Effect of ATP Depletion on Kinetics of Na/Ca Exchange-mediated Ca Influx in Na-loaded Heart Cells

Abstract

The kinetics of Ca influx by Na/Ca exchange into adult rat heart cells loaded with Na and depleted of ATP were investigated, to further elucidate how ATP regulates exchanger properties in the intact heart cell. We found an eight-fold reduction in Vmaxfor Ca uptake by ATP depletion, with no significant change in the Kmfor extracellular Ca or the Kifor inhibition by extracellular Na. Autoradiography of ATP depleted cells after45Ca uptake showed no gross heterogeneity of Ca uptake, as would occur if a small fraction of cells still retained some ATP. We conclude that the reduction in Vmaxis a property of all of the ATP depleted cells. These kinetics are consistent with regulation of the putative calmodulin binding site on the exchanger. They are also consistent with sequestration of the exchanger at the intracellular face of the sarcolemma by intracellular Na dependent inactivation, a property of the exchanger observed in membrane patches to be relieved by ATP. However, no effect of heptalysine was observed on exchange activity in cells with ATP; nor was the restoration of exchange activity to ATP depleted cells by ATP resynthesis blocked by heptalysine. This suggests that in intact cells the activation of exchange activity by ATP is not caused by phosphatidylserine translocation to the cytosolic leaflet of the sarcolemma. Also, no effect of cytochalasin D was observed on exchanger activity or kinetics, in contrast with observations in Chinese hamster ovary (CHO) cells transfected with bovine cardiac Na/Ca exchanger. These results suggest that there are multiple modes of regulation of the heart Na/Ca exchanger by ATP, and these are differently expressed depending on the exchanger environment.