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

基本信息

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
项目类别：
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

项目摘要

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.

概括每年在美国进行超过4000万麻醉剂进行外科手术。期间这些麻醉药，许多患者接受卤代麻醉药物，例如异氟烷，七氟醚或 Desflurane。但是，这些药物引起麻醉的药理机制尚不清楚。一个对这些机制的详细理解可能会促进开发更安全，更具选择性的麻醉药。较少的不想要的副作用（例如心肺抑郁症和认知功能障碍）和药物的副作用较少促进从全身麻醉中恢复。在酵母分子生物学，电生理学和生物化学和啮齿动物研究，我们将检验以下假设：卤化麻醉剂导致麻醉部分是由任务3钾通道的直接结合和激活。 Task-3是膜相关的串联孔钾通道蛋白在调节的大脑和脊髓中表达神经元兴奋性。 Task-3钾通道功能被异氟烷，Sevoflurane，desflurane，Desflurane激活和其他卤代麻醉药；缺少任务3基因的敲除小鼠需要明显更高卤代麻醉药剂量，以诱导纠正措施丧失和在有害的情况下保持不动刺激。在AIM 1中，我们将使用基于酵母的高吞吐量屏幕，该屏幕为4,693个任务3的错义突变体识别任务3氨基酸残基对其功能至关重要的氨基酸残基和卤代麻醉激活。在AIM 2中，我们将净化功能性野生型和抗麻醉的突变体任务-3蛋白，并测量其单一基线时的通道功能，并在使用脂质双层电压进行卤代麻醉治疗后夹具方法。最后，在AIM 3中，将对有效和选择性的任务-3拮抗剂化合物施用异氟烷麻醉大鼠。我们将量化任务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，有害时失去矫正反射和不动的刺激。以上研究将阐明麻醉与任务3通道相互作用的机制引起激活。他们还将揭示任务3对生命中异氟烷麻醉的贡献未修饰的动物。