Volatile anesthetic modulation of glutamate transporters

描述（由申请人提供）：谷氨酸转运蛋白（EAATs）在调节谷氨酸的细胞外水平中起重要作用，谷氨酸是中枢神经系统中主要的兴奋性神经递质。目前的数据表明，细胞外谷氨酸浓度的增加与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）的病理生理有关。因此，EAATs的正常功能对于维持有效的神经传递和防止神经元损伤至关重要。我们的长期研究目标是了解EAATs的调控及其在麻醉、突触可塑性和神经保护机制中的作用。异氟醚等挥发性麻醉剂可增加主要神经元EAAT3型（EAAT3）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，依赖于EAAT3中S465的磷酸化。异氟醚还增加了EAAT3蛋白的表达。本研究旨在：1)确定异氟醚诱导的EAAT3向质膜的再分布是否通过增加EAAT3从细胞质溶胶向质膜的递送介导；2)鉴定参与EAAT3循环的蛋白，确定S465磷酸化如何改变蛋白-蛋白相互作用从而改变EAAT3的分布；3)确定S465磷酸化诱导的EAAT3重分布在形成长时程增强和异氟醚诱导的神经保护中的作用；4)确定异氟醚增加EAAT3表达的机制。细胞培养，海马体切片和完整的动物（大鼠和小鼠）将用于研究。蛋白生物素化和免疫沉淀将用于研究EAAT3的运输和蛋白-蛋白相互作用。RNA干扰和显性阴性构建体将用于下调蛋白表达和功能。序列特异性肽抑制剂将用于研究EAAT3再分布到质膜的生物学功能。EAAT3启动子活性将在异氟醚存在或不存在的情况下进行测定。这些研究将为麻醉调节谷氨酸转运提供分子基础，并可能为神经保护或突触可塑性调节提供新的靶点。公共卫生相关性：异氟醚是临床常用的麻醉剂，它会影响谷氨酸转运体的功能和位置，谷氨酸转运体是参与多种脑功能的细胞蛋白。该项目旨在研究异氟醚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醚是如何产生诸如脑保护等生物效应的，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.