Stoichiometries of acetylcholine uptake, release, and drug inhibition in Torpedo synaptic vesicles: heterogeneity in acetylcholine transport and storage.
Highly purified Torpedo electric organ synaptic vesicles form a 49 nM suspension at 1 mg protein/ml. Under active transport conditions hundreds of molecules of [3H]acetylcholine ([3H]ACh) can be accumulated per vesicle, which requires the ACh transporter to undergo multiple turnovers. The transport blocker trans-2-(4-phenylpiperidino)cyclohexanol (AH5183) has no effect on storage of endogenous ACh by vesicles. In contrast, AH5183, other blocking drugs, and nonradioactive ACh caused a rapid release of at least 30-63 molecules of newly transported [3H]ACh per vesicle. Thus AH5183 distinguishes recently transported "new" vesicular ACh from "old" endogenous ACh. l-AH5183 inhibits transport of ACh with a half-inhibitory concentration of 16 +/- 7 nM at 12 nM vesicles and 115 +/- 34 nM at 120 nM vesicles. With the assumption that AH5183 acts on a receptor in an unamplified manner about 2.7 or fewer receptors per vesicle need to be occupied to cause inhibition of ACh transport. The apparent amplification in the number of [3H]ACh molecules per vesicle that are released by AH5183 suggests that AH5183 inhibits ACh storage by an indirect mechanism that distinguishes new from old ACh.
D000109:Acetylcholine / Q000378:metabolism*; D000818:Animals; D001693:Biological Transport, Active / Q000187:drug effects; D004557:Electric Organ / Q000378:metabolism*; D006426:Hemicholinium 3 / Q000494:pharmacology; D010622:Phencyclidine / Q000031:analogs & derivatives / Q000494:pharmacology; D010880:Piperidines; D011796:Quinacrine / Q000494:pharmacology; D013572:Synaptic Vesicles / Q000378:metabolism*; D014101:Torpedo / Q000378:metabolism*
D010880:Piperidines; D006426:Hemicholinium 3; C006189:vesamicol; D011796:Quinacrine; D010622:Phencyclidine; D000109:Acetylcholine