[ATP-dependent fusion of synaptic vesicles and synaptosomal plasma membrane in a cell-free system]

The final step in exocytosis is the fusion of synaptic vesicle membrane with the synaptosomal plasma membrane, leading to the release of the neurotransmitters. We have reconstituted this fusion event in vitro, using isolated synaptic vesicles and synaptosomal plasma membranes from the bovine brain. The membranes of synaptic vesicles were loaded with the lipid--soluble fluorescent probe octadecylrhodamine B at the concentration that resulted in self-quenching of its fluorescence. The vesicles were then incubated with synaptosomal plasma membranes at 37 degrees C and fusion was measured through the dilution-dependent de-quenching of the fluorescence of the probe. Synaptic vesicles by themselves did not fused with plasma membrane, only addition of ATP induced the fusion. W-7 and trifluoroperasine, the drugs reported to inhibit calmodulin-dependent events, were effective inhibitors of the ATP-induced fusion synaptic vesicles and synaptosomal plasma membranes. Our results indicate that the membrane fusion in the nerve terminals during exocytosis may be under direct control of calmodulin-dependent protein phosphorylation.