Active zone core proteins in interaction with synaptic Ca2+ channels

活性区核心蛋白与突触 Ca2 通道相互作用

• 批准号: 230148358
• 负责人: Professor Dr. Stephan J. Sigrist
• 金额: --
• 依托单位: Institut für Biologie
• 依托单位国家: 德国
• 项目类别: Research Grants
• 财政年份: 2012
• 资助国家: 德国
• 起止时间: 2011-12-31 至 2016-12-31
• 项目状态: 已结题
• 来源: https://gepris.dfg.de/gepris/projekt/230148358?language=en
• 关键词: Active; zone; core; proteins; interaction

At the presynaptic active zone, Ca2+ channels trigger the release of synaptic vesicles (SVs) to transmit and transform neuronal information. Active zones are now known to be composed of an evolutionarily conserved complex containing as primary constituents RIM, Munc13, RIM-binding protein (RBP), alpha-liprin, and ELKS proteins. RIM and RBP directly interact directly with the Ca2+ channel intracellular C-termini using highly conserved motifs. Same time, physiological analysis suggests that Ca2+ channels get into very close physical distance from Ca2+ sensors of the SVs (10-100 nm). This nanodomain coupling might increase the efficacy, speed of synaptic transmission and be important for information processing and coding. Mechanisms of active zone protein function and nanodomain coupling, however, remain largely enigmatic. Our recent work at Drosophila NMJ model synapses provided evidence that loss of either RBP or ELKS-family protein Bruchpilot (BRP) results in i) structural deficits at active zones including a loss of Ca2+-channels ii) deficits in base-line transmission iii) severe loss of SV release probability associated with atypical short-term facilitation. Whether these phenotypes reflect defects in defining physical proximity or in the assembly of biochemical scaffolds facilitating Ca2+ triggered SV release remained open. The Drosophila alpha-1 subunit Cacophony (Cac) is the only representative of the N-/ P/Q-type family and solely responsible for SV release at NMJ synapses. We here propose to systematically explore the role of protein interactions between the intracellular domains of Cac and active zone proteins. We start interfering with specific Cac interactions by introducing suitable point mutations into a large point mutated cac genomic constructs, which will be subjected to detailed electrophysiological analysis (SV release probability, numbers of releasable SVs, and short- term plasticity indicative of nanodomain coupling). The active zone protein architecture might directly define SV binding and release sites (slots) in tight proximity to the channels. This hypothesis we seek to test on ultrastructural level (acute Channelrhodopsin-mediated stimulation followed by electron microscopic tomography). Last not least, we seek to address whether direct contacts between Cac/RBP and the SV release machinery components (particularly unc-13 family) are important for effective SV release as well. Collectively, by focusing on discrete interaction surfaces we hope to genetically uncouple assembly from truly functional deficits at active zones, and gain generic insights into the functional principles of the active zone core.

在突触前活动区，钙通道触发突触小泡的释放，传递和转换神经元信息。现已知，活动区由进化上保守的复合体组成，主要成分包括RIM、Munc13、RIM结合蛋白(RBP)、α-脂蛋白和ELKS蛋白。RIM和RBP通过高度保守的基序直接与细胞内的钙通道C末端相互作用。同时，生理分析表明，钙通道与SVS的钙感受器之间有非常近的物理距离(10-100 nm)。这种纳米结构域耦合可能会提高突触传递的效率和速度，并对信息处理和编码具有重要意义。然而，活动区蛋白功能和纳米结构域耦合的机制在很大程度上仍然是个谜。我们最近对果蝇NMJ模型突触的研究提供了证据：RBP或ELKS家族蛋白BruchPilot(BRP)的丢失导致：1)活动区结构缺陷，包括钙通道的丢失；2)基线传递的缺陷；3)与非典型的短期促进相关的SV释放概率的严重丧失。无论这些表型是否反映了在定义物理接近方面的缺陷，还是反映在促进钙离子触发的SV释放的生化支架组装方面的缺陷，仍然是开放的。果蝇α-1亚单位杂音(CAC)是N-/P/Q型家族的唯一代表，是NMJ突触释放SV的唯一原因。我们建议系统地探索CAC细胞内结构域和活动区蛋白之间的蛋白质相互作用的作用。我们通过在大的点突变的CAC基因组结构中引入合适的点突变来干扰特定的CAC相互作用，这将受到详细的电生理分析(SV释放概率、可释放的SV数量和指示纳米结构域耦合的短期可塑性)。活动区蛋白结构可能直接定义紧邻通道的SV结合和释放位置(槽)。我们试图在超微结构水平上测试这一假设(急性通道视紫红质介导的刺激随后进行电子显微镜断层扫描)。最后，我们试图解决CAC/RBP与SV释放机械部件(特别是UNC-13系列)之间的直接接触是否对SV有效释放也很重要。总的来说，通过关注离散的相互作用面，我们希望从基因上将组装从活动区的真正功能缺陷中分离出来，并获得对活动区核心的功能原理的一般性见解。