Department of Brain and Cognitive Sciences, Massachusetts Institute of Technology, Cambridge, MA 02139, United States.
Mol Cell Neurosci. 2013 Jan;52:161-72. doi: 10.1016/j.mcn.2012.11.009. Epub 2012 Nov 16.
Complexins are small α-helical proteins that modulate neurotransmitter release by binding to SNARE complexes during synaptic vesicle exocytosis. They have been found to function as fusion clamps to inhibit spontaneous synaptic vesicle fusion in the absence of Ca(2+), while also promoting evoked neurotransmitter release following an action potential. Complexins consist of an N-terminal domain and an accessory α-helix that regulates the activating and inhibitory properties of the protein, respectively, and a central α-helix that binds the SNARE complex and is essential for both functions. In addition, complexins contain a largely unstructured C-terminal domain whose role in synaptic vesicle cycling is poorly defined. Here, we demonstrate that the C-terminus of Drosophila complexin (DmCpx) regulates localization to synapses and that alternative splicing of the C-terminus can differentially regulate spontaneous and evoked neurotransmitter release. Characterization of the single DmCpx gene by mRNA analysis revealed expression of two alternatively expressed isoforms, DmCpx7A and DmCpx7B, which encode proteins with different C-termini that contain or lack a membrane tethering prenylation domain. The predominant isoform, DmCpx7A, is further modified by RNA editing within this C-terminal region. Functional analysis of the splice isoforms showed that both are similarly localized to synaptic boutons at larval neuromuscular junctions, but have differential effects on the regulation of evoked and spontaneous fusion. These data indicate that the C-terminus of Drosophila complexin regulates both spontaneous and evoked release through separate mechanisms and that alternative splicing generates isoforms with distinct effects on the two major modes of synaptic vesicle fusion at synapses.
衔接蛋白是小的α-螺旋蛋白，通过在突触小泡胞吐过程中与 SNARE 复合物结合来调节神经递质的释放。已经发现它们作为融合夹在没有 Ca(2+)的情况下抑制自发的突触小泡融合，同时在动作电位后促进诱发的神经递质释放。衔接蛋白由一个 N 端结构域和一个辅助α-螺旋组成，分别调节蛋白的激活和抑制特性，以及一个中央α-螺旋，该螺旋结合 SNARE 复合物，对于这两种功能都是必需的。此外，衔接蛋白包含一个主要无结构的 C 端结构域，其在突触小泡循环中的作用尚未明确。在这里，我们证明果蝇衔接蛋白（DmCpx）的 C 端调节突触的定位，并且 C 端的选择性剪接可以差异调节自发和诱发的神经递质释放。通过 mRNA 分析对单个 DmCpx 基因的特征描述显示，存在两种不同表达的异构体，DmCpx7A 和 DmCpx7B，它们编码具有不同 C 端的蛋白质，这些 C 端含有或缺乏膜固定的异戊二烯化结构域。主要的异构体 DmCpx7A 在这个 C 端区域进一步被 RNA 编辑修饰。剪接异构体的功能分析表明，这两种异构体都相似地定位于幼虫神经肌肉接头的突触小泡，但对诱发和自发融合的调节有不同的影响。这些数据表明，果蝇衔接蛋白的 C 端通过独立的机制调节自发和诱发释放，并且选择性剪接产生对突触中两种主要的突触小泡融合模式具有不同影响的异构体。
