Multiple factors maintain assembled trans-SNARE complexes in the presence of NSF and αSNAP

Neurotransmitter release requires formation of trans-SNARE complexes between the synaptic vesicle and plasma membranes, which likely underlies synaptic vesicle priming to a release-ready state. It is unknown whether Munc18-1, Munc13-1, complexin-1 and synaptotagmin-1 are important for priming because they mediate trans-SNARE complex assembly and/or because they prevent trans-SNARE complex disassembly by NSF-αSNAP, which can lead to de-priming. Here we show that trans-SNARE complex formation in the presence of NSF-αSNAP requires both Munc18-1 and Munc13-1, as proposed previously, and is facilitated by synaptotagmin-1. Our data also show that Munc18-1, Munc13-1, complexin-1 and likely synaptotagmin-1 contribute to maintaining assembled trans-SNARE complexes in the presence of NSF-αSNAP. We propose a model whereby Munc18-1 and Munc13-1 are critical not only for mediating vesicle priming but also for precluding de-priming by preventing trans-SNARE complex disassembly; in this model, complexin-1 also impairs de-priming, while synaptotagmin-1 may assist in priming and hinder de-priming.

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A new life for an old pump: V-ATPase and neurotransmitter release

The Journal of Cell Biology ◽

10.1083/jcb.201403040 ◽

2014 ◽

Vol 205 (1) ◽

pp. 7-9 ◽

Cited By ~ 11

Author(s):

Stefano Vavassori ◽

Andreas Mayer

Keyword(s):

Plasma Membrane ◽

Complex Formation ◽

Membrane Fusion ◽

Synaptic Vesicle ◽

Neurotransmitter Release ◽

Synaptic Vesicles ◽

Snare Complex ◽

Spontaneous Release ◽

Synaptic Vesicle Fusion ◽

Snare Complex Formation

Neurons fire by releasing neurotransmitters via fusion of synaptic vesicles with the plasma membrane. Fusion can be evoked by an incoming signal from a preceding neuron or can occur spontaneously. Synaptic vesicle fusion requires the formation of trans complexes between SNAREs as well as Ca2+ ions. Wang et al. (2014. J. Cell Biol. http://dx.doi.org/jcb.201312109) now find that the Ca2+-binding protein Calmodulin promotes spontaneous release and SNARE complex formation via its interaction with the V0 sector of the V-ATPase.

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Conformational Change of Syntaxin-3b in Regulating SNARE Complex Assembly in the Ribbon Synapses

10.1101/2021.09.07.459295 ◽

2021 ◽

Author(s):

Claire Gething ◽

Joshua Ferrar ◽

Bishal Misra ◽

Giovanni Howells ◽

Ucheor B. Choi

Keyword(s):

Plasma Membrane ◽

Complex Formation ◽

Synaptic Vesicle ◽

Conformational Change ◽

Single Molecule ◽

Conformational Changes ◽

Snare Complex ◽

Complex Assembly ◽

Ribbon Synapses ◽

Snare Complex Formation

AbstractNeurotransmitter release of synaptic vesicles relies on the assembly of the soluble N-ethylmaleimide-sensitive factor attachment protein receptor (SNARE) complex, consisting of syntaxin and SNAP-25 on the plasma membrane and synaptobrevin on the synaptic vesicle. The formation of the SNARE complex progressively zippers towards the membranes, which drives membrane fusion between the plasma membrane and the synaptic vesicle. However, the underlying molecular mechanism of SNARE complex regulation is unclear. In this study, we investigate the syntaxin-3b isoform found in the retinal ribbon synapses using single-molecule fluorescence resonance energy transfer (smFRET) to monitor the conformational changes of syntaxin-3b that modulate the SNARE complex formation. We found that syntaxin-3b is predominantly in a self-inhibiting closed conformation, inefficiently forming the ternary SNARE complex. Conversely, a phosphomimetic mutation (T14E) at the N-terminal region of syntaxin-3b promoted the open conformation, similar to the constitutively open form of syntaxin LE mutant. When syntaxin-3b is bound to Munc18-1, SNARE complex formation is almost completely blocked. Surprisingly, the T14E mutation of syntaxin-3b partially abolishes Munc18-1 regulation, acting as a conformational switch to trigger SNARE complex assembly. Thus, we suggest a model where the conformational change of syntaxin-3b induced by phosphorylation initiates the release of neurotransmitters in the ribbon synapses.

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Functional synergy between the Munc13 C-terminal C1 and C2 domains

eLife ◽

10.7554/elife.13696 ◽

2016 ◽

Vol 5 ◽

Cited By ~ 45

Author(s):

Xiaoxia Liu ◽

Alpay Burak Seven ◽

Marcial Camacho ◽

Victoria Esser ◽

Junjie Xu ◽

...

