Transmembrane effects of intracellular chloride on the inhibitory potency of extracellular H2DIDS. Evidence for two conformations of the transport site of the human erythrocyte anion exchange protein.

Abstract

The ping-pong model for the red cell anion exchange system postulates that the transport protein band 3 can exist in 2 different conformations, 1 in which the transport site faces the cytoplasm (Ei) and another in which it faces the outside medium (Eo). This model predicts that an increase in intracellular Cl should increase the fraction of sites in the outward-facing, unloaded form (Eo). Since external H2DIDS [4,4''-diisothiocyano-1,2-diphenylethane-2,2''-disulfonic acid] is a competitive inhibitor of Cl exchange that does not cross the membrane, it must bind only to the E0 form. Thus, an increase in E0 should cause an increase in H2DIDS inhibition. When intracellular Cl was increased at constant extracellular Cl, the inhibitory potency of H2DIDS rose, as predicted by the ping-pong model. This increase was not due to the concomitant changes in intracellular pH or membrane potential. When the Cl gradient was reversed, the inhibitory potency of H2DIDS decreased, again in qualitative agreement with the ping-pong model. Thus, Cl gradients can be used to change the orientation of the transport protein.