Regulation of neurotransmitter release by C2-domain proteins

描述(申请人提供)：神经递质释放由钙离子强烈触发，并在突触前可塑性过程中受到调节，突触前可塑性过程是大脑某些形式信息处理的基础。因此，表征释放机制及其调控对于了解大脑功能至关重要，并将有助于开发突触前起源的神经疾病的治疗方法。控制释放的机制包含由Munc18-1和SNARE蛋白组成的核心，以及调节释放的专门蛋白。其中一些蛋白含有多个C2结构域，这些结构域是广泛存在的钙和磷脂结合模块，但Ca2+也显示出不依赖于钙的活性。这些蛋白质包括：i)synaptopagmin-1，触发快速释放的钙感受器；ii)Munc13-1及其相关的异构体，它们对释放是必不可少的，并介导各种形式的突触前可塑性；以及iii)？rims，它们是Rab3的效应器，在释放和突触前可塑性中也起着关键作用。所有这些蛋白质的C2结构域都是高度保守的，在本研究中假设通过它们的钙依赖和非钙离子相互作用在多个水平上调节神经递质的释放。在本申请中提出的研究的最终目标是检验这一假说，更广泛地说，是为了阐明神经递质释放的关键调控形式背后的分子机制。这项研究构成了一种综合方法的一部分，在该方法中，在PI的实验室中执行的结构和重建数据与在密切合作者的实验室中执行的遗传和生理实验相关联。我们提出了以下三个具体目标：1.突触素-1的作用机制；2.Munc13依赖的神经递质释放调节的结构基础；3.Munc13依赖的神经递质释放调节的重建。拟议的实验将建立在前一个资助期获得的令人兴奋的结果的基础上，包括synaptopagmin-1/SNARE复合体的初步结构，大Munc13-1片段结晶的关键进展，与释放机制最中心的八个组件的突触小泡融合的重建，以及通过分子内和分子间相互作用的迷人网络强化Munc13作为释放的主要调节器这一概念的初步数据。我们期待这项研究将为各种突触前可塑性过程中释放的调节提供关键的见解，有助于建立对大脑功能至关重要的神经元交流的基本原则。

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.