Keyword(s):

Neurotransmitter Release ◽

Plasma Membranes ◽

Snare Complex ◽

Complex Assembly ◽

C2 Domains ◽

Vesicle Docking ◽

Synaptotagmin 1 ◽

Primed State ◽

Multiple Domains ◽

Stimulation Of

Neurotransmitter release requires SNARE complexes to bring membranes together, NSF-SNAPs to recycle the SNAREs, Munc18-1 and Munc13s to orchestrate SNARE complex assembly, and Synaptotagmin-1 to trigger fast Ca2+-dependent membrane fusion. However, it is unclear whether Munc13s function upstream and/or downstream of SNARE complex assembly, and how the actions of their multiple domains are integrated. Reconstitution, liposome-clustering and electrophysiological experiments now reveal a functional synergy between the C1, C2B and C2C domains of Munc13-1, indicating that these domains help bridging the vesicle and plasma membranes to facilitate stimulation of SNARE complex assembly by the Munc13-1 MUN domain. Our reconstitution data also suggest that Munc18-1, Munc13-1, NSF, αSNAP and the SNAREs are critical to form a ‘primed’ state that does not fuse but is ready for fast fusion upon Ca2+ influx. Overall, our results support a model whereby the multiple domains of Munc13s cooperate to coordinate synaptic vesicle docking, priming and fusion.

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Synaptotagmin-1–, Munc18-1–, and Munc13-1–dependent liposome fusion with a few neuronal SNAREs

Proceedings of the National Academy of Sciences ◽

10.1073/pnas.2019314118 ◽

2021 ◽

Vol 118 (4) ◽

pp. e2019314118

Author(s):

Karolina P. Stepien ◽

Josep Rizo

Keyword(s):

Neurotransmitter Release ◽

Plasma Membranes ◽

Snare Complex ◽

Complex Assembly ◽

Synaptotagmin 1 ◽

Low Levels ◽

Liposome Fusion ◽

Membrane Anchoring ◽

Fusion Efficiency

Neurotransmitter release is governed by eight central proteins among other factors: the neuronal SNAREs syntaxin-1, synaptobrevin, and SNAP-25, which form a tight SNARE complex that brings the synaptic vesicle and plasma membranes together; NSF and SNAPs, which disassemble SNARE complexes; Munc18-1 and Munc13-1, which organize SNARE complex assembly; and the Ca2+ sensor synaptotagmin-1. Reconstitution experiments revealed that Munc18-1, Munc13-1, NSF, and α-SNAP can mediate Ca2+-dependent liposome fusion between synaptobrevin liposomes and syntaxin-1–SNAP-25 liposomes, but high fusion efficiency due to uncontrolled SNARE complex assembly did not allow investigation of the role of synaptotagmin-1 on fusion. Here, we show that decreasing the synaptobrevin-to-lipid ratio in the corresponding liposomes to very low levels leads to inefficient fusion and that synaptotagmin-1 strongly stimulates fusion under these conditions. Such stimulation depends on Ca2+ binding to the two C2 domains of synaptotagmin-1. We also show that anchoring SNAP-25 on the syntaxin-1 liposomes dramatically enhances fusion. Moreover, we uncover a synergy between synaptotagmin-1 and membrane anchoring of SNAP-25, which allows efficient Ca2+-dependent fusion between liposomes bearing very low synaptobrevin densities and liposomes containing very low syntaxin-1 densities. Thus, liposome fusion in our assays is achieved with a few SNARE complexes in a manner that requires Munc18-1 and Munc13-1 and that depends on Ca2+ binding to synaptotagmin-1, all of which are fundamental features of neurotransmitter release in neurons.

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Munc18-1 is crucial to overcome the inhibition of synaptic vesicle fusion by αSNAP

Nature Communications ◽

10.1038/s41467-019-12188-4 ◽

2019 ◽

Vol 10 (1) ◽

Cited By ~ 8

Author(s):

Karolina P. Stepien ◽

Eric A. Prinslow ◽

Josep Rizo

Keyword(s):

