Dissecting the Forward Trafficking of Presynaptic Voltage Gated Calcium Channels

剖析突触前电压门控钙通道的前向运输

• 批准号: 10601491
• 负责人: Morven Chin
• 金额: $ 3.56万
• 依托单位: HARVARD MEDICAL SCHOOL
• 依托单位国家: 美国
• 财政年份: 2023
• 资助国家: 美国
• 起止时间: 2023-01-01 至 2025-12-31
• 项目状态: 未结题
• 来源: https://reporter.nih.gov/project-details/10601491
• 关键词: Address; Affect; Axon; Axonal Transport; Biology; C-terminal; Calcium; Calcium Channel; Cell Line; Cell membrane; Cells; Chimera organism; Cytoplasm; Data; Dendrites; Destinations; EF Hand Motifs; Electrophysiology (science); Exhibits; Face; Failure; Goals; Golgi Apparatus; Healthcare; Hippocampus; Image; Individual; Maintenance; Mediating; Microscopy; Mus; Mutation; Neurons; Neuropathy; P-Q type voltage-dependent calcium channel; Pathway interactions; Phase; Positioning Attribute; Presynaptic Terminals; Protein Isoforms; Proteins; Role; Route; Site; Sorting; Source; Synaptic Membranes; Synaptic Transmission; Synaptic Vesicles; Testing; Transfection; Travel; Vesicle; Visualization; Whole-Cell Recordings; experimental study; insight; millimeter; mutant; neuronal cell body; neurotransmitter release; novel; postsynaptic; presynaptic; presynaptic neurons; scaffold; stem; superresolution microscopy; synaptogenesis; trafficking; voltage

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.

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， 它们直接驱动突触传递然而，突触前末梢内CaV的表达 呈现出相当大的贩运困境。突触前CaV在它们所在的细胞体中合成， 通常与最终目的地相隔数百毫米。CaV在前往突触前末梢的途中 因此，必须通过多个交通检查站，这些检查站限制被误导的货物通过， 从高尔基体输出，进入轴突室，在突触前末梢捕获货物，以及 最后锚定在活动区域内。此外，所有神经元CaV都表现出显著程度的序列， 结构同源性，包括那些只分类到体细胞和树突区室。在 因此，将CaV特异性靶向突触前末梢的机制通路仍然存在， 模糊，尽管它们对神经元活动至关重要。 在这篇论文中，我将重点分析CaV2.1s的运输，这是一种主要的突触前CaV亚型。最近 研究已经确定CaV2.1C-末端是靶向序列的关键位点。除了图案 将CaV2.1连接到活性区支架上，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的区室靶向主要由其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，我将确定贩运人口检查站， 近端C末端。我的初步数据表明近端C末端的突变 CaV2.1s的突触前定位。我的目标是确定被破坏的具体贩运检查站。我 将评估近端C末端突变如何影响高尔基体出口，轴突靶向，突触前捕获， 通过在神经元的胞体和轴突中可视化CaV2.1s， 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 定位和功能。在初步的实验中，我表明，关键靶向序列的转移， CaV 2.1 ~ CaV 1可以将这些通道从索马和树突重定向到突触前活动区。我会 产生进一步的嵌合CaV以分离单个CaV2.1序列的运输功能。我会 全面探索在重新表达CaV嵌合体的神经元中拯救突触传递， STED显微镜检查、钙成像和电生理学。总而言之，这一建议涉及一个基本问题， 我们对突触前CaV运输的理解存在差距。这些实验的结果将揭示新的 CaV运输中的调节机制，它们反过来可以为源于CaV的神经病变的医疗保健提供信息。 突触前CaV的异常表达和运输。