Mechanisms of neurotransmitter release and its regulation

神经递质释放机制及其调控

• 批准号: 10532693
• 负责人: Jose Rizorey
• 金额: $ 96.75万
• 依托单位: UT SOUTHWESTERN MEDICAL CENTER
• 依托单位国家: 美国
• 财政年份: 2016
• 资助国家: 美国
• 起止时间: 2016-12-01 至 2024-11-30
• 项目状态: 已结题
• 来源: https://reporter.nih.gov/project-details/10532693
• 关键词: Acute; Alzheimer' s Disease; Biological Assay; Blood Pressure; Brain; Cellular biology; Communication; Complement; Complex; Cryo-electron tomography; Cryoelectron Microscopy; Defect; Development; Disease; Docking; Electrophysiology (science); Goals; Heart Rate; Insulin; Intracellular Membranes; Knowledge; Membrane; Membrane Fusion; Molecular; NMR Spectroscopy; Nervous System; Neurons; Parkinson Disease; Pharmaceutical Preparations; Physiological; Process; Proteins; Regulation; Research; SNAP receptor; Schizophrenia; Synaptic Transmission; Synaptic Vesicles; Synaptic plasticity; Testing; X-Ray Crystallography; biophysical techniques; experimental study; information processing; insight; interdisciplinary approach; nervous system disorder; neurotransmitter release; novel; novel strategies; presynaptic; protein complex; reconstitution; success; synaptotagmin; therapy development; three dimensional structure

Neurotransmitter release is acutely triggered by Ca2+ and is regulated during presynaptic plasticity processes that underlie some forms of information processing in the brain. Characterization of the mechanisms of release and its regulation is thus critical to understand brain function and will facilitate the development of therapies for multiple neurological disorders (e.g. schizophrenia, Alzheimer's and Parkinson's) and for diseases involving defects in regulated secretion, which controls many important physiological functions (e.g. heart rate, blood pressure and insulin release). This research is also relevant to cell biology in general because of its importance to understand intracellular membrane fusion. The machinery that controls release contains a core formed by SNARE proteins, Munc18, Munc13, NSF and SNAP, and specialized proteins that regulate release including Munc13 itself, synaptotagmin, complexin, RIM, Rab3 and CAPS among others. The research proposed in this application involves an interdisciplinary approach integrating structural studies of these proteins, reconstitution assays and electrophysiological analyses of neurotransmitter release in neurons performed by collaborators. This research will build on the previous success of this approach, which has yielded many of the three-dimensional structures of the proteins that govern neurotransmitter release, has revealed crucial mechanistic concepts in the field and has allowed reconstitution of basic steps of Ca2+-evoked synaptic vesicle fusion with eight central components of the release machinery. However, despite these advances, the mechanism of release is still unclear and fundamental questions remain unanswered. The ultimate goals of this proposal are to develop a detailed picture of the mechanism of release that integrates the functions of all these proteins and to provide novel insights into how release is regulated in diverse presynaptic plasticity processes. For this purpose, structural studies of complexes formed by these proteins using a combination of biophysical techniques, including cryo-electron microscopy, cryo-electron tomography, NMR spectroscopy and X-ray crystallography, will be performed. A key aspect of these studies, which is essential to make major, definitive advances in this field, will be to analyze protein complexes between two membranes, as the membranes form intrinsic part of the fusion apparatus and thus are expected to have a strong influence on how the protein components are arranged to induce membrane fusion. The structural studies will be complemented with reconstitution experiments to understand how the different proteins control docking, priming and fusion, as well as with physiological analyses performed by collaborators that will test the relevance of the structural and reconstitution studies. This research is expected to establish fundamental principles on neuronal communication that are vital for brain function.

神经递质释放由 Ca2+ 急剧触发，并在突触前可塑性过程中受到调节 这是大脑中某些形式的信息处理的基础。释放机制的表征 因此，它的调节对于理解大脑功能至关重要，并将促进治疗方法的开发 用于多种神经系统疾病（例如精神分裂症、阿尔茨海默氏症和帕金森氏症）以及涉及以下疾病 调节分泌的缺陷，控制着许多重要的生理功能（例如心率、血液 压力和胰岛素释放）。由于其重要性，这项研究也与一般细胞生物学相关 了解细胞内膜融合。控制释放的机制包含一个由以下组成的核心 SNARE 蛋白、Munc18、Munc13、NSF 和 SNAP，以及调节释放的特殊蛋白，包括 Munc13 本身、突触结合蛋白、复合蛋白、RIM、Rab3 和 CAPS 等。本文提出的研究 应用涉及整合这些蛋白质的结构研究的跨学科方法， 神经元中神经递质释放的重建测定和电生理分析 合作者。这项研究将建立在这种方法先前成功的基础上，该方法已经产生了许多 控制神经递质释放的蛋白质的三维结构揭示了至关重要的 该领域的机械概念，并允许重建 Ca2+ 诱发的突触小泡的基本步骤 与释放机械的八个中心部件融合。然而，尽管取得了这些进步， 释放机制仍不清楚，基本问题仍未得到解答。此次活动的最终目标 建议制定一个详细的释放机制图，整合所有这些功能 蛋白质并提供关于如何在不同的突触前可塑性过程中调节释放的新见解。 为此，结合生物物理学对这些蛋白质形成的复合物进行结构研究 技术，包括冷冻电子显微镜、冷冻电子断层扫描、核磁共振波谱和 X 射线 将进行晶体学分析。这些研究的一个关键方面对于做出重大的、明确的研究至关重要 该领域的进展将是分析两个膜之间的蛋白质复合物，因为膜形成 融合装置的内在部分，因此预计会对蛋白质如何 组分被排列以诱导膜融合。结构研究将得到补充 重构实验以了解不同的蛋白质如何控制对接、引发和融合，例如 以及合作者进行的生理分析，以测试结构和结构的相关性 重建研究。这项研究有望建立神经元的基本原理 对大脑功能至关重要的沟通。