kan ^R ): after disruption, 223 nucleotides of the 5 'coding gene remain from the coding region Region and 88 nucleotides of the 3 'coding sequence (see also FIGS. 7A and 7B). Genomic DNA was isolated from G418 resistant colonies by standard methods (Rothstein, 1991).

1.3 Characterization of the positive strains by PCR

The correct integration of the trk1Δ51 :: lox-kanMX-lox, trk2Δ50 :: lox-kanMX-lox and tok1Δ50 :: lox-kanMX-lox deletion cassettes at the respective chromosomal TRK1, TRK2 and TOK1 locus of the yeast strains HLY39, HLY40 and HLY41 was checked with PCR Reactions demonstrated. The genomic DNA isolated from the yeast strains HLY39, HLY40 and HLY41 and the oligonucleotides (see also FIG. 2) were used as templates .

(SEQ. ID. NO. 7),

TRK1-START: 5 'CGT TCG GGG CTG ACA ACG CA3'

(SEQ. ID. NO. 8),

TRK2-START: 5 'CCC GTC CAT TGA GTG CCC GT 3'

and (SEQ. ID. NO. 9)

TOK1-START: 5 'CGC ATT CGC GTC TCG IJTA CC 3'

as 5 'sense start molecules against the 3' antisense start molecule (SEQ. ID. NO. 10),
KAN-antisense: 5 'CCT CAG TGG CAA ATC CTA AC 3'
which is 358 base pairs within the region coding for the aminoglycoside phosphotransferase (kan ^R , lox-kanMX-lox module) in plasmid pUG6.

Result

The yeast strains HLY39, HLY40 and HLY41 contain the trk1Δ51 :: lox-kanMX-lox, trk2Δ50 :: lox-kanMX-lox and tok1Δ50 :: lox-kanMX-lox modules on the correct chromosomal Locus of the corresponding genes TRK1, TRK2 and TOK1.

1.4 Excision of the biosynthetic marker gene for 6418 resistance

The isolated strains HLY39 (trk1Δ51 :: lox-kanMX-lox), HLY40 (trk2Δ50 :: lox-kanMX-lox) and HLY41 (tok1Δ50 :: lox-kanMX-lox) ( FIG. 3) were separated with the plasmid pSH47, which the nucleic acid sequence for the Cre-loxP recombinase under the control of the GAL1 promoter, as described in Güldener, U., Heck, S., Fiedler, T., Beinhauer, J. and Hegemann, J. (1996). Nucleic Acid Research 24: 2519-2524. Transformed yeast colonies were grown on culture media with galactose to induce the Cre-loxP recombinase promoter, and isolated single cell colonies sensitive to 6418 (kanamycin sulfate). In these yeast strains, the lox-kanMX-lox cassette was cut out specifically by the activity of the pSH47 plasmid-encoded Cre-loxP recombinase.

1.5 Isolation of non-plasmid-containing mutants

In order to remove the plasmid pSH47 from these yeast strains, the 6418 sensitive yeast strains were grown on culture media with 5-fluoro-orotinic acid (5FOA) as described in Rothstein, R. (1991). SFOA-resistant, single-cell colonies were isolated and grown, the isolated yeast strains (see also FIG. 3) HLY42 (trk1Δ51), HLY43 (trk2Δ50) and HLY44 (tok1Δd50) contain the unlabeled mutations. Figure 5 shows the deduced amino acid (SEQ. ID. NO. 11; 5A) and nucleic acid (SEQ. ID. NO. 12; 5B) sequences of the Saccharomyces cerevisiae potassium transport protein TRK1. The regions highlighted by the underscore correspond to the deleted sequence. The START and STOP codons are shown in bold in the nucleic acid sequence and the oligonucleotides used are shown in italics. In FIG. 6 is the deduced amino acid (SEQ NO 13 a;... 6A) and the nucleic acid sequence (SEQ ID NO 14;... 6B) of Saccharomyces cerevisiae potassium transport protein TRK2 shown. The regions highlighted by the underscore correspond to the deleted sequence. The START and STOP codons are shown in bold in the nucleic acid sequence and the oligonucleotides used are shown in italics. Figure 7 shows the deduced amino acid (SEQ. ID. NO. 15; 7A) and nucleic acid (SEQ. ID. NO. 16; 7B) sequences of the Saccharomyces cerevisiae potassium ion channel protein TOK1. The regions highlighted by the underscore correspond to the deleted sequence. In the nucleic acid sequence, the START and STOP codons are identified by boldface and the oligonucleotides used and indicated by italics.

