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 t o what extent phosphorylation is required for the activity of these ch annels, the patch-clamp method was applied to protoplasts from the leg ume 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 15 min. This decli ne could be reversed in excised, inside-out patches by restoring MgATP (1 mM) to the cytoplasmic side of the membrane. Mg2+ (1 mM) plus 5'-a denylylimidodiphosphate (1 mM), a nonhydrolyzable ATP analog, did not substitute for ATP. Mg2+ (1 mM) plus adenosine 5'-O-(3-thiotriphosphat e) (25 to <100 mu M), an irreversibly thiophosphorylating ATP analog, sustained channel activity irreversibly. 1-(5-isoquinolinesulphonyl)-2 -methylpiperazine (100 mu M), a broad-range kinase inhibitor, blocked the activity of K, channels in the presence of MgATP. These results st rongly suggest that the activation of the outward-rectifying K channel s by depolarization depends critically on phosphorylation by a kinase tightly associated with the K-D channel.