Reaction of internal forms of the choline carrier of erythrocytes with N-ethylmaleimide: Evidence for a carrier conformational change on complex formation

Summary The choline carrier of human erythrocyte membranes exists in distinguishable outward-facing and inward-facing conformations, and previous studies demonstrated that only the latter reacts with N-ethylmaleimide, producing an irreversible inhibition of transport. We now report experiments to determine the individual reaction rates for the two inward-facing forms: the free carrier and the complex. The pseudo-first-order rate constant for the complex with a substrate analog, di-n-butylaminoethanol, is found to be nearlydouble that for the free carrier, showing that the carrier conformation is altered following addition of a ligand (with 1mm N-ethylmaleimide at pH 6.8, 37°C, the constants are 0.57±0.05 min^–1 and 0.33±0.02 min^–1, respectively). Hence three different conformational states have been distinguished by experiment: (1) the inward-facing free carrier; (2) the inward-facing complex; and (3) the outward-facing carrier.