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

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

Research output: Contribution to journalArticlepeer-review

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 languageEnglish
JournalAmerican Journal of Physiology - Cell Physiology
Volume281
Issue number5 50-5
Publication statusPublished - 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)

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