Membrane Trafficking of the Cystic Fibrosis Gene Product, Cystic Fibrosis Transmembrane Conductance Regulator, Tagged with Green Fluorescent Protein in Madin-Darby Canine Kidney Cells

• Published in: The Journal of biological chemistry 1998-08, Vol.273 (34), p.21759-21768
• Main Authors: Moyer, Bryan D., Loffing, Johannes, Schwiebert, Erik M., Loffing-Cueni, Dominique, Halpin, Patricia A., Karlson, Katherine H., Ismailov, Iskandar I., Guggino, William B., Langford, George M., Stanton, Bruce A.
• Format: Article
• Language: English
• Subjects: 4,4'-Diisothiocyanostilbene-2,2'-Disulfonic Acid - pharmacology; Animals; Biotin - metabolism; Cell Line; Chlorides - metabolism; Cyclic AMP - metabolism; Cystic Fibrosis Transmembrane Conductance Regulator - metabolism; Dogs; Green Fluorescent Proteins; Kidney - metabolism; Lipid Bilayers - metabolism; Luminescent Proteins - metabolism; Microscopy, Fluorescence
• ISSN: 0021-9258; 1083-351X
• DOI: 10.1074/jbc.273.34.21759

The mechanism by which cAMP stimulates cystic fibrosis transmembrane conductance regulator (CFTR)-mediated chloride (Cl−) secretion is cell type-specific. By using Madin-Darby canine kidney (MDCK) type I epithelial cells as a model, we tested the hypothesis that cAMP stimulates Cl− secretion by stimulating CFTR Cl− channel trafficking from an intracellular pool to the apical plasma membrane. To this end, we generated a green fluorescent protein (GFP)-CFTR expression vector in which GFP was linked to the N terminus of CFTR. GFP did not alter CFTR function in whole cell patch-clamp or planar lipid bilayer experiments. In stably transfected MDCK type I cells, GFP-CFTR localization was substratum-dependent. In cells grown on glass coverslips, GFP-CFTR was polarized to the basolateral membrane, whereas in cells grown on permeable supports, GFP-CFTR was polarized to the apical membrane. Quantitative confocal fluorescence microscopy and surface biotinylation experiments demonstrated that cAMP did not stimulate detectable GFP-CFTR translocation from an intracellular pool to the apical membrane or regulate GFP-CFTR endocytosis. Disruption of the microtubular cytoskeleton with colchicine did not affect cAMP-stimulated Cl− secretion or GFP-CFTR expression in the apical membrane. We conclude that cAMP stimulates CFTR-mediated Cl− secretion in MDCK type I cells by activating channels resident in the apical plasma membrane.