Proton fluxes associated with the Ca pump in human red blood cells.

Ca fluxes and H fluxes were measured in human red blood cells at 37 degrees C to characterize the effects of extracellular protons (Hout) on the Ca pump and to determine the stoichiometry of Ca-H exchange. A pH-stat technique was used to measure the rate of H influx, and 45Ca was used to determine the rate of Ca efflux. 4,4'-Diisothiocyanostilbene-2,2'-disulfonic acid (DIDS) was used to reduce proton permeability. A La-sensitive H influx was observed in Ca-loaded cells (Ca = 2 mmol/l packed cells) and was not observed in the cells loaded with vanadate as well as Ca. Similar results were obtained in Ca-loaded ghosts. The La dose-response curves for H influx and for Ca efflux were similar [50% inhibitory concentration (IC50) = approximately 5 microM] in intact red blood cells. The stoichiometry of the La-sensitive fluxes among different experiments ranged from 1.7 to 2.1 H/Ca when extracellular pH (pHout) = 6.3. Thus the Ca pump in intact red blood cells mediates Ca-2H exchange at pHout = 6.3. A 100-fold decrease in Hout [from pH 6.5 to 8.5; intracellular pH (pHin) approximately 7.4] only decreased Ca efflux 1.5- to 3-fold, hence Hout had little effect on the overall rate under the conditions studied. The small effect of Hout was a surprising result for a Ca-H exchange system, since one would have expected a steep dependence of Ca pump on Hout at Hout less than the Michaelis constant (Km). However, no La-sensitive H influx was observed when pHout = 8. On the basis of these data, it is suggested that the Ca pump also mediates Ca efflux uncoupled from H influx (Ca2+/phi H+). Ca efflux in the presence of 11 mM extracellular Ca (Caout) was one-fifth the value obtained in the absence of Caout at pHout = 8.5; this inhibition was reversed by increasing Hout (to pH 6.1). These results are consistent with a model in which 1) the Ca pump mediates Ca2+/2H+ exchange at high Hout; 2) the Ca pump mediates Ca2+/phi H+ exchange at low pHout; 3) the rates of the two processes are less than or equal to 4-fold different; 4) Caout inhibits pump activity at low Hout; and 5) Caout competes with Hout for binding.