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

#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.

#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.

#Materials and Strains

#Media

#YPD (complete yeast medium): 1% Bacto yeast extract, 2% Bacto peptone, 2% Bacto agar, 2% glucose.