Calcium channels in synaptic vesicle recycling

描述（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。正如我们最近报道的那样，电压门控Ca2+通道（VGCCs）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。vgcc和亲内吞蛋白（endo）形成一个大分子复合物，用于将内吞机制招募到神经末梢。特别有趣的是发现通道内复合体的形成是Ca2+依赖的。Ca2+的影响是由一个新的Ca2+传感器驻留在内腔介导。Ca2+与内do结合使其构象由开放模式转变为封闭模式，从而为Ca2+调节SV内吞作用提供了一种机制。长期目标是了解我们在酵母双杂交筛选中发现的vgc及其新伴侣蛋白在调节SV释放和再循环中的独特作用。在本应用中，我们将重点研究vgc和嗜内噬蛋白在SV内吞作用中的相互作用。我们将使用生物化学、分子生物学、荧光成像、电生理学和x射线晶体学的结合方法来测试从我们的模型预测的几个假设。我们将讨论：(1)在嗜内蛋白中新型Ca2+传感器的分子组成；(2) Ca2+通道-内啡肽-动力蛋白复合物的生化表征；(3)体内Ca2+对嗜内啡肽通道相互作用的影响；(4)亲内蛋白结合对Ca2+通道功能的影响，以及亲内蛋白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Ca2+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，这些结果将揭示通过蛋白-蛋白相互作用建立的细胞信号网络是如何实现其特异性的，以及蛋白-蛋白相互作用是如何被生理因素（如Ca2+）调节的。

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+.