Cholinergic synaptic vesicle heterogeneity: evidence for regulation of acetylcholine transport

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Abstract
Crude cholinergic synaptic vesicles from a homogenate of the electric organ of Torpedo californica were centrifuged to equilibrium in an isosmotic sucrose density gradient. The classical VP1 synaptic vesicles banding at 1.055 g/mL actively transported [3H]acetylcholine (AcCh). An organelle banding at about 1.071 g/mL transported even more [3H]AcCh. Transport by both organelles was inhibited by the known AcCh storage blockers trans-2-(4-phenylpiperidino)cyclohexanol (vesamicol, formerly AH5183) and nigericin. Relative to VP1 vesicles the dense organelle was slightly smaller as shown by size-exclusion chromatography. It is concluded that the denser organelle corresponds to the recycling VP2 synaptic vesicle orginally described in intact Torpedo marmorata electric organ [Zimmermann, H., and Denston, C. R. (1977) Neuroscience (Oxford) 2, 695-714; Zimmermann, H., and Denston, C. R. (1977) Neuroscience (Oxford) 2, 715-730]. The properties of the receptor for vesamicol were studied by measuring binding of [3H]vesamicol, and the amount of SV2 antigen characteristic of secretory vesicles was assayed with a monoclonal antibody directed against it. Relative to VP1 vesicles the VP2 vesicles had a ratio of [3H]AcCh transport activity to vesamicol receptor concentration that typically was 4-7-fold higher, whereas the ratio of SV2 antigen concentration to vesamicol receptor concentration was about 2-fold higher. Based on an antibody standardization, in a typical preparation the VP1 vesicles contained 237 .+-. 15 pmol of receptor/mg of protein whereas VP2 vesicles contained 102 .+-. 3 pmol of receptor/mg of protein, and VP2 vesicles transported AcCh 2-3-fold more actively than VP1 vesicles. The Hill coefficients .alpha.H and equilibrium dissociation constants K for vesamicol binding to VP1 and VP2 vesicles were essentially the same, with .alpha.H = 2.0 and K = 19 nM. The positive Hill coefficient suggests that the vesamicol receptor exists as a homotropic oligomeric complex. The results demonstrate that VP1 and VP2 synaptic vesicles exhibit functional differences in the AcCh transport system, presumably as a result of regulatory phenomena.