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.
Gerona
R R
RR
Department of Biochemistry, University of Wisconsin, Madison, Wisconsin 53706, USA.
Larsen
E C
EC
Kowalchyk
J A
JA
Martin
T F
TF
D000818:Animals; D001905:Botulinum Toxins / Q000494:pharmacology; D019274:Botulinum Toxins, Type A / Q000494:pharmacology; D002118:Calcium / Q000494:pharmacology*; D002135:Calcium-Binding Proteins; D002462:Cell Membrane / Q000378:metabolism; D005089:Exocytosis; D008562:Membrane Glycoproteins / Q000378:metabolism*; D008565:Membrane Proteins / Q000378:metabolism*; D009419:Nerve Tissue Proteins / Q000737:chemistry / Q000378:metabolism*; D009498:Neurotoxins / Q000494:pharmacology; D009638:Norepinephrine / Q000378:metabolism; D016716:PC12 Cells; D011485:Protein Binding; D050683:R-SNARE Proteins; D051381:Rats; D050600:SNARE Proteins; D050825:Synaptosomal-Associated Protein 25; D050857:Synaptotagmins; D033921:Vesicular Transport Proteins
D002135:Calcium-Binding Proteins; D008562:Membrane Glycoproteins; D008565:Membrane Proteins; D009419:Nerve Tissue Proteins; D009498:Neurotoxins; D050683:R-SNARE Proteins; D050600:SNARE Proteins; C494210:Snap25 protein, rat; D050825:Synaptosomal-Associated Protein 25; D033921:Vesicular Transport Proteins; D050857:Synaptotagmins; D001905:Botulinum Toxins; D019274:Botulinum Toxins, Type A; D002118:Calcium; C096326:botulinum toxin type E; D009638:Norepinephrine