title: Complexin inhibits spontaneous release and synchronizes Ca2+-triggered synaptic vesicle fusion by distinct mechanisms.
abstract: Previously we showed that fast Ca(2+)-triggered vesicle fusion with reconstituted neuronal SNAREs and synaptotagmin-1 begins from an initial hemifusion-free membrane point contact, rather than a hemifusion diaphragm, using a single vesicle-vesicle lipid/content mixing assay (Diao et al., 2012). When complexin-1 was included, a more pronounced Ca(2+)-triggered fusion burst was observed, effectively synchronizing the process. Here we show that complexin-1 also reduces spontaneous fusion in the same assay. Moreover, distinct effects of several complexin-1 truncation mutants on spontaneous and Ca(2+)-triggered fusion closely mimic those observed in neuronal cultures. The very N-terminal domain is essential for synchronization of Ca(2+)-triggered fusion, but not for suppression of spontaneous fusion, whereas the opposite is true for the C-terminal domain. By systematically varying the complexin-1 concentration, we observed differences in titration behavior for spontaneous and Ca(2+)-triggered fusion. Taken together, complexin-1 utilizes distinct mechanisms for synchronization of Ca(2+)-triggered fusion and inhibition of spontaneous fusion.
authors.affiliation: Department of Molecular and Cellular Physiology, Stanford University, Stanford, United States Department of Neurology and Neurological Science, Stanford University, Stanford, United States Department of Structural Biology, Stanford University, Stanford, United States Department of Photon Science, Stanford University, Stanford, United States.
authors.forename: Ying
authors.initials: Y
authors.lastname: Lai
authors.affiliation: Department of Molecular and Cellular Physiology, Stanford University, Stanford, United States Department of Neurology and Neurological Science, Stanford University, Stanford, United States Department of Structural Biology, Stanford University, Stanford, United States Department of Photon Science, Stanford University, Stanford, United States Howard Hughes Medical Institute, Stanford University, Stanford, United States.
authors.forename: Jiajie
authors.initials: J
authors.lastname: Diao
authors.affiliation: Department of Molecular and Cellular Physiology, Stanford University, Stanford, United States Department of Neurology and Neurological Science, Stanford University, Stanford, United States Department of Structural Biology, Stanford University, Stanford, United States Department of Photon Science, Stanford University, Stanford, United States Howard Hughes Medical Institute, Stanford University, Stanford, United States.
authors.forename: Daniel J
authors.initials: DJ
authors.lastname: Cipriano
authors.affiliation: Department of Molecular and Cellular Physiology, Stanford University, Stanford, United States Department of Neurology and Neurological Science, Stanford University, Stanford, United States Department of Structural Biology, Stanford University, Stanford, United States Department of Photon Science, Stanford University, Stanford, United States.
authors.forename: Yunxiang
authors.initials: Y
authors.lastname: Zhang
authors.affiliation: Department of Molecular and Cellular Physiology, Stanford University, Stanford, United States Department of Neurology and Neurological Science, Stanford University, Stanford, United States Department of Structural Biology, Stanford University, Stanford, United States Department of Photon Science, Stanford University, Stanford, United States Howard Hughes Medical Institute, Stanford University, Stanford, United States.
authors.forename: Richard A
authors.initials: RA
authors.lastname: Pfuetzner
authors.affiliation: Department of Molecular and Cellular Physiology, Stanford University, Stanford, United States Department of Neurology and Neurological Science, Stanford University, Stanford, United States Department of Structural Biology, Stanford University, Stanford, United States Department of Photon Science, Stanford University, Stanford, United States Howard Hughes Medical Institute, Stanford University, Stanford, United States.
authors.forename: Mark S
authors.initials: MS
authors.lastname: Padolina
authors.affiliation: Department of Molecular and Cellular Physiology, Stanford University, Stanford, United States Department of Neurology and Neurological Science, Stanford University, Stanford, United States Department of Structural Biology, Stanford University, Stanford, United States Department of Photon Science, Stanford University, Stanford, United States Howard Hughes Medical Institute, Stanford University, Stanford, United States brunger@stanford.edu.
authors.forename: Axel T
authors.initials: AT
authors.lastname: Brunger
mesh_terms: D033942:Adaptor Proteins, Vesicular Transport / Q000737:chemistry / Q000235:genetics / Q000378:metabolism; D000818:Animals; D001681:Biological Assay; D001692:Biological Transport; D002118:Calcium / Q000737:chemistry* / Q000378:metabolism; D004926:Escherichia coli / Q000235:genetics / Q000378:metabolism; D005089:Exocytosis; D015870:Gene Expression; D008561:Membrane Fusion; D008954:Models, Biological; D009154:Mutation; D009419:Nerve Tissue Proteins / Q000737:chemistry / Q000235:genetics / Q000378:metabolism; D009474:Neurons / Q000737:chemistry / Q000166:cytology / Q000378:metabolism; D011485:Protein Binding; D058849:Protein Refolding; D017434:Protein Structure, Tertiary; D051381:Rats; D011994:Recombinant Proteins / Q000737:chemistry / Q000235:genetics / Q000378:metabolism; D013572:Synaptic Vesicles / Q000737:chemistry* / Q000378:metabolism; D050825:Synaptosomal-Associated Protein 25 / Q000737:chemistry / Q000235:genetics / Q000378:metabolism; D050863:Synaptotagmin I / Q000737:chemistry / Q000235:genetics / Q000378:metabolism; D050827:Syntaxin 1 / Q000737:chemistry* / Q000235:genetics / Q000378:metabolism; D050988:Vesicle-Associated Membrane Protein 2 / Q000737:chemistry* / Q000235:genetics / Q000378:metabolism
chemical_list: D033942:Adaptor Proteins, Vesicular Transport; D009419:Nerve Tissue Proteins; D011994:Recombinant Proteins; C494210:Snap25 protein, rat; C494213:Stx1a protein, rat; D050825:Synaptosomal-Associated Protein 25; D050863:Synaptotagmin I; D050827:Syntaxin 1; C494312:Syt1 protein, rat; C494706:Vamp2 protein, rat; D050988:Vesicle-Associated Membrane Protein 2; C095992:complexin I; D002118:Calcium
keywords: SNARE; complexin; membrane fusion; neurotransmitter release; synaptic vesicle fusion; synaptotagmin