Release of neurotransmitter occurs when synaptic vesicles fuse with the plasma membrane. This neuronal exocytosis is triggered by calcium and requires three SNARE (soluble-N-ethylmaleimide-sensitive factor attachment protein receptors) proteins: synaptobrevin (also known as VAMP) on the synaptic vesicle, and syntaxin and SNAP-25 on the plasma membrane1,2,3,4. Neuronal SNARE proteins form a parallel four-helix bundle that is thought to drive the fusion of opposing membranes5,6. As formation of this SNARE complex in solution does not require calcium, it is not clear what function calcium has in triggering SNARE-mediated membrane fusion. We now demonstrate that whereas syntaxin and SNAP-25 in target membranes are freely available for SNARE complex formation, availability of synaptobrevin on synaptic vesicles is very limited. Calcium at micromolar concentrations triggers SNARE complex formation and fusion between synaptic vesicles and reconstituted target membranes. Although calcium does promote interaction of SNARE proteins between opposing membranes, it does not act by releasing synaptobrevin from synaptic vesicle restriction. Rather, our data suggest a mechanism in which calcium-triggered membrane apposition enables syntaxin and SNAP-25 to engage synaptobrevin, leading to membrane fusion.
Subscribe to Journal
Get full journal access for 1 year
only $3.90 per issue
All prices are NET prices.
VAT will be added later in the checkout.
Rent or Buy article
Get time limited or full article access on ReadCube.
All prices are NET prices.
Sollner, T., Bennett, M. K., Whiteheart, S. W., Scheller, R. H. & Rothman, J. E. A protein assembly-disassembly pathway in vitro that may correspond to sequential steps of synaptic vesicle docking, activation, and fusion. Cell 75, 409–418 (1993).
Sudhof, T. C. The synaptic vesicle cycle: a cascade of protein–protein interactions. Nature 375, 645–653 (1995).
Lin, R. C. & Scheller, R. H. Mechanisms of synaptic vesicle exocytosis. Annu. Rev. Cell Dev. Biol. 16, 19–49 (2000).
Kelly, R. B. in Neurotransmitter Release (ed. Bellen, H. J.) 1–33 (Oxford Univ. Press, Oxford, 1999).
Sutton, R. B., Fasshauer, D., Jahn, R. & Brunger, A. T. Crystal structure of a SNARE complex involved in synaptic exocytosis at 2.4 Å resolution. Nature 395, 347–353 (1998).
Weber, T. et al. SNAREpins: minimal machinery for membrane fusion. Cell 92, 759–772 (1998).
Fatt, P. & Katz, B. Spontaneous subthreshold activity at motor nerve endings. J. Physiol. (Lond.) 117, 109–128 (1952).
Schikorski, T. & Stevens, C. F. Quantitative ultrastructural analysis of hippocampal excitatory synapses. J. Neurosci. 17, 5858–5867 (1997).
Elferink, L. A. & Scheller, R. H. Synaptic vesicle proteins and regulated exocytosis. Prog. Brain Res. 105, 79–85 (1995).
Fernandez-Chacon, R. & Sudhof, T. C. Genetics of synaptic vesicle function: toward the complete functional anatomy of an organelle. Annu. Rev. Physiol. 61, 753–776 (1999).
Fernandez-Chacon, R. et al. Synaptotagmin I functions as a calcium regulator of release probability. Nature 410, 41–49 (2001).
Popov, S. V. & Poo, M. M. Synaptotagmin: a calcium-sensitive inhibitor of exocytosis? Cell 73, 1247–1249 (1993).
Hua, S. Y. & Charlton, M. P. Activity-dependent changes in partial VAMP complexes during neurotransmitter release. Nature Neurosci. 2, 1078–1083 (1999).
Tolar, L. A. & Pallanck, L. NSF function in neurotransmitter release involves rearrangement of the SNARE complex downstream of synaptic vesicle docking. J. Neurosci. 18, 10250–10256 (1998).
Montecucco, C. & Schiavo, G. Structure and function of tetanus and botulinum neurotoxins. Q. Rev. Biophys. 28, 423–472 (1995).
Hayashi, T. et al. Synaptic vesicle membrane fusion complex: action of clostridial neurotoxins on assembly. EMBO J. 13, 5051–5061 (1994).
