Effects of the transport site conformation on the binding of external NAP-taurine to the human erythrocyte anion exchange system. Evidence for intrinsic asymmetry.

Abstract

External N-(4-azido-2-nitrophenyl)-2-aminoethylsulfonate (NAP-taurine) inhibits human red cell Cl exchange by binding to a site that is distinct from the Cl transport site. Increases in the intracellular Cl concentration (at constant external Cl) cause an increase in the inhibitory potency of external NAP-taurine. This effect is not due to the changes in pH or membrane potential that usually accompany a Cl gradient, since even when these changes are reversed or eliminated the inhibitory potency remains high. According to the ping-pong model for anion exchange, such transmembrane effects of intracellular Cl on external NAP-taurine can be explained if NAP-taurine only binds to its site when the transport site is in the outward-facing (Eo or EClo) form. Since NAP-taurine prevents the conformational change from EClo to ECli, it must lock the system in the outward-facing form. NAP-taurine can therefore be used just like the competitive inhibitor H2DIDS (4,4-diisothiocyano-1,2-diphenylethane-2,2''-disulfonic acid) to monitor the fraction of transport sites that face outward. A quantitative analysis of the effects of Cl gradients on the inhibitory potency of NAP-taurine and H2DIDS reveals that the transport system is intrinsically asymmetric, such that when Ci = Clo, most of the unloaded transport sites face the cytoplasmic side of the membrane.