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.

这个子项目是许多研究子项目中利用 资源由NIH/NCRR资助的中心拨款提供。子项目和 调查员(PI)可能从NIH的另一个来源获得了主要资金， 并因此可以在其他清晰的条目中表示。列出的机构是 该中心不一定是调查人员的机构。 突触素1是一种突触囊泡蛋白，已被证明是触发神经递质快速释放的钙感受器。突触素1的大部分细胞质区域是由两个C2结构域组成的，分别称为C2a结构域和C2b结构域。我们先前已经解决了这两个synaptopagmin1C2-结构域的三维结构，并表明它们通过β-三明治顶端的环与多个钙离子结合。这些环还介导了钙离子依赖的结合到带负电荷的磷脂上，这种活性很可能是突触素1触发释放功能的基础。这些结果导致了一个模型，其中C2a-结构域和C2b-结构域的顶环与膜接触并在磷脂结合中协同作用。 含有两个突触素1 C2结构域的片段(C2AB-片段)在蛋白质浓度接近1 mM的无钙或有钙存在的溶液中是单体。在这些研究过程中，动态光散射实验表明，C2AB-片段在钙依赖的结合上诱导囊泡聚集，这种聚集可以被EGTA逆转。我们的第一个目标是使用冷冻-EM来可视化突触素的这种囊泡聚集活动。 最近报道，C2AB-片段在钙依赖的磷脂结合上形成七聚体低聚物。对沉积在EM网格上的脂质单层结合的C2AB-片段样品进行电子显微镜(EM)阴性染色观察。然而，我们还没有通过化学交联或荧光共振能量转移实验观察到这种低聚物。该项目的第二个目标是了解突触素是否在磷脂膜上是单体，或者它是否如单层电子显微镜所观察到的那样形成七聚体环状低聚物。我们还想测量两个聚集的小泡之间的距离，以计算小泡之间的空间是否足够大，以容纳直径11 nm的突触素寡聚体。 该系统为研究神经功能、神经递质释放和膜融合提供了机会。