Regulation of neurotransmitter release by C2-domain proteins

描述（由申请人提供）：神经递质释放是由Ca2+急性触发的，并在突触前可塑性过程中受到调节，这是大脑中某些信息处理形式的基础。因此，表征释放机制及其调控对于理解脑功能至关重要，并将促进突触前起源的神经系统疾病的治疗发展。控制释放的机制包含一个由Munc18-1和SNARE蛋白以及调节释放的特殊蛋白组成的核心。其中一些蛋白质含有多个C2结构域，这些结构域是广泛存在的Ca和磷脂结合模块，但也可以表现出Ca2+独立的活性。这些蛋白质包括：i) Synaptotagmin-1，触发Ca2+快速释放的传感器；ii) Munc13-1及其相关异构体，它们对释放和介导多种形式的突触前可塑性至关重要；iii)¿RIMs，它们是Rab3效应物，在释放和突触前可塑性中也起关键作用。所有这些蛋白质的C2结构域都是高度保守的，并且在本提案中假设通过它们的Ca2+依赖性和Ca2+非依赖性相互作用在多个水平上调节神经递质释放。本申请中提出的研究的最终目标是验证这一假设，更一般地说，阐明神经递质释放调节关键形式的分子机制。这项研究是综合方法的一部分，在PI实验室进行的结构和重构数据与密切合作者的实验室进行的遗传和生理实验相关联。我们提出三个具体目标，重点关注以下几个方面：Synaptotagmin-1的作用机制2. munc13依赖性调节神经递质释放的结构基础和3。munc13依赖性神经递质释放调控的重构。拟议的实验将建立在先前资助期间获得的令人兴奋的结果的基础上，包括Synaptotagmin-1/SNARE复合物的初步结构，大型Munc13-1片段结晶的关键进展，突触囊泡融合与释放机制的八个最核心组件的重建。以及初步数据，这些数据强化了munc13通过分子内和分子间相互作用的迷人网络充当释放的主要调节剂的概念。我们期望这项研究将为不同突触前可塑性过程中的释放调节提供重要见解，有助于建立对大脑功能至关重要的神经元通信的基本原理。

DESCRIPTION (provided by applicant): 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 neurological disorders with a presynaptic origin. The machinery that controls release contains a core formed by Munc18-1 and SNARE proteins, and specialized proteins that regulate release. Several of these proteins contain multiple C2 domains, which are widespread Ca and phospholipids binding modules but can 2+ also exhibit Ca2+-independent activities. These proteins include: i) Synaptotagmin-1, the Ca2+ sensor that triggers fast release; ii) Munc13-1 and related isoforms, which are essential for release and mediates diverse forms of presynaptic plasticity; and iii) ¿RIMs, which are Rab3 effectors that also have key roles in release and presynaptic plasticity. The C2 domains of all these proteins are highly conserved and are hypothesized in this proposal to regulate neurotransmitter release at multiple levels through their Ca2+-dependent and Ca2+- independent interactions. The ultimate goals of the research proposed in this application are to test this hypothesis and, more generally, to elucidate the molecular mechanisms underlying key forms of regulation of neurotransmitter release. This research forms part of an integrated approach where the structural and reconstitution data performed in the PI's lab are correlated with genetic and physiological experiments performed in the laboratories of close collaborators. We propose three Specific Aims that focus on the following areas: 1. Mechanism of action of Synaptotagmin-1; 2. Structural basis for Munc13-dependent regulation of neurotransmitter release; and 3. Reconstitution of Munc13-dependent regulation of neurotransmitter release. The proposed experiments will build on exciting results obtained during the previous funding period, including a preliminary structure of a Synaptotagmin-1/SNARE complex, key advances in crystallization of large Munc13-1 fragments, the reconstitution of synaptic vesicle fusion with the eight most central components of the release machinery, and preliminary data that reinforce the notion that Munc13s act as master regulators of release through a fascinating network of intramolecular and intermolecular interactions. We expect that this research will provide critical insights into the regulation of release in varied presynaptic plasticity processes, helping to establish fundamental principles on neuronal communication that are vital for brain function.