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The C terminus of SNAP25 is essential for Ca(2+)-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 Ca(2+)-dependent step in regulated exocytosis, but their precise roles and regulation by Ca(2+) 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 cleaves 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 Ca(2+) concentrations. BoNT A treatment essentially increased the Ca(2+) concentration required to activate exocytosis, which suggested a role for the C terminus of SNAP25 in the Ca(2+) regulation of exocytosis. Synaptotagmin, a proposed Ca(2+) sensor for exocytosis, was found to bind SNAP25 in a Ca(2+)-stimulated manner. Ca(2+)-dependent binding was abolished by BoNT E treatment, whereas BoNT A treatment increased the Ca(2+) concentration required for binding. The C terminus of SNAP25 was also essential for Ca(2+)-dependent synaptotagmin binding to SNAP25. syntaxin and SNAP25.syntaxin.VAMP SNARE complexes. These results clarify classical observations on the Ca(2+) 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 Ca(2+)-dependent interactions between synaptotagmin and SNARE protein complexes.

Pubmed ID: 10692432

Authors

  • Gerona RR
  • Larsen EC
  • Kowalchyk JA
  • Martin TF

Journal

The Journal of biological chemistry

Publication Data

March 3, 2000

Associated Grants

  • Agency: NIDDK NIH HHS, Id: DK25861
  • Agency: NIDDK NIH HHS, Id: DK40428
  • Agency: NIGMS NIH HHS, Id: GM07215

Mesh Terms

  • Animals
  • Botulinum Toxins
  • Botulinum Toxins, Type A
  • Calcium
  • Calcium-Binding Proteins
  • Cell Membrane
  • Exocytosis
  • Membrane Glycoproteins
  • Membrane Proteins
  • Nerve Tissue Proteins
  • Neurotoxins
  • Norepinephrine
  • PC12 Cells
  • Protein Binding
  • R-SNARE Proteins
  • Rats
  • SNARE Proteins
  • Synaptosomal-Associated Protein 25
  • Synaptotagmins
  • Vesicular Transport Proteins