MOLECULAR INTERACTIONS OF SYNAPTOTAGMIN MEDIATING MEMBRANE FUSION

神经元突触的膜融合是一个由突触前细胞内微摩尔浓度的钙离子触发的高度调控的过程。尽管SNARE复合体的组装是融合所必需的，但SNARE蛋白本身并不直接受钙的调节。突触素I是一种囊泡相关的膜蛋白，被认为是神经元胞吐中的钙感受器。它通过N端的一个跨膜片段固定在囊泡膜上，并含有两个C2结构域，已被证明以钙离子依赖的方式与膜结合。据报道，突触素也能结合圈套。目前，介导突触聚集蛋白相互作用的本质 核聚变是不被理解的。目前尚不清楚synaptopagmin中的C2结构域是否仅通过与膜结合来触发融合，或者它们是否也与SNARE复合体的组件相互作用并进行调节。这项拟议的工作将利用磁共振方法，如定点自旋标记，来确定突触素与膜和蛋白质之间的相互作用。这些实验将确定synaptopagmin的两个C2结构域在提供靶膜时是否具有顺式或反式配置，并确定这些结构域是否与重组系统中的SNARs相互作用。拟议的实验将测试点突变对膜结合方向的影响 以及假设的突触聚集作用的机制。最后，将表征PI(4，5)P2与突触素和SNARS相互作用的强度和化学计量比。了解钙离子触发融合的机制将对理解突触传递和增强具有直接的神经生物学意义。一般来说，了解融合是如何被触发的，以及PI(4，5)P2所扮演的角色，可能会对理解其他与融合相关的过程，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)产生影响。

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).