Volatile anesthetic modulation of glutamate transporters

描述（由申请人提供）：谷氨酸转运蛋白（EAAT）在调节谷氨酸（中枢神经系统中的主要兴奋性神经递质）的细胞外水平中发挥重要作用。目前的数据表明，细胞外谷氨酸浓度增加涉及缺血性脑损伤和几种主要的人类神经退行性疾病，如阿尔茨海默病和肌萎缩侧索硬化症的病理生理学。因此，EAAT的正常功能对于维持有效的神经传递和防止神经元损伤是重要的。我们的长期研究目标是了解EAAT的调节及其在麻醉，突触可塑性和神经保护机制中的作用。挥发性麻醉剂，如异氟烷，增加EAAT 3型（EAAT 3），主要的神经元EAAT的活性和细胞表面表达。这种作用是蛋白激酶C（PKC）1依赖性的，并依赖于EAAT 3中S465的磷酸化。异氟烷还增加EAAT 3的蛋白表达。该提议旨在1）确定异氟烷诱导的EAAT 3向质膜的再分布是否由EAAT 3从胞质溶胶向质膜的递送增加介导; 2）鉴定参与EAAT 3再循环的蛋白质，并确定S465的磷酸化如何改变蛋白质-蛋白质相互作用以改变EAAT 3的分布; 3）确定S465磷酸化诱导的EAAT 3再分布在长时程增强和异氟烷诱导的神经保护作用的形成中的作用;和4）鉴定异氟烷增加EAAT 3表达的机制。研究中将使用细胞培养物、海马切片和完整动物（大鼠和小鼠）。将进行蛋白质生物素化和免疫沉淀以研究EAAT 3运输和蛋白质-蛋白质相互作用。RNA干扰和显性负性构建体将用于下调蛋白质表达和功能。序列特异性肽抑制剂将用于研究EAAT 3重新分布到质膜的生物学功能。将在存在或不存在异氟烷的情况下测定EAAT 3启动子活性。这些研究将为谷氨酸转运的麻醉调节提供分子基础，并可能为神经保护或调节突触可塑性提供新的靶点。公共卫生相关性：异氟烷是临床实践中常用的麻醉剂，影响谷氨酸转运体的功能和位置，谷氨酸转运体是参与广泛脑功能的细胞蛋白。该项目旨在研究异氟烷如何调节这些蛋白质的功能和位置以及这些调节的生物学后果，例如提供大脑适应和保护。这些研究可能有助于了解异氟烷如何发挥其生物学效应，如脑保护，也可能提出诱导这些效应的新方法。

DESCRIPTION (provided by applicant): Glutamate transporters (EAATs) play an important role in the regulation of extracellular levels of glutamate, a major excitatory neurotransmitter in the central nervous system. Current data suggest that increased extracellular glutamate concentration is involved in the pathophysiology of ischemic brain injury and several major human neurodegenerative disorders, such as Alzheimer's diseases and amyotrophic lateral sclerosis. Thus, normal functioning of EAATs is important for maintaining efficient neurotransmission and preventing neuronal injury. Our long-term research goals are to understand the regulation of EAATs and their role in the mechanisms of anesthesia, synaptic plasticity and neuroprotection. Volatile anesthetics, such as isoflurane, increase the activity and cell surface expression of EAAT type 3 (EAAT3), the major neuronal EAAT. This effect is protein kinase C (PKC) 1-dependent and relies on the phosphorylation of S465 in EAAT3. Isoflurane also increases the protein expression of EAAT3. This proposal aims to 1) determine whether isoflurane-induced EAAT3 redistribution to the plasma membrane is mediated by increased delivery of EAAT3 from cytosol to the plasma membrane; 2) identify proteins involved in the recycling of EAAT3 and determine how phosphorylation of S465 changes the protein-protein interaction to alter the distribution of EAAT3; 3) determine the role of S465 phosphorylation-induced EAAT3 redistribution in the formation of long-term potentiation and isoflurane-induced neuroprotection; and 4) identify the mechanisms for isoflurane to increase the EAAT3 expression. Cell cultures, hippocampal slices and intact animals (rats and mice) will be used in the studies. Protein biotinylation and immunoprecipitation will be performed to study EAAT3 trafficking and protein- protein interaction. RNA interference and dominant negative constructs will be used to down-regulate protein expression and functions. Sequence-specific peptide inhibitors will be used to investigate the biological functions of EAAT3 redistribution to the plasma membrane. EAAT3 promoter activity will be assayed in the presence or absence of isoflurane. These studies will provide a molecular basis for the anesthetic regulation of glutamate transport and may suggest new targets for neuroprotection or for regulation of synaptic plasticity. PUBLIC HEALTH RELEVANCE: Isoflurane, a commonly used anesthetic in clinical practice, affects the function and location of glutamate transporters, cellular proteins involved in a broad range of brain functions. This project is designed to investigate how isoflurane regulates the function and location of these proteins and the biological consequences of these regulations, such as providing brain adaptation and protection. These studies may help understand how isoflurane works to cause its biological effects, such as brain protection, and also may suggest novel methods to induce these effects.