Patent Application
20100167275
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+ (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
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 Mikroorganlsmen and Zellkulturen GmbH (Mascheroder Weg 1b. D-38124 Braunschweig) in compliance with the provisions of the Budapest Treaty on the International recognition of the deposit of microorganisms for the purposes of patent procedure; deposit number DSM 13197.
A particular embodiment of the invention relates to a mutated S. cerevisiae cell which heterologously expresses a eukaryotic potassium channel, the eukaryotic potassium channel preferably being a human potassium channel, for example a HERG1, Kv1.5 or gpIRK1 or a human Kv 4.3 [Genbank Accession Number AF 187963], TASK (Genbank Accession Number AF 006823] or ROMK2 [Genbank Accession Number U 12542] and where the potassium channel has the natural sequence or can be mutated.
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 to substances.
The invention also relates to a process for identifying activators of a eukaryotic potassium channel, in which
The invention furthermore relates to a process for identifying activators of a eukaryotic potassium channel, in which
The invention also relates to a process for the preparation of a medicament, in which
The invention also relates to a process for the preparation of a medicament, in which
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.
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.
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.
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.).
1: Growth of the triple mutant Δtrk1Δtrk2Δtok1 upon expression of the blank vector p423GPD as negative control. 2: Growth of the triple mutant Δtrk1Δtrk2Δtok1 upon expression of p423GPD-TRK1 as positive control. 3: Growth of the triple mutant Δtrk1Δtrk2Δtok1 upon expression of p423GPD-Kv1.5. 4: Expression of the double mutant Δtrk1Δtrk2 upon expression of p423GPD-Kv1.5. The vectors and constructs used are explained in the patent application (see pages 12 et seq. and 15 et seq.).
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.
YPD (complete yeast medium): 1% Bacto yeast extract, 2% Bacto peptone, 2% Bacto agar, 2% glucose.
SC (synthetic complete) Medium: 0.67% Bacto yeast nitrogen base, amino adds, 2% glucose.
Sporulation medium: 1% potassium acetate, amino acids.
5-FOA medium: 0.67% Bacto yeast nitrogen base, amino acids, Uracil (50 μg/ml), 2% sugar (galactose or glucose), 0.1% 5-FOA
All media are described in: (Fink, G. R. et al., 1991)
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.
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):
TE buffer: Tris/HCl (pH 7.5) 10 mM; EDTA (pH 8.0) 1 mM;
TAE buffer: Tris 40 mM; EDTA 1 mM; acetic acid 0.2 mM;
SSC buffer (20×): NaCl3 M; sodium citate*2H2O 0.3 M;
Gel loading buffer. Bromphenol Blue 0.05% (w/v); sucrose 40% (w/v); EDTA, pH 8.0 0.1 M; SDS 0.5% (w/v);
Hybridization buffer. SSC 5×; SDS 0.1% (w/v); dextran sulfate 5% (w/v); stop reagent 1:20;
Buffer A (sterile): Tris-HCl 100 mM; NaCl, pH 9.5 300 mM;
Depurination solution: HCl 0.25 M;
Denaturation solution: NaCl 1.5 M; NaOH 0.5 M;
Neutralization solution: NaCl 1.5 M; Tris, pH 8.0 0.5 M.
Oligonucleotides (PCR primers):
Bacterial strains: DH5Δ; One Shot™ TOP10 (Invitrogen)
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.
The following yeast strains were generated:
ROMK2 (see appendix “Sequence ROMK2”)
Protocol for Powerscript polymerase (PAN Biotech):
3 μl H2O; 2.5 μl 10× OptiPerform™ III buffer, pH 9.2; 10 μl 1.25 mM dNTPs (=200 μM);
1.5 μl forward primer (20 μmol/μl); 1.5 μl reverse primer (20 pmol/μl); 1.5 μl 50 mM MgCl2 (=1.25 mM); 5 μl 5× OptiZyme™ enhancer.
