Activity of Glutamate Transporter GLT1 in CA3 Neurons

描述（由申请人提供）：脑中的谷氨酸转运蛋白在兴奋性突触中起至少两个关键作用：维持低细胞外谷氨酸水平，其支持谷氨酸受体的正常激活并防止神经元的兴奋性毒性变性，以及调节细胞外空间中谷氨酸的浓度时程。已经鉴定了五种谷氨酸转运蛋白亚型，其中GLT-1（在人类中称为EAAT 2）占总谷氨酸转运活性的高达95%。在正常CNS中，GLT-1蛋白主要限于星形胶质细胞，在那里它在清除突触外谷氨酸中起主导作用。已经报道了GLT-1 mRNA的神经元表达的证据，以及表明突触前转运蛋白存在的生化和电生理数据，但是没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调控的生物学，我产生了GLT 1-BAC启动子转基因小鼠。使用这些动物，我们现在已经确定了GLT-1启动子活性，随后，谷氨酸转运体电流，选择性地在小鼠海马CAS神经元。 该提案的目标是建立CAS神经元中GLT-1转运蛋白的身份和亚细胞位置，研究其表达的调节，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.