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:
- 金额:$ 50.04万
- 依托单位:
- 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:
- 关键词:AbbreviationsAction PotentialsAddressBrain DiseasesCell membraneCommunicationComplexDataDefectDependenceDiseaseDissectionDockingElectrophysiology (science)ExocytosisFamilyGene FamilyGoalsGrantHealthHippocampusImageImpairmentIntuitionKnock-outKnockout MiceMediatingMembraneMicroscopyModelingMolecularMutateNerveNerve DegenerationNeuronsP-Q type voltage-dependent calcium channelPathologicPositioning AttributeProbabilityPropertyProtein FamilyProteinsRoleScaffolding ProteinSchizophreniaSiteSourceStructureSynapsesSynaptic ReceptorsSynaptic TransmissionSynaptic VesiclesTestingVesicleWorkaddictionautism spectrum disorderchemical releaseconditional knockoutexperimental studyinsightinterestmillisecondmutantnanoscalenervous system disorderneurotransmissionneurotransmitter releasepostsynapticpresynapticrecruitrole modelscaffoldsensorsuperresolution microscopysynaptic functionsynaptogenesistransmission 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,突触囊泡仅在活动区融合。活动区由一个
蛋白质支架,其锚定到质膜上并形成与细胞膜精确相对的释放位点。
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研究的中心。
兴趣Ca2+通过CaV2家族的通道流入触发释放,并且它们的精确定位支持
突触传输的亚毫秒定时并决定突触强度。有两个竞争对手
钙通道在突触和活动区组装中的作用和机制的模型。第一,Ca2+
通道可能是突触结构所必需的。第二,活动区可能募集Ca 2+通道释放
位点,这意味着突触结构是CaV2独立的。很难区分这些
模型,因为Ca2+通道基因家族及其辅助亚基的复杂性导致广泛的
冗余此外,精确定位Ca2+通道一直具有挑战性。
我们已经克服了这些障碍,通过产生条件性三重基因敲除小鼠来去除所有成孔a1
亚基的CaV2通道,并通过调整超分辨率显微镜,以评估Ca2+通道的本地化。
我们的数据证实,通过这些通道的Ca2+流量是必不可少的释放触发。基于我们
根据初步数据,我们假设活性区组装不依赖于CaV2通道,
活性区以纳米级精度靶向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,并剖析其内在机制。目标1:评估
竞争模型通过去除形成a1亚基的孔,然后评估突触和活性
区域结构和功能。然后,我们提出了救援实验,以评估哪些序列的CaV2通道
我们测试了哪些CaV2序列足以赋予活性区靶向作用,
非CaV2通道。在目标2中,我们确定了辅助亚基的精确突触前定位,
评估它们的突触前定位是否依赖于a1。然后,我们测试这些功能角色是否
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CaV 2的到达顺序,
在活性区组装过程中的蛋白质,然后我们确定了CaV2s及其
突变体中缺乏特定活性区蛋白的亚基。
这项资助将测试两个根本不同的模型之间的关系钙通道和活性
区,并剖析了Ca2+通道锚定在靶膜的机制。精确
了解这些机制对于了解健康和疾病中的突触是重要的。
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.
- DOI:10.1016/j.neuron.2016.07.005
- 发表时间:2016-08-17
- 期刊:
- 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:
- 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.
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Pascal Simon Kaeser其他文献
Pascal Simon Kaeser的其他文献
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{{ truncateString('Pascal Simon Kaeser', 18)}}的其他基金
Mechanisms for somatodendritic dopamine release in the midbrain
中脑体细胞树突多巴胺释放机制
- 批准号:
10604832 - 财政年份:2023
- 资助金额:
$ 50.04万 - 项目类别:
Architecture and function of striatal dopamine release machinery
纹状体多巴胺释放机制的结构和功能
- 批准号:
9402528 - 财政年份:2017
- 资助金额:
$ 50.04万 - 项目类别:
Architecture and function of striatal dopamine release machinery
纹状体多巴胺释放机制的结构和功能
- 批准号:
9528696 - 财政年份:2017
- 资助金额:
$ 50.04万 - 项目类别:
Architecture and function of striatal dopamine signaling machinery
纹状体多巴胺信号机制的结构和功能
- 批准号:
10464718 - 财政年份:2017
- 资助金额:
$ 50.04万 - 项目类别:
Dissecting the assembly of neurotransmitter release sites
剖析神经递质释放位点的组装
- 批准号:
10536772 - 财政年份:2017
- 资助金额:
$ 50.04万 - 项目类别:
Dissecting the assembly of neurotransmitter release sites
剖析神经递质释放位点的组装
- 批准号:
10682464 - 财政年份:2017
- 资助金额:
$ 50.04万 - 项目类别:
Architecture and Function of Striatal Dopamine Signaling Machinery
纹状体多巴胺信号传导机制的结构和功能
- 批准号:
10589076 - 财政年份:2017
- 资助金额:
$ 50.04万 - 项目类别:
Dissecting the assembly of vertebrate neurotransmitter release sites-Research Supplements to Promote Diversity in Health-Related Research
剖析脊椎动物神经递质释放位点的组装——促进健康相关研究多样性的研究补充
- 批准号:
9896449 - 财政年份:2017
- 资助金额:
$ 50.04万 - 项目类别:
Architecture and function of striatal dopamine release machinery
纹状体多巴胺释放机制的结构和功能
- 批准号:
9915988 - 财政年份:2017
- 资助金额:
$ 50.04万 - 项目类别:
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:
9275552 - 财政年份:2014
- 资助金额:
$ 50.04万 - 项目类别:
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