BETA-ADRENERGIC ACTIVATION OF A HEART CHLORIDE CHANNEL

这个项目广泛而长期的目标是了解

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.