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，这可能是学习和记忆的基础。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+依赖性磷脂结合模块，也表现出Ca 2+非依赖性活性。这些蛋白质包括：i）突触结合蛋白1，触发快速释放的Ca 2+传感器; ii）其他突触结合蛋白同种型，其可能充当替代Ca 2+传感器和/或调节释放的Ca 2+敏感性; iii）munc 13 -1，其对于突触囊泡引发是必需的并且介导佛波醇酯的释放增强;和iv）RIM 1，一种Rab效应物，其也参与囊泡引发并且是苔藓纤维长时程增强（LTP）所必需的。这些蛋白质的C2-结构域是高度保守的，并且可能通过其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。为了深入了解这些不同的功能的C2-结构域的释放，我们提出了一个综合的方法，涉及结构，生化，遗传和电生理实验。我们将详细研究突触结合蛋白1 C2-结构域与磷脂和被称为SNARE的膜融合机制的组件的相互作用，并将我们的结果与突触结合蛋白1在体内功能的电生理分析相关联，以阐明它如何触发释放。我们还将比较突触结合蛋白1和2 C2-域的Ca 2+，磷脂和SNARE结合特性，并将测试突触结合蛋白2作为触发脂肪释放的替代Ca 2+传感器的假设。我们还将分析这些属性的C2-域从其他synaptotagmin亚型，并将进行诱变实验，以深入了解生物物理基础的差异，可能是不同类型的Ca 2+调节释放。为了帮助理解RIM 1和munc 13 -1 C2-结构域的功能，它们大多是Ca 2+独立的，我们将确定它们的三维结构，并分析它们与不同靶分子的相互作用，特别强调可能是可塑性过程的基础的相互作用，如苔藓纤维LTP和佛波酯依赖性释放增强。

DESCRIPTION (provided by applicant): Neurotransmitter release is acutely triggered by Ca2+ and is regulated in presynaptic plasticity processes that may underlie learning and memory. 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 diseases with a presynaptic origin. Several proteins with critical roles in release contain multiple C2-domains, which are widespread Ca2+-dependent phospholipid-binding modules that also exhibit Ca2+-independent activities. These proteins include: i) synaptotagmin 1, the Ca2+ sensor that triggers fast release; ii) other synaptotagmin isoforms, which likely act as alternate Ca2+ sensors and/or regulate the Ca2+ sensitivity of release; iii) munc13-1, which is essential for synaptic vesicle priming and mediates augmentation of release by phorbol esters; and iv) RIM1, a Rab effector that is also involved in vesicle priming and is essential for mossy fiber long-term potentiation (LTP). The C2-domains of these proteins are highly conserved and likely regulate neurotransmitter release at multiple levels through their Ca2+-dependent and Ca2+-independent interactions. To gain insight into these diverse functions of C2-domains in release, we propose an integrated approach involving structural, biochemical, genetic and electrophysiological experiments. We will study in detail the interactions of the synaptotagmin 1 C2-domains with phospholipids and with components of the membrane fusion machinery known as SNAREs, and will correlate our results with electrophysiological analyses of synaptotagmin 1 function in vivo to shed light on how it triggers release. We will also compare the Ca2+, phospholipid and SNARE-binding properties of the synaptotagmin 1 and 2 C2-domains, and will test the hypothesis that synaptotagmin 2 acts as an alternate Ca2+-sensor in triggering fats release. We will also analyze these properties in the C2-domains from other synaptotagmin isoforms and will perform mutagenesis experiments to gain insight into the biophysical basis for differences that may underlie distinct types of Ca2+ regulation of release. To help understand the functions of the RIM1 and munc13-1 C2-domains, which are mostly Ca2+-independent, we will determine their three-dimensional structures and will analyze their interactions with diverse target molecules, with particular emphasis on interactions that likely underlie plasticity processes such as mossy fiber LTP and phorbol-ester dependent augmentation of release.