Calcium‐Dependent Dissociation of Synaptotagmin from Synaptic SNARE Complexes

The formation of the synaptic core (SNARE) complex constitutes a crucial step in synaptic vesicle fusion at the nerve terminal. The interaction of synaptotagmin I with this complex potentially provides a means of conferring Ca 2+ -dependent regulation of exocytosis. However, the subcellular compartments in which interactions occur and their modulation by Ca 2+ influx remain obscure. Sodium dodecyl sulfate (SDS)-resistant core complexes, associated with synaptotagmin I, were enriched in rat brain fractions containing plasma membranes and docked synaptic vesicles. Depolarization of synaptosomes triggered [ 3 H]GABA release and Ca 2+ -dependent dissociation of synaptotagmin from the core complex. In perforated synaptosomes, synaptotagmin dissociation was induced by Ca 2+ (30-300 μM) but not Sr 2+ (1 mM); it apparently required intact membrane bilayers but did not result in disassembly of trimeric SNARE complexes. Synaptotagmin was not associated with unstable v-SNARE/t-SNARE complexes, present in fractions containing synaptic vesicles and cytoplasm. These complexes acquired SDS resistance when N-ethylmaleimide-sensitive fusion protein (NSF) was inhibited with N-ethylmaleimide or adenosine 5'-O-(3-thiotriphosphate), suggesting that constitutive SNARE complex disassembly occurs in undocked synaptic vesicles. Our findings are consistent with models in which the Ca 2+ -triggered release of synaptotagmin precedes vesicle fusion. NSF may then dissociate ternary core complexes captured by endocytosis and recycle/prime individual SNARE proteins.