Calcineurin, a type 2B protein phosphatase, modulates the Ca2+-permeable slow vacuolar ion channel of stomatal guard cells

Abstract

The slowly activating vacuolar (SV) channel of plant vacuoles is gated open by cytosolic free Ca2+ and by cytosol-positive potentials. Using vacuoles isolated from broad bean guard cell protoplasts, SV-mediated currents could be measured in the whole-vacuole configuration of a patch clamp as the time-dependent increase in current at cytosol-positive voltages. Time-dependent deactivation of the SV currents when changing from activating to nonactivating voltages (tail currents) was used to calculate the selectivity of the channel to Ca2+ and Cl- with respect to K+. Changing the equilibrium potential for each permeant ion (Ca2+, Cl-, and K+) at least once for individual vacuoles allowed the relative permeabilities (P) of each of these ions to be calculated in a single experiment. The resulting PCa:PCl:PK ratio was close to 3:0.1:1. In accord with its characterization as a weakly selective Ca2+ channel, the SV-mediated current density decreased with increasing Ca2+ activity in the vacuole lumen. SV currents were potently modulated by the Ca2+-dependent, calmodulin-stimulated protein phosphatase 2B (calcineurin). At low concentrations (≤0.4 units per mL), calcineurin stimulated SV currents by ∼60%, whereas at higher concentrations the phosphatase was inhibitory, reaching ∼90°% inhibition at 3 units per mL. Bovine calmodulin had no direct effect on SV-mediated currents, although calcineurin stimulated by exogenous calmodulin inhibited SV currents at all concentrations tested with half-maximal inhibition for calcineurin at 0.16 units per mL. The inhibitory effect of calcineurin could be blocked by the pyrethroid deltamethrin, indicating inhibition of SV channels by calcineurin via dephosphorylation. A model is discussed in which vacuolar Ca2+ release through SV channels is subject to both positive feedforward and negative feedback control through cytosolic Ca2+ and dephosphorylation, respectively.