Synaptotagmin and C2-domains: structure and function

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。几种在神经递质释放中起关键作用的蛋白质含有多个C2结构域，这是广泛存在的Ca2+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)其他synaptotagmin亚型，在中枢神经系统和神经内分泌细胞的不同区域充当替代Ca2+传感器，并调节Ca2+释放的敏感性；iii) Munc13-1及其相关异构体，对突触囊泡启动至关重要，并介导多种形式的突触前可塑性；iv) RIMs，这是Rab3效应物，在囊泡启动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。为了验证这一假设，并从这些释放的蛋白质中深入了解C2结构域的不同功能，本申请提出了通过多种生物物理方法研究它们的结构和相互作用，包括核磁共振波谱、x射线晶体学、冷冻电子显微镜和荧光光谱。这项研究形成了一个综合方法的一部分，其中生物物理数据与密切合作者在实验室进行的遗传和功能实验相关联。提出了三个具体目标。Aim 1将继续正在进行的研究，旨在阐明synaptotagmin-1如何通过表征它们的相互作用，以Ca2+依赖的方式与SNARE蛋白一起触发膜融合和神经递质释放，并与复杂蛋白紧密相互作用。特别的重点将放在阐明由SNAREs， synaptotagmin-1， Ca2+和磷脂形成的四元复合物的结构，这很可能在Ca2+依赖的膜融合中起核心作用。Aim 2将继续致力于比较其他参与Ca2+诱发胞外分泌的synaptotagmin亚型的生化特性，并破译这些特性差异背后的序列决定因素。这些研究将揭示系统之间功能分化的基础，这可能是大脑功能的基础。目的3将通过表征这些相互作用以及它们如何影响MUN结构域活性，验证Munc13-1 C2结构域通过与c端MUN结构域的分子内相互作用控制多种形式的突触前可塑性的假设，后者在囊泡启动中起着至关重要的作用。RIM C2结构域在囊泡启动和突触前可塑性中的潜在相互作用也将被研究。公共卫生相关性：本申请中提出的研究将对突触传递和大脑中某些形式的信息处理的基本分子机制产生关键见解。这些知识对于理解大脑和神经系统的一般功能是至关重要的。此外，由于许多神经系统疾病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重要线索。

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.