Ca2+-triggered catecholamine exocytosis from chromaffin cells involves SNAP-25, synaptobrevin and syntaxin (known as SNAREs). Synaptotagmin I has been implicated as the Ca2+-sensor because it binds Ca2+, and this enhances its binding to syntaxin, SNAP-25 and phospholipids in vitro. However, most of these interactions are only mediated by [Ca2+]i two orders of magnitude higher than that needed to elicit secretion. Thus, the Ca2+ sensitivities of synaptotagmin I and the other SNAREs were quantified in situ. Secretion elicited from permeabilised cells by μM Ca2+ was accompanied,with almost identical Ca2+ dependencies, by changes in synaptotagmin I, SNAP-25, syntaxin and synaptobrevin that rendered them less susceptible to trypsin. The majority of the trypsin-resistant SNAREs were not associated with SDS-resistant complexes. None of these proteins acquired trypsin resistance in cells rendered incompetent for exocytosis by run-down. Removal of nine C-terminal residues from SNAP-25 by botulinum toxin A reduced both exocytosis and the SNAREs' acquisition of trypsin resistance but did not alter the Ca2+ sensitivity, except for synaptotagmin I. Even after synaptobrevin had been cleaved by botulinum toxin B, all the other proteins still responded to Ca2+. These data support a model whereby Ca2+ is sensed, probably by synaptotagmin I, and the signal passed to syntaxin and SNAP-25 before they interact with synaptobrevin.
