SYNAPTOTAGMIN AND C2 DOMAINS--STRUCTURE AND FUNCTION

神经递质的释放在非常快的反应（<1 ms）中由Ca 2+急性触发神经递质的释放也在突触可塑性过程中由Ca 2+在多个水平上调节，这可能是学习和记忆形成的基础。 因此，神经递质释放及其Ca 2+调节的分子基础的表征对于理解脑功能至关重要，并且将极大地促进具有突触前起源的神经系统疾病的治疗的发展。 许多参与神经递质释放的蛋白质都含有一个通用的钙离子结合模块，称为C2结构域，这表明C2结构域在神经递质释放的钙离子调节中起着多种作用。 这些蛋白质包括synaptotagmin、rabphilin、DOC 2s、munc 13、Rim、aczonin和intersectin。突触结合蛋白是一种突触囊泡蛋白，在其胞质区域中含有两个C2-结构域，由于其作为触发释放的Ca 2+传感器的假定作用，因此在这些蛋白质中研究最广泛。然而，synaptotagmin的功能在很大程度上仍不清楚。 为了阐明这一功能，提出了一个综合的方法，涉及结构，生化和遗传实验。 该方法的目的是表征突触结合蛋白的结构特征，可能是重要的，其功能和与突触胞吐机制的基本组成部分的相互作用，并在体内测试这些相互作用的重要性。 此外，该研究还旨在将突触结合蛋白的研究扩展到其他与神经递质释放有关但功能也不清楚的C2结构域蛋白。 在这方面的主要目标是有一个比较图片的突触前C2-域的钙离子和磷脂结合特性，了解这些属性的结构决定因素，并确定是否最有趣的这些C2-域的结构特征，可能是其功能的基础。 为实现这些目标，拟议的五个具体目标侧重于以下领域。 1. 突触结合蛋白胞质区的结构和钙结合特性。 2. 涉及突触结合蛋白的蛋白质相互作用的结构基础。 3. 突触结合蛋白在体内功能的突变分析。 4. Ca 2+和磷脂与C2-结构域的结合。 5. 来自munc 13、RIM和aczonin的C2-结构域的三维结构。 总体而言，这项研究将产生关键的信息，了解synaptotagmin的功能，并将揭示一般的Ca 2+和磷脂结合到C2-结构域的结构决定因素。这些特性的表征不仅将深入了解C2-结构域在神经递质释放中的功能，而且还将促进未来的研究，进一步阐明这些功能。

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.