1.6 Isolation of the Potassium Transport Defective Triple Mutant

For isolation of the trk1trk2tok1 triple mutant Saccharomyces cerevisiae strain and parallel preservation of auxotrophies which can be complemented by transformation the constructed yeast strains according to the Sherman, F. et al. (Methods in Yeast Genetics: A Laboratory Manual, Cold Spring Harbor Laboratory, Cold Spring Harbor, New York, 1981) described method crossed and diploid strains by micromanipulation isolated. Then the diploid tribes were sporulated and segregants with the desired potassium transport defects isolated.

The HLY38 strain (MATα his3-Δ200 leu2-3, 112 trp1-Δ901 urα3-52 suc2- Δ9) and the HLY42 strain (MATα his3-Δ200 leu2-3, 112 trp1-Δ901 urα3-52 suc2-Δ9 trk1Δ51) crossed, the resulting diploid strain sporulated and tetrads Culture media isolated with 50 mM KCl. Because the TRK1 gene for the highly affine Potassium transporter encoded in S. cerevisiae, the trk1 mutant yeast strain should Need to grow potassium. The crossed mutant trk1Δ51 allele segregated  as expected 2+: 2-, with trk1 mutant yeast strains due to poor growth Culture media with only 100 µM potassium were checked.

A yeast strain (HLY45) with MATα grown from the resulting trk1Δ51 spores Mating type was established with the HLY43 strain (MATα his3-Δ200 leu2-3, 112 trp1-Δ901 urα3-52 suc2-Δ9 trk2Δ50) crossed, the diploid strain sporulates and tetrads on culture media with 50 mM KCl isolated. The diploid yeast strain with one non-mutated allele each Potassium transporters TRK1 and TRK2 grew on all media tested. This growth was used to check recessive mutations. Recessive mutations are for complementation with heterologous genes is necessary. After sporulation and Tetrad analysis to obtain the haploids segregated the defective phenotype of the potassium intake as expected for two unconnected genes. Three phenotypes were related to Potassium needs expected from this crossing according to the four genotypes and observed: wild type without defects in the TRK1, TRK2 alleles, trk1TRK2 mutant with poor growth on 100 µM potassium, trk2TRK1 mutant with the same phenotype and trk1 trk2 mutant with very poor growth on 100 µM potassium. The trk1 trk2 phenotype could be suppressed by growth on culture media with 10 mM potassium.

A trk1 trk2 double mutant HLY46 strain (trk1Δ51 trk2Δ50) with MATα mating type was grown from one of the resulting spores and crossed with the HLY44 strain (MATα his3-Δ200 leu2-3.112 trp1-Δ901 urα3-52 suc2-Δ9 tok1Δ50), the diploid strain sporulated and tetrads isolated on culture media with 50 mM KCl. The diploid yeast strain, each with a non-mutated allele of the potassium transporters TRK1, TRK2 and TOK1, grew on all media tested. This growth was used to check the recessive mutations. Recessive mutations are necessary for complementation with heterologous genes. After sporulation and tetrad analysis to obtain the haploids, the three deletion alleles 2+: 2- segregated. As expected, a combination of trk1Δ51 and tok1Δ50 was observed: these genes are approximately 26 cM apart on chromosome X (46 parental dityp, 1 non-parental dityp and 48 tetratypes). In the trk1trk2tok1 triple mutant Saccharomyces cerevisiae yeast strain HLY47 (trk1Δ51 trk2Δ50 tok1Δ50) all previously described yeast-specific transport and channel proteins TRK1, TRK2 and TOK1 have been specifically deleted (see also Fig. 3). The phenotype of this strain is characterized in that growth occurs only on culture media with at least 50 mM potassium.

