EFFECTS OF PROTEIN PHOSPHATASE AND KINASE INHIBITORS ON THE CARDIAC L-TYPE CA CURRENT SUGGEST 2 SITES ARE PHOSPHORYLATED BY PROTEIN-KINASE-A AND ANOTHER PROTEIN-KINASE

We previously showed (Frace, A. M. and H. C. Hartzell. 1993. Journal o f Physiology. 472:305-326) that internal perfusion of frog atrial myoc ytes with the nonselective protein phosphatase inhibitors microcystin or okadaic acid produced an increase in the L-type Ca current (P-Ca) a nd a decrease in the delayed rectifier K current (I-K). We hypothesize d that microcystin revealed the activity of a protein kinase (PKX) tha t was basally active in the cardiac myocyte that could phosphorylate t he Ca and K channels or regulators of the channels. The present studie s were aimed at determining the nature of PKX and its phosphorylation target. The effect of internal perfusion with microcystin on I-Ca or I -K was not attenuated by inhibitors of protein kinase A (PKA). However , the effect of microcystin on I-Ca was largely blocked by the nonsele ctive protein kinase inhibitors staurosporine (10-30 nM), K252a (250 n M), and H-7 (10 mu M). Staurosporine and H-7 also decreased the stimul ation of I-Ca by isoproterenol, but K252a was more selective and block ed the ability of microcystin to stimulate I-Ca, without significantly reducing isoproterenol-stimulated current. Internal perfusion with se lective inhibitors of protein kinase C (PKC), including the autoinhibi tory pseudosubstrate PKC peptide (PKC19-31) and a myristoylated deriva tive of this peptide had no effect. External application of several PK C inhibitors had negative side effects that prevented their use as sel ective PKC inhibitors. Nevertheless, we conclude that PKX is not PKC. PKA and PKX phosphorylate sites with different sensitivities to the ph osphatase inhibitors calyculin A and microcystin. In contrast to the r esults with I-Ca, the effect of microcystin on I-K was not blocked by any of the kinase inhibitors tested, suggesting that the effect of mic rocystin on I-K may not be mediated by a protein kinase but may be due to a direct effect of microcystin on the I-K channel.