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

基本信息

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
项目类别：
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

项目摘要

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.

在神经末梢内，突触小泡仅在活动区域融合。活动区域由一个锚定在质膜上并形成与质膜精确相对的释放位点的蛋白质支架突触后受体。活性区蛋白和 Ca2+ 通道之间的相互作用长期以来一直是核心兴趣。 Ca2+ 通过 CaV2 家族通道的流入触发释放，并且它们的精确定位支持突触传递的亚毫秒时间并决定突触强度。有两个竞争者 Ca2+ 通道在突触和活动区组装中的作用和机制的模型。一、Ca2+ 通道可能对于突触结构至关重要。二、活性区可能招募Ca2+通道释放位点，这意味着突触结构与 CaV2 无关。很难区分这些由于 Ca2+ 通道基因家族及其辅助亚基的复杂性导致广泛的模型冗余。此外，精确定位 Ca2+ 通道一直具有挑战性。我们通过生成条件三重基因敲除小鼠来消除所有成孔 a1，从而克服了这些障碍 CaV2 通道的亚基，并通过采用超分辨率显微镜来评估 Ca2+ 通道定位。我们的数据证实，Ca2+ 通过这些通道的通量对于触发释放至关重要。基于我们的初步数据，我们假设活性区组装独立于 CaV2 通道，但相反活性区以纳米级精度瞄准 CaV2 通道以释放位点。我们的实验计划从三个独立的角度检验这一假设并剖析潜在的机制。在目标 1 中，我们评估通过去除孔形成 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，我们提出救援实验来评估 CaV2 通道的哪些序列是其靶向所必需的，我们测试哪些 CaV2 序列足以赋予活性区域靶向到非 CaV2 通道。在目标 2 中，我们确定辅助亚基的精确突触前定位和评估它们的突触前靶向是否取决于 a1。然后我们测试这些功能是否发挥作用辅助亚基需要a1的存在。在目标 3 中，我们解决了 CaV2 靶向的分子机制从活性区支架的角度来看。我们首先确定活动区和 CaV2 的到达顺序活性区组装过程中的蛋白质，然后我们确定 CaV2 及其它们的定位和功能突变体中缺乏特定活性区蛋白的亚基。这笔赠款将测试 Ca2+ 通道和活性物质之间关系的两种根本不同的模型区，并剖析了 Ca2+ 通道锚定在目标膜上的机制。精确的了解这些机制对于了解健康和疾病中的突触非常重要。

项目成果

期刊论文数量（4）

专著数量（0）

科研奖励数量（0）

会议论文数量（0）

专利数量（0）

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

具有融合能力的突触小泡在活动区破坏和小泡对接丢失时持续存在。

DOI：
发表时间：
2016-08-17
期刊：
影响因子：
16.2
作者：
Wang, Shan Shan H;Held, Richard G;Wong, Man Yan;Liu, Changliang;Karakhanyan, Aziz;Kaeser, Pascal S
通讯作者：
Kaeser, Pascal S

The intracellular C-terminus confers compartment-specific targeting of voltage-gated Ca2+ channels.

细胞内 C 末端赋予电压门控 Ca2 通道的区室特异性靶向。

DOI：
发表时间：
2023-12-23
期刊：
影响因子：
0
作者：
Chin, Morven;Kaeser, Pascal S
通讯作者：
Kaeser, Pascal S

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

远端轴突中用于控制多巴胺释放的动作电位启动机制。

DOI：
发表时间：
2022-03-25
期刊：
影响因子：
0
作者：
Liu, Changliang;Cai, Xintong;Ritzau;Kramer, Paul F;Li, Yulong;Khaliq, Zayd M;Hallermann, Stefan;Kaeser, Pascal S
通讯作者：
Kaeser, Pascal S

ELKS controls the pool of readily releasable vesicles at excitatory synapses through its N-terminal coiled-coil domains.

ELKS 通过其 N 端卷曲螺旋结构域控制兴奋性突触处易于释放的囊泡池。