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Updated: Aug 8, 2026
Functional Reconstitution and Channel Activity Measurements of Purified Wildtype and Mutant CFTR Protein
Published on: March 9, 2015
Cell-based assay for high-throughput quantitative screening of CFTR chloride transport agonists
L V Galietta1, S Jayaraman, A S Verkman
1Department of Medicine and Physiology, Cardiovascular Research Institute, University of California, San Francisco, California 94143-0521, USA.
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.
Insights
A new fluorescence assay enables rapid screening for compounds that restore chloride transport in cystic fibrosis (CF) cells. This method aids in discovering new therapies for CF by identifying CFTR protein activators.
Area of Science:
- Biochemistry
- Cell Biology
- Pharmacology
Background:
- Cystic fibrosis (CF) is a lethal genetic disease caused by mutations in the cystic fibrosis transmembrane conductance regulator (CFTR) protein.
- CFTR mutations lead to reduced or absent chloride (Cl-) permeability in epithelial cells, underlying CF pathology.
- Developing new therapies for CF requires identifying compounds that can restore CFTR function and cell Cl- transport.
Purpose of the Study:
- To develop a rapid, quantitative, and sensitive screening assay for identifying compounds that activate CFTR-mediated halide transport.
- To establish cell lines expressing wild-type or mutant CFTR along with a halide sensor for high-throughput screening.
- To demonstrate the utility of the assay in characterizing CFTR activators and alternative ion channels.
Main Methods:
- Generation of doubly transfected cell lines expressing either wild-type or mutant CFTR and a yellow fluorescent protein (YFP)-based halide sensor.
- Assay development using a conventional fluorescence plate reader to monitor time-course YFP fluorescence changes.
- Optimization of cell culture and assay conditions to minimize background halide transport and maximize sensitivity for detecting CFTR activation.
Main Results:
- The assay demonstrated reproducible halide permeabilities and reliably detected a 2% activation of CFTR-dependent halide transport.
- Successful application of the assay for comparative dose-dependent activation studies of known CFTR modulators (e.g., genistein, apigenin).
- The assay showed potential for characterizing the activation of alternative chloride channels and transporters.
Conclusions:
- The developed fluorescence assay and cell lines provide a robust platform for high-throughput screening of novel CFTR activators.
- This screening approach can accelerate the discovery of new therapeutic compounds for cystic fibrosis.
- The assay is also valuable for the detailed characterization of ion channel and transporter function.
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