Haigh_1994_Neuroreport_5_773

Uptake of acetylcholine was studied in a synaptic vesicle fraction isolated from rat brain. Hyposmotically treated P3 vesicles took up acetylcholine (ACh) in the presence of MgATP, and the uptake was inhibited by low temperature, ammonium ions, the protonophore carbonyl cyanide p-(trifluoromethoxy)phenylhydrazone (FCCP) and bafilomycin A1, a specific inhibitor of the vacuolar H(+)-ATPase. Uptake was also inhibited by drugs that bind allosterically to the ACh transporter, namely vesamicol (IC50 value of 170 nM) and 4-aminobenzovesamicol (IC50 value of 25 nM). KM and Vmax values for ACh active transport were estimated to be 5 mM and 4 nmol min-1 mg-1 of cholinergic vesciles, respectively. Active transport of ACh by synaptic vesicles partially purified from brain is mediated by a vesamicol-sensitive transporter and is dependent on a proton gradient generated by the vesicular H(+)-ATPase.