Department of Molecular and Cellular Physiology, Stanford University, Stanford, CA 94305.
Department of Neurology and Neurological Sciences, Stanford University, Stanford, CA 94305.
Proc Natl Acad Sci U S A. 2017 Aug 22;114(34):9110-9115. doi: 10.1073/pnas.1708492114. Epub 2017 Jul 24.
Neurotransmitter release is orchestrated by synaptic proteins, such as SNAREs, synaptotagmin, and complexin, but the molecular mechanisms remain unclear. We visualized functionally active synaptic proteins reconstituted into proteoliposomes and their interactions in a native membrane environment by electron cryotomography with a Volta phase plate for improved resolvability. The images revealed individual synaptic proteins and synaptic protein complex densities at prefusion contact sites between membranes. We observed distinct morphologies of individual synaptic proteins and their complexes. The minimal system, consisting of neuronal SNAREs and synaptotagmin-1, produced point and long-contact prefusion states. Morphologies and populations of these states changed as the regulatory factors complexin and Munc13 were added. Complexin increased the membrane separation, along with a higher propensity of point contacts. Further inclusion of the priming factor Munc13 exclusively restricted prefusion states to point contacts, all of which efficiently fused upon Ca triggering. We conclude that synaptic proteins have evolved to limit possible contact site assemblies and morphologies to those that promote fast Ca-triggered release.
神经递质的释放是由突触蛋白（如 SNAREs、突触结合蛋白和复合物素）协调的，但分子机制仍不清楚。我们通过带有 Volta 相板的电子晶体断层扫描，在天然膜环境中可视化功能活跃的重组到类脂体中的突触蛋白及其相互作用，以提高分辨率。这些图像揭示了在膜之间的融合前接触点处，单个突触蛋白和突触蛋白复合物的密度。我们观察到了单个突触蛋白及其复合物的独特形态。由神经元 SNAREs 和突触结合蛋白-1 组成的最小系统产生了点状和长接触融合前状态。随着调节因子复合物素和 Munc13 的加入，这些状态的形态和群体发生了变化。复合物素增加了膜分离，同时增加了点状接触的倾向。进一步加入启动因子 Munc13 则将融合前状态专门限制在点状接触上，所有这些状态在 Ca 触发时都能有效地融合。我们得出的结论是，突触蛋白已经进化到将可能的接触点组装和形态限制在那些促进快速 Ca 触发释放的范围内。
