Calcium channels in synaptic vesicle recycling

突触小泡回收中的钙通道

基本信息

批准号：
7471349
负责人：
JI-FANG ZHANG
金额：
$ 23.25万
依托单位：
THOMAS JEFFERSON UNIVERSITY
依托单位国家：
美国
项目类别：
财政年份：
2005
资助国家：
美国
起止时间：
2005-08-15 至 2010-07-31
项目状态：
已结题

来源：
https://reporter.nih.gov/project-details/7471349
关键词：
Address Affinity Amino Acids Binding Biochemical Biochemistry Biological Assay Calcium Calcium Channel Charge Chimeric Proteins Clathrin Complex Coupling Dynamin EF Hand Motifs Electrophysiology (science)Elements Endocytosis Equilibrium Exocytosis Figs - dietary Glutamates Goals Knowledge Light Macromolecular Complexes Mediating Modeling Modus Molecular Molecular Biology Molecular Conformation Mutagenesis Nerve Neurons Physiological Play Proline-Rich Domain Property Proteins Recruitment Activity Recycling Regulation Reporting Research Personnel Rest Role SH3 Domains SNAP receptor Screening procedure Signal Transduction Specificity Structure Synapses Synaptic Vesicles Testing Vesicle X-Ray Crystallography Yeasts base design fluorescence imaging in vitro Assay in vivo interest novel protein protein interaction research study sensor synaptojanin voltage voltage gated channel yeast two hybrid system

项目摘要

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+.

描述（由申请人提供）：突触囊泡胞吐和内吞作用之间的紧密平衡对于维持突触结构和功能至关重要。正如我们最近报道的那样，电压门控的Ca2+通道（VGCC）不仅是突触囊泡（SV）释放机械的组成部分，而且还是SV内吞作用机制的重要元素。 VGCC和内菲林（Endo）是网格蛋白介导的囊泡内吞作用的关键调节剂，形成了一种大分子复合物，可将内吞机械募集到神经末端。特别令人感兴趣的是，内通通道复合物的形成是Ca2+依赖性的。 Ca2+的作用是由驻留在Endo内的新型Ca2+传感器介导的。 Ca2+与Endo的结合将其构象从开放模式变为封闭模式，从而为CA2+提供了调节SV内吞作用的机制。长期目标是了解VGCC及其新型合作伙伴蛋白的独特作用，我们在调节SV释放和回收中确定了它们在酵母两杂种筛选中所确定的。在此应用中，我们将重点关注VGCC和内po蛋白之间在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射线晶体学的合并方法来检验模型预测的几种假设。我们将解决：（1）新型Ca2+传感器在内pophilin中的分子组成；（2）Ca2+通道 - 内丁基蛋白蛋白复合物的生化表征；（3）Ca2+对体内内pophilin通道相互作用的影响；（4）内菲尔蛋白结合对Ca2+通道功能的影响以及内pophilin的作案切换对网格蛋白介导的内吞作用的影响。结果将提高我们对VGCC和Ca2+涌入通过VGCC在耦合和协调SV外胞毒性和内吞作用中的独特作用的了解。在更广泛的范围内，结果将阐明如何通过蛋白质 - 蛋白质相互作用建立的细胞信号网络实现其特异性以及如何通过CA2+等生理因素来调节蛋白质 - 蛋白质相互作用。