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

• Published in: The Plant cell Vol. 7; no. 9; pp. 1473 - 1483
• Main Authors: Allen, G.J. (University of York, York, UK.), Sanders, D
• Format: Journal Article
• Language: English
• Published: United States 01.09.1995
• Subjects: ACTIVIDAD ENZIMATICA; ACTIVITE ENZYMATIQUE; CALCIO; CALCIUM; CATION; CATIONES; CELLULE; CELULAS; chlorides; CHLORURE; CLORUROS; electrophysiology; enzyme activity; ESTERASAS; ESTERASE; ESTOMA; guard cells; inorganic ions; ION; ion transport; IONES; MEMBRANAS CELULARES; MEMBRANE CELLULAIRE; phosphoprotein phosphatase; POTASIO; POTASSIUM; stomata; STOMATE; tonoplast; VACUOLA; VACUOLE; vacuoles; VICIA FABA
• ISSN: 1040-4651; 1532-298X; 1532-298X
• DOI: 10.1105/tpc.7.9.1473

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 P(Ca):P(Cl):P(K) 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 (less than or equal to 0.4 units per mL), calcineurin stimulated SV currents by approximately 60%, whereas at higher concentrations the phosphatase was inhibitory, reaching approximately 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