Active zone core proteins in interaction with synaptic Ca2+ channels

在突触前活跃区，Ca2+通道触发突触囊泡（SVs）的释放，以传递和转化神经元信息。目前已知的活性区是由一个进化上保守的复合体组成的，其主要成分包括RIM、Munc13、RIM结合蛋白（RBP）、α -脂质和ELKS蛋白。RIM和RBP使用高度保守的基序直接与细胞内Ca2+通道c端相互作用。同时，生理分析表明，Ca2+通道与SVs Ca2+传感器的物理距离非常近（10-100 nm）。这种纳米结构域耦合可能提高突触传递的效率和速度，对信息处理和编码具有重要意义。然而，活性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谜。我们最近在果蝇NMJ模型突触上的工作提供了证据，表明RBP或elks家族蛋白Bruchpilot （BRP）的缺失会导致i)活性区结构缺陷，包括Ca2+通道的缺失ii)基线传输缺陷iii)与非典型短期促进相关的SV释放概率严重丧失。这些表型是否反映了在定义物理接近性或在促进Ca2+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Cacophony （Cac）是N-/ P/ q -型家族的唯一代表，唯一负责NMJ突触的SV释放。我们建议系统地探索Cac胞内结构域和活性区蛋白之间的蛋白质相互作用的作用。我们通过在大型点突变的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）。活性区蛋白结构可能直接定义靠近通道的SV结合和释放位点（槽）。我们试图在超微结构水平（电子显微镜断层扫描后的急性通道视紫红质介导的刺激）上验证这一假设。最后，我们试图解决Cac/RBP与SV释放机械组件（特别是unc-13家族）之间的直接接触是否对有效释放SV也很重要。总的来说，通过关注离散的相互作用表面，我们希望从基因上解开活性区域真正功能缺陷的组装，并获得对活性区域核心功能原理的一般见解。

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.