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

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 mu M to 2 mM) changes in cytosolic Ca2+ (5 mu M to 2 mM) revealed an exponential dependence of the SV channel open probability on the electrochemical potential gradient for Ca2+ (Delta mu(Ca)). At the Ca2+ equilibrium potential (Delta mu(Ca) = 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.