Cell-based assay for high-throughput quantitative screening of CFTR chloride transport agonists.
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.
Galietta
L V
LV
Department of Medicine and Physiology, Cardiovascular Research Institute, University of California, San Francisco, California 94143-0521, USA.
Jayaraman
S
S
Verkman
A S
AS
D000818:Animals; D001426:Bacterial Proteins; D016227:Benzoquinones / Q000494:pharmacology; D016466:CHO Cells; D002460:Cell Line; D002478:Cells, Cultured; D065101:Chloride Channel Agonists; D005576:Colforsin / Q000494:pharmacology; D006224:Cricetinae; D019005:Cystic Fibrosis Transmembrane Conductance Regulator / Q000819:agonists*; D003584:Cytological Techniques; D004353:Drug Evaluation, Preclinical; D005419:Flavonoids / Q000494:pharmacology; D019833:Genistein / Q000494:pharmacology; D006863:Hydrogen-Ion Concentration; D008164:Luminescent Proteins; D008856:Microscopy, Fluorescence; D051381:Rats; D011916:Rats, Inbred F344
D001426:Bacterial Proteins; D016227:Benzoquinones; D065101:Chloride Channel Agonists; D005419:Flavonoids; D008164:Luminescent Proteins; C054562:yellow fluorescent protein, Bacteria; D019005:Cystic Fibrosis Transmembrane Conductance Regulator; D005576:Colforsin; D019833:Genistein