Activation of the Cystic Fibrosis Transmembrane Conductance Regulator by the Flavonoid Quercetin Potential Use as a Biomarker of ΔF508 Cystic Fibrosis Transmembrane Conductance Regulator Rescue

Therapies to correct the ΔF508 cystic fibrosis transmembrane conductance regulator (CFTR) folding defect require sensitive methods to detect channel activity in vivo. The β₂ adrenergic receptor agonists, which provide the CFTR stimuli commonly used in nasal potential difference assays, may not overc...

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Published in	American journal of respiratory cell and molecular biology Vol. 43; no. 5; pp. 607 - 616
Main Authors	Pyle, Louise C., Fulton, Jennifer C., Sloane, Peter A., Backer, Kyle, Mazur, Marina, Prasain, Jeevan, Barnes, Stephen, Clancy, J. P., Rowe, Steven M.
Format	Journal Article
Language	English
Published	United States American Thoracic Society 01.11.2010
Subjects	Animals Biomarkers - metabolism Cell Membrane - drug effects Cell Membrane - metabolism Chlorides - metabolism Cyclic AMP - metabolism Cystic Fibrosis Transmembrane Conductance Regulator - chemistry Cystic Fibrosis Transmembrane Conductance Regulator - metabolism Dose-Response Relationship, Drug Drug Evaluation, Preclinical Humans Ion Channel Gating - drug effects Ion Transport - drug effects Membrane Potentials - drug effects Mice Mutant Proteins - chemistry Mutant Proteins - metabolism Nasal Mucosa - drug effects Nasal Mucosa - metabolism NIH 3T3 Cells Phosphorylation - drug effects Protein Structure, Tertiary Quercetin - pharmacology Rats Receptors, Adrenergic, beta-2 - metabolism
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Summary:	Therapies to correct the ΔF508 cystic fibrosis transmembrane conductance regulator (CFTR) folding defect require sensitive methods to detect channel activity in vivo. The β₂ adrenergic receptor agonists, which provide the CFTR stimuli commonly used in nasal potential difference assays, may not overcome the channel gating defects seen in ΔF508 CFTR after plasma membrane localization. In this study, we identify an agent, quercetin, that enhances the detection of surface ΔF508 CFTR, and is suitable for nasal perfusion. A screen of flavonoids in CFBE41o⁻ cells stably transduced with ΔF508 CFTR, corrected to the cell surface with low temperature growth, revealed that quercetin stimulated an increase in the short-circuit current. This increase was dose-dependent in both Fisher rat thyroid and CFBE41o⁻ cells. High concentrations inhibited Cl⁻ conductance. In CFBE41o⁻ airway cells, quercetin (20 μg/ml) activated ΔF508 CFTR, whereas the β₂ adrenergic receptor agonist isoproterenol did not. Quercetin had limited effects on cAMP levels, but did not produce detectable phosphorylation of the isolated CFTR R-domain, suggesting an activation independent of channel phosphorylation. When perfused in the nares of Cftr(+) mice, quercetin (20 μg/ml) produced a hyperpolarization of the potential difference that was absent in Cftr(-/-) mice. Finally, quercetin-induced, dose-dependent hyperpolarization of the nasal potential difference was also seen in normal human subjects. Quercetin activates CFTR-mediated anion transport in respiratory epithelia in vitro and in vivo, and may be useful in studies intended to detect the rescue of ΔF508 CFTR by nasal potential difference.
Bibliography:	ObjectType-Article-1 SourceType-Scholarly Journals-1 ObjectType-Feature-2 content type line 23 This research was funded by National Institutes of Health grants 1K23DK075788–01 (S.M.R.), 1R03DK084110-01 (S.M.R.), and 1P30DK072482–01A1 (to Eric J Sorscher; see Acknowledgments), and Cystic Fibrosis Foundation grants ROWE08XX (S.M.R.), R464-CR07 (to Eric J Sorscher), and CLANCY02YO (J.P.C.). This study also benefited from National Institutes of Health funding to the Purdue University-University of Alabama at Birmingham Botanicals Center for Age-Related Disease from the National Center for Complementary and Alternative Medicine, and b funding from the National Institutes of Health Office of Dietary Supplements (P50 AT00477, C. Weaver, principal investigator). This article has an online supplement, which is accessible from this issue's table of contents at www.atsjournals.org These authors contributed equally to this manuscript. Originally Published in Press as DOI: 10. 1165/rcmb.2009-0281OC on December 30, 2009 Author Disclosure: L.C.P. has received sponsored grants from the National Heart, Lung, and Blood Institute of the National Institutes of Health (in excess of $100,000). S.M.R. has served on the advisory boards for Vertex Pharmaceuticals (up to $1,000) and PTC Therapeutics ($1,001–$5,000), has received industry-sponsored grants from Novartis Pharmaceuticals (in excess of $100,000), Vertex Pharmaceuticals (in excess of $100,000), and PTC Therapeutics (in excess of $100,000), and has received sponsored grants from the Cystic Fibrosis Foundation ($50,000–$100,000) and the National Institutes of Health (in excess of $100,000). None of the other authors has a financial relationship with a commercial entity that has an interest in the subject of this manuscript. The contents of this article are solely the responsibility of the authors, and do not necessarily represent the official views of the National Institute of Diabetes and Digestive and Kidney Diseases, the National Heart, Lung, and Blood Institute, or the National Institutes of Health.
ISSN:	1044-1549 1535-4989 1535-4989
DOI:	10.1165/rcmb.2009-0281OC