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.

除其他因素外，神经递质释放受八种中心蛋白控制：神经元 SNAREsyntaxin-1、synaptobrevin 和 SNAP-25，它们形成一个紧密的 SNARE 复合物，将突触小泡和质膜结合在一起；NSF 和 SNAP，分解 SNARE 复合物；Munc18-1 和 Munc13-1，它们组织 SNARE 复合体组装；和 Ca ²⁺传感器 synaptotagmin-1。重组实验表明 Munc18-1、Munc13-1、NSF 和 α-SNAP 可以介导 Ca ²⁺synaptobrevin 脂质体和 syntaxin-1-SNAP-25 脂质体之间的依赖脂质体融合，但由于不受控制的 SNARE 复合物组装导致的高融合效率不允许研究 synaptotagmin-1 在融合中的作用。在这里，我们表明将相应脂质体中的突触蛋白与脂质的比例降低到非常低的水平会导致融合效率低下，并且突触结合蛋白-1 在这些条件下会强烈刺激融合。这种刺激依赖于 Ca ²⁺与 synaptotagmin-1的两个 C _{2结构域的结合。}我们还表明，将 SNAP-25 锚定在 syntaxin-1 脂质体上可显着增强融合。此外，我们发现了 synaptotagmin-1 和 SNAP-25 的膜锚定之间的协同作用，这允许有效的 Ca ²⁺具有非常低的 synaptobrevin 密度的脂质体和含有非常低的 synaptobrevin-1 密度的脂质体之间的依赖性融合。因此，在我们的分析中，脂质体融合是通过一些 SNARE 复合物以需要 Munc18-1 和 Munc13-1 并且依赖于 Ca ²⁺与突触结合蛋白-1 结合的方式实现的，所有这些都是神经元中神经递质释放的基本特征.