Abstract
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.
Original language | English |
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Journal | American Journal of Physiology - Cell Physiology |
Volume | 281 |
Issue number | 5 50-5 |
Publication status | Published - 2001 |
Keywords
- Cystic fibrosis
- Cystic fibrosis transmembrane conductance regulator
- Epithelia
- Fluorescence
- High-throughput screening
ASJC Scopus subject areas
- Clinical Biochemistry
- Cell Biology
- Physiology
- Physiology (medical)