期刊
AMERICAN JOURNAL OF PHYSIOLOGY-CELL PHYSIOLOGY
卷 281, 期 5, 页码 C1734-C1742出版社
AMER PHYSIOLOGICAL SOC
DOI: 10.1152/ajpcell.2001.281.5.C1734
关键词
cystic fibrosis; fluorescence; high-throughput screening; epithelia; cystic fibrosis transmembrane conductance regulator
资金
- NHLBI NIH HHS [HL-60288, HL-59198] Funding Source: Medline
- NIDDK NIH HHS [DK-35124, DK-43840] Funding Source: Medline
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.
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