BETA-ADRENERGIC ACTIVATION OF A HEART CHLORIDE CHANNEL

心脏氯离子通道的 β-肾上腺素能激活

• 批准号: 3473400
• 负责人: KENNETH B WALSH
• 金额: $ 1.74万
• 依托单位: UNIVERSITY OF SOUTH CAROLINA AT COLUMBIA
• 依托单位国家: 美国
• 财政年份: 1992
• 资助国家: 美国
• 起止时间: 1992-05-01 至 1996-04-30
• 项目状态: 已结题
• 来源: https://reporter.nih.gov/project-details/3473400
• 关键词: active sites; alpha adrenergic agent; beta adrenergic agent; beta adrenergic receptor; chloride channels; clofibrate; cyclic AMP; enzyme inhibitors; enzyme substrate analog; guinea pigs; heart cell; heart metabolism; heart pharmacology; isoproterenol; laboratory rat; molecular cloning; protein kinase A; protein kinase C; site directed mutagenesis; voltage /patch clamp; voltage gated channel

The broad, long-term objective of this project is to understand the mechanism(s) by which drugs and hormones regulate cardiac ion channels by stimulating and inhibiting cellular protein kinases. The specific goal for this grant period is to characterize a beta-adrenergic activated chloride channel present in cardiac ventricular cells. Whole-cell and single channel chloride currents will be recorded using the patch clamp technique in isolated guinea pig and rat ventricular cells during stimulation by the beta-agonist isoproterenol. The voltage-dependence, ion selectivity and conductance of the chloride current will be determined. The time course for isoproterenol-induced activation of the chloride current will be determined using a light flash-activated derivative of this beta-agonist. The ability of cAMP-dependent protein kinase (protein kinase A) and various isozymes of calcium/diacylglycerol- dependent protein kinase (protein kinase C) to regulate the chloride channel will be examined. The effects of inhibitors of protein kinase A (the Walsh inhibitor) and protein kinase C (the pseudosubstrate of protein kinase C) will be monitored during both alpha and beta-adrenergic stimulation. Using chemical reagents such as trypsin and N- bromoacetamide, the amino acid residues associated with the channel that represent possible sites for protein kinase-mediated phosphorylation will be characterized. The effect of various chloride channel ligands including anthracene-9-carboxylic acid, 4,4'-dinitrostilbene-2,2'-disulphonic acid, 5-nitro-2(3-phenylpropylamino)-benzoate and analogues of clofibric acid will be determined on this current. The role that this beta-adrenergic activated chloride current plays in the genesis of the cardiac action potential will be investigated. An appropriate expression system to be used in the eventual cloning of the chloride ion channel protein and other ion channels will be developed. These experiments represent the first step in the long-term goal of locating the sites of phosphorylation and ligand binding on the protein and developing high affinity labels for the channel. Overall, this project will provide new information concerning changes that occur in cardiac excitability during periods of increased sympathetic tone and may led to important advances in treating cardiac arrhythmias that develop during this stimulation.

该项目的广泛、长期目标是了解 药物和激素调节心脏离子通道的机制(S) 刺激和抑制细胞蛋白激酶。具体目标 对于这一授权期的特征是一种β-肾上腺素能激活 氯离子通道存在于心肌细胞中。全蜂窝和 使用膜片钳记录单通道氯电流 体外培养豚鼠和大鼠心室肌细胞技术 由β-激动剂异丙肾上腺素刺激。电压依赖性， 氯电流的离子选择性和电导将为 下定决心。异丙肾上腺素激活心肌细胞的时程 氯电流将使用闪光灯激活来确定 这种β-激动剂的衍生物。CAMP依赖蛋白的能力 蛋白激酶A和各种钙/二酰甘油同工酶- 依赖蛋白激酶(蛋白激酶C)调节氯离子 将对频道进行检查。蛋白激酶A抑制剂的作用 (沃尔什抑制物)和蛋白激酶C(假底物 蛋白激酶C)在α-和β-肾上腺素能 刺激。使用化学试剂，如胰酶和N- 溴乙酰胺，与通道有关的氨基酸残基 代表蛋白激酶介导的磷酸化的可能位点 被刻画出来。不同氯离子通道配体的作用 包括菲-9-羧酸， 4，4‘-二硝基-2，2’-二磺酸， 5-硝基-2-(3-苯丙氨基)-苯甲酸及其类似物 将在这股洋流中确定。这种β-肾上腺素能 激活氯电流在心脏活动发生中的作用 我们将对其潜力进行调查。一个合适的表达系统有待于 用于最终克隆氯离子通道蛋白和 还将开发其他离子通道。这些实验代表了 定位磷酸化位点的长期目标的第一步 和配体结合在蛋白质上，并开发出高亲和力的标记 频道。总体而言，该项目将提供新的信息 关于心脏兴奋性在以下期间发生的变化 增加交感神经的音调，并可能导致治疗的重要进展 在这种刺激过程中发生的心律失常。