Synaptotagmin C2B Domain as a Ca2+-sensing module

描述（由申请人提供）： 神经元之间的交流依赖于神经递质的释放， 突触前神经末梢释放是由增加 细胞内[Ca 2 +]和介导的融合的transmitter填充 突触囊泡与质膜。的分子机制 Ca 2+与胞吐作用的偶联尚不清楚。Synaptotagmin是一种钙离子结合蛋白， 一种蛋白质，被认为是一种Ca 2+传感器， release.我们建议研究这种蛋白质的功能，主要是在其 两个推定的Ca 2+敏感结构域，C2 A和C2B。结构和Ca 2+结合 C2 A-结构域的性质先前已被详细研究。然而，在这方面， 关于C2B结构域的功能知之甚少。我们假设 C2B是一种钙离子敏感模块，在钙离子触发的细胞凋亡中起着关键作用。 胞吐作用初步数据支持这一假设 这表明C2B必须结合Ca 2+，改变构象并寡聚化， 对接的突触囊泡在体内对刺激的反应中融合的顺序。 为了确定C2B在胞吐中的功能，提出了三个具体目标。 (1)一系列的生化研究将检查Ca 2 +-C2B的动力学 相互作用，突触结合蛋白寡聚化的Ca 2+要求，以及 C2B在促进SNARE复合物组装中的作用。(2)时间分辨 电流分析法，以及果蝇的遗传操作，将被用来 阐明C2B在融合孔动力学和兴奋分泌中的作用 偶合器.一个关键的初步发现是，突触结合蛋白调节 融合孔的开放和扩张动力学，将synaptotagmin放置在 由SNARE介导的融合反应的最后阶段。这一发现 支持突触结合蛋白和SNARE是 Ca 2+调节的胞吐融合孔。(3)将进行生化研究 确定这种复合物的亚基化学计量，其形态， 这些界面是其组装的媒介。 释放机制对Ca 2+的反应能力取决于 调制，并可能包括一个重要的位点，为突触 可塑性。因此，更好地理解发布过程将提供 深入了解突触可塑性的新模式，并最终提供 用于治疗其中突触传递被破坏的疾病的靶点 受损

DESCRIPTION (provided by applicant): Communication between neurons relies on the release of neurotransmitters from pre-synaptic nerve terminals. Release is triggered by increases in intracellular [Ca2+] and is mediated by the fusion of transmitter-filled synaptic vesicles with the plasma membrane. The molecular mechanism that couples Ca2+ to exocytosis is not known. Synaptotagmin is a Ca2+ binding protein that has been proposed to function as a Ca2+ sensor that triggers release. We propose to examine the function of this protein, primarily on its two putative Ca2+-sensing domains, C2A and C2B. The structure and Ca2+-binding properties of the C2A-domain have been previously studied in detail. However, little is known concerning the function of the C2B-domain. We hypothesize that C2B is a Ca2+-sensing module that plays a critical role in Ca2+-triggered exocytosis. Support for this hypothesis is provided by preliminary data indicating that C2B must bind Ca2+, change conformation and oligomerize in order for docked synaptic vesicles to fuse in response to stimulation in vivo. To determine how C2B functions in exocytosis, three Specific Aims are proposed. (1) A series of biochemical studies will examine the kinetics of Ca2+-C2B interactions, the Ca2+ requirements for synaptotagmin oligomerization, and the role of C2B in the facilitation of SNARE complex assembly. (2) Time resolved amperometry, as well as genetic manipulations of Drosophila, will be used to address the role of C2B in fusion pore dynamics and in excitation-secretion coupling. A critical preliminary finding is that synaptotagmin regulates the opening and dilation kinetics of fusion pores, placing synaptotagmin at the final stages of the fusion reaction that is mediated by SNAREs. This finding supports the hypothesis that synaptotagmins and SNAREs are constituents of Ca2+-regulated exocytotic fusion pores. (3) Biochemical studies will be carried out to determine the subunit stoichiometry of this complex, its morphology, and the interfaces that mediate its assembly. The ability of the release machinery to respond to Ca2+ is subject to modulation and is likely to comprise an important locus for synaptic plasticity. Thus, a better understanding of the release process will provide insights into novel modes of synaptic plasticity and should ultimately provide targets for the treatment of diseases in which synaptic transmission is impaired.