SNARE proteins contribute to calcium cooperativity of synaptic transmission (original) (raw)
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eLife
The synaptic vesicle Ca2+ sensor Synaptotagmin binds Ca2+ through its two C2 domains to trigger membrane interactions. Beyond membrane insertion by the C2 domains, other requirements for Synaptotagmin activity are still being elucidated. To identify key residues within Synaptotagmin required for vesicle cycling, we took advantage of observations that mutations in the C2B domain Ca2+-binding pocket dominantly disrupt release from invertebrates to humans. We performed an intragenic screen for suppressors of lethality induced by expression of Synaptotagmin C2B Ca2+-binding mutants in Drosophila. This screen uncovered essential residues within Synaptotagmin that suggest a structural basis for several activities required for fusion, including a C2B surface implicated in SNARE complex interaction that is required for rapid synchronization and Ca2+ cooperativity of vesicle release. Using electrophysiological, morphological and computational characterization of these mutants, we propose a s...
2001
The cytoplasmic H3 helical domain of syntaxin is implicated in numerous protein-protein interactions required for the assembly and stability of the SNARE complex mediating vesicular fusion at the synapse. Two specific hydrophobic residues (Ala-240, Val-244) in H3 layers 4 and 5 of mammalian syntaxin1A have been suggested to be involved in SNARE complex stability and required for the inhibitory effects of syntaxin on N-type calcium channels. We have generated the equivalent double point mutations in Drosophila syntaxin1A (A243V, V247A; syx 4 mutant) to examine their significance in synaptic transmission in vivo. The syx 4 mutant animals are embryonic lethal and display severely impaired neuronal secretion, although nonneuronal secretion appears normal. Synaptic transmission is nearly abolished, with residual transmission delayed, highly variable, and nonsynchronous, strongly reminiscent of transmission in null synaptotagmin I mutants. However, the syx 4 mutants show no alterations in synaptic protein levels in vivo or syntaxin partner binding interactions in vitro. Rather, syx 4 mutant animals have severely impaired hypertonic saline response in vivo, an assay indicating loss of fusion-competent synaptic vesicles, and in vitro SNARE complexes containing Syx 4 protein have significantly compromised stability. These data suggest that the same residues required for syntaxin-mediated calcium channel inhibition are required for the generation of fusion-competent vesicles in a neuronal-specific mechanism acting at synapses.
Proceedings of the National Academy of Sciences, 1994
Since the demonstration that Ca2+ influx into the presynaptic terminal is essential for neurotransmitter release, there has been much speculation about the Ca2+ receptor responsible for initiating exocytosis. Numerous experiments have shown that the protein, or protein complex, binds multiple Ca2+ ions, resides near the site of Ca2+ influx, and has a relatively low affinity for Ca2+. Synaptotagmin is an integral membrane protein of synaptic vesicles that contains two copies of a domain known to be involved in Ca(2+)-dependent membrane interactions. Synaptotagmin has been shown to bind Ca2+ in vitro with a relatively low affinity. In addition, synaptotagmin has been shown to bind indirectly to Ca2+ channels, positioning the protein close to the site of Ca2+ influx. Recently, a negative regulatory role for synaptotagmin has been proposed, in which it functions as a clamp to prevent fusion of synaptic vesicles with the presynaptic membrane. Release of the clamp would allow exocytosis. ...
