Dissecting the Forward Trafficking of Presynaptic Voltage Gated Calcium Channels

摘要 电压门控钙通道(CAV)是突触前活动区不可或缺的组成部分，因为 它们直接驱动突触传递。然而，CAV在突触前终末的表达 造成了相当大的人口贩运困境。突触前突触是在它们所在的细胞体中合成的 通常与它们的最终目的地相隔数百毫米。骑兵在前往突触前终端的途中 因此，必须导航多个限制误导货物通过的贩运检查站，包括 从高尔基体输出，进入轴突隔室，在突触前终末捕获货物，以及 最后锚定在活动区内。此外，所有神经元腔表现出显著程度的序列和 结构同源性，包括那些只分成体细胞和树突的隔室。在……里面 结果，针对CAV的特定突触前终末的机械性路径仍然存在 鲜为人知，尽管它们对神经元活动至关重要。 在这项提案中，我将重点剖析CaV2.1s的贩运，CaV2.1是一种主要的突触前Cav亚型。近期 研究已经确定CaV2.1 C-末端是靶向序列的关键位置。除了图案之外 将CaV2.1连接到活动区支架上，我的初步数据显示CaV2.1的近端区域 C末端包含突触前表达所必需的关键序列。此外，我还展示了 CAV的隔室靶向主要由其C-末端序列决定。因此我假设 CaV2.1近端C末端是CaV2.1活性区靶向和功能的关键决定因素。我 将通过两个具体目标来检验这一假设。在目标1中，我将确定需要以下条件的贩运检查站 近端的C-末端。我的初步数据表明，近端C-末端的突变可以消除 CaV2.1s的突触前定位我的目标是确定被扰乱的特定人口贩运检查站。我 将评估近端C末端突变如何影响高尔基体输出、轴突靶向、突触前捕获、 和通过可视化CaV2.1在神经元胞体和轴突中以及在 细胞系中的质膜。在目标2中，我将定义活动区的最小CaV2.1序列 本土化和功能化。在初步实验中，我证明了关键靶向序列从 CaV2.1到CaV1可以将这些通道从胞体和树突重定向到突触前活动区。这就做 产生更多的嵌合体Cavs以分离单个CaV2.1序列的运输功能。这就做 全面探讨通过多种手段挽救重新表达CAV嵌合体的神经元的突触传递 STED显微镜、钙成像和电生理学。总而言之，这项提议解决了一个根本问题 我们对突触前CAV交易的理解存在差距。这些实验的发现将揭示出新的 CAV贩运中的监管机制，它们反过来可能会向医疗保健部门通报源于 突触前Cavs的异常表达和运输。

ABSTRACT Voltage-gated calcium channels (CaVs) are indispensable components of the presynaptic active zone because they directly drive synaptic transmission. However, the expression of CaVs within the presynaptic terminal presents a considerable trafficking dilemma. Presynaptic CaVs are synthesized at the cell body where they are often separated from their final destinations by hundreds of millimeters. CaVs en route to the presynaptic terminal must therefore navigate multiple trafficking checkpoints that restrict passage of misdirected cargoes including export from the Golgi apparatus, entry into the axon compartment, cargo capture at presynaptic terminals, and finally anchoring within the active zone. Further, all neuronal CaVs exhibit a significant degree of sequence and structural homology, including those that sort exclusively into somatic and dendritic compartments. In consequence, the mechanistic pathways that target CaVs specifically to the presynaptic terminals remain obscure, despite their central importance to neuronal activity. In this proposal I will focus on dissecting the trafficking of CaV2.1s, a major presynaptic CaV isoform. Recent studies have established the CaV2.1 C-terminus as a critical locus of targeting sequences. In addition to motifs that tether CaV2.1s to the active zone scaffold, my preliminary data reveals that the proximal region of the CaV2.1 C-terminus contains key sequences that are together necessary for presynaptic expression. Further, I show the compartment targeting of CaVs is determined primarily by its C-terminal sequences. I therefore hypothesize that the CaV2.1 proximal C-terminus is a key determinant for CaV2.1 active zone targeting and function. I will test this hypothesis through two specific aims. In aim 1, I will identify the trafficking checkpoints that require the proximal C-terminus. My preliminary data establish that mutations within the proximal C-terminus abolish presynaptic localization of CaV2.1s. My goal is to identify the specific trafficking checkpoint that is disrupted. I will evaluate how proximal C-terminus mutations affect the Golgi export, axon targeting, presynaptic capture, and active zone anchoring of CaV2.1 cargoes by visualizing CaV2.1s in the somas and axons of neurons and at the plasma membrane in cell lines. In aim 2, I will define the minimal CaV2.1 sequences for active zone localization and function. In preliminary experiments, I show that the transfer of key targeting sequences from CaV2.1 to CaV1 can redirect these channels from the soma and dendrites to the presynaptic active zone. I will generate further chimeric CaVs to isolate the trafficking functions of individual CaV2.1 sequences. I will comprehensively probe for rescue of synaptic transmission in neurons that re-express CaV chimeras by means STED microscopy, calcium imaging and electrophysiology. In summary, this proposal addresses a fundamental gap in our understanding of presynaptic CaV trafficking. The findings from these experiments will reveal novel regulatory mechanisms in CaV trafficking and they may in turn inform healthcare of neuropathies that stem from the aberrant expression and trafficking of presynaptic CaVs.