MOLECULAR INTERACTIONS OF SYNAPTOTAGMIN MEDIATING MEMBRANE FUSION

突触结合蛋白介导膜融合的分子相互作用

• 批准号: 7036466
• 负责人: DAVID S CAFISO
• 金额: $ 16.75万
• 依托单位: UNIVERSITY OF VIRGINIA
• 依托单位国家: 美国
• 财政年份: 2004
• 资助国家: 美国
• 起止时间: 2004-12-01 至 2010-03-31
• 项目状态: 已结题
• 来源: https://reporter.nih.gov/project-details/7036466
• 关键词: calcium flux; exocytosis; intermolecular interaction; long term potentiation; magnetic resonance imaging; membrane fusion; membrane proteins; neural transmission; point mutation; protein binding; protein protein interaction; protein structure function; stoichiometry; synapses; synaptotagmin

Membrane fusion at neuronal synapses is a highly regulated process that is triggered by micromolar concentrations of Ca 2+ in the presynaptic cell. Although assembly of the SNARE complex is necessary for fusion, the SNARE proteins themselves are not directly regulated by Ca 2+. Synaptotagmin I is a vesicle associated membrane protein that is believed to function as the Ca 2+ sensor in neuronal exocytosis. It is anchored to the vesicle membrane by a single transmembrane segment at its N-terminus and contains two C2 domains that have been shown to bind membranes in a Ca2+-dependent fashion. Synaptotagmin is also reported to bind SNAREs. At the present time, the nature of the synaptotagmin interactions that mediate fusion are not understood. It is not known whether the C2 domains in synaptotagmin trigger fusion by binding to membranes alone, or whether they also interact with and modulate components of the SNARE complex. The proposed work will utilize magnetic resonance methods, such as site-directed spin labeling, to determine the membrane and protein interactions made by synaptotagmin. These experiments will determine whether synaptotagmin's two C2 domains assume cis or trans configurations when presented with target membranes, and determine whether these domains interact with SNAREs in reconstituted systems. Proposed experiments will test the effect of point mutations on the membrane bound orientation of synaptotagmin as well as hypothesized mechanims for synaptotagmin action. Finally, both the strength and stoichiometry of PI(4,5)P2 interactions with synaptotagmin and SNAREs will be characterized. Understanding the mechanism of Ca2+-triggered fusion will have direct consequences in neurobiology for understanding synaptic transmission and potentiation. In general, understanding how fusion is triggered, and the role played by PI(4,5)P2, may have consequences for understanding other fusion related processes, such as intracellular transport, host defense (killing of microorganisms, immune response), and human physiology and disease (e.g., glucose regulation/diabetes, allergic response).

神经元突触时的膜融合是一个高度调节的过程，在突触前细胞中由Ca 2+触发。尽管SNARE复合物的组装对于融合是必需的，但SNARE蛋白本身并非由Ca 2+直接调节。 Synaptotagmin I是一种囊泡相关的膜蛋白，据信在神经元胞胞菌病中充当Ca 2+传感器。它通过其N末端的单个跨膜段固定在囊泡膜上，并包含两个C2结构域，这些域已显示出以Ca2+依赖性方式结合膜。据报道，SynaptoTagmin结合了鼻。目前，介导的突触毒素相互作用的性质 融合不了解。尚不清楚突触毒素中的C2结构域是否仅通过与膜结合，或者它们是否也与SNARE复合物的相互作用并调节组件。拟议的工作将利用磁共振方法（例如位于位置的自旋标记）来确定突触量抗蛋白产生的膜和蛋白质相互作用。这些实验将确定突触磁蛋白的两个C2域在用目标膜呈现时是否假定顺式或反式构型，并确定这些域是否与重构系统中的SNARE相互作用。提出的实验将测试点突变对膜结合方向的影响 Synaptotagmin以及突触策略作用的假设机制。最后，将表征PI（4,5）P2的强度和化学计量法与突触抗体和幼虫的相互作用。了解Ca2+触发的融合的机制将在神经生物学中有直接的后果，以理解突触传播和增强。通常，了解融合的触发方式以及PI（4,5）P2的作用可能会影响理解其他相关过程，例如细胞内运输，宿主防御（杀死微生物，免疫反应）以及人类的生理学和疾病（例如，glucose and glucose and glucose consemant/diabetes/diabetes，extragic Respress）。