Volatile anesthetic modulation of glutamate transporters

描述(申请人提供)：谷氨酸转运体(EAATs)在调节细胞外谷氨酸水平方面发挥重要作用，谷氨酸是中枢神经系统中的一种主要兴奋性神经递质。目前的数据表明，细胞外谷氨酸浓度的增加参与了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过程。因此，EAATs的正常功能对于维持有效的神经传递和预防神经元损伤具有重要意义。我们的长期研究目标是了解EAATs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，如异氟醚，增加了EAAT3型(EAAT3)的活性和细胞表面表达，EAAT3是主要的神经元EAAT。这种作用依赖于蛋白激酶C(PKC)1，并依赖于EAAT3中S465的磷酸化。异氟醚还可增加EAAT3的蛋白表达。该建议旨在1)确定异氟烷诱导的EAAT3重分布是否通过增加EAAT3从胞浆到质膜的转运来介导；2)识别参与EAAT3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EAAT3的分布；3)确定S465磷酸化诱导的EAAT3重分布在形成长期增强和异氟醚诱导的神经保护中的作用；以及4)确定异氟烷增加EAAT3表达的机制。研究中将使用细胞培养、海马片和完整的动物(大鼠和小鼠)。蛋白质生物素化和免疫沉淀将被用来研究EAAT3的转运和蛋白质之间的相互作用。RNA干扰和显性负结构将被用来下调蛋白质的表达和功能。序列特异性多肽抑制剂将被用来研究EAAT3重新分布到质膜的生物学功能。EAAT3启动子的活性将在存在或不存在异氟烷的情况下进行检测。这些研究将为麻醉剂调节谷氨酸运输提供分子基础，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：异氟醚是临床上常用的麻醉剂，它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大脑功能的细胞蛋白质。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，如提供大脑适应和保护。这些研究可能有助于理解异氟烷是如何产生生物效应的，例如大脑保护，也可能提出新的方法来诱导这些效应。

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.