Single-Molecule Studies of Synaptic Vesicles

突触小泡的单分子研究

基本信息

批准号：
7504654
负责人：
Daniel T Chiu
金额：
$ 30.12万
依托单位：
UNIVERSITY OF WASHINGTON
依托单位国家：
美国
项目类别：
财政年份：
2008
资助国家：
美国
起止时间：
2008-07-01 至 2012-06-30
项目状态：
已结题

来源：
https://reporter.nih.gov/project-details/7504654
关键词：
3-phosphoglycerate ATP Synthesis Pathway Address Affect Amphetamines Antibodies Antiepileptic Agents Automobile Driving Back Binding Binding Sites Biochemical Biogenesis Biological Models Caliber Calibration Carrier Proteins Categories Cells Chromosome Pairing Cisplatin/Doxorubicin/Melphalan/Teniposide Class Complex Condition Count Coupled Coupling Crowding Depth Diffusion Dimensions Disruption Electron Microscopy Endocytosis Endosome Proton Environment Enzymes Eukaryotic Cell Event Exhibits Exocytosis Expenditure Extravasation Floor Fluorescence Fluorescence Microscopy Fluorescent Antibody Technique Gel Glass Glutamate Transporter Glutamates Glyceraldehyde 3-Phosphate Green Fluorescent Proteins Hand Hypoglycemia Individual Intake Integral Membrane Protein Investigation Keppra Kinetics Knowledge Label Lasers Learning Length Levetiracetam Lipid Bilayers Lipids Location Lysosome Proton Macromolecular Complexes Maps Measurement Measures Membrane Membrane Fusion Membrane Protein Traffic Membrane Proteins Microscope Modeling Modification Molecular Molecular Neurobiology Monitor Motion Movement Nature Neurologic Neurons Neurotransmitters Nicotinamide adenine dinucleotide Nigericin Numbers Oregon Organelles Oxidoreductase Pathway interactions Peripheral Personal Satisfaction Pharmaceutical Preparations Phosphoglycerate Kinase Physiology Play Positioning Attribute Presynaptic Terminals Procedures Process Protein Isoforms Protein Sortings Proteins Proteomics Proton Pump Proton-Translocating ATPases Pump Rate Recycling Relative (related person)Reporting Research Reserpine Resolution Ribosomes Role Role playing therapy Rose Bengal Rotation Running SNAP receptor Sampling Signal Transduction Site Solutions Sorting - Cell Movement Spatial Distribution Staging Study Section Surface Synapses Synaptic Cleft Synaptic Receptors Synaptic Transmission Synaptic Vesicles Synaptic plasticity Synaptophysin System Techniques Testing Thick Thinking Time Transgenic Mice Transmembrane Domain Trypan Blue Valinomycin Vesicle Viscosity Water Work base carbohydrate transport dimer driving force gamma-Aminobutyric Acid human VAPA protein inhibitor/antagonist inorganic phosphate insight interest monoamine nervous system disorder neurotransmission neurotransmitter release neurotransmitter uptake numb protein pH gradient postsynaptic presynaptic prevent protein function rab GTP-Binding Proteins receptor research study response sensor single molecule size small molecule statistics stoichiometry synaptic function synaptogyrin synaptotagmin trafficking uptake vesicle-associated membrane protein

项目摘要

DESCRIPTION (provided by applicant): Gaining detailed knowledge on the workings of the vesicular transmitter uptake machinery is important in understanding synaptic function and plasticity, because this uptake process governs directly the number of transmitters released into the synaptic cleft and thus the degree of activation of the postsynaptic receptors. Determining the mechanism and rates of vesicular transmitter uptake also is important towards understanding the kinetics of vesicle recycling at the synapse and the energy requirements for loading and maintaining the filled vesicle at the presynaptic terminal. Given synaptic transmission is dependent on the uptake and storage of neurotransmitters within vesicles, it is not surprising that they are sites of action for many drugs, and thus understanding the functioning of this important molecular machinery will also have important pharmacological implications. Our proposed single-molecule and single-vesicle experiments are targeted towards deciphering the molecular organization and mechanism by which neurotransmitters are loaded into the synaptic vesicle. Towards this end, our specific aims are: Aim 1: Rotational measurements of synaptic vesicle integral membrane proteins and of small molecules contained within the vesicle Aim 2: Single-molecule studies of the vesicular H+ATPase Aim 3: Single-molecule studies of the vesicular glutamate transporters Aim 4: Investigation of the coupling of transmitter uptake to the glycolytic enzymes on synaptic vesicles and the spatial organization of the uptake machinery. From these experiments, we will develop an in-depth understanding of the workings of this complex molecular machinery with single-molecule resolutions, and offer new insight into how malfunctioning caused by neurological diseases or targeted disruption of this machinery with drugs can affect synaptic transmission. Project Narrative: To orchestrate neurotransmission, more than one thousand proteins are present at the presynaptic terminal and which either directly or indirectly interact with the synaptic vesicle, with the end result being the regulated release of neurotransmitters into the synaptic cleft. This high degree of convergence of presynaptic functions onto the synaptic vesicle has led to a "vesicocentric" view of neurotransmission that focuses on the synaptic vesicle as the central organelle in synaptic function. This proposal describes plans to study the synaptic vesicle with single-molecule resolutions, because discerning the detailed workings of this important organelle is critical in achieving the next level of quantitative understanding of synaptic transmission and malfunctioning caused by neurological diseases.

描述（由申请人提供）：获得有关囊泡递质摄取机制工作原理的详细知识对于理解突触功能和可塑性非常重要，因为这种摄取过程直接控制释放到突触间隙的递质数量，从而控制突触间隙的递质激活程度。突触后受体。确定囊泡递质摄取的机制和速率对于理解突触囊泡再循环的动力学以及在突触前末端加载和维持填充囊泡的能量需求也很重要。鉴于突触传递依赖于囊泡内神经递质的摄取和储存，因此它们是许多药物的作用位点也就不足为奇了，因此了解这一重要分子机制的功能也将具有重要的药理学意义。我们提出的单分子和单囊泡实验旨在破译神经递质加载到突触囊泡中的分子组织和机制。为此，我们的具体目标是：目标 1：突触囊泡整合膜蛋白和囊泡内小分子的旋转测量目标 2：囊泡 H+ATP 酶的单分子研究目标 3：囊泡 H+ATP 酶的单分子研究囊泡谷氨酸转运蛋白目标 4：研究突触上递质摄取与糖酵解酶的耦合囊泡和摄取机制的空间组织。通过这些实验，我们将深入了解这种具有单分子分辨率的复杂分子机制的工作原理，并提供新的见解来了解神经系统疾病引起的功能障碍或药物对这种机制的有针对性的破坏如何影响突触传递。项目叙述：为了协调神经传递，突触前末端存在一千多种蛋白质，它们直接或间接与突触小泡相互作用，最终结果是神经递质受调节释放到突触间隙。突触前功能在突触小泡上的高度收敛导致了神经传递的“以囊泡为中心”的观点，该观点将突触小泡作为突触功能的中心细胞器。该提案描述了以单分子分辨率研究突触小泡的计划，因为辨别这一重要细胞器的详细运作对于实现对神经系统疾病引起的突触传递和功能障碍的下一阶段的定量理解至关重要。