Cholinergic synaptic vesicle heterogeneity: evidence for regulation of acetylcholine transport

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 denser organelle was slightly smaller as shown by size-exclusion chromatography. It is concluded that the denser organelle corresponds to the recycling VP2 synaptic vesicle originally described in intact Torpedo marmorata electric organ [Zimmermann, H., & Denston, C.R. (1977) Neuroscience (Oxford) 2, 695-714; Zimmermann, H., & 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.(ABSTRACT TRUNCATED AT 250 WORDS)

Acetylcholine recycling and release at rat motor nerve terminals studied using (‐)‐vesamicol and troxpyrrolium.

“…This suggestion is supported by the finding (Gracz, Wang & Parsons, 1988) that VP1 and VP2 vesicles at the Torpedo electric organ, as well as having different ACh and ATP contents and transport activities, also express a different number of (-)-vesamicol binding sites. As it is unlikely that the integral protein to which (-)-vesamicol binds is actually removed and replaced during either vesicle exocytosis, recycling or refiling, Gracz et al (1988) suggested that the protein containing the (-)-vesamicol binding site plays a role in the regulation of the vesicular transport of ACh. It therefore seems possible that (-vesamicol inhibits the uptake of ACh into vesicles by acting on this regulatory system.…”

Motor neuron-specific overexpression of the presynaptic choline transporter: impact on motor endurance and evoked muscle activity

“…Increasing the ratio of reserve pool to releasable pool vesicles would reduce the amount of ACh available for release during lighter periods of stimulation and thereby reduce overall stimulation if the mobilization of reserve pool vesicles is too slow to compensate. The two hypotheses we present to explain high and low CMAP values may interrelate as newly synthesized ACh is thought to preferentially load more rapidly recycling vesicles (Gracz et al, 1988, Bonzelius and Zimmermann, 1990). Possibly, only in mice where there is a sufficiently large population of recycling vesicles can elevated ACh synthesis “win out” and result in an elevation in cholinergic signaling.…”

Transport mechanisms in acetylcholine and monoamine storage

“…The lead compound vesamicol binds to the cytoplasmic side (14) with an equilibrium dissociation constant ( K D ) value of ~5nM (15). Binding sometimes exhibits positive cooperativity, but this is a variable observation (16, 17). Vesamicol analogs inhibit transport noncompetitively (16, 18), but they apparently need bind only a fraction of VAChT in isolated vesicles to inhibit fully (19).…”