23 μl H2O; 3.5 μl 10× OptiPerform™ III buffer, 1.5 μl 50 mM MgCl2; 0.5 μl PowerScript DNA polymerase; 7 μl 5× OptiZyme™ enhancer.
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.
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).
42 μl H2O; 5 μl 10× buffer II; 1 μl AmpliTaq polymerase; 2 μl template.
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 pellet or the DNA in 150 μl of TE buffer and dissolve for 10 minutes at 65° C.
DNA cloning techniques: All DNA cloning techniques were carried out following standard protocols.
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 1 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 PRISM™red. protokoll/AmpliTaq®FS ¼ BigDyeTerminator
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×
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.
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
Base unit, vacuum platform, porous vacuum slab, fitter 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 UV stratalinker and start crosslinking (1200000 J→0); membrane may be stored in cling-film or between Whatman filter paper at room temperature or 4° C.
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.
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
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 (Hyperfilm™ MP, Amersham), expose for 0.5 2 hours in a film cassette (BioMax, Kodak), develop and scan; the blot can be stored in cling-film at 4° C.
All deletions were carried out by standard methods (Fink, G. R. et al., 1991; Wall, A. et al., 1994; Guldener, U. et al., 1996; Goldstein, A. L et al., 1999).
Fragments of about 500 by each, each of which represents the region at the beginning and the end of the gene, was amplified by PCR with the primers TRK1-FL-BamHI-Fo, TRK1-FL-Pstl-Re. TRK1-FL-Pstl-Fo and TRK1-FL-XhoI-Re for TRK1 or TRK2-DEL-5-Fo-B. TRK2-DEL-5-Re, TRK2-DEL-3-Fo and TRK2-DEL-3-Re for TRK2 and TOK1-DEL-5-Fo, TOK1-DEL-5-Re, TOK1-DEL-3-Fo and TOK1-DEL-3-Re for TOK1 (see Chapter 2.3). The amplified termini later allow correct integration into the yeast genome. The yeast strain w303 a/α or w303 a/α Δtrk1 acted as DNA template.
The constructed deletion cassettes for TRK1, TRK2 and TOK1 were each transformed into the diploid yeast strain YM 96 (MATa/MATα). Integration of the deletion cassettes to the genome was verified by growing the trk1 mutants (YM123/124) on (−)URA/Glc and the trk2- (YM 158-161) and tok1 mutants (YM154-157) on YPD/geniticin, since the URA3 marker in the TRK1 deletion cassette allows growth on (−)URA medium and the KAN marker in the TRK2 or TRK1 deletion cassette allows growth on geneticin (Fink, G. R. et al., 1991). The positive colonies were transferred to a sporulation plate by replica plating, whereupon MATa/MATα diploid cells sporulate after 18-24 hours without vegetative growth. After they were treated with Zymolase and regrown on YPD, tetrads of some colonies were then divided into 4 individual spores with the aid of a dissecting microscope.
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.
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 were selected which had eliminated the URA3 marker and a hisG repeat from the TRK1 deletion cassette (Fink, G. R. et al., 1991). Accordingly, no colonies which lacked the URA3 gene (in TRK1) for uracil synthesis grew on (−)URA/Glc, while all colonies survived on YPD/gen owing to the resistance gene in the TOK1 deletion cassette. To remove the Kan marker from the genome, the (−)URA3 mutants were transformed with plasmid pSH47, on which the genes for Cre recombinase and uracil synthesis (URA3) are located. Positive transformants grew on (−)URA/Glc and it was then possible to induce Cre recombinase by incubation in (−)URA/Gal liquid medium. In this process, the Kan marker together with one loxP repeat is eliminated, and one loxP remains.
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).
Overnight cultures in YPD were set up with single Δtrk1 Δtok1 single colonies (YM162 and YM164), and, next day, transformed with the Bs/WI/SpeI-digested TRK2 deletion cassette and plated onto YPD/KCl/geneticin. After a yeast DNA preparation, the triple knock-out was verified by diagnostic PCR and Southern blot.
