A critical histidine in the vesicular acetylcholine transporter

The role of proton binding sites in the vesicular acetylcholine transporter was investigated by characterization of the pH dependence for the binding of [H-3]vesamicol [(-)-trans-2-(4-phenylpiperidino)cyclohexanol] to Torpedo synaptic vesicles. A single proton binds to a site with pK(a) 7.1 +/- 0.1, which is characteristic of histidine, to competitively inhibit vesamicol b inding. The histidine-selective reagent diethylpyrocarbonate causes time-de pendent inhibition of [H-3]vesamicol binding with a rate constant only abou t 20-fold lower than for reaction with free histidine. Because its pH titra tion has a simple, ideal shape, this residue probably controls all pH effec ts in the transporter between pH 6-8. Inhibition of [H-3]vesamicol binding by diethylpyrocarbonate was slowed by vesamicol but not acetylcholine, whic h binds to a separate site. The data suggest that a critical histidine with a pK(a) of 7.1 is unhindered when reacting with diethylpyrocarbonate. A co nformational model for the histidine is proposed to explain why acetylcholi ne competes with protons but not with diethylpyrocarbonate. A conserved his tidine in transmembrane helix VIII possibly is the histidine detected here. (C) 2000 Published by Elsevier Science Ltd. All rights reserved.