Example 2 Construction of the HERG integration plasmid 2.1 Construction of plasmid p679 / HERG

The gene for the human erg potassium ion channel (HERG) was by cleavage with restriction endonucleases Bam HI and XbaI as a 3.9 kilobase pair (kb) fragment the plasmid pcDNAHERG (obtained from Dr. G. Robertson, University of Wisonsin, Madison, USA) and separation by agarose gel electrophoresis.

For the transcription of a human gene in the yeast Saccharomyces cerevisiae the yeast-own promoter of the plasma membrane ATPase PMA1 used. The one in Saccharomyces cerevisiae constitutively active pmal promoter was identified as an Eco RI / Bam HI 0.9 kb fragment plasmid pRS408 (obtained from Dr. A. Goffeau, Universite Catholique de Louvain-1a- Neuve, Belgium) after separation by agarose gel electrophoresis.

In a triple ligation the 0.9 kb pma1 Eco RI / Bam HI and 3.9 kb HERG Bam HI XbaI fragments ligated with the Eco RI / Xbal digested plasmid vector pUC 18. The plasmid pmalHerg obtained was obtained by restriction mapping and sequencing approved. The 4.8 kb pma1Herg composite fragment was cleaved from this plasmid with the restriction endonucleases Eco RI / Sal I and after separation by agarose obtained gel electrophoresis.

In the following plasmid construction, plasmid p679 (obtained from Dr. P. Ljungdahl, Ludwig Institute of Cancer Research, Stockholm, Sweden) was cleaved with the restriction endonucleases Eco RI / Sal I. This linear vector was ligated to the 4.9 kb pma1Herg composite fragment. The desired plasmid was identified by restriction mapping. FIG. 4 shows a schematic representation of the plasmid construct which was used for the expression of the human erg potassium ion channel. Using the vector p679pma1HERG, the human erg gene was stably integrated into the gene locus for the biosynthetic marker LEU2 of the Saccharomyces cerevisiae yeast host strain. The pmal (Saccharomyces cerevisiae plasma membrane ATPase) promoter sequence is shown as a hatched arrow and the nucleic acid sequence for the human erg potassium ion channel (HERG) as a filled box.

Example 3 Construction of the HERG-integrated yeast host strain 3.1 Construction of the HERG-expressing Saccharomyces cerevisiae strain

In the plasmid p679pma1HERG (see 2.1, FIG. 4) for the homologous recombination of the cloned insert at the chromosomal locus of the LEU2 gene in Saccharomyces cerevisiae 5 'and 3' flanking regions of the LEU2 gene are inserted. For the directed integration of the total plasmid including the 4.9 kb pmalHerg composite fragment at the chromosomal locus of the LEU2 gene, a linearization of the plasmid p679pma1HERG at position 2189 was introduced with the restriction endonuclease Not I between the flanking LEU2 regions.

The linear 9.1 Not I fragment obtained was used after separation by agarose gel electrophoresis to transform the trk1trk2tok1 triple mutant Saccharomyces cerevisiae yeast strain in which all of the yeast-specific transport and channel proteins TRK1, TRK2 and TOK1 described so far have been specifically deleted. For this purpose, transformants were selected for the biosynthetic marker HIS3 (HIS3 ⁺ protrophy), which is also contained in the plasmid p679. Colonies from single cells were further selected on culture media with potassium concentrations less than 10 mM.