The human genes HERG, HCN2, Kv1.5 and, as positive controls, TRK1 and IRK1 (guinea pig) were excised from the plasmids harboring them (HERG between BamHI in pcDNA; HCN2 between NcoI/XhoI in pTLN; Kv1.5 between NheI/EcoRI in pcDNA3.1(−); IRK1 between BamHI/EcoRI in pSGEM) by cleavage with restriction enzymes, separated by gel electrophoresis and extracted from the gel. The individual human potassium channels were ligated into the yeast vector p423-GPD3 (Mumberg, D. et al., 1995; Ronicke, V. et al., 1997) and transformed into E. coli. Control digestion of the plasmid preparations and sequencing permitted the identification of the clones which had integrated the human gene. The plasmids were subsequently transformed into the Δtrk2 Δtrk2 double knock-out (YM 168) and into the Δtrk1 Δtrk2 Δtok1 triple knock-out (YM 182) and plated onto (−)HIS/80 mM KCl.
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).
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.
Each of the strains YM168 (Δtrk1 Δtrk2) and YM182 (Δtrk1 Δtrk2 Δtok1) was transformed with the human potassium channels Kv1.5 ((Fedida, D. et al, 1998); YM190 and YM195) and HERG1 ((Fedida, D. et al., 1998); YM191 and YM196) in p423-GPD3, respectively, as yeast expression vector. gpIRK1 ((Tang, W. et al., 1995); YM193 and YM198) acted as positive control in p423-GPD3 as yeast expression vector (Mumberg, D. et al., 1995; Ronicke, V. et al., 1997). The blank vector p423-GPD3 (YM189 and YM194) acted as negative control. The transformed yeast strains were plated onto (−)HIS/80 mM KCl medium. After this, patches of single colonies were replica-plated onto DPM/5 mM KCl (pH 6.5) to check the capacity of complementing the potassium deficiency.
These experiments demonstrated that the positive control gpIRK1 (YM193 and YM198) in p423-GPD3 fully complemented growth deficiency of double and triple knock-outs. The blank vector p423-GPD3 (YM189 and YM194) as negative control is not capable of complementing the growth deficiency. While the human potassium channel Kv1.5 complements the growth deficiency of triple knock-out, it does so significantly less effectively than the positive control gpIRK1. It was also observed that the human potassium channel Kv 1.5 does not complement the double knock-out Δtrk1 Δtrk2. Under the given experimental conditions, the HERG1 channel does not complement the growth deficiency of double and triple knock-outs.
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 (
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 (
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.
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.
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-αphenylbenzyl)-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-piperidinecarboxytate; HMR) and sertindole (1-(2-[4-[5-chloro-1-(4-fluorophenyl)-1H-indol-3-yl]-1-piperidinyl]ethyl)-2-imidazolidinone; HMR) (Richelson, E. 1996; Richelson, E. 1999; Delpon, E. et al., 1999; Kobayashi, T. et al., 2000; Drici, M. D. et al., 2000). Diphenyhydramine (Sigma, Cat. No. D3630) and fexofenadine (4-[hydroxy-4-[4-(hydroxydiphenylmethyl)-1-piperidinyl]butyl]-α,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
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 (
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 (
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
The human potassium channels HERG1 and Kv1.5 do not complement the growth deficiency of the double mutant Δtrk1Δtrk2 (
The human potassium channel ROMK2 ((Shuck, M. E. et al., 1994; Bock, J. H. et al., 1997); Sequence SEQ ID NO. 31 hROMK2) was subcloned into the yeast vector p423GPD and transformed into the triple mutant Δtrk1Δtrk2Δtok1. The studies demonstrated that this human potassium channel too is capable of complementing the growth deficiency of the triple mutant Δtrk1Δtrk2Δtok1.
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
| Number | Date | Country | Kind |
|---|---|---|---|
| 100 00 651 | Jan 2000 | DE | national |
| Number | Date | Country | |
|---|---|---|---|
| Parent | 09758036 | Jan 2001 | US |
| Child | 12121900 | US |
