Mode-specific inhibition of sodium-calcium exchange during protein phosphatase blockade

The effects of the protein phosphatase inhibitors calyculin A and okadaic acid on Na+/Ca2+ exchange activity were examined in transfected Chinese hamster ovary cells expressing the bovine cardiac Na+/Ca2+ exchanger. Incubating the cells for 5-10 min with 100 nM calyculin A reduced exchange-mediated Ca-45(2+) uptake or Ba2+ influx by 50-75%. Half-maximal inhibition of Ca-45(2+) up-take was observed at 15 nM calyculin A. The nonselective protein kinase inhibitors K252a and staurosporine provided partial protection against the effects of calyculin A. Okadaic acid, another protein phosphatase inhibitor, nearly completely blocked exchange-mediated Ba2+ influx. Chinese hamster ovary cells expressing a mutant exchanger in which 420 out of 520 amino acid residues were deleted from the central hydrophilic domain of the exchanger remained sensitive to the inhibitory effects of calyculin A and okadaic acid. Surprisingly, Na-o(+)-dependent Ca2+ efflux appeared to be only modestly inhibited, if at all, by calyculin A or okadaic acid. We conclude that protein hyperphosphorylation during protein phosphatase blockade selectively inhibits the Ca2+ influx mode of Na+/Ca2+ exchange, probably by an indirect mechanism that does not involve phosphorylation of the exchanger itself.