MECHANISM OF AMINO ACID NEUROTRANSMITTER RELEASE

这项研究的目的是了解 谷氨酸以一种钙依赖的方式从神经末梢释放，在 特别是为了研究一种蛋白质类胞浆因子 最佳释放。谷氨酸被广泛认为是最有力的候选者 中枢神经系统中的主要兴奋性神经递质。 现在有充分的证据表明谷氨酸积聚到突触中 以能量依赖的方式产生囊泡，支持神经递质 角色。这种摄取系统对谷氨酸有很高的专一性 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是很清楚。 最近，我们设计了一种方法来研究谷氨酸的释放 无细胞系统和囊泡谷氨酸释放的证据 以钙依赖的方式从通透性突触体内释放。我们的 有证据表明，需要一种胞浆蛋白质类物质 最佳释放。在本申请中，建议(A)提纯 刺激性(或强制性)蛋白质因子 层析和高压液相色谱；(B)表征 通透性突触体内谷氨酸的释放与(I) 对该因素的敏感性，(Ii)对钙的敏感性，(Iii)需求 对于质膜，以及(Iv)金属离子专一性；(C)确定 十二烷基硫酸钠-聚丙烯酰胺凝胶纯化因子的理化性质 电泳法、凝胶过滤法、蔗糖法确定其作用部位 通过检验因子与钙和钙的相互作用来确定因子 通透性突触体中的特定蛋白；和(E)确定 该因子是否需要释放其他氨基酸 通过检测该因子对神经递质释放的影响 从通透性突触体内提取的GABA和甘氨酸。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 谷氨酸异常的神经病理生理学类型 牵涉到神经传递。

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.