Molecular Dissection of Active Zone Functions in Neurotransmitter Release

神经递质释放中活性区功能的分子剖析

• 批准号: 10613501
• 负责人: Pascal Simon Kaeser
• 金额: $ 50.04万
• 依托单位: HARVARD MEDICAL SCHOOL
• 依托单位国家: 美国
• 财政年份: 2014
• 资助国家: 美国
• 起止时间: 2014-07-01 至 2024-04-30
• 项目状态: 已结题
• 来源: https://reporter.nih.gov/project-details/10613501
• 关键词: Abbreviations; Action Potentials; Address; Brain Diseases; Cell membrane; Communication; Complex; Data; Defect; Dependence; Disease; Dissection; Docking; Electrophysiology (science); Exocytosis; Family; Gene Family; Goals; Grant; Health; Hippocampus; Image; Impairment; Intuition; Knock-out; Knockout Mice; Mediating; Membrane; Microscopy; Modeling; Molecular; Mutate; Nerve; Nerve Degeneration; Neurons; P-Q type voltage-dependent calcium channel; Pathologic; Positioning Attribute; Probability; Property; Protein Family; Proteins; Role; Scaffolding Protein; Schizophrenia; Site; Source; Structure; Synapses; Synaptic Receptors; Synaptic Transmission; Synaptic Vesicles; Testing; Vesicle; Work; addiction; autism spectrum disorder; chemical release; conditional knockout; experimental study; insight; interest; millisecond; mutant; nanoscale; nervous system disorder; neurotransmission; neurotransmitter release; postsynaptic; presynaptic; recruit; role model; scaffold; sensor; superresolution microscopy; synaptic function; synaptogenesis; transmission process

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.

在神经末端，突触囊泡仅在活性区域融合。活动区包括一个 固定在质膜上并形成释放位点的蛋白质支架恰好相反 突触后受体。活跃区蛋白与Ca2+通道之间的相互作用长期以来属于中心 兴趣。 CA2+通过CAV2家族触发器释放的通道及其确切的定位支撑 突触传播的亚毫秒时间并确定突触强度。有两个竞争 Ca2+通道在突触和活动区组装中的角色和机制的模型。首先，Ca2+ 通道可能对于突触结构至关重要。其次，活动区可以募集CA2+通道释放 站点，这意味着突触结构与CAV2无关。很难区分这些 模型是因为Ca2+通道基因家族及其辅助亚基的复杂性导致广泛 冗余。此外，精确定位Ca2+通道一直是具有挑战性的。 我们通过产生有条件的三重基因敲除小鼠来克服这些障碍以去除所有孔形成A1 CAV2通道的亚基，并通过调整超分辨率显微镜来评估Ca2+通道定位。 我们的数据证实，通过这些通道的Ca2+通量对于释放触发至关重要。基于我们 初步数据，我们假设活动区组件独立于CAV2通道，而是 活动区针对具有纳米级精度的CAV2通道释放位点。我们的实验计划 从三个独立角度来检验这一假设，并剖析了基本机制。在AIM 1中，我们评估 竞争模型通过去除形成A1亚基的孔，然后评估突触和主动 区域结构和功能。然后，我们提出救援实验，以评估哪些CAV2通道序列 它们的靶向需要，我们测试哪些CAV2序列足以赋予活动区域的靶向 在非CAV2通道上。在AIM 2中，我们确定辅助亚基的精确突触前定位和 评估其突触前靶向是否取决于A1。然后，我们测试这些功能的作用是否 辅助亚基需要A1。在AIM 3中，我们解决了CAV2靶向的分子机制 从主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 活跃区组装期间的蛋白质，然后我们确定Cav2s及其其定位和功能 缺乏特定活性区蛋白的突变体中的亚基。 该赠款将测试CA2+通道与活动之间关系的两个根本不同的模型 区域，并剖析基于CA2+通道锚定在目标膜的机制。精确的 了解这些机制对于理解健康和疾病中的突触很重要。

项目成果

Fusion Competent Synaptic Vesicles Persist upon Active Zone Disruption and Loss of Vesicle Docking.

• DOI: 10.1016/j.neuron.2016.07.005
• 发表时间: 2016-08-17
• 期刊: Neuron
• 影响因子: 16.2
• 作者: Wang SSH;Held RG;Wong MY;Liu C;Karakhanyan A;Kaeser PS
• 通讯作者: Kaeser PS

An action potential initiation mechanism in distal axons for the control of dopamine release.

• DOI: 10.1126/science.abn0532
• 发表时间: 2022-03-25
• 期刊: SCIENCE
• 影响因子: 56.9
• 作者: Liu, Changliang;Cai, Xintong;Ritzau-Jost, Andreas;Kramer, Paul F.;Li, Yulong;Khaliq, Zayd M.;Hallermann, Stefan;Kaeser, Pascal S.
• 通讯作者: Kaeser, Pascal S.