Divalent cation block of inward currents and low-afinity K+ uptake in Saccharomyces cerevisiae

We have used the patch clamp technique to characterize whole-cell currents in spheroplasts isolated from a trk1 Delta trk2 Delta strain of Saccharomyc es cerevisiae which lacks high- and moderate-affinity K+ uptake capacity. I n solutions in which extracellular divalent cation concentrations were 0.1 mM, cells exhibited a large inward current. This current was not the result of increasing leak between the glass pipette and membrane, as there was no effect on the outward current. The inward current comprised both instantan eous and time-dependent components. The magnitude of the inward current inc reased,vith increasing extracellular K+ and negative membrane potential but was insensitive to extracellular anions. Replacing extracellular K+ with R b+, Cs+, or Na+ only slightly modulated the magnitude of the inward current , whereas replacement,vith Li+ reduced the inward current by approximately 50%, and tetraethylammonium (TEA(+)) and choline were relatively impermeant . The inward current was blocked by extracellular Ca2+ and Mg2+ with appare nt K(i)s (at -140 mV) of 363 +/- 78 and 96 +/- 14 mu M, respectively. Furth ermore, decreasing cytosolic K+ increased the magnitude of the inward curre nt independently of the electrochemical driving force for K+ influx, consis tent with regulation of the inward current by cytosolic K+. Uptake of Rb-86 (+) by intact trk1 Delta trk2 Delta cells was inhibited by extracellular Ca 2+ with a K-i within the range observed for the inward current. Furthermore , increasing extracellular Ca2+ from 0.1 to 20 mM significantly inhibited t he growth of these cells. These results are consistent with those of the pa tch clamp experiments in suggesting that low-affinity uptake of alkali cati ons in yeast is mediated by a transport system sensitive to divalent cation s.