VESICULAR TRANSPORT OF AMINO ACID NEUROTRANSMITTERS

中枢神经系统的信息处理依赖于 快速精确地释放氨基酸神经递质γ- 氨基丁酸（GABA）和谷氨酸。 尽管我们对 对GABA和谷氨酸的突触后反应的分子基础， 涉及释放的突触前机制还不太清楚。 由于经典的神经递质在细胞质中合成， 需要转运到突触囊泡中进行胞吐释放。 先前的研究已经证明GABA的能量依赖性摄取 和谷氨酸进入突触囊泡，但是负责 这些活动仍然不为人知。 这一长期目标 建议是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，C。elegans基因 UNC-47在释放GABA中的作用 我们最近证明， unc-47的脊椎动物同源物编码囊泡GABA转运。 的 该大鼠囊泡GABA转运蛋白（VGAT）的氨基酸序列显示 与以前鉴定的神经递质转运蛋白没有相似性， 这表明它定义了一个新的蛋白质家族， 氨基酸神经递质的囊泡转运， 包括谷氨酸的囊泡转运蛋白。 我们已经分离出 与unc-47和VGAT序列同源的cDNA。 初步 表征显示这种推定的大鼠的限制性表达 通过北方的研究， 用原位杂交法检测脑内多巴胺能神经元 杂交方法 为了评估预测的蛋白质在囊泡细胞中的作用， 谷氨酸转运，我们提出以下具体目标：1）产生 VT 2抗体，并确定其组织分布和亚细胞 定位; 2）表征表达的VT 2的生化功能 在哺乳动物细胞系中; 3）分离与VGAT和VT 2相关cDNA;和 4)靶向破坏小鼠的VT 2基因。 的关键作用 调节胞吐释放的神经递质包装表明 这些研究的结果将与正常的 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.