CONTROL OF IONIC CURRENTS IN GUARD-CELL VACUOLES BY CYTOSOLIC AND LUMINAL CALCIUM

Activity of vacuolar ion channels can be regulated by the cytosolic fr ee Ca2+ concentration ([Ca2+](cyt)). Using the whole-vacuole mode of p atch-clamp with Vicia faba guard cell vacuoles, three distinct cation currents were apparent that were differentially regulated by [Ca2+](cy t). At 'zero' to 100 nM [Ca2+](cyt), instantaneous currents typical of Fast Vacuolar (FV) channels were activated. A 10 fold KCl gradient di rected out of the vacuole increased FV currents (up to fivefold) at ne gative potentials compared with the currents in symmetrical KCI. At [C a2+](cyt) higher than 100 nM, instantaneous currents became smaller an d voltage-independent (non-rectifying) and were typical of Vacuolar K-selective (VK) channels. These currents were less sensitive to a KCI gradient than were the FV currents, being stimulated less than twofold at negative potentials. Reversal potentials measured in the presence of a KCl gradient indicated a high K+ permeability of both FV and VK c urrents. At [Ca2+](cyt) higher than 600 nM time-dependent currents eli cited by positive potentials were typical of Slow Vacuolar (SV) channe l activation. When the Ca2+ mole fraction in the cytosolic or luminal solution was varied the reversal potential of SV currents (determined by tail current analysis) passed through maximum or minimum values. Th e resultant calculated apparent permeability ratios varied with ionic conditions but indicated high Ca2+ and K+ permeabilities. If a Cl- per meability was assumed then the apparent P-Ca was lower. However, subst itution of Cl- by the larger (impermeant) anion gluconate had no effec t on the reversal potential of SV tail currents in the presence of Ca2 + and a K+ gradient, demonstrating that the assumption of Cl- permeabi lity of the SV channel is invalid. Single-channel SV currents also dec reased with increasing cytosolic Ca2+ mole fraction. These data indica te that the SV channel is highly cation selective, shows characteristi cs typical of a multi-ion pore and derives ion selectivity by Ca2+ bin ding. The SV channel currents could also be Mg2+-activated and were de monstrated to be Mg2+-permeable in the absence of Ca2+. The apparent p ermeability ratio (P-Mg:P-K) also varied under different ionic conditi ons. The results indicate not only that FV, VK and SV channels are all present in a single cell type, but also that each is differentially r egulated by [Ca2+](cyt). The respective roles of these channels in vac uolar ion release are discussed, and possible conditions are presented in which these channels could be activated by disparate signalling pa thways during stomatal closure.