Molecular Dissection of Active Zone Functions in Neurotransmitter Release

在神经末梢内，突触小泡专门在活动区融合。活动区由一个 一种固定在质膜上的蛋白质支架，形成与之完全相反的释放部位 突触后受体。活动区蛋白与钙离子通道之间的相互作用长期以来一直是 利息。Ca~(2+)通过CaV2家族的通道内流触发释放，其精确定位支持 突触传递的亚毫秒计时，并决定突触的强度。有两个竞争对手 钙离子通道在突触和活动区组装中的作用和机制的模型。第一，钙离子 通道对突触结构可能是必不可少的。第二，活跃区可能招募钙离子通道释放 这意味着突触结构不依赖于CaV2。很难将它们区分开来 模型因为钙离子通道基因家族及其辅助亚基的复杂性导致了广泛的 冗余性。此外，准确定位钙离子通道一直是一个挑战。 我们已经克服了这些障碍，通过产生有条件的三重敲除小鼠来移除所有形成毛孔的A1 CaV2通道的亚基，并采用超分辨显微镜来评估钙通道的定位。 我们的数据证实，通过这些通道的钙离子流量对于释放触发是必不可少的。基于我们的 初步数据，我们假设活动区组装独立于CaV2通道，但相反 活动区以纳米级的精度靶向CaV2通道以释放位置。我们的实验计划 从三个独立的角度检验了这一假设，并剖析了潜在的机制。在目标1中，我们评估 通过去除形成孔的A1亚基，然后评估突触和活性来竞争模型 区域结构和功能。然后，我们提出了救援实验，以评估哪些CaV2通道序列 是它们靶向所必需的，我们测试哪些CaV2序列足以提供活动区靶向 传输到非CAV2频道。在目标2中，我们确定了辅助亚基和辅助亚基的精确突触前定位 评估他们的突触前靶点是否依赖于A1。然后，我们测试这些功能角色是否 辅助亚单位需要A1的存在。在目标3中，我们解决了CaV2靶向的分子机制 从活动区支架的角度来看。我们首先确定活动区和CaV2的到达顺序 然后我们确定了CaV2及其受体的定位和功能。 缺乏特定活性区带蛋白的突变体中的亚基。 这项资助将测试两种根本不同的钙通道和活性钙通道之间的关系模型。 区域，并剖析了钙离子通道锚定在靶膜下的机制。精确 了解这些机制对于了解突触在健康和疾病中的作用是很重要的。

Within a nerve terminal, synaptic vesicles exclusively fuse at the active zone. The active zone consists of a protein scaffold that is anchored to the plasma membrane and forms release sites precisely opposed to postsynaptic receptors. Interactions between active zone proteins and Ca2+ channels have long been of central interest. Ca2+ influx through channels of the CaV2 family triggers release, and their exact positioning supports the sub-millisecond timing of synaptic transmission and determines synaptic strength. There are two competing models for roles and mechanisms of Ca2+ channels in synapse and active zone assembly. First, Ca2+ channels may be essential for synapse structure. Second, the active zone may recruit Ca2+ channels to release sites, implying that synapse structure is CaV2 independent. It has been difficult to distinguish between these models because the complexity of the Ca2+ channel gene family and their auxiliary subunits leads to extensive redundancy. Furthermore, precisely localizing Ca2+ channels has been challenging. We have overcome these hurdles by generating conditional triple knockout mice to remove all pore-forming a1 subunits of CaV2 channels, and by adapting superresolution microscopy to assess Ca2+ channel localization. Our data confirm that Ca2+ flux through these channels is essential for release triggering. Based on our preliminary data, we hypothesize that active zone assembly is independent of CaV2 channels, but instead the active zone targets CaV2 channels with nanoscale precision to release sites. Our experimental plan tests this hypothesis from three independent angles and dissects underlying mechanisms. In aim 1, we assess the competing models by removing the pore forming a1 subunits, followed by assessment of synapse and active zone structure and function. We then propose rescue experiments to assess which sequences of CaV2 channels are required for their targeting, and we test which CaV2 sequences are sufficient to confer active zone targeting onto non-CaV2 channels. In aim 2, we determine the precise presynaptic localization of auxiliary subunits and assess whether their presynaptic targeting depends on a1. We then test whether functional roles of these auxiliary subunits require the presence of a1. In aim 3, we address molecular mechanisms for CaV2 targeting from the perspective of active zone scaffolds. We first determine the order of arrival of active zone and CaV2 proteins during active zone assembly, and we then determine localization and function of CaV2s and their subunits in mutants that lack specific active zone proteins. This grant will test two fundamentally different models of the relationship between Ca2+ channels and the active zone, and dissects the mechanisms that underlie Ca2+ channel anchoring at the target membrane. Precise understanding of these mechanisms is important for understanding synapses in health and disease.