REGULATION OF DYNAMIN I IN SYNAPTIC TRANSMISSION

描述（来自申请人摘要）动作电位到达 突触前神经终末伸长膜去极化和突触后神经元凋亡 游离Ca 2+浓度增加。 这些事件触发释放 神经递质从突触囊泡胞吐，其次是 通过内吞作用恢复融合的突触囊泡膜。 这 囊泡膜的再循环对于释放过程及其 损伤可能导致瘫痪和精神失常。 在 目前只有一种酶，即GT3发动蛋白I，已明确显示 在突触膜修复中发挥重要作用。 如果GTTT 发动蛋白I的活性被抑制，或者通过突变，或者通过使用 不可水解的GTP类似物，深深陷入网格蛋白包被的囊泡 仍然附着在神经元质膜上，显然是在晚期冻结的。 就在内化之前的内吞作用阶段。 这项提案的目标是确定发动蛋白I如何活动， 延伸，突触前囊泡再循环，在细胞中受到调节。 在 具体而言，将探讨两种监管模式：刺激依赖型 磷酸化和去磷酸化以及与特异性 磷酸肌醇。 发动蛋白I是主要的神经元蛋白， 突触去极化后的去磷酸化。 该项目将确定 在发动蛋白一级结构的调控位点，测定其重要性 体外蛋白质的功能特性，并确定在 这些类型的调节通过突变相应的 通过同源重组在小鼠中产生氨基酸。 工作假设是 发动蛋白I的磷酸化状态决定了它对 蛋白质和/或脂质靶向于网格状体包被的脂肪，而GT3蛋白质和/或脂质靶向于网格状体包被的脂肪， 活性受磷酸肌醇结合控制。 的 磷脂相互作用位点将通过定点分析 发动蛋白I突变体GT3刺激的结构和动力学基础 将检查磷酸肌醇。 并且，如上所述， 磷酸肌醇结合的重要性将通过同源 小鼠中的重组技术。

DESCRIPTION (from applicants abstract) The arrival of an action potential at the presynaptic nerve terminal elicits membrane depolarization and an increase in free Ca2+ concentration. These events trigger the release of neurotransmitters from synaptic vesicles by exocytosis, followed by retrieval of fused synaptic vesicle membranes by endocytosis. This recycling of vesicle membranes is critical for the release process and its impairment is likely to result in paralysis and mental disorders. At present only one enzyme, the GTPase dynamin I, has been shown unambiguously to have an essential function in synaptic membrane retrieval. If the GTPase activity of dynamin I is inhibited, either by mutation or through the use of non-hydrolyzable GTP analogs, deeply invaginated clathrin-coated vesicles remain attached to the neuronal plasma membrane, apparently frozen at a late stage of endocytosis just prior to internalization. The goal of this proposal is to determine how dynamin I activity and, by extension, presynaptic vesicle recycling, is regulated in cells. In particular, two modes of regulation will be explored: stimulus-dependent phosphorylation and dephosphorylation and interaction with specific phosphoinositides. Dynamin I is the major neuronal protein to undergo dephosphorylation upon synaptic depolarization. This project will identify regulatory sites in the dynamin primary structure, assay their importance for the functional properties of the protein in vitro and determine the in vivo significance of these types of regulation by mutating the corresponding amino acids in mice by homologous recombination. The working hypothesis is that the phosphorylation state of dynamin I determines its affinity for protein and/or lipid targets at the clathria-coated put, whereas GTPase activity is controlled by phosphoinositide binding. The phospholipid-interaction sites will be mapped by analysis of site-directed mutants of dynamin I. The structural and kinetic basis of GTPase stimulation by phosphoinositides will be examined. And, as above, the in vivo significance of phosphoinositide binding will be determined by homologous recombination techniques in mice.