Activity of Glutamate Transporter GLT1 in CA3 Neurons

描述(申请人提供)：大脑中的谷氨酸转运体在兴奋性突触中至少扮演两个关键角色：维持细胞外低水平的谷氨酸，以支持谷氨酸受体的正常激活和防止神经元的兴奋性毒性退化；以及调节细胞外间隙中谷氨酸的浓度时间进程。目前已鉴定出5种谷氨酸转运蛋白亚型，其中GLT-1(人称EAAT2)占谷氨酸转运蛋白总活性的95%。在正常的中枢神经系统中，GLT-1蛋白主要局限于星形胶质细胞，在清除突触外谷氨酸方面起主导作用。已有证据表明神经元表达GLT-1mRNA，以及生化和电生理数据表明存在突触前转运体，但尚未有强有力的证据表明神经元中存在功能性GLT-1蛋白。为了研究细胞特异性转运蛋白调控的生物学，我培育了GLT1-BAC-启动子转基因小鼠。利用这些动物，我们现在已经鉴定了GLT-1启动子的活性，并随后在小鼠海马CAS神经元中选择性地鉴定了谷氨酸转运体电流。本研究的目的是确定GLT-1转运体在CAS神经元中的定位和亚细胞定位，研究其表达的调控，并评估其在突触传递中的潜在作用。这些实验将开始直接测试GLT-1在神经元中的功能意义。

DESCRIPTION (provided by applicant): Glutamate transporters in the brain play at least two critical roles at excitatory synapses: maintenance of low extracellular glutamate levels, which supports normal activation of glutamate receptors and prevents excitotoxic degeneration of neurons, and modulation of the concentration time course of glutamate in the extracellular space. Five glutamate transporter isoforms have been identified, of which GLT-1 (termed EAAT2 in human) accounts for up to 95% of total glutamate transport activity. In the normal CNS, GLT-1 protein is largely restricted to astrocytes where it plays a dominant role in clearing extrasynaptic glutamate. Evidence for neuronal expression of GLT-1 mRNA, as well as biochemical and electrophysiological data suggesting the presence of pre-synaptic transporters, has been reported, but there has been no strong evidence of functional GLT-1 protein in neurons. To examine the biology of cell-specific transporter regulation, I generated GLT1-BAC-promoter transgenic mice. Using these animals, we have now identified GLT-1 promoter activity, and, subsequently, glutamate transporter currents, selectively in mouse hippocampal CAS neurons. The goals of this proposal are to establish the identity and subcellular location of the GLT-1 transporter in CAS neurons, to investigate regulation of its expression, and to assess its potential role in synaptic transmission. These experiments will begin to directly test the functional significance of GLT-1 in neurons.