Complexin inhibits spontaneous release and synchronizes Ca2+-triggered synaptic vesicle fusion by distinct mechanisms

Elife. 2014 Aug 13:3:e03756. doi: 10.7554/eLife.03756.

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.

Keywords: SNARE; complexin; membrane fusion; neurotransmitter release; synaptic vesicle fusion; synaptotagmin.

Publication types

  • Research Support, N.I.H., Extramural
  • Research Support, Non-U.S. Gov't

MeSH terms

  • Adaptor Proteins, Vesicular Transport / chemistry
  • Adaptor Proteins, Vesicular Transport / genetics
  • Adaptor Proteins, Vesicular Transport / metabolism
  • Animals
  • Biological Assay
  • Biological Transport
  • Calcium / chemistry*
  • Calcium / metabolism
  • Escherichia coli / genetics
  • Escherichia coli / metabolism
  • Exocytosis
  • Gene Expression
  • Membrane Fusion*
  • Models, Biological
  • Mutation
  • Nerve Tissue Proteins / chemistry
  • Nerve Tissue Proteins / genetics
  • Nerve Tissue Proteins / metabolism
  • Neurons / chemistry
  • Neurons / cytology
  • Neurons / metabolism
  • Protein Binding
  • Protein Refolding
  • Protein Structure, Tertiary
  • Rats
  • Recombinant Proteins / chemistry
  • Recombinant Proteins / genetics
  • Recombinant Proteins / metabolism
  • Synaptic Vesicles / chemistry*
  • Synaptic Vesicles / metabolism
  • Synaptosomal-Associated Protein 25 / chemistry
  • Synaptosomal-Associated Protein 25 / genetics
  • Synaptosomal-Associated Protein 25 / metabolism
  • Synaptotagmin I / chemistry
  • Synaptotagmin I / genetics
  • Synaptotagmin I / metabolism
  • Syntaxin 1 / chemistry*
  • Syntaxin 1 / genetics
  • Syntaxin 1 / metabolism
  • Vesicle-Associated Membrane Protein 2 / chemistry*
  • Vesicle-Associated Membrane Protein 2 / genetics
  • Vesicle-Associated Membrane Protein 2 / metabolism

Substances

  • Adaptor Proteins, Vesicular Transport
  • Nerve Tissue Proteins
  • Recombinant Proteins
  • Snap25 protein, rat
  • Stx1a protein, rat
  • Synaptosomal-Associated Protein 25
  • Synaptotagmin I
  • Syntaxin 1
  • Syt1 protein, rat
  • Vamp2 protein, rat
  • Vesicle-Associated Membrane Protein 2
  • complexin I
  • Calcium