REGULATION OF EAAC1, A NEURONAL GLUTAMATE TRANSPORTER

神经谷氨酸转运蛋白 EAAC1 的调控

• 批准号: 6078161
• 负责人: KAREN D SIMS
• 金额: $ 3.11万
• 依托单位: UNIVERSITY OF PENNSYLVANIA
• 依托单位国家: 美国
• 财政年份: 1999
• 资助国家: 美国
• 起止时间: 1999-09-30 至
• 项目状态: 未结题
• 来源: https://reporter.nih.gov/project-details/6078161
• 关键词: brain cell; cell line; confocal scanning microscopy; glutamate transporter; intracellular transport; laboratory rat; neuroregulation; neurotoxins; second messengers; voltage /patch clamp

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.

这项建议的目标是研究活动和细胞 神经元型谷氨酸转运体EAAC1亚型的定位 由第二信使通路调节。在最近的几年里，情况变得很明显 多年来，谷氨酸不仅在正常情况下作为一种神经递质 哺乳动物的大脑，但谷氨酸水平升高参与了 癫痫发作以及由此发生的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。EAAC1是一种 神经元特异性谷氨酸转运体在脑内高水平存在 大脑皮层和海马体。更好地理解TH的监管 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 兴奋毒性损伤和癫痫发作活动。用C6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 评估过了。手法前后3[H]-谷氨酸摄取活性的变化 这些带有激活剂和抑制剂的途径将被执行。变化 转运蛋白在细胞中的分布可能是另一种机制 快速增加谷氨酸摄取，并将进行平行评估 利用细胞表面蛋白的生物素化进行摄取研究 随后进行Western分析和共聚焦显微镜观察。为了更多的控制权 细胞膜生理、电生理的微妙变化 用其处理的电压钳制电池的记录 将获得药理药剂。为了更好地联系C6胶质瘤 研究结果表明，EAAC1在体内的功能、转运特性 神经元丰富的大鼠海马区培养将被分析以寻找 调节的相似或不同以及神经元的影响 EAAC1对这些培养物中谷氨酸毒性的上调 检查过了。希望对EAAC1规则有更好的理解 可能为大脑如何应对过量的谷氨酸提供线索 在病理状态下释放，如果转运体故障导致 神经损伤，以及如何使用治疗策略来 在兴奋性应激时增强转运蛋白功能，如 中风、创伤或慢性病。