Measurements of plasma membrane potential changes inSaccharomyces cerevisiaecells reveal the importance of the Tok1 channel in membrane potential maintenance

Abstract

K+ is one of the cations (besides protons) whose transport across the plasma membrane is believed to contribute to the maintenance of membrane potential. To ensure K+ transport, Saccharomyces cerevisiae cells possess several types of active and passive transporters mediating the K+ influx and efflux, respectively. A diS-C3(3) assay was used to compare the contributions of various potassium transporters to the membrane potential changes of S. cerevisiae cells in the exponential growth phase. Altogether, the contributions of six K+ transporters to the maintenance of a stable membrane potential were tested. As confirmed by the observed hyperpolarization of trk1 trk2 deletion strains, the diS-C3(3) assay is a suitable method for comparative studies of the membrane potential of yeast strains differing in the presence/absence of one or more cation transporters. We have shown that the presence of the Tok1 channel strongly influences membrane potential: deletion of the TOK1 gene results in significant plasma membrane depolarization, whereas strains overexpressing the TOK1 gene are hyperpolarized. We have also proved that plasma membrane potential is not the only parameter determining the hygromycin B sensitivity of yeast cells, and that the role of intracellular transporters in protecting against its toxic effects must also be considered.

New applications of pHluorin—measuring intracellular pH of prototrophic yeasts and determining changes in the buffering capacity of strains with affected potassium homeostasis

“…Despite their simplicity, unicellular yeast cells are similar to higher eukaryotes in their cell structure and physiological processes. S. cerevisiae has been used as a model organism for studying the role of alkali-metal-cation transporters in many physiological processes, including cation detoxification (Fukuda et al, 2004), cell cycle regulation (Simon et al, 2003), maintaining plasma membrane electrochemical potential (Maresova et al, 2006), etc. Also, adaptation to alterations in extracellular and intracellular pH has been widely studied in yeast (e.g.…”

Alkali Metal Cation Transport and Homeostasis in Yeasts

“…What we know is that Tok1 is activated by plasma membrane depolarization (26) and that K ϩ accumulated in the cells is probably released in order to regenerate the membrane potential (24). In fact, the relationship between membrane potential regulation and Tok1 has been more recently confirmed by the observation that tok1 mutants are depolarized and cells overexpressing TOK1 are hyperpolarized (154).…”

“…Overexpression of NHA1 results in a rescue of the G 1 /S cell cycle blockage in cells with conditional sit4 hal3 mutations, implying that Nha1 plays a role in some aspect of cell cycle regulation (262). Nha1 participates, together with other K ϩ transporters (Tok1, Trk1, Trk2, and Ena1), in the maintenance of stable plasma membrane potential (122,154), and it is involved in the rapidrescue mechanism during the immediate cell response to osmotic or saline shock (119, 120) (see below). Nha1 also acts as a safety valve upon sudden alkalinization of the cytosol, when the outward gradient of potassium cations can serve as a driving force for proton influx (17,120,198).…”

“…The coordination of potassium uptake and efflux systems is necessary for the maintenance of the potential across the plasma membrane. Whereas the absence of potassium uptake systems leads to plasma membrane hyperpolarization (148), the deletion of efflux systems results in depolarization (122,154).…”

Activation of an Essential Calcium Signaling Pathway in Saccharomyces cerevisiae by Kch1 and Kch2, Putative Low-Affinity Potassium Transporters

“…cerevisiae cells grow best in acidic media, and they employ an electrogenic H ϩ -ATPase for pH homeostasis and maintenance of a large resting voltage, estimated to be close to Ϫ200 mV (19)(20)(21). K ϩ also contributes to the resting membrane voltage (22,23), and K ϩ uptake by proliferating S. cerevisiae cells largely depends on the related K ϩ transporters Trk1 and Trk2 (24). In addition, wholecell patch recordings from protoplasts of trk1 trk2 mutant cells have demonstrated an ensemble of additional K ϩ -permeable "channels" that can be blocked by divalent cations (25,26).…”