MOLECULAR PHYSIOLOGY OF GLUTAMATE TRANSPORT IN THE BRAIN

目的是确定结构-功能关系和 高亲和力谷氨酸的细胞和亚细胞表达 中枢神经系统中的转运蛋白。 我们将专注于高亲和力的神经元和 神经胶质谷氨酸转运蛋白在脑缺血过程中起重要作用， 通过从细胞中清除释放的谷氨酸， 突触间隙 这个项目是基于我们最近在 通过在非洲爪蟾卵母细胞中功能性表达克隆EAAC 1高 来自兔、大鼠和人的亲和谷氨酸转运蛋白。 为了 详细了解高亲和力谷氨酸的作用 这些研究还将包括 胶质细胞高亲和力谷氨酸转运体GLT-1。 EAAC 1消息是 在整个CNS的多巴胺能神经元中强烈表达， 一些非多巴胺能神经元 为了研究细胞和 EAAC 1的亚细胞分布，并确定其是否作为 我们提出， 制备一组针对不同亲水性的多克隆抗体， EAAC 1区域，并将其用于光下的免疫细胞化学， 电镜水平。 功能和药理学特性 EAAC 1和GLT-1在非洲爪蟾卵母细胞中的表达将被广泛地 使用电生理学方法表征。 电压跳变 实验将提供一个详细的了解谷氨酸的动力学 运输 此外，EAAC 1和GLT-1的基因工程应 允许识别参与的单个残留物 谷氨酸的结合和转运，直接影响速率 运输过程各个步骤的常数。 为了进一步评估 EAAC 1的生理和病理生理学意义， GLT-1研究旨在确定缺血或海马 长时程增强改变EAAC 1和GLT-1的表达水平。 我们 还建议评估过氧化物对谷氨酸摄取的作用。 的 后者是重要的，因为受损的谷氨酸摄取和无氧 自由基参与了家族性肌萎缩性 侧索硬化症（ALS）。 这些研究的结果可能会导致新的 局部缺血和神经退行性疾病的治疗策略 例如ALS，通过调节EAAC 1和/或GLT-1的转运功能。

The goal is to determine the structure-function relationship and the cellular and subcellular expression of high affinity glutamate transporters in the CNS. We will focus on the high affinity neuronal and glial glutamate transporters which play an important role during the glutamatergic transmission process by removing released glutamate from the synaptic cleft. This project is based on our recent success in the cloning by functional expression in Xenopus oocytes of the EAAC1 high affinity glutamate transporters from rabbit, rat and human. In order to develop a detailed understanding of the role of high affinity glutamate uptake in the transmission process these studies will also include the glial high affinity glutamate transporter GLT-1. EAAC1 message is strongly expressed in glutamatergic neurons throughout the CNS and also in some non-glutamatergic neurons. In order to study the cellular and subcellular distribution of EAAC1 and to determine whether it functions as a presynaptic uptake carrier in glutamatergic terminals we propose to prepare a panel of polyclonal antibodies against different hydrophilic regions of EAAC1 and to use them for immunocytochemistry at the light and electronmicroscopy levels. The functional and pharmacological properties of EAAC1 and GLT-1 expressed in Xenopus oocytes will be extensively characterized using electrophysiological methods. Voltage jump experiments will provide a detailed insight into the kinetics of glutamate transport. In addition, genetic engineering of EAAC1 and GLT-1 should permit the identification of individual residues which are involved in the binding and translocation of glutamate and which directly affect the rate constants of individual steps of the transport process. To further assess the physiological and pathophysiological significance of EAAC1 and also GLT-1 studies are designed to determine whether ischemia or hippocampal long-term potentiation alter the expression levels of EAAC1 and GLT-1. We also propose to evaluate the action of peroxide on glutamate uptake. The latter is important because both impaired glutamate uptake and oxygen free radicals were implicated in the pathogenesis of familial amyotrophic lateral sclerosis (ALS). The results from these studies may lead to new strategies for the treatment of ischemia and neurodegenerative diseases such as ALS by modulating the transport functions of EAAC1 and/or GLT-1.