Calakos, N. & Scheller, R. H. Vesicle-associated membrane protein and synaptophysin are associated on the synaptic vesicle. J. Biol. Chem. 269, 24534–24537 (1994).
McMahon, H. T. et al. Synaptophysin, a major synaptic vesicle protein, is not essential for neurotransmitter release. Proc. Natl Acad. Sci. USA 93, 4760–4764 (1996).
Quetglas, S., Leveque, C., Miquelis, R., Sato, K. & Seagar, M. Ca2+-dependent regulation of synaptic SNARE complex assembly via a calmodulin- and phospholipid-binding domain of synaptobrevin. Proc. Natl Acad. Sci. USA 97, 9695–9700 (2000).
Bai, J., Earles, C. A., Lewis, J. L. & Chapman, E. R. Membrane-embedded synaptotagmin penetrates cis or trans target membranes and clusters via a novel mechanism. J. Biol. Chem. 275, 25427–25435 (2000).
Viguera, A. R., Mencia, M. & Goni, F. M. Time-resolved and equilibrium measurements of the effects of poly(ethylene glycol) on small unilamellar phospholipid vesicles. Biochemistry 32, 3708–3713 (1993).
Meyuhas, D., Nir, S. & Lichtenberg, D. Aggregation of phospholipid vesicles by water-soluble polymers. Biophys. J. 71, 2602–2612 (1996).
Rosenmund, C. & Stevens, C. F. Definition of the readily releasable pool of vesicles at hippocampal synapses. Neuron 16, 1197–1207 (1996).
Capogna, M., McKinney, R. A., O'Connor, V., Gahwiler, B. H. & Thompson, S. M. Ca2+ or Sr2+ partially rescues synaptic transmission in hippocampal cultures treated with botulinum toxin A and C, but not tetanus toxin. J. Neurosci. 17, 7190–7202 (1997).
McCammon, J. A. A speed limit for protein folding. Proc. Natl Acad. Sci. USA 93, 11426–11427 (1996).
Wittung-Stafshede, P., Lee, J. C., Winkler, J. R. & Gray, H. B. Cytochrome b562 folding triggered by electron transfer: approaching the speed limit for formation of a four-helix-bundle protein. Proc. Natl Acad. Sci. USA 96, 6587–6590 (1999).
Perin, M. S. et al. Structural and functional conservation of synaptotagmin (p65) in Drosophila and humans. J. Biol. Chem. 266, 615–622 (1991).
Davletov, B., Perisic, O. & Williams, R. L. Calcium-dependent membrane penetration is a hallmark of the C2 domain of cytosolic phospholipase A2 whereas the C2A domain of synaptotagmin binds membranes electrostatically. J. Biol. Chem. 273, 19093–19096 (1998).
Zhang, X., Rizo, J. & Sudhof, T. C. Mechanism of phospholipid binding by the C2A-domain of synaptotagmin I. Biochemistry 37, 12395–12403 (1998).
Davletov, B. A. et al. Vesicle exocytosis stimulated by α-latrotoxin is mediated by latrophilin and requires both external and stored Ca2+. EMBO J. 17, 3909–3920 (1998).
We thank H. McMahon for syntaxin and synaptobrevin plasmids and H. Hirling for advice on anti-SNAP-25 immunochromatography. K.H. was supported in part by the University of Cambridge MB/PhD Programme. A.S. and S.F. were supported by postdoctoral fellowships from the Royal Society and Wellcome Trust, respectively.
The authors declare no competing financial interests.
About this article
Cite this article
Hu, K., Carroll, J., Fedorovich, S. et al. Vesicular restriction of synaptobrevin suggests a role for calcium in membrane fusion. Nature 415, 646–650 (2002). https://doi.org/10.1038/415646a
Journal of Neuroscience Methods (2019)
Effects of haloperidol and clozapine on synapse-related gene expression in specific brain regions of male rats
European Archives of Psychiatry and Clinical Neuroscience (2018)
International Journal of Molecular Sciences (2018)
Cell Calcium (2017)
Linking kindling to increased glutamate release in the dentate gyrus of the hippocampus through the STXBP5/tomosyn-1 gene
Brain and Behavior (2017)