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

The effects of the protein phosphatase inhibitors calyculin A and okadaic a cid on Na+/Ca2+ exchange activity were examined in transfected Chinese hams ter ovary cells expressing the bovine cardiac Na+/Ca2+ exchanger. Incubatin g 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 ki nase inhibitors K252a and staurosporine provided partial protection against the effects of calyculin A. Okadaic acid, another protein phosphatase inhi bitor, nearly completely blocked exchange-mediated Ba2+ influx. Chinese ham ster ovary cells expressing a mutant exchanger in which 420 out of 520 amin o acid residues were deleted from the central hydrophilic domain of the exc hanger remained sensitive to the inhibitory effects of calyculin A and okad aic acid. Surprisingly, Na-o(+)-dependent Ca2+ efflux appeared to be only m odestly inhibited, if at all, by calyculin A or okadaic acid. We conclude t hat protein hyperphosphorylation during protein phosphatase blockade select ively inhibits the Ca2+ influx mode of Na+/Ca2+ exchange, probably by an in direct mechanism that does not involve phosphorylation of the exchanger its elf.