Probing the extracellular release site of the plasma membrane calcium pump

The plasma membrane Ca2+ pump is known to mediate Ca2+/H+ exchange. Extracellular protons activated Ca-45(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 IC50 for extracellular Ca2+ from similar to 33 to similar to 6 mM. Changing the membrane potential by >54 mV had no effect on the IC50 for extracellular Ca2+. This argues against Ca2+ release through a high-field access channel. Extracellular Ni2+ inhibited Ca2+ efflux with an IC50 Of 11 mM. Extracellular Cd2+ inhibited with an IC50 of 1.5 mM, >10 times better than Ca2+. The Cd2+ IC50 also decreased when the pH was raised from 7.1 to 8.2, consistent with Ca2+, Cd2+, and Hi competing for the same site. The higher affinity for inhibition by Ni2+ and Cd2+ is consistent with a histidine or cysteine as part of the release site. The cysteine reagent 2-(trimethylammonium)ethyl methanethiosulfonate did not inhibit Ca2+ efflux. Our results are consistent with the notion that the release site contains a histidine.