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从 突触前神经末梢。发布的触发因素是 细胞内[Ca~(2+)]，并由充满递质的 与质膜相连的突触小泡。其分子机制是 钙离子对胞吐作用的影响尚不清楚。突触素是一种钙离子结合蛋白 一种蛋白质，被认为是一种钙离子感受器，可以触发 放手。我们建议研究这种蛋白质的功能，主要是通过它的 两个可能的钙敏感结构域，C2a和C2b。结构与钙离子结合 C2a-结构域的性质已经被详细研究过了。然而， 关于C2B结构域的功能知之甚少。我们假设 C2B是一种钙离子感应模块，在钙离子触发过程中起着关键作用 胞吐。初步数据支持这一假设 提示C2B必须结合Ca~(2+)，改变构象和寡聚。 体内对接的突触小泡在刺激下融合的顺序。 为了确定C2B在胞吐作用中的作用，提出了三个具体目标。 (1)一系列生化研究将考察钙-C2B的动力学 相互作用，突触素齐聚所需的钙离子，以及 C2B在促进SNARE复合体组装中的作用。(2)时间已解决 安培法以及对果蝇的遗传操作将被用于 阐述C2B在融合孔动力学和刺激分泌中的作用 耦合。一个关键的初步发现是，突触素调节 融合孔的开放和扩张动力学，将突触素放置在 由圈套介导的融合反应的最后阶段。这一发现 支持突触素和陷阱是 钙离子调控的胞吐融合毛孔。(3)进行生化研究 来确定这个复合体的亚单位化学计量、它的形态和 协调其组装的接口。 释放机制对钙离子的反应能力受 并可能构成突触的一个重要基因位点 可塑性。因此，对发布过程的更好理解将提供 对突触可塑性的新模式的洞察，并最终应提供 突触传递异常的疾病的治疗靶点 受伤了。

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.