Membrane trafficking of the cystic fibrosis gene product, cystic fibrosis transmembrane conductance regulator, tagged with green fluorescent protein in Madin-Darby canine kidney cells

Bryan D. Moyer, Johannes Loffing, Erik M. Schwiebert, Dominique Loffing-Cueni, Patricia A. Halpin, Katherine H. Karlson, Iskandar I. Ismailov, William B. Guggino, George M. Langford, Bruce A. Stanton

Research output: Contribution to journalArticle

132 Scopus citations

Abstract

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 patchclamp 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.

Original languageEnglish (US)
Pages (from-to)21759-21768
Number of pages10
JournalJournal of Biological Chemistry
Volume273
Issue number34
DOIs
StatePublished - Aug 21 1998

ASJC Scopus subject areas

  • Biochemistry
  • Molecular Biology
  • Cell Biology

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