The transformation of the trk1trk2tok1 triple mutant Saccharomyces cerevisiae Yeast strain with the human potassium ion channel erg gene under control of the pmal Promoter led to the isolation of a Saccharomyces cerevisiae yeast host strain in which all yeast-owned transport and channel proteins TRK1, TRK2 and TOK1 are specifically deleted, and the human erg gene (HERG) at the chromosomal locus of the biosynthetic LEU2 gene stably integrated and under the control of yeast's own pmal- Promoter is expressed. Expression of the human potassium ion channel erg gene complements the potassium transport defective phenotype of trk1trk2tok1 in triplicate Saccharomyces cerevisiae mutant on culture media with potassium concentrations lower 10 mM.

Instead of the yeast-specific potassium transport and channel proteins, this Saccharomyces cerevisiae strain (genotype MATα his3-Δ200 leu2-3, 112 trp1-Δ901 urα3-52 suc2-Δ9 trk1Δ51trk2Δ50 tok1Δ50 leu2 :: pma1HERG HIS3) (see also Fig. 3) only the human HERG potassium ion channel is expressed.

3.2 Characterization of the HERG-expressing Saccharomyces cerevisiae strain through RNA analysis

The expression of the human potassium ion channel erg gene (HERG) introduced by homologous recombination at the Saccharomyces cerevisiae LEU2 locus was confirmed by RNA analysis ( FIG. 9). In FIG. 8 is the nucleic acid (SEQ ID NO 17;... 8A) and deduced amino acid sequence (SEQ NO 18 a;... 8B) shown of the human ERG potassium ion channel gene (HERG). The sequence corresponds to that in the original publication by Trudeau, MC, Warmke, JW, Ganetzky, B. and Robertson, G. (1995). Science 269: 92-95. The oligonucleotides used for RNA analysis

(SEQ. ID. NO. 19)
H1119 5 'CAG CGG CTT GCT CAA CTC CA 3 and

(SEQ. ID. NO. 20)
H 1557 5 'GAT GAG GTC CAC CAC AGC CA 3'

are indicated by italics and underscores.

In Fig. 9, the results are a "reverse transcription" polymerase chain reaction shown. For this purpose, RNA was isolated from various positive clones expressing the human potassium ion channel erg gene (clones No. 5, 6, 7 and 8) after selective growth in the culture medium with 10 mM potassium. The total RNA was separated in order to check the integrity and quantitative ratios in a 1% denturizing agarose gel (left part in FIG. 9). This total RNA was transcribed into complementary DNA using reverse transcriptase and in a subsequent PCR analysis with the help of erg specific oligonucleotides (SEQ. ID. NO. 19) / (SEQ. ID. NO. 20) a fragment of 440 base pairs in length was amplified . To check for possible DNA contamination in the RNA preparation, non-rewritten RNA from clone No. 6 was used. 20 pg of the plasmid p679pma1HERG was used to check the correct amplification product. To control the reaction mixture, a reaction was carried out without a template (water control). The fragments were separated in a 0.8% agarose gel.

Result

The expression of the was confirmed by RNA analysis with the RT-PCR homologous recombination introduced human potassium ion channel erg gene (HERG) in the trk1trk2tok1 triple mutant Saccharomyces cerevisiae yeast strain.  

3.3 Characterization of the HERG-expressing Saccharomyces cerevisiae strain through growth tests on potassium-limited media

Cultures of Saccharomyces cerevisiae wild strain, of the trk1trk2tok1 triple mutant yeast strain of a HERG-expressing strain were grown in the full medium YPD (2% yeast extract, 1% peptone) with 2% D-glucose, pH 5.0 at 30 ° C. overnight with shaking. 1 × 10 ⁵ cells were spread on full-medium agar plates, incubated at 30 ° C. overnight and on selective agar plates (0.67% yeast nitrogen base with amino acids, 0.5% NH ₄ SO ₄ , 2% D-glucose) with 100 or 10 mM KCl plated at pH 5.5 and pH 5.0. These plates were incubated at 30 ° C for 48 hours. In Fig. 10 the growth of the isolated trkltrk2tok1 Saccharomyces cerevisiae triple mutant is shown with broken potassium transport in response to the potassium concentration in the culture medium. The Saccharomyces cerevisiae yeast strain with defects in all potassium translocation systems TRK1, TRK2 and TOK1 (trk1trk2tok1, triple mutant) does not grow with low potassium concentrations (10 mM) in the medium at pH 5.0 and pH 5.5. In contrast to the Saccharomyces cerevisiae triple mutant, the strain expressing the human potassium ion channel erg gene (HERG) is able to grow to 10 mM potassium in the medium at pH 5.0 and pH 5.5, comparable to the Saccharomyces cerevisiae wild strain.

