BETA-ADRENERGIC ACTIVATION OF A HEART CHLORIDE CHANNEL

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， 刺激和抑制细胞蛋白激酶。 具体目标 在这个授权期内，是为了表征β-肾上腺素能激活 存在于心室细胞中的氯离子通道。 全细胞和 将使用膜片钳记录单通道氯电流 分离豚鼠和大鼠心室肌细胞 β-激动剂异丙肾上腺素的刺激。 电压依赖性， 离子选择性和电导的氯电流将是 测定 异丙肾上腺素诱导激活的时间过程 将使用闪光灯激活的 这种β-激动剂的衍生物 cAMP依赖性蛋白 激酶（蛋白激酶A）和钙/二酰基甘油的各种同工酶- 依赖性蛋白激酶（蛋白激酶C）调节氯化物 渠道将被检查。 蛋白激酶A抑制剂的作用 (the沃尔什抑制剂）和蛋白激酶C（ 蛋白激酶C）将在α和β-肾上腺素能 刺激. 利用胰蛋白酶和N- 溴乙酰胺，与通道相关的氨基酸残基， 代表蛋白激酶介导的磷酸化的可能位点， 被定性。 各种氯离子通道配体的作用 包括蒽-9-羧酸， 4，4 '-二硝基芪-2，2'-二磺酸， 5-硝基-2-（3-苯基丙基氨基）-苯甲酸酯和氯贝酸的类似物 将取决于当前的情况。 这种β-肾上腺素能神经 激活的氯电流在心脏活动的发生中起作用 潜力将被调查。 一个合适的表达系统 用于氯离子通道蛋白的最终克隆， 将开发其它离子通道。 这些实验代表了 定位磷酸化位点的长期目标的第一步 和蛋白质上的配体结合，并开发高亲和力标记， 频道。 总体而言，该项目将提供新的信息， 关于心脏兴奋性的变化， 增加交感神经张力，并可能导致治疗的重要进展 在这种刺激过程中产生的心律失常。

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.