REGULATION OF EAAC1, A NEURONAL GLUTAMATE TRANSPORTER

该提案的目的是检查活性和细胞如何 神经元谷氨酸转运蛋白亚型 EAAC1 的定位是 受第二信使途径调节。 最近已经很清楚了 多年来，谷氨酸不仅在正常情况下充当神经递质 哺乳动物的大脑，但谷氨酸水平升高参与 癫痫发作以及发生的兴奋性神经元损伤 中风、急性头部创伤和神经退行性疾病。 EAAC1 是 神经元特异性谷氨酸转运蛋白，其高水平存在于 皮质和海马体。 更好地理解监管 EAAC1 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活动。 使用 C6 神经胶质瘤细胞系 内源性表达 EAAC1，第二信使的贡献 运输活动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 这些途径将通过激活剂和抑制剂进行。 变化 转运蛋白的细胞分布可能是一种额外的机制 快速增加谷氨酸的吸收，并将同时进行评估 利用细胞表面蛋白的生物素化进行研究 随后进行蛋白质印迹分析和共聚焦显微镜检查。 控制更多 细胞膜生理学、电生理学的微妙变化 来自用相同处理的电压钳制细胞的记录 将获得药剂。 为了更好地关联 C6 神经胶质瘤 体内功能的发现，EAAC1 的转运特性 将分析富含神经元的大鼠海马培养物以寻找 调节的相似性或差异，以及神经元的影响 EAAC1 对这些培养物中谷氨酸毒性的上调将是 检查了。 希望对EAAC1监管有更好的了解 可能提供有关大脑如何应对过量谷氨酸的线索 在病理状态下释放，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，例如 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.