Membrane-delimited phosphorylation enables the activation of the outward-rectifying K channels in motor cell protoplasts of Samanea saman

Outward-rectifying K channels activated by membrane depolarization (K(out) or K(D) channels) control K⁺ efflux from plant cells. To find out to what extent phosphorylation is required for the activity of these channels, the patch-clamp method was applied to protoplasts from the legume Samanea saman in both whole-cell and isolated-patch configurations. In the absence of either Mg²⁺ or ATP in the 'cytosolic' solution, the K(D) channel activity declined completely within 75 min. This decline could be reversed in excised, inside-out patches by restoring MgATP (7 mM) to the cytoplasmic side of the membrane. Mg²⁺ (1 mM) plus 5'-adenylylimidodiphosphate (1mM), a nonhydrolyzable ATP analog, did not substitute for ATP. Mg²⁺ (1 mM) plus adenosine 5'-O-(3-thiotriphosphate) (25 to <100 μM), an irreversibly thiophosphorylating ATP analog, sustained channel activity irreversibly. 1- (5-Isoquinolinesulphonyl)-2-methylpiperazine (100 μM), a broad-range kinase inhibitor, blocked the activity of K(D) channels in the presence of MgATP. These results strongly suggest that the activation of the outward-rectifying K channels by depolarization depends critically on phosphorylation by a kinase tightly associated with the K(D) channel.