MOLECULAR INTERACTIONS OF SYNAPTOTAGMIN MEDIATING MEMBRANE FUSION

神经元突触的膜融合是一个高度调节的过程，由突触前细胞中微摩尔浓度的Ca 2+触发。尽管SNARE复合物的组装是融合所必需的，但SNARE蛋白本身不受Ca 2+的直接调节。突触结合蛋白I是一种囊泡相关的膜蛋白，被认为在神经元胞吐中起Ca 2+传感器的作用。它通过其N-末端的单个跨膜片段锚定到囊泡膜上，并含有两个已显示以Ca 2+依赖性方式结合膜的C2结构域。据报道，突触结合蛋白也结合SNARE。目前，介导突触结合蛋白相互作用的性质 融合是不被理解的。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复合物的组分相互作用并调节SNARE复合物的组分。拟议的工作将利用磁共振方法，如定点自旋标记，以确定突触结合蛋白的膜和蛋白质的相互作用。这些实验将确定是否synaptotagmin的两个C2结构域假设顺式或反式配置时，与目标膜，并确定这些域是否与SNARE在重建系统中相互作用。提出的实验将测试点突变对膜结合取向的影响， synaptotagmin以及synaptotagmin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Ca 2+触发融合的机制将在神经生物学中对理解突触传递和增强产生直接影响。一般而言，理解融合是如何触发的，以及P1（4，5）P2所起的作用，可能对理解其他融合相关过程，如细胞内转运、宿主防御（杀死微生物、免疫应答）和人类生理学和疾病（例如，葡萄糖调节/糖尿病、过敏反应）。

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).