3.4 Characterization of the HERG-expressing Saccharomyces cerevisiae strain by Growth tests in the presence of inhibitors

Cultures of Saccharomyces cerevisiae wild strain, the trk1trk2tok1 triple mutant yeast strain and the HERG-expressing strain were grown in the full medium YPD (2% yeast extract, 1% peptone) with 2% D-glucose, pH 5.0 at 30 ° C. overnight with shaking. 1 × 10 ⁵ cells were streaked on full-medium agar plates, incubated at 30 ° C. overnight and on selective agar plates (0.67% yeast nitrogen base with amino acids, 0.5% NH ₄ SO ₄ , 2% D-glucose, pH 5.5) 10 mM KCl and 1 mM barium, 1 mM cesium, 40 µM lanthanum and 20 nM astemisole plated. These plates were incubated at 30 ° C for 48 hours.

Result

The restoration of the potassium uptake wild-type phenotype depends on the heterologous expression of the HERG potassium ion channel, such as on the basis of the inhibition of the expressed HERG on agar plates containing 10 mM potassium in the presence of 1 mM barium, 1 mM cesium, 40 μM lanthanum and 20 nM astemisole was confirmed ( Fig. 11).

By inhibiting the heterologously expressed HERG potassium ion channel, the trk1trk2tok1 triple mutant Saccharomyces cerevisiae strain with defects in all Potassium translocation systems no longer grow to 10 mM potassium concentration.  

List of publications

Adelmann, JP, Bond, CT, Pessia, M. and Mylie, J. (1995). Episodic ataxia results from voltage-dependent potassium channels with altered fiinctions. Neuron 15: 1449-1454
Anderson, JA, Huprikar, SS, Kochian, LV, Lucas, WJ and RF Gaber (1992). Functional expression of a probable Arabidopsis thaliana potassium channel in Saccharomyces cerevisiae. Proc. Natl. Acad. Science USA 89: 3736-3740
Ashcroft, FM (1996). Sweet news for hyperglycemic babies. Nature Medicine 2: 1301-1302
Basson, ME, Thorsness, M., Finer-Moore, J., Stroud, RM, and Rine, J. (1988). Structural and functional conservation between yeast and human 3-hydroxy-3-methylglutaryl coenzyme A reductases, the rate-limiting enzyme of sterol biosynthesis. Mol. Cell. Biol. 8: 3797-3808
Blachly-Dyson, E., Brygida Zambronicz, E., Hong Yu, W., Adams, V., McCabe, ERB, Adelman, J., Colombini, M. and Forte, M. (1993). Cloning and functional expression in yeast of two human isoforms of the outer mitochondrial membrane channel, the voltage-dependent anion channel. J. Bio. Chem. 268: 1835-1841
Broach, JR (1991). RAS genes in Saccharomyces cerevisiae: signal transduction in search of a pathway. Trends Genet. 7: 28-33
Curran, ME, Splaski, L, Timothy, KW, Vincent, GM, Green, ED and Keating, MT (1995). A molecular basis for cardiac arrythmia: HERG mutations cause long QT syndrome. Cell 80: 795-803
Defarias, FP, Stevens, SP and Leonard, RD (1995). Stable expression of human Kv 1.3 potassium channels resets the resting membrane potential of cultured mammalian cells. Receptors and Channels 3: 273-281
Fairman, C., Zhou, X. and Kung, C. (1999). Potassium uptake tbrough the TOK1 K + channel in the budding yeast. J Membr Biol. 168: 149-57.
Foor, F., Parent, SA, Morin, N., Dahl, AM, Ramadan, N., Cherbet, G., Bostian, KA and Nielsen JB (1992). Calcineurin mediates inhibition by FK506 and cyclosporin of recovery from alpha-factor arrest in yeast. Nature 360: 682-684
Gomez-Lagungs, F., Olamendi-Portugal, T., Zamudio, FZ and Oossani, LD (1996). Two novel toxins from the venom of the scorpion Pandinus imperator show that the N-terminal amino acid sequence is improtant for their affinities towards shaker BK ⁺

