Kinetic properties of sodium-proton antiport in Escherichia coli membrane vesicles: effects of imposed electrical potential, proton gradient, and internal pH

Abstract

Modifications of the kinetic properties of the E. coli (RA11) Na+-H+ antiport system by imposedpH gradients (.DELTA.pH, interior alkaline) and membrane potential (.DELTA..psi. interior negative) were studied by looking at the accelerating effects of .DELTA.pH and .DELTA..psi. on downhill Na+ efflux from membrane vesicles incubated at different external pH. Variations of the Na+ efflux rate (VNa) as a function of imposed .DELTA.pH appear to be strongly dependent on the external pH value. The individual VNa vs. .DELTA.pH relationships observed between pH 5.5 and pH 6.6 are all nonlinear and indicate the existence of a threshold .DELTA.pH above which VNa increases steeply as the .DELTA.pH magnitude increases; threshold .DELTA.pH values progressively decrease as the pH is raised from 5.5 to 6.6. At or above neutrality, VNa acceleration is linearly related to .DELTA.pH amplitude. It is shown that the .DELTA.pH-dependent variations in the Na+ efflux rate measured in vesicles incubated at different external pH can be accounted for by variations of internal pH; the observed relationship suggests that a high internal H+ concentration inhibits the Na+-H+ antiport activity. This inhibition results from a drastic increase in the apparent Km of the Na+ efflux reaction as the internal H+ concentration increases. Imposed .DELTA..psi. increases the Na+ efflux rate linearly by a selective modification of the Vmax value of the Na+ efflux. Evidently the internal H+ concentration controls the Na+-H+ antiport activity and the chemical and electrical proton gradients affect 2 different kinetic steps of the Na+-H+ exchange reaction.