Genetic analysis of synaptotagmin C2 domain specificity in regulating spontaneous and evoked neurotransmitter release.
Genetic analysis of synaptotagmin C2 domain specificity in regulating spontaneous and evoked neurotransmitter release.
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DOI：
10.1523/jneurosci.3214-12.2013
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发表时间：
2013-01-02
期刊：
影响因子：
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通讯作者：
Littleton JT
中科院分区：
文献类型：
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作者：
Lee J;Guan Z;Akbergenova Y;Littleton JT
突触囊泡融合介导神经元之间的通讯，并由钙离子的快速内流触发。融合的钙触发步骤受突触小泡跨膜蛋白突触素1(Syt1)的调节。Syt1含有两个胞质C2结构域，分别称为C2a和C2b，它们协调钙结合。尽管C2a和C2b具有相似的拓扑结构，但钙离子与C2b结构域的结合被认为是诱发囊泡释放的唯一关键触发因素。C2a结构域功能是否以及如何与C2b协调仍不清楚。在这项研究中，我们在果蝇中构建了一组Syt1嵌合结构，以描绘每个C2结构域在调节突触小泡融合中的独特和共享功能。仅含有单个C2结构域的Syt1转基因表达，或双重C2a-C2a或C2b-C2b嵌合体，在Syt1−/−缺失突变背景下未能恢复Syt1功能，表明C2a和C2b都是支持快速同步释放所特需的。破坏与这两个C2结构域结合的钙离子结合的突变未能挽救诱发的释放，但支持突触小泡对接和内吞作用，表明Syt1的这些功能不依赖于钙离子。双C2结构域钙结合突变体还促进了自发融合，同时显著增加了与天然Syt1共表达时的诱发性释放。综上所述，这些数据表明，突触传递可以通过Syt1的多聚体来调节，并且Syt1的两个C2结构域都是调节钙非依赖性自发融合和钙依赖性同步释放所唯一需要的。
Synaptic vesicle fusion mediates communication between neurons and is triggered by rapid influx of Ca2+. The Ca2+-triggering step for fusion is regulated by the synaptic vesicle transmembrane protein Synaptotagmin 1 (Syt1). Syt1 contains two cytoplasmic C2 domains, termed C2A and C2B, which coordinate Ca2+ binding. Although C2A and C2B share similar topology, binding of Ca2+ ions to the C2B domain has been suggested as the only critical trigger for evoked vesicle release. If and how C2A domain function is coordinated with C2B remains unclear. In this study, we generated a panel of Syt1 chimeric constructs in Drosophila to delineate the unique and shared functions of each C2 domain in regulation of synaptic vesicle fusion. Expression of Syt 1 transgenes containing only individual C2 domains, or dual C2A-C2A or C2B-C2B chimeras, failed to restore Syt1 function in a syt1−/− null mutant background, indicating both C2A and C2B are specifically required to support fast synchronous release. Mutations that disrupted Ca2+ binding to both C2 domains failed to rescue evoked release, but supported synaptic vesicle docking and endocytosis, indicating these functions of Syt1 are Ca2+-independent. The dual C2 domain Ca2+-binding mutant also enhanced spontaneous fusion, while dramatically increasing evoked release when co-expressed with native Syt1. Taken together, these data indicate that synaptic transmission can be regulated by Syt1 multimerization, and that both C2 domains of Syt1 are uniquely required for modulating Ca2+-independent spontaneous fusion and Ca2+-dependent synchronous release.