Gating behaviors of a voltage-dependent and Ca2+-activated cation channel of yeast vacuolar membrane incorporated into planar lipid bilayer

Abstract

A voltage-dependent and Ca2+-activated cation channel found in the vacuolar membrane of the yeast, Saccharomyces cerevisiae, was incorporated into planar lipid bilayer and its gating characteristics were studied at the macroscopic and single-channel levels. The open-channel probability at steady state, which was estimated by the macroscopic current measurement, gave a maximum value at -10 mV and decreased in a graded fashion as the voltage became more positive or more negative. The steady-state voltage dependence was explained by assuming two independent gates, which had different rate constants and opposite voltage dependence. The fast-responding gate opened when the voltage of the cis side (the side to which the vesicles were added) was made more negative and the slow-responding gate behaved in the opposite direction. Relatively high concentrations of Ca2+, about 1 mM, were required on the cis side for opening the slow gate in a voltage-dependent manner. DIDS increased the open-channel probability of the fast gate when added to the cis side, but was ineffective on the slow gate.