MOLECULAR PHYSIOLOGY OF GLUTAMATE TRANSPORT IN THE BRAIN

目标是确定结构-功能关系和 高亲和力谷氨酸的细胞和亚细胞表达 中枢神经系统的运输者。我们将重点关注高亲和力神经元和 神经胶质谷氨酸转运体在脑出血过程中发挥重要作用 谷氨酸能传递过程中清除细胞内释放的谷氨酸 突触裂隙。这个项目是基于我们最近在 EAAC1 High基因在非洲爪哇卵母细胞中的功能表达克隆 兔、大鼠和人的谷氨酸亲和转运蛋白。为了 详细了解高亲和力谷氨酸的作用 在传播过程中的吸收这些研究还将包括 胶质细胞高亲和力谷氨酸转运蛋白GLT-1。EAAC1报文为 在整个中枢神经系统的谷氨酸能神经元中有强表达，在 一些非谷氨酸能神经元。为了研究细胞和 EAAC1的亚细胞分布并确定其是否具有 谷氨酸能终末突触前摄取载体的研究 制备一组针对不同亲水性的多克隆抗体 EAAC1的区域，并将其用于在光和 电子显微镜水平。其功能和药理作用 EAAC1和GLT-1在非洲爪哇卵母细胞中的表达 用电生理学方法来表征的。电压跳跃 实验将提供对谷氨酸动力学的详细了解。 运输。此外，EAAC1和GLT-1的基因工程应该 允许鉴定涉及到的单个残留 谷氨酸的结合和转运及其直接影响速率 运输过程的各个步骤的常量。进一步评估 EAAC1和EAAC1的生理和病理生理意义 GLT-1研究旨在确定脑缺血或海马区 长时程增强改变了EAAC1和GLT-1的表达水平。我们 还建议评估过氧化氢对谷氨酸摄取的作用。这个 后者很重要，因为谷氨酸摄取和氧自由都受到损害 自由基参与家族性肌营养不良的发病机制 侧索硬化症(ALS)这些研究的结果可能会导致新的 缺血性和神经退行性疾病的治疗策略 例如通过调制EAAC1和/或GLT-1的运输功能的ALS。

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.