Department of Biomedical Sciences, Molecular, Cellular, Integrative Neurosciences Program, Colorado State University, Fort Collins, CO, United States of America.
PLoS One. 2020 Feb 7;15(2):e0228348. doi: 10.1371/journal.pone.0228348. eCollection 2020.
The synaptic vesicle protein, synaptotagmin, is the principle Ca2+ sensor for synaptic transmission. Ca2+ influx into active nerve terminals is translated into neurotransmitter release by Ca2+ binding to synaptotagmin's tandem C2 domains, triggering the fast, synchronous fusion of multiple synaptic vesicles. Two hydrophobic residues, shown to mediate Ca2+-dependent membrane insertion of these C2 domains, are required for this process. Previous research suggested that one of its tandem C2 domains (C2B) is critical for fusion, while the other domain (C2A) plays only a facilitatory role. However, the function of the two hydrophobic residues in C2A have not been adequately tested in vivo. Here we show that these two hydrophobic residues are absolutely required for synaptotagmin to trigger vesicle fusion. Using in vivo electrophysiological recording at the Drosophila larval neuromuscular junction, we found that mutation of these two key C2A hydrophobic residues almost completely abolished neurotransmitter release. Significantly, mutation of both hydrophobic residues resulted in more severe deficits than those seen in synaptotagmin null mutants. Thus, we report the most severe phenotype of a C2A mutation to date, demonstrating that the C2A domain is absolutely essential for synaptotagmin's function as the electrostatic switch.
突触小泡蛋白突触融合蛋白，是突触传递的主要钙离子传感器。钙离子流入活跃的神经末梢，通过与突触融合蛋白串联的 C2 结构域结合将钙离子转化为神经递质释放，从而触发多个突触小泡的快速、同步融合。两个疏水性残基介导这些 C2 结构域的钙离子依赖性膜插入，是该过程所必需的。先前的研究表明，其串联的 C2 结构域之一（C2B）对融合至关重要，而另一个结构域（C2A）仅起促进作用。然而，C2A 中两个疏水性残基的功能尚未在体内得到充分测试。在这里，我们证明这两个疏水性残基对于突触融合蛋白触发囊泡融合是绝对必需的。使用在果蝇幼虫神经肌肉接点的体内电生理记录，我们发现这些两个关键的 C2A 疏水性残基的突变几乎完全消除了神经递质的释放。重要的是，这两个疏水性残基的突变导致的缺陷比在突触融合蛋白缺失突变体中观察到的更严重。因此，我们报告了迄今为止 C2A 突变的最严重表型，证明了 C2A 结构域是作为静电开关的突触融合蛋白功能所必需的。
