REGULATION OF EAAC1, A NEURONAL GLUTAMATE TRANSPORTER

这项建议的目的是研究如何活动和细胞 神经元谷氨酸转运子亚型EAAC 1的定位是 由第二信使途径调节。 近年来， 多年来，谷氨酸不仅作为一种神经递质， 哺乳动物的大脑，但谷氨酸水平的升高涉及 癫痫发作以及兴奋性毒性神经元损伤， 中风、急性头部创伤和神经退行性疾病。 EAAC 1是一个 神经元特异性谷氨酸转运蛋白，它在神经元中以高水平存在， 大脑皮层和海马体 更好地理解 EAAC 1可能有助于解释为什么大脑的这些区域更容易受到影响 兴奋性毒性损伤和癫痫发作 使用C6胶质瘤细胞系 内源性表达EAAC 1，第二信使的贡献 转运活性和细胞定位的途径将是 评估。 操作后3[H]-谷氨酸摄取活性的变化 将进行具有活化剂和抑制剂的这些途径。 变化 在转运蛋白的细胞分布中可能是另外的机制 快速增加谷氨酸摄取，并将平行评估 涉及使用细胞表面蛋白的生物素化的摄取研究， 随后进行Western分析和共聚焦显微镜检查。 控制更多 细胞膜生理学、电生理学 来自用相同物质处理的电压钳位细胞的记录 将获得药理学试剂。 为了更好地将C6神经胶质瘤 在体内功能的研究结果，EAAC 1的转运特性， 神经元富集的大鼠海马培养物将被分析以寻找 调节的相似性或差异，以及神经元的作用 在这些培养物中，EAAC 1对谷氨酸毒性的上调将被证实。 考察 希望能更好地了解EAAC 1调节 可能会为大脑如何处理过量的谷氨酸提供线索 病理状态下释放，如果转运蛋白故障导致 神经损伤，以及如何使用治疗策略， 在兴奋性毒性应激时增强转运蛋白功能， 中风、创伤或慢性疾病。

The objective of this proposal is to examine how the activity and cellular localization of the neuronal glutamate transporter subtype EAAC1 is regulated by second messenger pathways. It has become clear in recent years that glutamate acts not only as a neurotransmitter in the normal mammalian brain, but that elevated levels of glutamate are involved in epileptic seizures as well as the excitotoxic neuronal damage that occurs in stroke, acute head trauma, and neurodegenerative disease. EAAC1 is a neuron specific glutamate transporter which is present in high levels in the cortex and the hippocampus. Better understanding of th regulation of EAAC1 may help explain why these areas of the brain are more susceptible to excitotoxic damage and seizure activity. Using the C6 glioma cell line that endogenously expresses EAAC1, the contribution of second messenger pathways to both transport activity and cellular localization will be assessed. Changes in 3[H]- glutamate uptake activity after manipulating these pathways with activators and inhibitors will be performed. Changes in cellular distribution of the transporter may be an additional mechanism for rapidly increasing glutamate uptake, and will be assessed in parallel to uptake studies using biotinylation of cell surface proteins with subsequent Western analysis and confocal microscopy. To control for more subtle changes in cellular membrane physiology, electrophysiological recordings from voltage clamped cells treated with the same pharmacological agents will be obtained. To better relate the C6 glioma findings to in vivo function, the EAAC1 transport characteristics of neuron enriched rat hippocampal cultures will be analyzed to look for similarities or differences of regulation, and the effects of neuronal EAAC1 upregulation on glutamate toxicity in these cultures will be examined. It is hoped that a better understanding of EAAC1 regulation might provide clues as to how the brain copes with excessive glutamate release in pathological states, if transporter malfunction contributes to neurological damage, and how therapeutic strategies might be used to enhance transporter function in times of excitotoxic stress such as stroke, trauma or chronic disease.