The C Terminus of SNAP25 Is Essential for Ca2+-dependent Binding of Synaptotagmin to SNARE Complexes

• Published in: The Journal of biological chemistry 2000-03, Vol.275 (9), p.6328-6336
• Main Authors: Gerona, Roy R.L., Larsen, Eric C., Kowalchyk, Judith A., Martin, Thomas F.J.
• Format: Article
• Language: English
• ISSN: 0021-9258; 1083-351X
• DOI: 10.1074/jbc.275.9.6328

The plasma membrane solubleN-ethylmaleimide-sensitive factor attachment protein receptor (SNARE) proteins syntaxin and synaptosome-associated protein of 25 kDa (SNAP25) and the vesicle SNARE protein vesicle-associated membrane protein (VAMP) are essential for a late Ca2+-dependent step in regulated exocytosis, but their precise roles and regulation by Ca2+ are poorly understood. Botulinum neurotoxin (BoNT) E, a protease that cleaves SNAP25 at Arg180-Ile181, completely inhibits this late step in PC12 cell membranes, whereas BoNT A, which cleaves SNAP25 at Gln197-Arg198, is only partially inhibitory. The difference in toxin effectiveness was found to result from a reversal of BoNT A but not BoNT E inhibition by elevated Ca2+ concentrations. BoNT A treatment essentially increased the Ca2+ concentration required to activate exocytosis, which suggested a role for the C terminus of SNAP25 in the Ca2+ regulation of exocytosis. Synaptotagmin, a proposed Ca2+ sensor for exocytosis, was found to bind SNAP25 in a Ca2+-stimulated manner. Ca2+-dependent binding was abolished by BoNT E treatment, whereas BoNT A treatment increased the Ca2+concentration required for binding. The C terminus of SNAP25 was also essential for Ca2+-dependent synaptotagmin binding to SNAP25·syntaxin and SNAP25·syntaxin·VAMP SNARE complexes. These results clarify classical observations on the Ca2+ reversal of BoNT A inhibition of neurosecretion, and they suggest that an essential role for the C terminus of SNAP25 in regulated exocytosis is to mediate Ca2+-dependent interactions between synaptotagmin and SNARE protein complexes.