VESICULAR TRANSPORT OF AMINO ACID NEUROTRANSMITTERS

中枢神经系统的信息处理依赖于 快速准确地释放氨基酸类神经递质-- 氨基丁酸(GABA)和谷氨酸。尽管很多人都知道 突触后对GABA和谷氨酸反应的分子基础， 涉及释放的突触前机制还不是很清楚。 由于经典的神经递质是在细胞质中合成的，它们 需要运输到突触小泡中以进行胞吐释放。 以前的研究表明，GABA的摄取依赖于能量 和谷氨酸进入突触小泡，但负责 这些活动仍然不为人知。这个项目的长期目标是 建议确定囊泡运输的分子基础 氨基酸递质及其在突触传递中的作用。 遗传和药理学分析表明线虫基因 UNC-47中的GABA释放。我们最近展示了一种 脊椎动物的UNC-47同源物编码囊泡GABA运输。这个 大鼠囊泡状GABA转运体(VGAT)的氨基酸序列显示 与之前发现的神经递质转运体没有相似之处， 这表明它定义了一个新的蛋白质家族，参与了 氨基酸神经递质的囊泡运输也可能 包括谷氨酸的囊泡转运体。我们现在已经隔离了 一个与UNC-47和VGAT序列同源的cDNA。初步 鉴定显示这只假定的大鼠有限制性表达 Northern在脑和肾脏中的囊泡转运蛋白2(VT2) 脑内谷氨酸能神经元的原位分析 杂交。评估预测蛋白在囊泡中的作用 谷氨酸转运，我们提出了以下具体目标：1)生成 抗VT2抗体及其组织分布和亚细胞定位 定位；2)表达的VT2的生化功能 在哺乳动物细胞系中；3)分离与VGAT和VT2相关的cDNA； 4)靶向VT2基因在小鼠体内的破坏。的关键作用 神经递质包装用于调节胞吐释放表明 这些研究的结果将与正常 突触功能、行为和人类神经精神疾病。

Information processing in the central nervous system depends on the rapid and precise release of amino acid neurotransmitters gamma- aminobutyric acid (GABA) and glutamate. Although much is known about the molecular basis of the postsynaptic response to GABA and glutamate, the presynaptic mechanisms involved in release are less well understood. Since classical neurotransmitters are synthesized in the cytoplasm, they require transport into synaptic vesicles for exocytotic release. Previous studies have demonstrated the energy - dependent uptake of GABA and glutamate into synaptic vesicles, but the proteins responsible for these activities remain unknown. The long-term objectives of this proposal are to determine the molecular basis for vesicular transport of amino acid transmitters and its role in synaptic transmission. Genetic and pharmacological analysis has implicated the C. elegans gene unc-47 in the release of GABA. WE have recently demonstrated that a vertebrate homologue of unc-47 encodes vesicular GABA transport. The amino acid sequence of this rat vesicular GABA transporter (VGAT) shows no similarity to previously identified neurotransmitter transporters, indicating that it defines a novel family of proteins involved in the vesicular transport of amino acid neurotransmitters that may also include the vesicular transporter for glutamate. We have now isolated a cDNA with sequence homology to unc-47 and VGAT. Preliminary characterization shows restricted expression of this putative rat vesicular transporter 2 (VT2) in the brain and the kidney by Northern analysis and in glutamatergic neurons in the brain by in situ hybridization. To assess the role of the predicted protein in vesicular glutamate transport, we propose the following specific aims: 1) generate antibodies to VT2 and determine its tissue distribution and subcellular localization; 2) characterize the biochemical function of VT2 expressed in mammalian cell lines; 3)isolate cDNAs related to VGAT and VT2; and 4)target the disruption of the VT2 gene in mice. The crucial role of neurotransmitter packaging for regulated exocytotic release indicates that the results of these studies will have relevance for normal synaptic function, behavior and human neuropsychiatric disease.