Probing the extracellular release site of the plasma membrane calcium pump

The plasma membrane Ca(2+) pump is known to mediate Ca(2+)/H(+) exchange. Extracellular protons activated (45)Ca(2+) efflux from human red blood cells with a half-maximal inhibition constant of 2 nM when the intracellular pH was fixed. An increase in pH from 7.2 to 8.2 decreased the IC(50) for extracellular Ca(2+) from approximately 33 to approximately 6 mM. Changing the membrane potential by >54 mV had no effect on the IC(50) for extracellular Ca(2+). This argues against Ca(2+) release through a high-field access channel. Extracellular Ni(2+) inhibited Ca(2+) efflux with an IC(50) of 11 mM. Extracellular Cd(2+) inhibited with an IC(50) of 1. 5 mM, >10 times better than Ca(2+). The Cd(2+) IC(50) also decreased when the pH was raised from 7.1 to 8.2, consistent with Ca(2+), Cd(2+), and H(+) competing for the same site. The higher affinity for inhibition by Ni(2+) and Cd(2+) is consistent with a histidine or cysteine as part of the release site. The cysteine reagent 2-(trimethylammonium)ethyl methanethiosulfonate did not inhibit Ca(2+) efflux. Our results are consistent with the notion that the release site contains a histidine.