The role of lipid raft disruption in the activation of TREK-1 channels by anesthetics

项目摘要 吸入麻醉药在全身麻醉中的分子机制仍然是一个重要的未解之谜 神经科学和人类健康的问题。自从乙醚诱导麻醉的第一次演示以来， 160多年前，麻醉理论试图了解膜在麻醉中的作用， 行动上本申请寻求将质膜确立为吸入给药的相关靶点。 麻醉剂脂质膜自发地划分为有序和不协调的脂质区域，称为 脂筏蛋白质的棕榈酰化使蛋白质进入有序相。我们已经证明 麻醉剂破坏活细胞膜中的这些结构域，但破坏与麻醉剂无关。 离子通道 我们将检验麻醉剂对脂筏的破坏释放脂质修饰酶以激活脂筏的假设。 钾离子通道通过化学信号。具体而言，GM 1筏螯合磷脂酶D（PLD）， 筏的破坏释放PLD，允许酶找到其底物并产生阴离子脂质 磷脂酸（PA）。PA然后调节双孔结构域钾通道（K2 P）TREK-1。TREK- 1也是麻醉敏感通道。我们的目的是描述麻醉药对脂筏的影响 膜中的结构。在第二个目标中，我们将阐明TREK-1激活的机制， 麻醉破坏膜，并在这样做时将膜建立为一个真正的靶点， 麻醉作用在第三个目标中，我们将开发更好的成像工具和活细胞荧光检测， 监测细胞中筏的破坏。

PROJECT SUMMARY The molecular mechanism of inhaled anesthetics in general anesthesia remains an important unanswered question in neuroscience and human health. Since the first demonstration of ether-induced anesthesia more than 160 years ago, theories of anesthesia have sought to understand the role of the membrane in anesthetic action. This application seeks to establishes the plasma membrane as a relevant target for inhaled anesthetics. Lipid membranes spontaneously partition into regions of ordered and discorded lipids known as lipid rafts. Palmitoylation of proteins drives the proteins into the ordered phase. We have shown that anesthetics disrupt these domains in live cell membranes, but disruption has not been linked to anesthetic ion channels. We will test the hypothesis that anesthetics disruption of lipid rafts releases lipid modifying enzymes to activate potassium channels through a chemical signal. Specifically, GM1 rafts sequester phospholipase D (PLD), disruption of the raft releases PLD allowing the enzyme to find its substrate and generate anionic lipid phosphatidic acid (PA). The PA then regulates the two-pore domain potassium channel (K2P) TREK-1. TREK- 1 is also an anesthetic sensitive channel. We aim to characterize the effects of anesthetics on lipid raft structure in the membrane. In a second aim, we will elucidate the mechanism of TREK-1 activation through anesthetic disruption of the membrane and in so doing establish the membrane as a bon a fide target of anesthetic action. In a third aim we will develop better imaging tools and a live cell fluorescent assay for monitoring raft disruption in cells.