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.

总结 在美国，每年有超过4000万的麻醉剂用于外科手术。期间 对于这些麻醉剂，许多患者接受卤化麻醉剂药物，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 对这些机制的详细了解可能有助于开发更安全、更具选择性的麻醉剂， 较少的不希望的副作用（例如，心肺抑制和认知功能障碍），以及 促进从全身麻醉中恢复。利用酵母分子生物学、电生理学和 生物化学以及啮齿动物研究，我们将测试卤化麻醉剂引起的假设， 麻醉部分是通过直接结合和激活ASK-3钾通道。Task-3是一种膜 在脑和脊髓中表达的相关串联孔钾通道蛋白， 神经元兴奋性ASK-3钾通道功能被异氟烷、七氟烷、地氟烷激活， 和其他卤代麻醉剂;和敲除小鼠缺乏的任务-3基因需要显着更高的 用于诱导翻正能力丧失和在有害的麻醉期间维持不动的卤代麻醉剂剂量 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ASK-3错义突变体， 鉴定对于其功能和卤代麻醉剂活化至关重要的ASK-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药抗性突变型的task-3蛋白，并测量它们的单个 使用脂质双层电压在基线和卤代麻醉剂治疗后的通道功能 钳位法最后，在目标3中，将施用有效的和选择性的ASK-3拮抗剂化合物， 异氟烷麻醉大鼠。我们将量化的任务-3拮抗剂对大鼠脑电图的影响 (EEG)以及麻醉的行为终点，翻正反射的丧失和在伤害性 刺激。上述研究将阐明麻醉药与ASK-3通道相互作用的机制， 导致激活。他们还将揭示ASK-3对异氟烷麻醉的作用， 未经改造的动物