Protein phosphorylation activates the guard cell Ca 2+ channel and is a prerequisite for gating by abscisic acid

Summary Protein phosphorylation and cytosolic‐free [Ca 2+ ] ([Ca 2+ ] i ) contribute to signalling cascades evoked by the water‐stress hormone abscisic acid (ABA) that lead to stomatal closure in higher‐plant leaves. ABA activates an inward‐rectifying Ca 2+ channel at the plasma membrane of stomatal guard cells, promoting Ca 2+ entry by shifting the voltage‐sensitivity of the channels. Because many of these effects could be mediated by kinase/phosphatase action at the membrane, we examined a role for protein (de‐)phosphorylation in plasma membrane patches from Vicia guard cells. Ca 2+ channel activity decayed rapidly in excised patches, and recovered on adding ATP (K 1/2 , 1.3 ± 0.7 m m ) but not the non‐hydrolyzable analog ATPγS. ABA activation of the channel required the presence of ATP and like ABA, the 1/2 A‐type protein phosphatase antagonists okadaic acid (OA) and calyculin A (CA) enhanced Ca 2+ channel activity by increasing the open probability and number of active channels. Neither ATP nor the antagonists affected the mean open lifetime of the channel, suggesting an action through changes in closed lifetime distributions. Like ABA, OA and CA shifted the voltage‐sensitivities of the Ca 2+ current and [Ca 2+ ] i increases in intact guard cells towards positive voltages. OA and CA also augmented the [Ca 2+ ] i rise evoked by hyperpolarization and delayed its recovery. These results demonstrate a membrane‐delimited interaction between 1/2 A‐type protein phosphatase(s) and the Ca 2+ channel or associated proteins, and they are consistent with a role for protein (de‐)phosphorylation in ABA signalling mediated directly through Ca 2+ channel gating that leads to [Ca 2+ ] i increases in the guard cells.