Structural determinants for SNARE-mediated neurosecretion
2010
14 synaptobrevin2) and the synaptic plasma membrane proteins syntaxin1A and SNAP-25 [44-47]. A molecular model of SNARE-mediated vesicle exocytosis has emerged within the last 30 years [2, 48] (Figure 4). This model of regulated exocytosis consists of a series of transition steps that are controlled by additional late regulatory proteins including synaptotagmin, complexin, tomosyn, Munc-13, syntaphilin and snapin [49-55]. The process begins when syntaxin1A and SNAP-25, which are organized in clusters at the plasma membrane [56, 57], assemble together to form a binary complex called acceptor complex [58-60]. The acceptor complex provides a binding interface for the vesicular SNARE VAMP2, thus forming a ternary complex. Figure 4. Model for SNARE-mediated neuronal exocytosis. The neuronal t-SNAREs SNAP-25 and syntaxin1A (labelled in green and red, respectively), assemble together to form the acceptor complex followed by binding of the v-SNARE partner VAMP2 (in blue). The three SNARE proteins form the trans-SNARE complex that brings opposing membranes into close proximity awaiting a Ca 2+ signal. Additional proteins such as synaptotagmin (purple) and complexin (pink) bind to this trans-SNARE complex with possibly distinct outcomes. Ca 2+ entry triggers membrane fusion, followed by the generation of cis-SNARE complexes, which are disassembled by NSF and α-SNAP upon ATP-hydrolysis This ternary complex proceeds from a loose state (in which only the Nterminal part is assembled) as studied in vivo [61-63] and in vitro [64] to a tight Ca + Calcium influx Membrane fusion Synaptobrevin/Vamp
Synaptotagmin-1 may be a distance regulator acting upstream of SNARE nucleation
Nature Structural & Molecular Biology, 2011
Synaptotagmin-1 triggers Ca 2+ -sensitive, rapid neurotransmitter release by promoting the interaction of SNARE proteins between the synaptic vesicles and the plasma membrane. How synaptotagmin-1 promotes this interaction is controversial, and the massive increase in membrane fusion efficiency of Ca 2+ -synaptotagmin-1 has not been reproduced in vitro. However, previous experiments have been performed at relatively high salt concentrations, screening potentially important electrostatic interactions. Using functional reconstitution in liposomes, we show here that at low ionic strength SNARE-mediated membrane fusion becomes strictly dependent on both Ca 2+ and synaptotagmin-1. Under these conditions, synaptotagmin-1 functions as a distance regulator: tethering the liposomes too far for SNARE nucleation in the absence of Ca 2+ , but brings the liposomes close enough for membrane fusion in the presence of Ca 2+ . These results may explain how the relatively weak electrostatic interactions of synaptotagmin-1 with membranes substantially accelerate fusion.
Molecular biology of the cell, 2007
Synaptotagmins contain tandem C2 domains and function as Ca(2+) sensors for vesicle exocytosis but the mechanism for coupling Ca(2+) rises to membrane fusion remains undefined. Synaptotagmins bind SNAREs, essential components of the membrane fusion machinery, but the role of these interactions in Ca(2+)-triggered vesicle exocytosis has not been directly assessed. We identified sites on synaptotagmin-1 that mediate Ca(2+)-dependent SNAP25 binding by zero-length cross-linking. Mutation of these sites in C2A and C2B eliminated Ca(2+)-dependent synaptotagmin-1 binding to SNAREs without affecting Ca(2+)-dependent membrane binding. The mutants failed to confer Ca(2+) regulation on SNARE-dependent liposome fusion and failed to restore Ca(2+)-triggered vesicle exocytosis in synaptotagmin-deficient PC12 cells. The results provide direct evidence that Ca(2+)-dependent SNARE binding by synaptotagmin is essential for Ca(2+)-triggered vesicle exocytosis and that Ca(2+)-dependent membrane binding...
Syntaxin and synaptobrevin function downstream of vesicle docking in drosophila
Neuron, 1995
In synaptic transmission, vesicles are proposed to dock at presynaptic active zones by the association of synaptobrevin (v-SNARE) with syntaxin (t-SNARE). We test this hypothesis in Drosophila strains lacking neural synaptobrevin (n-synaptobrevin) or syntaxin. We showed previously that loss of either protein completely blocks synaptic transmission. Here, we attempt to establish the level of this blockade. Ultrastructurally, vesicles are still targeted to the presynaptic membrane and dock normally at specialized release sites. These vesicles are mature and functional since spontaneous vesicle fusion persists in the absence of n-synaptobrevin and since vesicle fusion is triggered by hyperosmotic saline in the absence of syntaxin. We conclude that the SNARE hypothesis cannot fully explain the role of these proteins in synaptic transmission. Instead, both proteins play distinct roles downstream of docking.
