Calcium channels in synaptic vesicle recycling

描述（由申请人提供）：突触囊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。电压门控性钙通道（VGCC）不仅是突触囊泡（SV）释放机制的重要组成部分，也是SV胞吞机制的重要组成部分。VGCCs和内皮素（endo），网格蛋白介导的囊泡内吞作用的关键调节因子，形成大分子复合物，用于将内吞机制募集到神经末梢。特别感兴趣的是发现内通道复合物的形成是Ca 2+依赖性的。Ca 2+的影响是由一种新的Ca 2+传感器，驻留在endo。Ca ~（2+）与endo的结合改变了endo的构象，使其从开放模式变为封闭模式，从而为Ca ~（2+）调节SV内吞提供了一种机制。长期目标是了解VGCC及其新型伴侣蛋白在调节SV释放和回收中的独特作用，我们在酵母双杂交筛选中鉴定了这些蛋白。在本申请中，我们将集中在SV内吞作用中VGCC和内亲蛋白之间的相互作用。我们将使用生物化学，分子生物学，荧光成像，电生理学和X射线晶体学的综合方法来测试从我们的模型预测的几个假设。我们将处理：（1）新型Ca 2+传感器的分子组成;（2）Ca 2+通道-内啡肽-动力蛋白复合物的生化表征;（3）体内Ca 2+对内啡肽-通道相互作用的影响;（4）内啡肽结合对Ca 2+通道功能的影响以及内啡肽的模式转换对网格蛋白介导的内吞作用的影响。这些结果将促进我们对VGCC和通过VGCC的Ca 2+内流在耦合和协调SV胞吐和胞吞中的独特作用的认识。在更广泛的范围内，这些结果将揭示通过蛋白质-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Ca 2+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+.