AJP - Cell Physiology, Vol 258, Issue 3 C552-C562, Copyright © 1990 by American Physiological Society
Proton fluxes associated with the Ca pump in human red blood cells
M. A. Milanick
Department of Physiology, University of Missouri School of Medicine, Columbia 65212.
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.