The primed SNARE–complexin–synaptotagmin complex for neuronal exocytosis
Nature, 2017
Synaptotagmin, complexin and neuronal SNARE proteins mediate evoked synchronous neurotransmitter release, but the molecular mechanisms mediating the cooperation between these molecules remain unclear. Here, we determined crystal structures of the primed pre-fusion SNARE-complexin-synaptotagmin-1 complex. These structures reveal an unexpected tripartite interface between synaptotagmin-1 and both the SNARE complex and complexin. Simultaneously, a second synaptotagmin-1 molecule interacted with the other side of the SNARE complex via the previously identified primary interface. Mutations that disrupt either interface in solution also severely impaired evoked synchronous release in neurons, suggesting that both interfaces are essential for the primed pre-fusion state. Ca 2+ binding to the synaptotagmin-1 molecules unlocks the complex, allows full zippering of the SNARE complex, and triggers membrane fusion. The tripartite SNARE-complexin-synaptotagmin-1 complex at a synaptic vesicle docking site has to be unlocked for triggered fusion to commence, explaining the cooperation between complexin and synaptotagmin-1 in synchronizing evoked release on the sub-millisecond timescale. During synaptic transmission, Ca 2+ influx into a presynaptic terminal triggers fusion of neurotransmitter-filled synaptic vesicles with the presynaptic plasma membrane 1,2. The SNARE (for Soluble N-ethylmaleimide sensitive factor Attachment protein REceptor) proteins synaptobrevin-2/VAMP2 on the synaptic vesicle and syntaxin-1A and SNAP-25 on the plasma membrane initiate vesicle fusion by forming a trans-SNARE complex before Ca 2+ triggering 3,4. In addition to SNAREs, synaptotagmin-1 (Syt1) is vital for Ca 2+triggered synaptic vesicle fusion 5,6. Syt1 contains a single transmembrane-spanning domain Reprints and permissions information is available at www.nature.com/reprints.
SNARE Protein Recycling by αSNAP and βSNAP Supports Synaptic Vesicle Priming
Neuron, 2012
Neurotransmitter release proceeds by Ca 2+-triggered, SNARE-complex-dependent synaptic vesicle fusion. After fusion, the ATPase NSF and its cofactors aand bSNAP disassemble SNARE complexes, thereby recycling individual SNAREs for subsequent fusion reactions. We examined the effects of genetic perturbation of aand bSNAP expression on synaptic vesicle exocytosis, employing a new Ca 2+ uncaging protocol to study synaptic vesicle trafficking, priming, and fusion in small glutamatergic synapses of hippocampal neurons. By characterizing this protocol, we show that synchronous and asynchronous transmitter release involve different Ca 2+ sensors and are not caused by distinct releasable vesicle pools, and that tonic transmitter release is due to ongoing priming and fusion of new synaptic vesicles during high synaptic activity. Our analysis of aand bSNAP deletion mutant neurons shows that the two NSF cofactors support synaptic vesicle priming by determining the availability of free SNARE components, particularly during phases of high synaptic activity.
The Journal of Comparative Neurology, 2006
Synaptotagmin I is a synaptic vesicle protein postulated to mediate vesicle docking, vesicle recycling, and the Ca 2ϩ sensing required to trigger vesicle fusion. Analysis of synaptotagmin I knockouts (sytI NULL mutants) in both Drosophila and mice led to these hypotheses. Although much research on the mechanisms of synaptic transmission in Drosophila is performed at the third instar neuromuscular junction, the ultrastructure of this synapse has never been analyzed in sytI NULL mutants. Here we report severe synaptic vesicle depletion, an accumulation of large vesicles, and decreased vesicle docking at sytI NULL third instar neuromuscular junctions. Mutations in synaptotagmin I's C 2 B Ca 2ϩ -binding motif nearly abolish synaptic transmission and decrease the apparent Ca 2ϩ affinity of neurotransmitter release. Although this result is consistent with disruption of the Ca 2ϩ sensor, synaptic vesicle depletion and/or redistribution away from the site of Ca 2ϩ influx could produce a similar phenotype. To address this question, we examined vesicle distributions at neuromuscular junctions from third instar C 2 B Ca 2ϩ -binding motif mutants and transgenic wild-type controls. The number of docked vesicles and the overall number of synaptic vesicles in the vicinity of active zones was unchanged in the mutants. We conclude that the near elimination of synaptic transmission and the decrease in the Ca 2ϩ affinity of release observed in C 2 B Ca 2ϩ -binding motif mutants is not due to altered synaptic vesicle distribution but rather is a direct result of disrupting synaptotagmin I's ability to bind Ca 2ϩ . Thus, Ca 2ϩ binding by the C 2 B domain mediates a postdocking step in fusion.