Synaptotagmin and C2-domains: structure and function

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。一些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，这些C2结构域是广泛存在的钙和磷脂结合模块，但也可以显示出钙离子不依赖的活性。这些蛋白质包括：i)SNAPTOAGMIN-1，触发快速释放的钙离子感受器；ii)其他SNAPTOAGMIN异构体，在中枢神经系统和神经内分泌细胞的不同区域作为钙离子感受器，调节钙释放的敏感性；iii)Munc13-1及其相关异构体，对于突触小泡启动是必不可少的，并介导各种形式的突触前可塑性；iv)RIMS，它是Rab3的效应器，在囊泡启动和突触前可塑性中也有关键作用。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。为了验证这一假说并深入了解这些蛋白质C2结构域在释放过程中的不同功能，本申请提出了用不同的生物物理方法研究它们的结构和相互作用，包括核磁共振光谱、X射线结晶学、低温电子显微镜和荧光光谱。这项研究是一种综合方法的一部分，在这种方法中，生物物理数据与在密切合作者的实验室中进行的遗传和功能实验相关联。提出了三个具体目标。目的1将继续正在进行的研究，通过研究突触素-1与SNARE蛋白和复合蛋白的相互作用，阐明突触素-1如何以钙离子依赖的方式与SNARE蛋白一起触发膜融合和神经递质释放。重点将放在阐明由SNARS、突触素-1、钙和磷脂形成的四元复合体的结构，该复合体很可能在依赖钙的膜融合中发挥中心作用。目的2将继续致力于比较与钙离子引起的胞吐作用有关的其他突触素亚型的生化特性，并破译这些特性差异背后的序列决定因素。这些研究将阐明系统之间的功能分化的基础，这可能是大脑功能的基础。目的3通过研究Munc13-1 C2结构域与在囊泡启动中起关键作用的C端MUN结构域的分子内相互作用及其对MUN结构域活动的影响，验证Munc13-1 C2结构域控制多种形式突触前可塑性的假设。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中发挥作用，也将被研究。与公共健康相关：这项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的见解。这一知识对于了解大脑和神经系统的总体功能至关重要。此外，由于许多神经疾病是用改变突触传递的药物治疗的，这项研究有望为开发理解和治疗这些疾病的新策略提供关键线索。

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.