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

Academic Article


  • 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.
  • Authors

  • Moyer, BD
  • Loffing, J
  • Schwiebert, EM
  • Loffing-Cueni, D
  • Halpin, Patricia
  • Karlson, KH
  • Ismailov, II
  • Guggino, WB
  • Langford, GM
  • Stanton, BA
  • Status

    Publication Date

  • August 21, 1998
  • Published In


  • 4,4'-Diisothiocyanostilbene-2,2'-Disulfonic Acid
  • Animals
  • Biotin
  • Cell Line
  • Chlorides
  • Cyclic AMP
  • Cystic Fibrosis Transmembrane Conductance Regulator
  • Dogs
  • Green Fluorescent Proteins
  • Kidney
  • Lipid Bilayers
  • Luminescent Proteins
  • Microscopy, Fluorescence
  • Digital Object Identifier (doi)

    Pubmed Id

  • 9705313
  • Start Page

  • 21759
  • End Page

  • 21768
  • Volume

  • 273
  • Issue

  • 34