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

TASK-3串联孔钾通道的麻醉激活：分子机制和行为效应

• 批准号: 9900029
• 负责人: Joseph F Cotten
• 金额: $ 36.25万
• 依托单位: MASSACHUSETTS GENERAL HOSPITAL
• 依托单位国家: 美国
• 财政年份: 2018
• 资助国家: 美国
• 起止时间: 2018-04-01 至 2022-03-31
• 项目状态: 已结题
• 来源: https://reporter.nih.gov/project-details/9900029
• 关键词: 3-Dimensional; Amino Acids; Anesthesia procedures; Anesthetics; Animals; Behavioral; Binding; Biochemistry; Biological Assay; Brain; Cell Line; Codon Nucleotides; DNA sequencing; Development; Dose; Electroencephalogram; Electrophysiology (science); Essential Amino Acids; Fostering; General Anesthesia; Genes; Growth; Homology Modeling; Impaired cognition; Individual; Intravenous; Isoflurane; Knockout Mice; Lipid Bilayers; Mammalian Cell; Measures; Membrane; Mental Depression; Methods; Molecular; Molecular Biology; Mutate; Operative Surgical Procedures; Patients; Pharmaceutical Preparations; Pharmacology; Phenotype; Pichia; Population; Potassium; Potassium Channel; Probability; Property; Protein Structural Homology; Proteins; Rattus; Recovery; Reflex action; Regulation; Resistance; Rodent; Saccharomyces cerevisiae; Spinal Cord; Stimulus; Surveys; System; Terminator Codon; Testing; Time; United States; Work; Yeasts; base; cDNA Library; clinically relevant; desflurane; fitness; high throughput screening; in vivo; mutant; neuronal excitability; next generation; novel; pressure; response; sevoflurane; side effect; tribromoethanol; voltage clamp

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.

概括 在美国，每年有超过 4000 万次手术需要使用麻醉剂。期间 在使用这些麻醉剂时，许多患者接受卤化麻醉药物，例如异氟烷、七氟烷或 地氟烷。然而，这些药物引起麻醉的药理学机制尚不清楚。一个 对这些机制的详细了解可能会促进开发更安全、更具选择性的麻醉剂 更少的不良副作用（例如，心肺抑制和认知功能障碍），以及药物 促进从全身麻醉中恢复。使用酵母分子生物学、电生理学和 生物化学以及啮齿动物研究，我们将检验卤化麻醉剂引起的假设 麻醉部分是通过直接结合和激活 TASK-3 钾通道来实现的。 TASK-3是一种膜 相关串联孔钾通道蛋白在大脑和脊髓中表达，调节 神经元的兴奋性。 TASK-3钾通道功能由异氟烷、七氟烷、地氟烷、 和其他卤化麻醉剂；而缺乏 TASK-3 基因的敲除小鼠则需要显着更高的 卤化麻醉剂的剂量，用于诱导失去翻正能力并在有害的情况下保持不动 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 鉴定对其功能和卤代麻醉剂激活至关重要的 TASK-3 氨基酸残基。在目标 2 中， 我们将纯化功能性野生型和麻醉抗性突变体 TASK-3 蛋白并测量它们的单 使用脂质双层电压在基线和卤化麻醉治疗后的通道功能 夹钳法。最后，目标 3 将给予有效且选择性的 TASK-3 拮抗剂化合物 异氟烷麻醉大鼠。我们将量化 TASK-3 拮抗剂对大鼠脑电图的影响 （脑电图）以及麻醉的行为终点，在有害的情况下失去翻正反射和不动 刺激。上述研究将阐明麻醉药与 TASK-3 通道相互作用的机制 引起激活。他们还将揭示 TASK-3 在活体遗传性麻醉中对异氟烷麻醉的贡献 未经改造的动物。