Roles of glutamate transporters in the nervous system

谷氨酸转运体在突触传递终止和维持细胞外谷氨酸浓度低于神经毒性水平中起关键作用。在本项目中，我们利用分子生物学方法解决了谷氨酸转运体在神经系统中的作用问题。对谷氨酸转运体EAAC1的化学计量分析表明，一个谷氨酸与两个Na^+共转运，与一个K^+和OH-反转运。根据这一化学计量，估计谷氨酸转运体具有较高的谷氨酸浓缩能力，证实了转运体维持细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碱盐敏感性差异的嵌合体分析定位了谷氨酸转运蛋白区域，该区域可能是谷氨酸结合位点。此外，Na^+依赖性中性氨基酸转运蛋白的克隆和功能鉴定为谷氨酸和Na^+结合位点的研究提供了有用的信息。为了了解谷氨酸转运体基因的表达调控机制，我们分离了15kb的基因组DNA片段，该片段可能包含EAAC1的启动子区域。目前正在进行核苷酸序列分析和CAT分析，以研究该基因的调控区域。原位杂交研究表明，EAAC1仅在大鼠中枢神经系统神经元中表达。采用反义寡核苷酸脑室给药技术进行基因敲除实验，发现敲除神经元谷氨酸转运蛋白EAAC1可引起大鼠癫痫发作，而敲除神经胶质转运蛋白可引起神经元细胞死亡。本研究的结果将引发谷氨酸转运障碍所涉及的疾病的病理生理学研究以及特异性修饰谷氨酸转运蛋白功能的药物的开发。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