Calcium channels in synaptic vesicle recycling

描述(申请人提供)：突触小泡胞吐和胞吞之间的紧密平衡是维持突触结构和功能的基础。正如我们最近报道的那样，电压门控钙通道(VGCC)不仅是突触囊泡(SV)释放机制的组成部分，也是SV内吞机制的重要组成部分。VGCCs和亲内素(Endo)是网状蛋白介导的囊泡内吞作用的关键调节因子，形成一个大分子复合体，用于将内吞机械重新招募到神经末梢。特别令人感兴趣的是，内通道复合体的形成是钙依赖的。钙离子的作用是由一种新型的钙传感器介导的，该传感器位于Endo内。Ca~(2+)与Endo的结合使其构象从开放模式转变为闭合模式，从而为Ca~(2+)调节SV内吞作用提供了机制。我们的长期目标是了解VGCC及其新的伙伴蛋白在调节SV释放和循环中的独特作用，我们在酵母双杂交筛选中发现了这些蛋白。在这个应用中，我们将重点研究在SV内吞作用中VGCC和内嗜素之间的相互作用。我们将使用生物化学、分子生物学、荧光成像、电生理学和X射线结晶学的组合方法来检验从我们的模型预测的几个假说。我们将讨论：(1)内亲素中新型钙传感器的分子组成；(2)钙通道-内亲素-动力蛋白复合体的生化特征；(3)体内钙离子对内亲素-通道相互作用的影响；(4)内亲和素结合对钙通道功能的影响以及内亲素对胞内蛋白介导的内吞作用的影响。这些结果将加深我们对VGCCs和通过VGCCs内流在耦合和协调SV胞吐和内吞作用中的独特作用的认识。在更广泛的范围内，这些结果将阐明通过蛋白质-蛋白质相互作用建立的细胞信号网络如何实现其特异性，以及蛋白质-蛋白质相互作用如何受到生理因素(如钙离子)的调节。

DESCRIPTION (provided by applicant): A tight balance between synaptic vesicle exocytosis and endocytosis is fundamental to maintaining synaptic structure and function. As we reported recently, voltage-gated Ca2+ channels (VGCCs) are not only an integral part of the synaptic vesicle (SV) release machinery but also an essential element of SV endocytosis machinery. VGCCs and endophilin (endo), a key regulator of clathrin-mediated vesicle endocytosis, form a macromolecular complex that serves to recruit the endocytic machinery into the nerve terminal. Of particular interest is the finding that formation of the endo-channel complex is Ca2+-dependent. The effects of Ca2+ are mediated by a novel Ca2+ sensor that resides within endo. Binding of Ca2+ to endo changes its conformation from the open mode to the closed mode, thus providing a mechanism for Ca2+ to regulate SV endocytosis. The long term goal is to understand the unique role of VGCCs and their novel partner proteins, which we identified in yeast two-hybrid screening, in regulating SV release and recycling. In this application, we will focus on the interaction between VGCCs and endophilin in SV endocytosis. We will test several hypotheses predicted from our model using the combined approaches of biochemistry, molecular biology, fluorescence imaging, electrophysiology and X-ray crystallography. We will address: (1) the molecular composition of the novel Ca2+ sensor in endophilin; (2) biochemical characterization of the Ca2+ channel-endophilin-dynamin complex; (3) the effects of Ca2+ on the endophilin-channel interaction in vivo; and (4) the effects of endophilin binding on the Ca2+ channel functions and the effects of modus switching of endophilin on clathrin-mediated endocytosis. The results will advance our knowledge of the unique role of VGCCs and Ca2+ influx through VGCCs in coupling and coordinating SV exocytosis and endocytosis. On a broader scale, the results will shed light on how the cellular signaling network, established via protein-protein interactions, achieves its specificity and how protein-protein interactions can be regulated by physiological factors such as Ca2+.