Toulmé Estelle, Lázaro Andrea Salazar, Trimbuch Thorsten, Rizo Josep, Rosenmund Christian
Charité - Universitätsmedizin Berlin, corporate member of Freie Universität Berlin and Humboldt-Universität zu Berlin, Institute of Neurophysiology, Berlin, Germany.
NeuroCure Cluster of Excellence, Berlin, Germany.
bioRxiv. 2024 Jun 21:2024.06.17.599435. doi: 10.1101/2024.06.17.599435.
The Ca sensor synaptotagmin-1 triggers neurotransmitter release together with the neuronal SNARE complex formed by syntaxin-1, SNAP25 and synaptobrevin. Moreover, synaptotagmin-1 increases synaptic vesicle priming and impairs spontaneous vesicle release. The synaptotagmin-1 CB domain binds to the SNARE complex through a primary interface via two regions (I and II), but how exactly this interface mediates distinct functions of synaptotagmin-1, and the mechanism underlying Ca-triggering of release is unknown. Using mutagenesis and electrophysiological experiments, we show that region II is functionally and spatially subdivided: binding of C2B domain arginines to SNAP-25 acidic residues at one face of region II is crucial for Ca-evoked release but not for vesicle priming or clamping of spontaneous release, whereas other SNAP-25 and syntaxin-1 acidic residues at the other face mediate priming and clamping of spontaneous release but not evoked release. Mutations that disrupt region I impair the priming and clamping functions of synaptotagmin-1 while, strikingly, mutations that enhance binding through this region increase vesicle priming and clamping of spontaneous release, but strongly inhibit evoked release and vesicle fusogenicity. These results support previous findings that the primary interface mediates the functions of synaptotagmin-1 in vesicle priming and clamping of spontaneous release, and, importantly, show that Ca-triggering of release requires a rearrangement of the primary interface involving dissociation of region I, while region II remains bound. Together with modeling and biophysical studies presented in the accompanying paper, our data suggest a model whereby this rearrangement pulls the SNARE complex to facilitate fast synaptic vesicle fusion.
钙离子传感器突触结合蛋白-1与由 syntaxin-1、SNAP25 和突触小泡蛋白形成的神经元 SNARE 复合体共同触发神经递质释放。此外，突触结合蛋白-1 可增加突触小泡的预激活，并损害自发小泡释放。突触结合蛋白-1 的 C2B 结构域通过两个区域（I 和 II）经由一个主要界面与 SNARE 复合体结合，但该界面究竟如何介导突触结合蛋白-1 的不同功能以及钙离子触发释放的机制尚不清楚。通过诱变和电生理实验，我们发现区域 II 在功能和空间上是可细分的：C2B 结构域精氨酸与区域 II 一侧的 SNAP-25 酸性残基结合对于钙离子诱发的释放至关重要，但对于小泡预激活或自发释放的钳制则并非如此，而另一侧的其他 SNAP-25 和 syntaxin-1 酸性残基介导自发释放的预激活和钳制，但不介导诱发释放。破坏区域 I 的突变会损害突触结合蛋白-1 的预激活和钳制功能，而引人注目的是，通过该区域增强结合的突变会增加小泡预激活和自发释放的钳制，但会强烈抑制诱发释放和小泡融合能力。这些结果支持了先前的发现，即主要界面介导突触结合蛋白-1 在小泡预激活和自发释放钳制中的功能，并且重要的是，表明钙离子触发释放需要主要界面的重排，涉及区域 I 的解离，而区域 II 保持结合状态。连同随附论文中呈现的建模和生物物理研究，我们的数据提出了一个模型，据此这种重排拉动 SNARE 复合体以促进快速突触小泡融合。