Nmr Spectroscopy ◽

Complex Formation ◽

Synaptic Vesicle ◽

Active Zone ◽

Neurotransmitter Release ◽

Snare Complex ◽

Inhibitory Activities ◽

Liposome Fusion ◽

Synaptic Vesicle Fusion ◽

Snare Complex Formation

Abstract Munc18-1 and Munc13-1 orchestrate assembly of the SNARE complex formed by syntaxin-1, SNAP-25 and synaptobrevin, allowing exquisite regulation of neurotransmitter release. Non-regulated neurotransmitter release might be prevented by αSNAP, which inhibits exocytosis and SNARE-dependent liposome fusion. However, distinct mechanisms of inhibition by αSNAP were suggested, and it is unknown how such inhibition is overcome. Using liposome fusion assays, FRET and NMR spectroscopy, here we provide a comprehensive view of the mechanisms underlying the inhibitory functions of αSNAP, showing that αSNAP potently inhibits liposome fusion by: binding to syntaxin-1, hindering Munc18-1 binding; binding to syntaxin-1-SNAP-25 heterodimers, precluding SNARE complex formation; and binding to trans-SNARE complexes, preventing fusion. Importantly, inhibition by αSNAP is avoided only when Munc18-1 binds first to syntaxin-1, leading to Munc18-1-Munc13-1-dependent liposome fusion. We propose that at least some of the inhibitory activities of αSNAP ensure that neurotransmitter release occurs through the highly-regulated Munc18-1-Munc13-1 pathway at the active zone.

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Dual inhibition of SNARE complex formation by tomosyn ensures controlled neurotransmitter release

The Journal of Cell Biology ◽

10.1083/jcb.200805150 ◽

2008 ◽

Vol 183 (2) ◽

pp. 323-337 ◽

Cited By ~ 52

Author(s):

Toshiaki Sakisaka ◽

Yasunori Yamamoto ◽

Sumiko Mochida ◽

Michiko Nakamura ◽

Kouki Nishikawa ◽

...

Keyword(s):

Complex Formation ◽

Neurotransmitter Release ◽

Snare Complex ◽

Protein Receptor ◽

Repeat Domain ◽

Dual Inhibition ◽

Presynaptic Nerve Terminals ◽

Synaptic Vesicle Fusion ◽

Deficient Mice ◽

Snare Complex Formation

Neurotransmitter release from presynaptic nerve terminals is regulated by soluble NSF attachment protein receptor (SNARE) complex–mediated synaptic vesicle fusion. Tomosyn inhibits SNARE complex formation and neurotransmitter release by sequestering syntaxin-1 through its C-terminal vesicle-associated membrane protein (VAMP)–like domain (VLD). However, in tomosyn-deficient mice, the SNARE complex formation is unexpectedly decreased. In this study, we demonstrate that the N-terminal WD-40 repeat domain of tomosyn catalyzes the oligomerization of the SNARE complex. Microinjection of the tomosyn N-terminal WD-40 repeat domain into neurons prevented stimulated acetylcholine release. Thus, tomosyn inhibits neurotransmitter release by catalyzing oligomerization of the SNARE complex through the N-terminal WD-40 repeat domain in addition to the inhibitory activity of the C-terminal VLD.

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A Stimulation Function of Synaptotagmin-1 in Ternary SNARE Complex Formation Dependent on Munc18 and Munc13

Frontiers in Molecular Neuroscience ◽

10.3389/fnmol.2017.00256 ◽

2017 ◽

Vol 10 ◽

Cited By ~ 9

Author(s):

Yun Li ◽

Shen Wang ◽

Tianzhi Li ◽

Le Zhu ◽

Yuanyuan Xu ◽

...

Keyword(s):

Complex Formation ◽

Snare Complex ◽

Synaptotagmin 1 ◽

Snare Complex Formation

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BETA- AND GAMMA-SYNUCLEINS MODULATE SYNAPTIC VESICLE-BINDING OF ALPHA-SYNUCLEIN

10.1101/2020.11.19.390419 ◽

2020 ◽

Author(s):

Kathryn E. Carnazza ◽

Lauren Komer ◽

André Pineda ◽

Yoonmi Na ◽

Trudy Ramlall ◽

...

Keyword(s):

Nervous System ◽

Synaptic Vesicle ◽

Neurotransmitter Release ◽

Synaptic Vesicles ◽

Direct Interaction ◽

Alpha Synuclein ◽

Snare Complex ◽

Complex Assembly ◽

Mutual Compensation ◽

Conflicting Evidence

SUMMARYα-Synuclein (αSyn), β-synuclein (βSyn), and γ-synuclein (γSyn) are abundantly expressed in the vertebrate nervous system. αSyn functions in neurotransmitter release via binding to and clustering synaptic vesicles and chaperoning of SNARE-complex assembly. The functions of βSyn and γSyn are unknown. Functional redundancy of the three synucleins and mutual compensation when one synuclein is deleted have been proposed, but with conflicting evidence. Here, we demonstrate that βSyn and γSyn have a reduced affinity towards membranes compared to αSyn, and that direct interaction of βSyn or γSyn with αSyn results in reduced membrane binding of αSyn. Our data suggest that all three synucleins affect synapse function, but only αSyn mediates the downstream function of vesicle clustering and SNARE-complex assembly, while βSyn and γSyn modulate the activity of αSyn through regulating its binding to synaptic vesicles.

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