Synaphin/complexin is a cytosolic protein that preferentially binds to syntaxin within the SNARE complex. We find that synaphin promotes SNAREs to form precomplexes that oligomerize into higher order structures. A peptide from the central, syntaxin binding domain of synaphin competitively inhibits these two proteins from interacting and prevents SNARE complexes from oligomerizing. Injection of this peptide into squid giant presynaptic terminals inhibited neurotransmitter release at a late prefusion step of synaptic vesicle exocytosis. We propose that oligomerization of SNARE complexes into a higher order structure creates a SNARE scaffold for efficient, regulated fusion of synaptic vesicles.

The dynamics of exocytosis are diverse and have been optimized for the functions of synapses and a wide variety of cell types. For example, the kinetics of exocytosis varies by more than five orders of magnitude between ultrafast exocytosis in synaptic vesicles and slow exocytosis in large dense-core vesicles. However, in all cases, exocytosis is mediated by the same fundamental mechanism, i.e., the assembly of soluble N-ethylmaleimide-sensitive factor attachment protein receptor (SNARE) proteins. It is often assumed that vesicles need to be docked at the plasma membrane and SNARE proteins must be preassembled before exocytosis is triggered. However, this model cannot account for the dynamics of exocytosis recently reported in synapses and other cells. For example, vesicles undergo exocytosis without prestimulus docking during tonic exocytosis of synaptic vesicles in the active zone. In addition, epithelial and hematopoietic cells utilize cAMP and kinases to trigger slow exocytosis of nondocked vesicles. In this review, we summarize the manner in which the diversity of exocytosis reflects the initial configurations of SNARE assembly, including trans-SNARE, binary-SNARE, unitary-SNARE, and cis-SNARE configurations. The initial SNARE configurations depend on the particular SNARE subtype (syntaxin, SNAP25, or VAMP), priming proteins (Munc18, Munc13, CAPS, complexin, or snapin), triggering proteins (synaptotagmins, Doc2, and various protein kinases), and the submembraneous cytomatrix, and they are the key to determining the kinetics of subsequent exocytosis. These distinct initial configurations will help us clarify the common SNARE assembly processes underlying exocytosis and membrane trafficking in eukaryotic cells.

Neurotransmitter release relies on a series of synaptic vesicle trafficking reactions. We have determined the molecular basis of these reactions by microinjecting, into 'giant' nerve terminals of squid, probes that interfere with presynaptic proteins. These probes affect neurotransmitter release and disrupt nerve terminal structure. From the nature of these lesions, it is possible to deduce the roles of individual proteins in specific vesicle trafficking reactions. This approach has revealed the function of more than a dozen presynaptic proteins and we hypothesize that neurotransmitter release requires the coordinated action of perhaps 50-100 proteins.

It remains unclear how readiness for Ca2+-dependent exocytosis depends on varying degrees of SNARE complex assembly. Here we directly investigate the SNARE assembly using two-photon fluorescence lifetime imaging (FLIM) of Förster resonance energy transfer (FRET) between three pairs of neuronal SNAREs in presynaptic boutons and pancreatic β cells in the islets of Langerhans. These FRET probes functionally rescue their endogenous counterparts, supporting ultrafast exocytosis. We show that trans-SNARE complexes accumulated in the active zone, and estimate the number of complexes associated with each docked vesicle. In contrast, SNAREs were unassembled in resting state, and assembled only shortly prior to insulin exocytosis, which proceeds slowly. We thus demonstrate that distinct states of fusion readiness are associated with SNARE complex formation. Our FRET/FLIM approaches enable optical imaging of fusion readiness in both live and chemically fixed tissues.

Although the binding of synaphin (also called complexin) to the soluble N-ethylmaleimide-sensitive factor attachment protein receptor (SNARE) complex is critical for synaptic vesicle exocytosis, the exact role of synaphin remains unclear. Here, we show that synaphin directly binds to synaptotagmin 1, a major Ca(2+) sensor for fast neurotransmitter release, in a 1:1 stoichiometry. Mapping of the synaphin site involved in synaptotagmin 1 binding revealed that the C-terminal region is essential for the interaction between these two proteins. Binding was sensitive to ionic strength, suggesting the involvement of charged residues in the C-terminus region. Mutation of the seven consecutive glutamic acid residues (residues 108-114) at the C-terminal region of synaphin to alanines or glutamines resulted in a dramatic reduction in synaptotagmin 1 binding activity. Furthermore, a peptide from the C-terminus of synaphin (residues 91-124) blocked the binding of synaptotagmin 1 to synaphin, an effect that was abolished by mutating the consecutive glutamic acid residues to alanine. Immunoprecipitation experiments with brain membrane extracts showed the presence of a complex consisting of synaphin, synaptotagmin 1, and SNAREs. We propose that synaphin recruits synaptotagmin 1 to the SNARE-based fusion complex and synergistically functions with synaptotagmin 1 in mediating fast synaptic vesicle exocytosis.

Genetic studies in mice, worms and flies indicate that the synaptic protein UNC-13 plays a central role in the regulation of vesicle fusion.

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