Slowly activating vacuolar channels can not mediate Ca²⁺‐induced Ca²⁺ release

Abstract

In order to test the hypothesis that slowly activating vacuolar (SV) channels mediate Ca 2+ ‐induced Ca 2+ release the voltage‐ and Ca 2+ ‐dependence of these K + and Ca 2+ ‐ 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 Ca 2+ whereas an increase in vacuolar Ca 2+ decreased the unitary current in a voltage‐dependent manner. The SV channel open‐probability increased with positive potentials and elevated cytosolic Ca 2+ , but not with elevated cytosolic Mg 2+ . An increase of cytosolic Ca 2+ shifted the half‐activation potential to more negative voltages, whereas an increase of vacuolar Ca 2+ shifted the half‐activation potential to more positive voltages. At physiological vacuolar Ca 2+ activities (50 μM to 2 mM) changes in cytosolic Ca 2+ (5 μM to 2 mM) revealed an exponential dependence of the SV channel open‐probability on the electrochemical potential gradient for Ca 2+ (Δμ Ca ). At the Ca 2+ equilibrium potential (Δμ Ca = 0) the open‐probability was as low as 0.4%. Higher open‐probabilities required net Ca 2+ motive forces which would drive Ca 2+ influx into the vacuole. Under conditions favouring Ca 2+ 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 Ca 2+ ‐induced Ca 2+ release from the vacuole.