Synaptotagmin and C2-domains: structure and function

描述（由申请人提供）：神经递质释放由Ca 2+急性触发，并在突触前可塑性过程中受到调节，该过程是大脑中某些形式的信息处理的基础。因此，释放及其调节机制的表征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方法的发展。几种在神经递质释放中具有关键作用的蛋白质含有多个C2结构域，它们是广泛的Ca 2+和磷脂结合模块，但也可以表现出Ca 2+非依赖性活性。这些蛋白质包括：i）synaptotagmin-1，触发快速释放的Ca 2+传感器; ii）其他synaptotagmin同种型，其在中枢神经系统和神经内分泌细胞的不同区域中充当交替的Ca 2+传感器，并调节释放的Ca 2+敏感性; iii）Munc 13 -1和相关同种型，其对于突触囊泡引发是必需的，并介导多种形式的突触前可塑性; iv）RIM，其是Rab 3效应物，其在囊泡引发和突触前可塑性中也具有关键作用。所有这些蛋白质的C2结构域都是高度保守的，并且在该提议中假设通过其Ca 2+依赖性和Ca 2+非依赖性相互作用在多个水平上调节神经递质释放。为了验证这一假设并深入了解这些蛋白质释放过程中C2结构域的不同功能，本申请提出通过多种生物物理方法研究其结构和相互作用，包括NMR光谱学、X射线晶体学、冷冻电子显微镜和荧光光谱学。这项研究是综合方法的一部分，其中生物物理数据与密切合作者实验室中进行的遗传和功能实验相关。提出了三个具体目标。目标1将继续进行中的研究，旨在阐明突触结合蛋白-1如何触发膜融合和神经递质释放的Ca 2+依赖性的方式与SNARE蛋白，并在一个紧密的相互作用与复合蛋白，通过表征它们的相互作用。一个特别的重点将放在阐明由SNARE，突触结合蛋白-1，Ca 2+和磷脂形成的四元复合物的结构，这很可能在Ca 2+依赖的膜融合中起着核心作用。目标2将继续研究致力于比较其他synaptotagmin异构体的生化特性参与Ca 2+诱发的胞吐，并在破译这些属性的差异的基础上的序列决定因素。这些研究将揭示Syts之间的功能分化的基础，这可能是大脑功能的基础。目的3将测试的假设，Munc 13 -1 C2结构域控制不同形式的突触前可塑性，通过分子内相互作用与C-末端MUN结构域，起着至关重要的作用，在囊泡启动，通过表征这些相互作用，以及它们如何影响MUN结构域的活动。RIM C2结构域的潜在相互作用，可能在囊泡启动和突触前可塑性的基础作用也将进行研究。公共卫生相关性：在这项申请中提出的研究将产生关键的见解，基本的分子机制，突触传递和某些形式的信息处理在大脑中。这些知识对于理解大脑和神经系统的一般功能至关重要。此外，由于许多神经系统疾病都是用改变突触传递的药物治疗的，因此这项研究有望为开发新的策略来理解和治疗这些疾病提供关键线索。

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. Several proteins with crucial roles in neurotransmitter release contain multiple C2 domains, which are widespread Ca2+ and phospholipids binding modules but can also exhibit Ca2+-independent activities. These proteins include: i) synaptotagmin-1, the Ca2+ sensor that triggers fast release; ii) other synaptotagmin isoforms, which act as alternate Ca2+ sensors in different regions of the central nervous system and neuroendochrine cells, and modulate the Ca2+ sensitivity of release; iii) Munc13-1 and related isoforms, which are essential for synaptic vesicle priming and mediates diverse forms of presynaptic plasticity; iv) RIMs, which are Rab3 effectors that also have key roles in vesicle priming and presynaptic plasticity. The C2 domains of all these proteins are highly conserved and are hypothesizes in this proposal to regulate neurotransmitter release at multiple levels through their Ca2+-dependent and Ca2+- independent interactions. To test this hypothesis and gain insight into the diverse functions of C2 domains from these proteins in release, this application proposes studies of their structures and interactions by diverse biophysical methods, including NMR spectroscopy, X-ray crystallography, cryo-electron microscopy and fluorescence spectroscopy. This research forms part of an integrated approach where the biophysical data are correlated with genetic and functional experiments performed in the laboratories of close collaborators. Three Specific Aims are proposed. Aim 1 will continue ongoing studies directed at elucidating how synaptotagmin-1 triggers membrane fusion and neurotransmitter release in a Ca2+-dependent manner together with SNARE proteins and in a tight interplay with complexins, by characterizing their interactions. A particular focus will be placed at elucidating the structure of a quaternary complex formed by SNAREs, synaptotagmin-1, Ca2+ and phospholipids, which most likely plays a central role inCa2+-dependent membrane fusion. Aim 2 will continue studies devoted to compare the biochemical properties of other synaptotagmin isoforms involved in Ca2+- evoked exocytosis, and at deciphering the sequence determinants that underlie differences in these properties. These studies will shed light on the basis for functional differentiation between Syts, which is likely fundamental for brain function. Aim 3 will test the hypothesis that the Munc13-1 C2 domains control diverse forms of presynaptic plasticity through intramolecular interactions with the C-terminal MUN domain that plays a crucial role in vesicle priming, by characterizing these interactions and how they influence MUN domain activity. Potential interaction of RIM C2 domains that may underlie roles in vesicle priming and presynaptic plasticity will also be investigated. PUBLIC HEALTH RELEVANCE: The research proposed in this application will yield key insights into fundamental molecular mechanisms that underlie synaptic transmission and some forms of information processing in the brain. This knowledge is critical to understand how the brain and the nervous system in general function. Moreover, since many neurological disorders are treated with drugs that alter synaptic transmission, this research is expected to provide crucial clues for the development of novel strategies to understand and treat these disorders.