Anesthetic activation of TASK-3 tandem pore potassium channels: molecular mechanisms and behavioral effects

摘要 在美国，每年有超过4000万种麻醉剂用于外科手术。在.期间 这些麻醉剂，许多患者接受卤化麻醉药，如异氟醚，七氟醚，或 地氟醚。然而，这些药物导致麻醉的药理机制尚不清楚。一个 对这些机制的详细了解可能会促进更安全、更有选择性的麻醉药的开发 更少的不良副作用(如心肺抑制和认知功能障碍)，以及 促进全身麻醉恢复。利用酵母菌分子生物学、电生理学和 生物化学和啮齿动物研究，我们将检验卤化麻醉剂引起的假说 麻醉部分是通过直接结合和激活TASK-3钾通道实现的。任务3是一种膜 相关串联孔钾通道蛋白在脑和脊髓中表达，调节 神经元兴奋性。任务3钾通道功能可被异氟醚、七氟醚、地氟烷、 和其他卤化麻醉剂；以及缺乏TASK-3基因的敲除小鼠需要显著更高的 用于诱导伤害性呼吸暂停时失去翻正和保持静止的卤化麻醉剂的剂量 刺激。在目标1中，我们将使用基于酵母的高通量筛选4,693个TASK-3错义突变体来 确定TASK-3氨基酸残基对其功能和卤化麻醉剂活性至关重要。在目标2中， 我们将纯化具有功能的野生型和耐麻醉突变体TASK-3蛋白，并测定其单个 应用脂质双分子层电压测定基线和卤化麻醉剂治疗后的通道功能 钳位法。最后，在目标3中，将使用有效和选择性的TASK-3拮抗剂化合物来 异氟醚麻醉大鼠。我们将量化TASK-3拮抗剂对大鼠脑电的影响 (EEG)和在麻醉的行为终点，翻正反射的丧失和在有害的 刺激。上述研究将阐明麻醉剂与TASK-3通道相互作用的机制 导致激活。他们还将揭示TASK-3对活体基因异氟醚麻醉的贡献。 未经改造的动物。

Summary Over 40 million anesthetics are administered for surgical procedures in the United States every year. During these anesthetics, many patients receive halogenated anesthetic drugs such as isoflurane, sevoflurane, or desflurane. However, the pharmacologic mechanisms by which these drugs cause anesthesia are unclear. A detailed understanding of these mechanisms may foster development of safer, more selective anesthetics with fewer undesired side effects (e.g., cardiorespiratory depression and cognitive dysfunction), and drugs that facilitate recovery from general anesthesia. Using methods in yeast molecular biology, electrophysiology, and biochemistry as well as rodent studies, we will test the hypothesis that halogenated anesthetics cause anesthesia in part by direct binding and activation of TASK-3 potassium channels. TASK-3 is a membrane associated tandem pore potassium channel protein expressed in the brain and spinal cord that regulates neuronal excitability. TASK-3 potassium channel function is activated by isoflurane, sevoflurane, desflurane, and other halogenated anesthetics; and knockout mice lacking the TASK-3 gene require significantly higher doses of halogenated anesthetics for induction of loss of righting and to maintain immobility during a noxious stimulus. In Aim 1, we will use a yeast-based, high throughput screen of 4,693 TASK-3 missense mutants to identify TASK-3 amino acid residues critical for its function and for halogenated anesthetic activation. In Aim 2, we will purify functional wild-type and anesthetic-resistant mutant TASK-3 protein and measure their single channel function at baseline and following halogenated anesthetic treatment using the lipid bilayer voltage clamp method. Finally, in Aim 3 will administer potent and selective TASK-3 antagonist compounds to isoflurane anesthetized rats. We will quantify the TASK-3 antagonists' effects on the rat electroencephalogram (EEG) and on behavioral end points of anesthesia, loss of righting reflex and immobility during a noxious stimulus. The above studies will clarify the mechanism by which anesthetics interact with TASK-3 channels to cause activation. They will also reveal the contribution of TASK-3 to isoflurane anesthesia in a live, genetically unmodified animal.