SYNAPTOTAGMIN 1

突触标记1

• 批准号: 7598603
• 负责人: Jose Rizorey
• 金额: $ 1.63万
• 依托单位: BAYLOR COLLEGE OF MEDICINE
• 依托单位国家: 美国
• 财政年份: 2006
• 资助国家: 美国
• 起止时间: 2006-12-01 至 2007-11-30
• 项目状态: 已结题
• 来源: https://reporter.nih.gov/project-details/7598603
• 关键词: Binding; C2 Domain; Caliber; Charge; Chemicals; Computer Retrieval of Information on Scientific Projects Database; Cryoelectron Microscopy; Deposition; Egtazic Acid; Electron Microscopy; Fluorescence Resonance Energy Transfer; Funding; Grant; Institution; Ions; Lipids; Measures; Mediating; Membrane; Membrane Fusion; Modeling; Negative Staining; Neurophysiology - biologic function; Phospholipids; Proteins; Reporting; Research; Research Personnel; Resources; Sampling; Solutions; Source; Synaptic Vesicles; System; United States National Institutes of Health; Vesicle; complement C2a; complement C2b; crosslink; light scattering; monolayer; neurotransmitter release; research study; sensor; synaptotagmin; synaptotagmin I; three dimensional structure

This subproject is one of many research subprojects utilizing the resources provided by a Center grant funded by NIH/NCRR. The subproject and investigator (PI) may have received primary funding from another NIH source, and thus could be represented in other CRISP entries. The institution listed is for the Center, which is not necessarily the institution for the investigator. Synaptotagmin 1 is a synaptic vesicle protein that has been shown to act as the Ca2+ sensor that triggers fast neurotransmitter release. Most of the cytoplasmic region of synaptotagmin 1 is formed by two C2-domains that are referred to as C2A-domain and C2B-domain. We have previously solved the three-dimensional structures of both synaptotagmin 1 C2-domains and shown that they bind multiple Ca2+ ions through loops at the tip of a beta-sandwich. These loops also mediate Ca2+-dependent binding to negatively charged phospholipids, the activity that most likely underlies the function of synaptotagmin 1 in triggering release. These results have led to a model whereby the top loops of the C2A-domain and C2B-domain are in contact with the membrane and cooperate in phospholipid binding. A fragment containing both synaptotagmin 1 C2-domains (C2AB-fragment) is monomeric in solution in the absence or presence of Ca2+ at protein concentrations close to 1 mM. In the course of these studies, dynamic light scattering experiments suggested that the C2AB-fragment induces vesicle clustering upon Ca2+-dependent binding, and that such clustering can be reversed by addition of EGTA. Our first aim is to visualize this vesicle clustering activity of synaptotagmin using cryo-EM. It was recently reported that the C2AB-fragment forms heptameric oligomers upon Ca2+-dependent phospholipid binding. These oligomers were observed by electron microscopy (EM) performed with negative staining on samples of C2AB-fragment bound to lipid monolayers deposited on EM grids. However, we have not been able to observe such oligomers by chemical cross-linking or fluorescence resonance energy transfer experiments. The second aim of this project is to see whether synaptotagmin is monomeric on the phospholipid membranes or if it forms heptameric ring-like oligomers as observed in electron microscopy of monolayers. We would also like to measure the distance between two clustered vesicles to calculate whether the space between the vesicles is big enough to accommodate synaptotagmin oligomers of 11 nm diameter. This system afford the opportunity to study aspects of neural functioning, neurotransmitter release and membrane fusion in general.

该子项目是利用该技术的众多研究子项目之一 资源由 NIH/NCRR 资助的中心拨款提供。子项目和 研究者 (PI) 可能已从 NIH 的另一个来源获得主要资金， 因此可以在其他 CRISP 条目中表示。列出的机构是 对于中心来说，它不一定是研究者的机构。 Synaptotagmin 1 是一种突触小泡蛋白，已被证明可充当 Ca2+ 传感器，触发快速神经递质释放。突触结合蛋白 1 的大部分细胞质区域由两个 C2 结构域（称为 C2A 结构域和 C2B 结构域）形成。我们之前已经解析了突触结合蛋白 1 C2 结构域的三维结构，并表明它们通过 β 三明治尖端的环结合多个 Ca2+ 离子。这些环还介导 Ca2+ 依赖性与带负电荷的磷脂的结合，这种活性很可能是突触结合蛋白 1 触发释放功能的基础。这些结果产生了一个模型，其中 C2A 结构域和 C2B 结构域的顶部环与膜接触并协同磷脂结合。 含有两个突触结合蛋白 1 C2 结构域（C2AB 片段）的片段在存在或不存在 Ca2+ 的情况下，在蛋白质浓度接近 1 mM 的情况下在溶液中呈单体。在这些研究过程中，动态光散射实验表明，C2AB 片段在 Ca2+ 依赖性结合上诱导囊泡聚集，并且可以通过添加 EGTA 来逆转这种聚集。我们的首要目标是使用冷冻电镜可视化突触结合蛋白的囊泡聚集活性。 最近有报道称，C2AB 片段在 Ca2+ 依赖性磷脂结合后形成七聚体寡聚体。通过电子显微镜 (EM) 观察这些寡聚体，对与沉积在 EM 网格上的脂质单层结合的 C2AB 片段样品进行负染色。然而，我们尚未能够通过化学交联或荧光共振能量转移实验观察到此类低聚物。该项目的第二个目的是了解突触结合蛋白在磷脂膜上是否是单体，或者是否形成单层电子显微镜中观察到的七聚环状低聚物。我们还想测量两个聚集的囊泡之间的距离，以计算囊泡之间的空间是否足够大以容纳直径为 11 nm 的突触结合蛋白寡聚体。 该系统提供了研究神经功能、神经递质释放和膜融合等方面的机会。