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Am J Physiol Cell Physiol 281: C1734-C1742, 2001;
0363-6143/01 $5.00
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Vol. 281, Issue 5, C1734-C1742, November 2001

SPECIAL COMMUNICATION
Cell-based assay for high-throughput quantitative screening of CFTR chloride transport agonists

Luis V. J. Galietta, Sujatha Jayaraman, and A. S. Verkman

Departments of Medicine and Physiology, Cardiovascular Research Institute, University of California, San Francisco, California 94143-0521

Drug discovery by high-throughput screening is a promising approach to develop new therapies for the most common lethal genetic disease, cystic fibrosis. Because disease-causing mutations of the cystic fibrosis transmembrane conductance regulator (CFTR) protein produce epithelial cells with reduced or absent Cl- permeability, the goal of screening is to identify compounds that restore cell Cl- transport. We have developed a rapid, quantitative screening procedure for analysis of CFTR-mediated halide transport in cells with the use of a conventional fluorescence plate reader. Doubly transfected cell lines were generated that express wild-type or mutant CFTR together with a yellow fluorescent protein (YFP)-based halide sensor. CFTR function was assayed from the time course of cell fluorescence in response to extracellular addition of 100 mM I- followed by forskolin, resulting in decreased YFP fluorescence due to CFTR-mediated I- entry. Cell lines were chosen, and conditions were optimized to minimize basal halide transport to maximize assay sensitivity. In cells cultured on 96-well plastic dishes, the assay gave reproducible halide permeabilities from well to well and could reliably detect a 2% activation of CFTR-dependent halide transport produced by low concentrations of forskolin. Applications of the assay are shown, including comparative dose-dependent CFTR activation by genistein, apigenin, 8-cyclopentyl-1,3-dipropylxanthine, IBMX, 8-methoxypsoralen, and milrinone as well as activation of alternative Cl- channels. The fluorescence assay and cell lines should facilitate the screening of novel CFTR activators and the characterization of alternative Cl- channels and transporters.

cystic fibrosis; fluorescence; high-throughput screening; epithelia; cystic fibrosis transmembrane conductance regulator


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