RESUMEN
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.
Asunto(s)
Proteínas Munc18/fisiología , Proteínas Solubles de Unión al Factor Sensible a la N-Etilmaleimida/fisiología , Vesículas Sinápticas/metabolismo , Animales , Bovinos , Cricetulus , Escherichia coli/genética , Fusión de Membrana , Proteínas Munc18/química , Proteínas Munc18/metabolismo , Conformación Proteica , Ratas , Proteínas SNARE/metabolismo , Proteínas SNARE/fisiología , Proteínas Solubles de Unión al Factor Sensible a la N-Etilmaleimida/genética , Proteínas Solubles de Unión al Factor Sensible a la N-Etilmaleimida/metabolismo , Sintaxina 1/química , Sintaxina 1/metabolismoRESUMEN
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.
Asunto(s)
Proteínas Adaptadoras del Transporte Vesicular/química , Proteínas Munc18/química , Proteínas Sensibles a N-Etilmaleimida/química , Proteínas del Tejido Nervioso/química , Proteína 25 Asociada a Sinaptosomas/química , Sinaptotagminas/química , Animales , Células CHO , Calcio/química , Cricetinae , Cricetulus , Microscopía por Crioelectrón , Citoplasma/química , Transferencia Resonante de Energía de Fluorescencia , Cinética , Mutación , Proteínas R-SNARE/química , Ratas , Sintaxina 1/químicaRESUMEN
Neurotransmitter release depends on the SNARE complex formed by syntaxin-1, synaptobrevin and SNAP-25, as well as on complexins, which bind to the SNARE complex and play active and inhibitory roles. A crystal structure of a Complexin-I fragment bearing a so-called 'superclamp' mutation bound to a truncated SNARE complex lacking the C-terminus of the synaptobrevin SNARE motif (SNAREΔ60) suggested that an 'accessory' α-helix of Complexin-I inhibits release by inserting into the C-terminus of the SNARE complex. Previously, isothermal titration calorimetry (ITC) experiments performed in different laboratories yielded apparently discrepant results in support or against the existence of such binding mode in solution (Trimbuch et al., 2014; Krishnakumar et al., 2015). Here, ITC experiments performed to solve these discrepancies now show that the region containing the Complexin-I accessory helix and preceding N-terminal sequences does interact with SNAREΔ60, but the interaction requires the polybasic juxtamembrane region of syntaxin-1 and is not affected by the superclamp mutation within the experimental error of these experiments.
Asunto(s)
Proteínas Adaptadoras del Transporte Vesicular/metabolismo , Calorimetría/métodos , Complejos Multiproteicos/metabolismo , Proteínas del Tejido Nervioso/metabolismo , Proteínas SNARE/metabolismo , Proteínas Adaptadoras del Transporte Vesicular/química , Animales , Membrana Celular/metabolismo , Humanos , Complejos Multiproteicos/química , Proteínas del Tejido Nervioso/química , Unión Proteica , Estructura Secundaria de Proteína , Proteínas R-SNARE/metabolismo , Ratas , Proteínas SNARE/química , Proteína 25 Asociada a Sinaptosomas/metabolismo , Sintaxina 1/metabolismoRESUMEN
Rapid neurotransmitter release depends on the Ca2+ sensor Synaptotagmin-1 (Syt1) and the SNARE complex formed by synaptobrevin, syntaxin-1 and SNAP-25. How Syt1 triggers release has been unclear, partly because elucidating high-resolution structures of Syt1-SNARE complexes has been challenging. An NMR approach based on lanthanide-induced pseudocontact shifts now reveals a dynamic binding mode in which basic residues in the concave side of the Syt1 C2B-domain ß-sandwich interact with a polyacidic region of the SNARE complex formed by syntaxin-1 and SNAP-25. The physiological relevance of this dynamic structural model is supported by mutations in basic residues of Syt1 that markedly impair SNARE-complex binding in vitro and Syt1 function in neurons. Mutations with milder effects on binding have correspondingly milder effects on Syt1 function. Our results support a model whereby dynamic interaction facilitates cooperation between Syt1 and the SNAREs in inducing membrane fusion.
