In order to test the hypothesis that slowly activating vacuolar (SV) channels mediate Ca2+‐induced Ca2+ release the voltage‐ and Ca2+‐dependence of these K+ and Ca2+‐ permeable channels were studied in a quantitative manner. The patch‐clamp technique was applied to barley (Hordeum vulgare L.) mesophyll vacuoles in the whole vacuole and vacuolar‐free patch configuration. Under symmetrical ionic conditions the current‐voltage relationship of the open SV channel was characterized by a pronounced inward rectification. The single channel current amplitude was not affected by changes in cytosolic Ca2+ whereas an increase in vacuolar Ca2+ decreased the unitary current in a voltage‐dependent manner. The SV channel open‐probability increased with positive potentials and elevated cytosolic Ca2+, but not with elevated cytosolic Mg2+. An increase of cytosolic Ca2+ shifted the half‐activation potential to more negative voltages, whereas an increase of vacuolar Ca2+ shifted the half‐activation potential to more positive voltages. At physiological vacuolar Ca2+ activities (50 μM to 2 mM) changes in cytosolic Ca2+ (5 μM to 2 mM) revealed an exponential dependence of the SV channel open‐probability on the electrochemical potential gradient for Ca2+ (ΔμCa). At the Ca2+ equilibrium potential (ΔμCa = 0) the open‐probability was as low as 0.4%. Higher open‐probabilities required net Ca2+ motive forces which would drive Ca2+ influx into the vacuole. Under conditions favouring Ca2+ release from the vacuole, however, the open‐probability further decreased. Based on quantitative analysis, it was concluded that the SV channel is not suited for Ca2+‐induced Ca2+ release from the vacuole.

SummaryIn contrast to the vacuolar ion channels which are gated open by an increase of cytosolic Ca 2+ the vacuolar ion currents at resting cytosolic Ca2+are poorly explored. Therefore, this study was performed to investigate the properties of the so-called fast-activating vacuolar (FV) current which dominates the electrical characteristics of the tonoplast at physiological free Ca 2+ concentrations. Patch-clamp measurements were performed on whole barley (Hordeum vulgare) mesophyll vacuoles and on excised tonoplast patches. Single ion channels were identified, which, based on their selectivity, activation kinetics, Ca 2+-and voltage-dependence, carry the whole-vacuole FV current. Reversal potential determinations indicated a K ÷ over CI-permeability ratio of about 30. Both inward and outward whole-vacuole currents as well as the activity of single FV channels were inhibited by an increase of cytosolic Ca 2 +, with a Kd ~ 6 IxM. At physiological vacuolar Ca 2+ activities, the FV channel is an outward-rectifying potassium channel. The FV channel was activated in less than a few milliseconds both by negative and positive potential steps, having a minimal activity that is 40 mV negative of the K + equilibrium potential. It is proposed that transport of K + through this cation channel controls the electrical potential difference across the tonoplast.