The TRK proteins-Trk1p and Trk2p- are the main agents responsible for "active" accumulation of potassium by the yeast Saccharomyces cerevisiae. In previous studies, inward currents measured through those proteins by whole-cell patch-clamping proved very unresponsive to changes of extracellular potassium concentration, although they did increase with extracellular proton concentration-qualitatively as expected for H(+) coupling to K(+) uptake. These puzzling observations have now been explored in greater detail, with the following major findings: a) the large inward TRK currents are not carried by influx of either K(+) or H(+), but rather by an efflux of chloride ions; b) with normal expression levels for Trk1p and Trk2p in potassium-replete cells, the inward TRK currents are contributed approximately half by Trk1p and half by Trk2p; but c) strain background strongly influences the absolute magnitude of these currents, which are nearly twice as large in W303-derived spheroplasts as in S288c-derived cells (same cell-size and identical recording conditions); d) incorporation of mutations that increase cell size (deletion of the Golgi calcium pump, Pmr1p) or that upregulate the TRK2 promoter, can further substantially increase the TRK currents; e) removal of intracellular chloride (e.g., replacement by sulfate or gluconate) reveals small inward currents that are K(+)-dependent and can be enhanced by K(+) starvation; and f) finally, the latter currents display two saturating kinetic components, with preliminary estimates of K(0.5) at 46 micro M [K(+)](out) and 6.8 m M [K(+)](out), and saturating fluxes of approximately 5 m M/min and approximately 10 m M/min (referred to intracellular water). These numbers are compatible with the normal K(+)-transport properties of Trk1p and Trk2p, respectively.

• PMID: 15216418
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Journal Article | Research Support, Non-U.S. Gov't | Research Support, U.S. Gov't, P.H.S.

Authors

Kuroda T, Bihler H, Bashi E, Slayman CL, Rivetta A

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