SNARE complex formation is triggered by Ca2+ and drives membrane fusion

Neurotransmitter exocytosis, a process mediated by a core complex of syntax in, SNAP-25, and VAMP (SNAREs), is inhibited by SNARE-cleaving neurotoxins. Botulinum neurotoxin E inhibition of norepinephrine release in permeabiliz ed PC12 cells can be rescued by adding a 65 aa C-terminal fragment of SNAP- 25 (S25-C). Mutations along the hydrophobic face of the S25-C helix result in SNARE complexes with different thermostabilities, and these mutants resc ue exocytosis to different extents. Rescue depends on the continued presenc e of both S25-C and Ca2+ and correlates with complex formation. The data su ggest that Ca2+ triggers S25-C binding to a low-affinity site, initiating t rans-complex formation. Pairing of SNARE proteins on apposing membranes lea ds to bilayer fusion and results in a high-affinity cis-SNARE complex.