channels. J. Membrane Biol. 152: 49-56
Güldener, U., Heck, S., Fiedler, T., Beinhauer, J. and Hegemann, J. (1996). A new efficient gene disruption cassette for repeated use in budding yeast. Nucleic Acid Research 24: 2519-2524
Kane, C., Shepherd, RM, Squires, PE, Johnson, PRV, James, RFL, Milla, PJ, Aynsley-Green, A., Lindley, KJ and Dunne, MJ (1996). Loss of ftinctional K _ATP

channels in pancreatic β-cells causes persistent hyperinsulinemic hypoglycemia of infancy. Nature Medicine 2: 1344-1347
Keating, MT and Sanguinetti, MC (1996). Molecular genetic insights into cardiovascular disease. Science 272: 681-685
Ketchum, KA, Joiner, WJ, Sehers, AJ, Kaczmarek, LK and Goldstein, SAN (1995). A new family of outwardly rectifying potassium channel proteins with two pore domains in tandern. Nature 376: 690-695
King, K. Dohlmann, HG, Thorner, J., Caron, MG and Lefkowitz, RJ (1990). Control of yeast mating signal transduction by a mammalian beta 2-adrenergic receptor and Gs alpha subunit. Science 250: 121-123
Ko, C. and Gaber, RF (1991). TRK1 and TRK2 encode structurally related K ⁺

transporters in Saccharomyces cerevisiae. Mol. Cell. Biol. 11, 4266-4273
Lawson, K. (1996). Is there a therapeutic future for "potassium channel openers"? Clinical Science 91: 651-663
Lees-Miller, JP, Kondo, C., and Wang, L. (1997). Electrophysiological characterization of an alternatively processed ERG K ⁺

channel in mouse and human hearts. Circ. Res. 81: 719-723
Metzger, D., White, JH, Chambon, P. (1988). The human estrogen receptor functions in yeast. Nature 334: 31-36
Mosczydlowski, E., Lucchesi, K. and Ravindran, A. (1988). An emerging pharmacology of peptide toxins targeted against potassium channels. J. Membrane Biol. 105: 95-111
Pongs, O. (1992). Molecular biology of voltage-dependent potassium channels. Physiological Rev. 72: 69-87
Raymond, M., Gros, P., Whiteway, M. and Thomas, DY (1992). Functional complementation of yeast ste6 by a mammalian multidrug resistance mdr gene. Science 256: 232-234
Reid, JD, Lukas, W., Shafaatian, R., Bertl, A., Scheuermann-Kettner, C., Guy, HR and North, RA (1996). The S. cerevisiae outwardly rectifying potassium channel (DUK1) identifies a new family of channels with duplicated pore domains. Receptors and Channels 4: 51-61
Rodriguez-Navarro, A. and Ramos, J. (1984). Dual system for potassium transport in Saccharomyces cerevisiae. J. Bacteriol. 159, 940-945
Rothstein, R. (1991). Targeting, disruption, replacement, and allele rescue: Integrative DNA transformation in yeast. In: Guide to Yeast Genetics and Molecular Biology. C. Guthrie and GR Fink, eds., Pp. 281-302; Methods Enzymol. 194, Academic Press
Rudy, B. (1988). Diversity and ubiquity of K ⁺

