The C terminus of SNAP25 is essential for Ca2+-dependent binding of synaptotagmin to SNARE complexes

The plasma membrane soluble N-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 Arg(180)-Ile(181), completely inhibits this late step in PC12 cell membranes, whereas BoNT A, which cleat es SNAP25 at Gln(197)-Arg(198), 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.