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).

神经元突触的膜融合是一个高度调控的过程，由突触前细胞中微摩尔浓度的 Ca 2+ 触发。尽管 SNARE 复合物的组装对于融合是必要的，但 SNARE 蛋白本身并不直接受 Ca 2+ 调节。 Synaptotagmin I 是一种囊泡相关膜蛋白，被认为在神经元胞吐作用中充当 Ca 2+ 传感器。它通过 N 端的单个跨膜片段锚定在囊泡膜上，并包含两个 C2 结构域，这些结构域已被证明以 Ca2+ 依赖性方式结合膜。据报道，突触结合蛋白也能结合 SNARE。目前，介导突触结合蛋白相互作用的性质 融合不理解。目前尚不清楚突触结合蛋白中的 C2 结构域是否通过单独与膜结合来触发融合，或者它们是否也与 SNARE 复合物的成分相互作用并对其进行调节。拟议的工作将利用磁共振方法（例如定点自旋标记）来确定突触结合蛋白产生的膜和蛋白质相互作用。这些实验将确定突触结合蛋白的两个 C2 结构域在与靶膜一起出现时是否呈现顺式或反式构型，并确定这些结构域是否与重构系统中的 SNARE 相互作用。拟议的实验将测试点突变对膜结合方向的影响 突触结合蛋白以及突触结合蛋白作用的假设机制。最后，将表征 PI(4,5)P2 与突触结合蛋白和 SNARE 相互作用的强度和化学计量。了解 Ca2+ 触发融合的机制将对神经生物学中理解突触传递和增强产生直接影响。一般来说，了解融合如何触发以及 PI(4,5)P2 所发挥的作用可能会对理解其他融合相关过程产生影响，例如细胞内运输、宿主防御（杀死微生物、免疫反应）以及人类生理和疾病（例如葡萄糖调节/糖尿病、过敏反应）。