Calcium control of Neurotransmitter Release

钙控制神经递质释放

• 批准号: 10718229
• 负责人: SHYAM S KRISHNAKUMAR
• 金额: $ 36.93万
• 依托单位: YALE UNIVERSITY
• 依托单位国家: 美国
• 财政年份: 2023
• 资助国家: 美国
• 起止时间: 2023-07-15 至 2028-05-31
• 项目状态: 未结题
• 来源: https://reporter.nih.gov/project-details/10718229
• 关键词: Affect; Affinity; Behavior; Binding; Biochemical; Biochemistry; Biological Assay; Brain; Calcium; Calcium Signaling; Calcium ion; Cells; Closure by clamp; Communication; Complement; Complex; Coupling; Disease; Docking; Dyes; Genetic; Glutamates; Goals; Health; Image; In Vitro; Individual; Kinetics; Learning; Measurement; Measures; Mediating; Memory; Mental disorders; Modeling; Molecular; Mutation; Nerve; Neurons; Pattern; Physiological; Physiology; Presynaptic Terminals; Process; Property; Protein Isoforms; Proteins; Protocols documentation; Regulation; Research; Role; Shapes; Signal Transduction; Site; Synapses; Synaptic Membranes; Synaptic Vesicles; System; Testing; Time; Titrations; Vesicle; Work; controlled release; crosslink; experience; in vivo; insight; loss of function; millisecond; molecular modeling; nervous system disorder; neural; neurotransmission; neurotransmitter release; novel; presynaptic; reconstitution; response; sensor; single-molecule FRET; synaptotagmin; synaptotagmin I; temporal measurement; tool; vesicular release

Project Summary Neurons communicate with each other by the quantal release of neurotransmitters stored in synaptic vesicles (SVs), and the strength and efficacy of neurotransmitter release are dynamically altered during physiological activity. This process is essential for learning and memory and is disrupted in many neurological disorders. The neurotransmitter release occurs from a pool of SVs docked at the presynaptic active zone and is tightly controlled by activity-dependent changes in the presynaptic calcium ions concentration ([Ca2+]). At nerve terminals, Ca2+- sensing proteins (Synaptotagmins) couple vesicular release machinery (SNAREs) to Ca2+ signals, thus orchestrating neuronal communication. Despite years of research, there remains a substantial gap between a qualitative description of how the vesicle fusion machinery operates and the millisecond-precision and Ca2+- dependent kinetics of neurotransmitter release observed at the neuronal synapses. In this proposal, we describe the first systematic and comprehensive effort to reconcile the `molecular biochemistry' of vesicle fusion with the `physiology' of Ca2+-evoked neurotransmitter release in the neuronal synapses using a combination of complementary in vitro and in vivo experimental systems. Specifically, we aim to resolve whether Synaptotagmin isoforms with distinct biochemical properties (Syt1 and Syt7), along with the synaptic SNAREs represent the minimal protein machinery for different modes of neurotransmitter release and short-term plasticity. We also propose to quantitatively test the hypothesis that the `dual-binding' of Syt1 and Syt7 to SNAREs allows for synergistic regulation of vesicular release kinetics. For in vitro analysis, we will deploy a biochemically-defined, high-throughput fusion system capable of tracking individual vesicle docking and Ca2+ triggered fusion on a millisecond timescale, which is integrated with fast Ca2+-uncaging protocols to generate [Ca2+] transients mimicking presynaptic calcium dynamics. This will be complemented by physiological analysis in cultured neurons utilizing fast, fluorescent glutamate sensor (iGluSnFR) and Ca2+ dyes to image quantal glutamate release and presynaptic Ca2+ dynamics in individual presynaptic boutons with 2-4 millisecond temporal resolution. We anticipate that this project will provide important insights into the molecular mechanisms underlying neurotransmitter release and build toward a detailed mechanistic model of Ca2+-evoked synaptic vesicle fusion. Overall, this will greatly enhance our understanding of neurotransmission and of how it is tuned across different nerve cells allowing specific yet diverse communication in neuronal networks.

项目摘要 神经元通过储存在突触囊泡中的神经递质的数量释放相互通信 （SVS）以及神经递质释放的强度和功效在生理过程中动态改变 活动。这个过程对于学习和记忆至关重要，并且在许多神经系统疾病中受到破坏。这 神经递质释放发生在突触前活跃区域停靠的SVS池中，并受到严格控制 通过活动依赖性钙离子浓度的变化（[Ca2+]）。在神经终端，Ca2+ - 传感蛋白（突触量）夫妇囊泡释放机械（SNARES）到Ca2+信号，因此 编排神经元交流。尽管进行了多年的研究，但在 囊泡融合机械如何运行以及毫秒前的定性描述和Ca2+ - 在神经元突触上观察到的神经递质释放的依赖动力学。 在此提案中，我们描述了调和分子的第一个系统和全面的努力 囊泡融合的生物化学与Ca2+诱发神经递质释放的“生理学”在神经元中的释放 使用互补的体外和体内实验系统的突触。具体来说，我们的目标 解决具有不同生化特性（SYT1和SYT7）的突触量同工型是否以及 突触网罗代表不同模式神经递质释放的最小蛋白质机制和 短期可塑性。我们还建议定量检验SYT1和 SYT7至SNARES允许对囊泡释放动力学的协同调节。对于体外分析，我们将部署 能够跟踪单个囊泡对接和Ca2+的生化定义的高通量融合系统 在毫秒的时间表上触发融合，该融合与快速的Ca2+uncanged协议集成在一起 [Ca2+]模仿突触前钙动力学的瞬态。这将通过生理分析来补充 在使用快速的荧光谷氨酸传感器（IGLUSNFR）和Ca2+染料的培养神经元中 谷氨酸释放和突触前Ca2+动力学在单个突触前胸子中具有2-4毫秒的时间 解决。我们预计该项目将为分子机制提供重要的见解 基础神经递质释放，并朝着Ca2+诱发的突触的详细机械模型建立 囊泡融合。总体而言，这将大大增强我们对神经传递的理解及其如何调整 在不同的神经细胞中，可以在神经元网络中进行特定但多样化的沟通。