Synaptic Vesicles Are Constitutively Active Fusion Machines that Function Independently of Ca2+

SummaryBackgroundIn neurons, release of neurotransmitter occurs through the fusion of synaptic vesicles with the plasma membrane. Many proteins required for this process have been identified, with the SNAREs syntaxin 1, SNAP-25, and synaptobrevin thought to constitute the core fusion machinery. However, there is still a large gap between our understanding of individual protein-protein interactions and the functions of these proteins revealed by perturbations in intact synaptic preparations. To bridge this gap, we have used purified synaptic vesicles, together with artificial membranes containing coreconstituted SNAREs as reaction partners, in fusion assays.ResultsBy using complementary experimental approaches, we show that synaptic vesicles fuse constitutively, and with high efficiency, with proteoliposomes containing the plasma membrane proteins syntaxin 1 and SNAP-25. Fusion is inhibited by clostridial neurotoxins and involves the formation of SNARE complexes. Despite the presence of endogenous synaptotagmin, Ca2+ does not enhance fusion, even if phosphatidylinositol 4,5-bisphosphate is present in the liposome membrane. Rather, fusion kinetics are dominated by the availability of free syntaxin 1/SNAP-25 acceptor sites for synaptobrevin.ConclusionsSynaptic vesicles are constitutively active fusion machines, needing only synaptobrevin for activity. Apparently, the final step in fusion does not involve the regulatory activities of other vesicle constituents, although these may be involved in regulating earlier processes. This is particularly relevant for the calcium-dependent regulation of exocytosis, which, in addition to synaptotagmin, requires other factors not present in the vesicle membrane. The in vitro system described here provides an ideal starting point for unraveling of the molecular details of such regulatory events