Reconstitution of the vital functions of Munc18 and Munc13 in neurotransmitter release.
Reconstitution of the vital functions of Munc18 and Munc13 in neurotransmitter release.
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Munc18 和 Munc13 在神经递质释放中重要功能的重建
DOI：
10.1126/science.1230473
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发表时间：
2013-01-25
期刊：
影响因子：
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通讯作者：
Rizo J
中科院分区：
文献类型：
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作者：
Ma C;Su L;Seven AB;Xu Y;Rizo J
膜融合反应在体外已被重建，但重建的反应往往不能直接反映体内突触囊泡融合的要求。以往的研究一般只使用n -乙基丙烯酰亚胺敏感因子（NSF）附着蛋白SNAP受体（SNAREs）和一种或两种附加成分，无法解释为什么Munc18-1或Munc13的缺失会完全消除神经递质释放，导致敲除小鼠突触囊泡释放严重中断。Ma等人（第421页，12月20日在线发表；参见Hughson的观点）现在提出了突触囊泡融合的忠实重建。当重组实验包含所有八个关键成分（三个SNAREs， Munc18-1, Munc13, synaptotagmin-1， NSF和α-SNAP）时，膜融合需要Munc18-1和Munc13。神经递质释放的一个模型解释了为什么在体外不需要膜融合的两种蛋白质在体内是需要的。神经递质释放主要依赖于Munc18-1、Munc13、Ca2+传感器synaptotagmin-1和可溶性n-乙基甲酰亚胺敏感因子（NSF）附着蛋白（SNAP）受体（SNAREs） syntaxin-1、synaptobrevin和SNAP-25。体外重构表明syntaxin-1-SNAP-25脂质体在synaptotagin -1 - ca2 +存在下与synaptobrevin脂质体有效融合，但体内神经递质释放也需要Munc18-1和Munc13。我们发现Munc18-1可以取代syntaxin-1中的SNAP-25，并且syntaxin-1 - Munc18-1脂质体与synaptobrevin脂质体的融合需要Munc13，以及SNAP-25和synaptotagmin-1-Ca2+。此外，当从syntaxin-1-SNAP-25脂质体开始时，NSF -α-SNAP会分解syntaxin-1-SNAP-25异源二聚体并取消融合，然后需要Munc18-1和Munc13。我们认为融合不通过syntaxin-1-SNAP-25异源二聚体进行，而是从syntaxin-1-Munc18-1复合物开始；然后Munc18-1和Munc13以抗NSF和snap的方式与SNAREs和synaptotagmin-1-Ca2+一起协调膜融合。
Reconstituting Synaptic Vesicle Fusion Membrane fusion reactions have been reconstituted in vitro, but often the reconstituted reactions have not directly mirrored the requirements for synaptic vesicle fusion in vivo. Previous work generally used only N-ethylmaleimide–sensitive factor (NSF) attachment protein SNAP receptors (SNAREs) and one or two additional components and could not explain why deletion of Munc18-1 or Munc13 abolishes neurotransmitter release completely, yielding the severe disruptions of synaptic vesicle release in knockout mouse. Ma et al. (p. 421, published online 20 December; see the Perspective by Hughson) now present a faithful reconstitution of synaptic vesicle fusion. Membrane fusion required Munc18-1 and Munc13 when the reconstitution experiments included all eight key components (three SNAREs, Munc18-1, Munc13, synaptotagmin-1, NSF, and α-SNAP). A model of neurotransmitter release explains why two proteins not needed for membrane fusion in vitro are needed in vivo. [Also see Perspective by Hughson] Neurotransmitter release depends critically on Munc18-1, Munc13, the Ca2+ sensor synaptotagmin-1, and the soluble N-ethylmaleimide–sensitive factor (NSF) attachment protein (SNAP) receptors (SNAREs) syntaxin-1, synaptobrevin, and SNAP-25. In vitro reconstitutions have shown that syntaxin-1–SNAP-25 liposomes fuse efficiently with synaptobrevin liposomes in the presence of synaptotagmin-1–Ca2+, but neurotransmitter release also requires Munc18-1 and Munc13 in vivo. We found that Munc18-1 could displace SNAP-25 from syntaxin-1 and that fusion of syntaxin-1–Munc18-1 liposomes with synaptobrevin liposomes required Munc13, in addition to SNAP-25 and synaptotagmin-1-Ca2+. Moreover, when starting with syntaxin-1–SNAP-25 liposomes, NSF–α-SNAP disassembled the syntaxin-1–SNAP-25 heterodimers and abrogated fusion, which then required Munc18-1 and Munc13. We propose that fusion does not proceed through syntaxin-1–SNAP-25 heterodimers but starts with the syntaxin-1–Munc18-1 complex; Munc18-1 and Munc13 then orchestrate membrane fusion together with the SNAREs and synaptotagmin-1-Ca2+ in an NSF- and SNAP-resistant manner.