channels. Neuroscience 25: 729-749
Sambrook, J., Fritsch, EF, and Maniatis, T. (1989) In: Molecular cloning: A laboratory manual. Cold Spring Harbor Laboratory Press, Cold Spring Harbor, New York.
Sanguinetti, MC, Jiang, C., Curran, ME, and Keating, MT (1995). A mechanistic link between an inherited and an aquired cardiac arrythmia: HERG encodes the I _Kr

potassium channel. Cell 81: 299-307
Schachtman, DP and Schroeder, JI (1994). Structure and transport mechanism of a high affinity potassium uptake transporter from higher plants. Nature 370: 655-658
Schena, M, and Yamamoto, KR (1988). Mammalian glucocorticoid receptor derivatives enhance transcription in yeast. Science 251: 965-967
Sentenac, H., Bonneaud, N., Minet, M., Lacroute, F., Salmon, JM., Gaymard, F. and Grignon, C. (1992). Cloning and expression in yeast of a plant potassium ion transport system. Science 256: 663-665
Sherman, F. et al. (1981). Methods in Yeast Genetics: A Laboratory Manual, Cold Spring Harbor Laboratory, Cold Spring Harbor, New York.
Tang, W., Ruknudin, A., Yang, WP., Shaw, S.-Y., Knickerbocker, A. and Kurtz, S. (1995). Functional expression of a vertebrate inwardly rectifying K ⁺

channel in yeast. Mol. Biol. Cell 6: 1231-1240
Trudeau, MC, Warmke, JW, Ganetzky, B. and Robertson, GA (1995). HERG, a human inward rectifier in the voltage-gated potassium channel family. Science 269: 92-95
Wang, Q., ME Curran, I. Splawski, TC Burn, JM Miliholland, TJ VanRaay, J. Shen, KW Timothy, GM Vincent, T. de Jager, PJ Schwartz, JA Toubin, AJ Moss, DL Atkinson, GM Landes , TD Connors & MT Keating (1996). Positional cloning of a novel potassium channel gene: KVLQT1 mutations cause cardiac arrhythmias. Nat Genet 12: 17-23

Sequence list

Claims (9)

1. A genetically modified Saccharomyces cerevisiae yeast strain, its own Potassium translocation systems TRK1, TRK2 and TOK1 are specifically deleted and in which the nucleic acid sequence for the human erg potassium ion channel (HERG) is stable in the yeast genome is integrated and expressed. 2. Saccharomyces cerevisiae mutants with defects in the potassium uptake available are by mutation of the genes for the potassium translocation systems TRK1, TRK2 and TOK1, by introducing one or more selective markers (auxotrophies and / or Resistances). 3. Saccharomyces cerevisiae trk1trk2tok1 mutants. 4. Use of the Saccharomyces cerevisiae trk1trk2tok1 mutants according to claim 1 or 2 as a host organism for the homologous or heterologous expression of human Potassium ion channels. 5. The process of stable integration of human potassium ion channel genes to heterologous Expression in the genome of a Saccharomyces cerevisiae trk1trk2tok1 Hefewirtsstammes. 6. A method of detecting specific HERG potassium ion channel modulators, including:

• a) The treatment of the Saccharomyces cerevisiae yeast host strain which expresses the nucleic acid sequence for the human erg potassium ion channel (HERG) or a functional derivative or a mutation form of this potassium ion channel but not the yeast-specific TRK1, TRK2 and TOK1 potassium translocation systems with test substances,
• b) growth determinations in the presence or after application of a test substance,
• c) measurements of the increase or decrease in the potassium transport of those strains in the presence or after application of a test substance.

7. A method according to claim 4 for the detection of anti-arrhythmic substances. 8. A method according to claim 4 for the detection of anti-fibrillatory substances. 9. A method according to claim 4 for the detection of anti-inflammatory substances.