Roles of glutamate transporters in the nervous system

谷氨酸转运体在终止突触传递和维持细胞外谷氨酸浓度低于神经毒性水平方面起着关键作用。本研究利用分子生物学方法对谷氨酸转运蛋白EAAC1在神经系统中的作用进行了研究，通过对谷氨酸转运蛋白EAAC1的化学计量分析，发现一个谷氨酸转运蛋白与两个Na+离子共转运，一个K++和一个OH-离子反向转运。根据这一化学计量比，推测谷氨酸转运蛋白具有很高的浓缩谷氨酸的能力，证实了转运蛋白维持胞外谷氨酸浓度的作用。基于EAAC1和GLT-1对谷氨酸摄取抑制剂二氢海人酸差异敏感性的嵌合体分析，定位了谷氨酸转运蛋白的区域，似乎是谷氨酸结合部位。此外，…的克隆和功能鉴定更多新的依赖于Na+的中性氨基酸转运蛋白亚型为谷氨酸和Na+结合位点提供了有用的信息。为了了解谷氨酸转运蛋白基因表达的调控机制，我们克隆了似乎包含EAAC1启动子区域的15kb基因组DNA片段。EAAC1的核苷酸序列分析和CAT实验正在研究该基因的调节区。原位杂交研究表明，EAAC1仅在大鼠中枢神经系统的神经元中表达。利用反义寡核苷酸脑室给药技术进行的基因敲除实验表明，神经元谷氨酸转运蛋白EAAC1基因敲除可引起大鼠癫痫发作，而胶质细胞转运蛋白基因敲除可导致神经细胞死亡。这一研究结果将为谷氨酸转运蛋白功能障碍的病理生理学研究和特异性谷氨酸转运蛋白功能修饰药物的开发提供依据。较少

It has been proposed that glutamate transporters play critical roles in the termination of synaptic transmission and the maintenance of extracellular glutamate concentration below neurotoxic level. In this project, we have addressed the questions on the role of glutamate transporters in the nervous system utilizing molecular biology methodologies.Stoichiometric analyzes of the glutamate transporter EAAC1 revealed that one glutamate is cotransported with two Na^+ and countertransported with one K^+ and OH-. Based on this stoichiometry, it was estimated that glutamate transporter has a high capacity to concentrate glutamate, confirming the role of the transporters maintaining extracellular glutamate concentration.Chimera analysis based on the differential sensitivity of EAAC1 and GLT-1 to glutamate uptake inhibitor dihydrokainate addressed the region of glutamate transporter proteins which seems to be a glutamate binding site. In addition, the cloning and functional characterization of a … More new isoform of Na^+-dependent neutral amino acid transporter provided useful information on the sites for glutamate and Na^+ bindings.To understand the mechanisms of regulation of expression of glutamate transporter genes, we isolated 15kb genomic DNA fragment which seems to contain the promotor region of EAAC1. The analysis of the nucleotide sequence and a CAT assay to study the regulatory region of the gene are in process.The in situ hybridization study indicated that EAAC1 is expressed exclusively in neurons in rat central nervous system. The gene knockout experiment using an antisense oligo DNA intracerebroventricular administration technique revealed that the knockout of neuronal glutamate transporter EAAC1 elicits epileptic seizure on rat, which is in contrast with the glial transporter knockouts inducing neuronal cell death.The results of this projected research will trigger the researches addressing the pathophysiology of disorders in which glutamate transport disturbance is involved and the development of drugs which specifically modify glutamate transporter function. Less