Asymmetric effects of divalent cations and protons on active Ca2+ efflux and Ca2+-ATPase in intact red blood cells

The influence of the asymmetric addition of various divalent cations and protons on the properties of active Ca2+ transport have been examined in intact human red blood cells. Active Ca2+ efflux was determined from the initial rate of 45Ca2+ loss after CoCl2 was added to block Ca2+ loading via the ionophore A23187. Ca2+-ATPase activity was measured as phosphate production over 5 min in cells equilibrated with EGTA-buffered free Ca2+ in the presence of A23187. The apparent Ca affinity of active Ca2+ efflux (K0.5 = 30-40 mumol/liter cells) was significantly lower than that measured by the Ca2+-ATPase assay (K0.5 = 0.4 microM). Possible reasons for this apparent difference are considered. Both active Ca2+ efflux and Ca2+-ATPase activity were reduced to less than 5% of maximal levels (20 mmol/liter cells.hr) in Mg2+-depleted cells, and completely restored by reintroduction of intracellular Mg2+. Active Ca2+ efflux was inhibited almost completely by raising external CaCl2 (but not MgCl2) to 20 mM, probably by interaction of Ca2+ at the externally oriented E2P conformation of the pump. Cd2+ was more potent than Ca2+ in this inhibition, while Mn2+ was less potent and 10 mM Ba2+ was without effect. A Ca2+: proton exchange mechanism for active Ca2+ efflux was supported by the results, as external protons (pH 6-6.5) stimulated active Ca2+ efflux at least twofold above the efflux rate at pH 7.8 Ca2+ transport was not affected by decreasing the membrane potential across the red cell.