REGULATION OF ADENOSINE TRIPHOSPHATE-SENSITIVE POTASSIUM CHANNELS FROM RABBIT VENTRICULAR MYOCYTES BY PROTEIN-KINASE-C AND TYPE 2A PROTEIN PHOSPHATASE

Myocytes from rabbit ventricle were enzymatically dissociated and the effects of protein kinase C (PKC) on the properties of single ATP-sensitive (K-ATP) channels were studied using excised inside-out membrane patches. Application of a purified, constitutively active form of PKC (20 nM) to the intracellular surface of inside-out patches caused a 48% +/- 4% (n = 18) reduction in the open probability of single K-ATP channels. In the presence of the PKC inhibitors peptide PKC(19-31) or chelerythrine chloride, PKC had no effect on K-ATP channel properties. Heat-inactivated PKC had no effect on channel properties. K-ATP channel activity returned spontaneously after removal of PKC. However, application of okadaic acid, at a concentration (5 nM) appropriate for specific inhibition of type 2A protein phosphatase (PP-2A), after removal of PKC, prevented spontaneous recovery of channel activity. Treatment with purified PP-2A during the PKC-mediated inhibition of K-ATP channel activity caused a partial or full restoration of activity. The Hill coefficient for ATP binding was reduced from 2.2 (control) to 1.2 in the presence of PKC. The apparent inhibition constant (K-i) for ATP was unaffected by PKC [K-i(control) = 21 mu M; K-i(PKC) = 20 mu M]. PKC is, therefore, capable of inhibiting cardiac K-ATP channel activity, and the extent to which the channels remain phosphorylated appears to be dependent on membrane-associated PP-2A activity. These enzymes may, therefore, be involved in signal transduction mechanisms which serve to regulate the activity of cardiac K-ATP channels.