Active zone core proteins in interaction with synaptic Ca2+ channels

在突触前活动区，Ca~（2+）通道触发突触囊泡（SV）的释放以传递和转换神经元信息。现在已知活性区由进化上保守的复合物组成，所述复合物含有作为主要成分的RIM、Munc13、RIM结合蛋白（RBP）、α-liprin和ELKS蛋白。RIM和RBP使用高度保守的基序直接与Ca2+通道细胞内C-末端直接相互作用。同时，生理分析表明，Ca2+通道与SV的Ca2+感受器之间的物理距离很近（10 - 100 nm）。这种纳米结构域耦合可能会增加突触传递的效率和速度，并对信息处理和编码非常重要。然而，活性区蛋白功能和纳米结构域偶联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，证明RBP或ELKS家族蛋白Bruchpilot（BRP）的缺失导致i）活动区的结构缺陷，包括Ca 2+通道的缺失ii）基线传递的缺陷iii）与非典型短期易化相关的SV释放概率的严重丧失。这些表型是否反映了定义物理接近或生化支架组装促进Ca2+触发SV释放的缺陷仍然是开放的。果蝇α-1亚基Cacophony（Cac）是N-/P/Q-型家族的唯一代表，并且单独负责NMJ突触处的SV释放。在这里，我们建议系统地探讨蛋白质之间的相互作用的作用的细胞内结构域的钙离子和活性区蛋白。我们通过将合适的点突变引入大的点突变的cac基因组构建体中来开始干扰特异性Cac相互作用，所述大的点突变的cac基因组构建体将经受详细的电生理学分析（SV释放概率、可释放SV的数量和指示纳米结构域偶联的短期可塑性）。活性区蛋白质结构可能直接限定紧密靠近通道的SV结合和释放位点（狭槽）。我们试图在超微结构水平上测试这一假设（急性视紫红质介导的刺激，然后进行电子显微镜断层扫描）。最后同样重要的是，我们寻求解决是否直接接触之间的钙/RBP和SV释放机械组件（特别是unc-13家庭）是重要的有效SV释放以及。总的来说，通过专注于离散的相互作用表面，我们希望从基因上将组件与活性区的真正功能缺陷分离，并获得对活性区核心功能原理的通用见解。

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.