MECHANISM OF AMINO ACID NEUROTRANSMITTER RELEASE

本研究的目的是了解 谷氨酸以钙依赖的方式从神经末梢释放， 特别是研究蛋白质胞质因子所需的 最佳释放 谷氨酸被广泛认为是最强的候选者 中枢神经系统中主要的兴奋性神经递质。 现在有很好的证据表明，谷氨酸积累到突触 以能量依赖的方式支持神经递质 作用 该摄取系统对谷氨酸具有高度特异性， 被认为在谷氨酸突触传递中起重要作用。 然而，谷氨酸释放的机制还不清楚。 最近，我们设计了一种研究谷氨酸释放的方法， 无细胞系统，并获得了囊泡谷氨酸释放的证据 以钙依赖的方式从透化的突触体中分离。 我们 有证据表明，需要一种细胞溶质蛋白质样物质， 最佳释放 在本申请中，提出（a）纯化所述化合物， 刺激性（或强制性）蛋白质因子 色谱法和高压液相色谱法;（B）表征 从透化的突触体释放的谷氨酸，关于（i） 对因素的敏感性，（ii）对钙的敏感性，（iii）需求 （iv）金属离子特异性;（c）确定 SDS-聚丙烯酰胺凝胶纯化因子的理化性质 电泳、凝胶过滤、蔗糖测定作用位点 通过检查该因子与钙的相互作用， 透化突触体中的特异性蛋白质;以及（e）确定 该因子是否是释放其他氨基酸所必需的 神经递质的影响，通过检查的因素对释放 GABA和甘氨酸来自透化突触体。 这项研究不会 只会加深我们对谷氨酸分子机制的理解 但最终也可能为调查提供新的见解 这些类型的神经病理生理学， 神经传递是有牵连的。

The objective of this research is to understand the mechanism by which glutamate is released from nerve endings in a calcium-dependent manner, in particular to investigate a proteinaceous cytosolic factor required for optimal release. Glutamate is widely recognized as the strongest candidate for the major excitatory neurotransmitter in the central nervous system. There is now good evidence that glutamate is accumulated into synaptic vesicles in an energy-dependent manner, supporting the neurotransmitter role. This uptake system is highly specific for glutamate and is considered to play an important role in glutamate synaptic transmission. However, the mechanism underlying glutamate release is not well understood. Recently, we have devised an approach to studying glutamate release in a cell-free system and obtained evidence that vesicular glutamate is released in a calcium-dependent manner from permeabilized synaptosomes. Our evidence indicates that a cytosolic-proteinaceous substance is required for optimal release. In this application, it is proposed to (a) purify the stimulatory (or obligatory) proteinaceous factor by classical chromatography and high pressure liquid chromatography; (b) characterize the glutamate release from permeabilized synaptosomes with respect to (i) sensitivity to the factor, (ii) sensitivity to calcium, (iii) requirement for the plasma membrane, and (iv) metal ion specificity; (c) determine physicochemical properties of the purified factor by SDS-polyacrylamide gel electrophoresis, gel filtration, sucrose determine the site of action of the factor by examining the interaction of the factor with calcium and specific proteins in the permeabilized synaptosomes; and (e) determine whether the factor is required for release of other amino acid neurotransmitters by examining the effect of the factor on the release of GABA and glycine from permeabilized synaptosomes. This research will not only advance our understanding of the molecular mechanism of glutamate release but also may ultimately provide new insights into the investigation of those types of neuropathophysiology where abnormal glutamate neurotransmission is implicated.