MODULATION OF AN INACTIVATING HUMAN CARDIAC K+ CHANNEL BY PROTEIN-KINASE-C

The transient outward current (I-TO) is an important repolarizing component of the cardiac action potential. In native cardiac myocytes, I-TO is modulated after activation of protein kinase C, although the molecular nature of this effect is not well understood. A channel recently cloned from human ventricular myocardium (Kv1.4, HK1) produces a rapidly inactivating K+ current, which has phenotypic similarities to the 4-aminopyridine-sensitive component of I-TO. Therefore, we examined whether this recombinant channel was also modulated by protein kinase C activation by investigating the effects of the diacylglycerol analogue phorbol 12-myristate 13-acetate (PMA) on Kv1.4 K+ current expressed in Xenopus oocytes. At a concentration of 10 nmol/L, PMA caused a biphasic response with an initial increase (14 +/- 4%, mean +/- SEM) in current, which peaked in 14 minutes. This was followed by a significant reduction (40 +/- 11%) in the current within 30 minutes. There was no significant change in cell membrane electrical capacitance with 10 nmol/L PMA (1 +/- 1% decline in 30 minutes), demonstrating that loss of cell membrane surface area did not explain the reduction in K+ current, although cell capacitance did decrease when using a higher concentration of PMA (81 nmol/L). The inactive stereoisomer, 4 alpha-PMA, had no effect on Kv1.4 current, whereas preincubation with the protein kinase inhibitor staurosporine or protein kinase C-selective chelerythrine prevented the effects of PMA. When purified from a stably transfected mammalian cell line by using immunoprecipitation, the channel protein was readily phosphorylated in vitro by purified protein kinase C. These results indicate that human Kv1.4-induced current is modulated by protein kinase C activation and suggest a role for direct K+ channel phosphorylation as the molecular mechanism of this effect.