SYNAPTOTAGMIN AND C2 DOMAINS--STRUCTURE AND FUNCTION

神经递质的释放是在非常快的反应中(1ms)由钙离子强烈触发的，在突触可塑性过程中，神经递质的释放也受到钙离子在多个水平上的调节，这可能是学习和记忆形成的基础。因此，表征神经递质释放的分子基础及其对钙离子的调节对于了解大脑功能是至关重要的，并将极大地促进突触前起源的神经疾病的治疗。许多与神经递质释放有关的蛋白质都含有一个被称为C2-结构域的通用钙结合模块，这表明C2-结构域在神经递质释放的钙调节中发挥着多种作用。这些蛋白包括突触素、RabPhilin、DOC2s、Munc13、Rim、acztin和intersectin。突触囊泡蛋白是一种突触囊泡蛋白，其胞质区域含有两个C2结构域，是这些蛋白中研究最广泛的，因为它被认为是触发释放的钙感受器。然而，突触素的功能在很大程度上仍然不清楚。为了阐明这一功能，提出了一种涉及结构、生化和遗传实验的综合方法。该方法旨在表征突触聚集蛋白的结构特征，这些结构特征可能对其功能以及与突触胞吐机制的基本组件的相互作用至关重要，并测试这些相互作用在体内的重要性。此外，拟议的研究旨在将对突触素的研究扩展到其他C2结构域蛋白，这些蛋白与神经递质释放有关，但其功能尚不清楚。这一领域的主要目标是比较突触前C2结构域的钙离子和磷脂结合特性，了解这些特性的结构决定因素，并确定这些C2结构域中最有趣的是否具有可能支持其功能的结构特征。为实现这些目标，提出的五个具体目标侧重于以下几个方面。1.突触素胞浆区的结构和钙结合特性。2.涉及突触素的蛋白质-蛋白质相互作用的结构基础。3.体内突触素功能突变分析。4.钙离子和磷脂与C2结构域的结合。5.Munc13、RIM和Aczatin的C2结构域的三维结构。总体而言，这项研究将为理解突触素的功能提供关键信息，并将揭示钙离子和磷脂与C2-结构域结合的结构决定因素。对这些性质的表征不仅有助于深入了解C2结构域在神经递质释放中的功能，还将有助于未来进一步阐明这些功能的研究。

Neurotransmitters are released in a very fast reaction (<1ms) acutely triggered by Ca2+ Neurotransmitter release is also regulated by Ca2+ at multiple levels in processes of synaptic plasticity that may underlie learning and memory formation. Characterization of the molecular basis of neurotransmitter release and its Ca2+ regulation is thus crucial to understand brain function and would greatly facilitate the development of therapies for neurological diseases with a presynaptic origin. The observation that a variety of proteins implicated in neurotransmitter release contain a universal Ca2+-binding module known as the C2-domain suggests that C2-domains play multiple roles in Ca2+ regulation of neurotransmitter release. These proteins include synaptotagmin, rabphilin, DOC2s, munc13, Rim, aczonin and intersectin. Synaptotagmin, a synaptic vesicle protein that contains two C2-domains in its cytoplasmic region, is the most extensively studied among these proteins because of its putative role as the Ca2+ sensor that triggers release. However, the function of synaptotagmin remains largely unclear. To shed light on this function, an integrated approach involving structural, biochemical and genetic experiments is proposed. The approach is intended to characterize structural features of synaptotagmin that may be important for its function and for interactions with essential components of the synaptic exocytotic machinery, and to test the importance of these interactions in vivo. In addition, the research proposed is intended to extend the studies on synaptotagmin to other C2-domain proteins that have been implicated in neurotransmitter release but whose functions are also unclear. The primary goals in this area are to have a comparative picture of the Ca2+ and phospholipid binding properties of presynaptic C2-domains, to understand the structural determinants of these properties, and to determine whether the most interesting among these C2-domains have structural features that may underlie their functions. To achieve these goals, the five specific aims proposed focus on the following areas. 1. Structure and Ca2+-binding properties of the cytoplasmic region of synaptotagmin. 2. Structural basis of protein-protein interactions involving synaptotagmin. 3. Mutational analysis of synaptotagmin function in vivo. 4. Ca2+ and phospholipid binding to C2-domains. 5. Three dimensional structures of the C2-domains from munc13, RIM and aczonin. Overall, this research will yield critical information to understand the function of synaptotagmin and will reveal the structural determinants for Ca2+ and phospholipid binding to C2-domains in general. Characterization of these properties will not only yield insights into the functions of C2-domains in neurotransmitter release but will also facilitate future studies that will further clarify these functions.