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 [3H]vesamicol [(-)-trans-2-(4-phenylpiperidino)cyclohexanol] to Torpedo synaptic vesicles. A single proton binds to a site with pKa 7.1 +/- 0.1, which is characteristic of histidine, to competitively inhibit vesamicol binding. The histidine-selective reagent diethylpyrocarbonate causes time-dependent inhibition of [3H]vesamicol binding with a rate constant only about 20-fold lower than for reaction with free histidine. Because its pH titration has a simple, ideal shape, this residue probably controls all pH effects in the transporter between pH 6-8. Inhibition of [3H]vesamicol binding by diethylpyrocarbonate was slowed by vesamicol but not acetylcholine, which binds to a separate site. The data suggest that a critical histidine with a pKa of 7.1 is unhindered when reacting with diethylpyrocarbonate. A conformational model for the histidine is proposed to explain why acetylcholine competes with protons but not with diethylpyrocarbonate. A conserved histidine in transmembrane helix VIII possibly is the histidine detected here.
Keller
J E
JE
Department of Chemistry and The Neuroscience Research Institute, University of California, Santa Barbara, CA 93106, USA.
Parsons
S M
SM
D000818:Animals; D001665:Binding Sites; D002352:Carrier Proteins / Q000737:chemistry* / Q000378:metabolism*; D003545:Cysteine / Q000378:metabolism; D004047:Diethyl Pyrocarbonate / Q000494:pharmacology; D004557:Electric Organ; D006639:Histidine / Q000378:metabolism*; D006863:Hydrogen-Ion Concentration; D026901:Membrane Transport Proteins; D009467:Neuromuscular Depolarizing Agents / Q000378:metabolism; D010880:Piperidines / Q000378:metabolism; D011522:Protons; D013572:Synaptic Vesicles / Q000378:metabolism*; D014101:Torpedo; D014316:Tritium; D050494:Vesicular Acetylcholine Transport Proteins; D033921:Vesicular Transport Proteins
D002352:Carrier Proteins; D026901:Membrane Transport Proteins; D009467:Neuromuscular Depolarizing Agents; D010880:Piperidines; D011522:Protons; D050494:Vesicular Acetylcholine Transport Proteins; D033921:Vesicular Transport Proteins; D014316:Tritium; C006189:vesamicol; D006639:Histidine; D003545:Cysteine; D004047:Diethyl Pyrocarbonate