Although Cl- channels have been the subject of investigations in the heart for nearly thirty years, it is only within the last five years that any consensus has emerged regarding the identification of such channels and their functional role in mammalian heart. The most extensively studied Cl- channel in heart is activated through the cAMP-protein kinase A pathway. Evidence suggests that under normal physiological conditions, activation of this channel plays a key role in autonomic regulation of resting membrane potential and the duration of the cardiac action potential. Cardiac Cl- channels, therefore, may represent new potentially important target sites for the development of class III antiarrhythmic agents. Other types of Cl- conductances which have recently been identified in mammalian heart include those activated by Ca2+i, protein kinase C, ATP, membrane stretch and anion channels activate under basal conditions. Little data is currently available on the molecular structure of any of these putative Cl- channels in heart. Very recent molecular studies from this laboratory, however, suggest that the cardiac cAMP-dependent Cl- channel is encoded by an alter-natively spliced isoform of the cystic fibrosis transmembrane conductance regulator (CFTR) Cl- channel which is found in various epithelial cells and believed to be defective in patients with cystic fibrosis. Included in this revised application is the full length sequence of the rabbit cardiac CFTR Cl- channel, which is shown to exhibit over 90% homology (excluding the alternatively spliced region) to the human epithelial CFTR gene product. This data presently represents the only molecular information currently available on the structure of any putative cardiac Cl- channel. This revised application requests support of electrophysiological and molecular biological studies aimed at characterizing the properties of Cl- channels in guinea-pig, rabbit and human heart.
The specific aims i nclude: isolation and expression of full length cDNAs which encode each type of cardiac Cl- channel, comparison of the biophysical properties of these channels in native cardiac myocytes and in heterologous expression systems, and finally an examination of the mechanism responsible for the previously observed Na+ sensitivity of the cardiac CFTR Cl- channel. These studies will specifically test the hypothesis that some of the recently described Cl- channels in native cardiac cells may be encoded by a family of genes related to CFTR. We will also examine whether or not these cardiac Cl- channels are structurally related to other types of recently cloned voltage-dependent Cl- channels. The proposed experiments should help fill the existing gap of current knowledge regarding the properties, physiological role and molecular structure of Cl- channels in the heart.
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