The choline carrier of erythrocytes: location of the NEM-reactive thiol group in the inner gated channel.

Choline transport across the human erythrocyte membrane is irreversibly inhibited when N-ethylmaleimide (NEM) reacts with a carrier SH group which is located outside the substrate site, and which is exposed in the inward-facing form of the carrier but prevented from reacting in the outward-facing form. The location of the SH group with respect to the membrane has now been determined by studying the dependence of the NEM-alkylation rate on the intracellular and extracellular pH. The results show that the reactive SH group equilibrates with hydrogen ions in the cytoplasm, but is completely isolated from hydrogen ions in the external medium. With this added evidence it becomes possible to conclude that the SH group is located in the inner gated channel of the choline carrier.