RESUMO
The characteristics of the Ca(2+)/H(+) exchange were directly investigated in functionally inverted (inside-out) plasma membrane vesicles isolated from yeast using an aqueous two-phase partitioning method. Results showed that following the generation of an inside-acid pH gradient (fluorescence quenching), addition of Ca(2+) caused movement of H(+) out of the vesicles (fluorescence recovery). The Ca(2+)/H(+) exchange displayed saturation kinetics with respect to extravesicular Ca(2+) and ATP concentrations in the plasma membrane, and showed specificity for Ca(2+). The protonophore FCCP (carbonyl cyanide p-(trifluoromethoxy) phenylhydrazone), abolished the fluorescence quenching and consequently inhibited Ca(2+)/H(+) exchange in plasma membrane vesicles. Vanadate, which is known to inhibit the plasma membrane H(+)-ATPase, significantly decreased the Ca(2+)-dependent transport of H(+) out of vesicles. When the electrical potential across the plasma membrane was dissipated with valinomycin and potassium, the rate of Ca(2+)/H(+) exchange increased compared to that of the control without valinomycin, indicating that the stoichiometric ratio for this exchange is greater than 2H(+):Ca(2+). These data suggest that Ca(2+) is transported out of yeast cells through a Ca(2+)/H(+) exchange system that is driven by the proton-motive force generated by the plasma membrane H(+)-ATPase.