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Ca²⁺-induced changes in SNAREs and synaptotagmin I correlate with triggered exocytosis from chromaffin cells: insights gleaned into the signal transduction using trypsin and botulinum toxins

Centre for Neurobiochemistry, Department of Biological Sciences, Imperial College of Science, Technology and Medicine, South Kensington, London SW7 2AY, UK

^* Author for correspondence (e-mail: o.dolly{at}ic.ac.uk )

Accepted 15 April 2002

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.

Key words: Secretion, Large dense-core granules, SNAP-25, Synaptobrevin, Clostridial neurotoxins

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