REGULATION OF DYNAMIN I IN SYNAPTIC TRANSMISSION

描述(摘自申请者摘要)动作电位在 突触前神经末梢引起膜去极化和 游离钙离子浓度升高。这些事件会触发释放 通过胞吐作用来自突触小泡的神经递质，随后 通过内吞作用恢复融合突触囊泡膜。这 囊泡膜的回收对于释放过程和其 损伤很可能导致瘫痪和精神障碍。在… 目前只有一种酶，GTP酶动力蛋白I，已经被明确地显示出来 在突触膜修复中具有重要作用。如果GTP酶 动力素I的活性受到抑制，要么是通过突变，要么是通过使用 不可水解的GTP类似物，深度内陷的笼蛋白包裹的囊泡 仍然附着在神经元质膜上，显然是在晚期冻结 内吞就在内化之前的内吞阶段。 这项提案的目标是确定Dynamin I活动如何，并通过 延伸，即突触前囊泡循环，在细胞中受到调节。在……里面 特别是，将探索两种监管模式：依赖刺激 磷酸化和去磷酸化及其与特异性蛋白的相互作用 磷脂酰肌醇。动力蛋白I是主要的神经元蛋白。 突触去极化时的去磷酸化。该项目将确定 动力蛋白一级结构中的调控位点，分析它们的重要性 对该蛋白的体外功能性质进行研究，并测定其在 这些类型通过相应的突变调节的活体意义 小鼠体内的氨基酸通过同源重组。工作假说是 动力蛋白I的磷酸化状态决定了它与 蛋白质和/或脂类靶标在笼罩的Put上，而GTPase 活性受肌醇磷脂结合控制。这个 磷脂相互作用位点将通过定点分析来绘制 动力蛋白突变体I.GTP酶刺激的结构和动力学基础 将对磷脂酰肌醇进行检测。而且，如上所述，体内的 肌醇磷脂结合的意义将由同源基因决定 小鼠体内的重组技术。

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.