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Membrane trafficking of the cystic fibrosis gene product, cystic fibrosis transmembrane conductance regulator, tagged with green fluorescent protein in madin-darby canine kidney cells
The Journal of Biological Chemistry (1998)
  • Brian D Moyer
  • Johannes Loffing
  • Eric M Schwiebert
  • Dominique Loffing-Cueni
  • Patricia A Halpin
  • Katherine H Karlson
  • Iskandar I Ismailov
  • William B Guggino
  • George M Langford, Syracuse University
  • Bruce A Stanton
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 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.

Keywords
  • 4,
  • 4'-Diiothiocyanostilbene-2,
  • 2'-Disulfonic acid,
  • Animals,
  • Biotin,
  • Cell line,
  • Chlorides,
  • Cyclic AMP,
  • Cystic fibrosis transmembrance conductance regulator,
  • Dogs,
  • Gree fluorescent protiens,
  • Kidney,
  • Lipid Bilayers,
  • Luminescent protiens,
  • Fluorescence microscopy
Disciplines
Publication Date
August 21, 1998
Publisher Statement
Moyer, B. D., J. Loffing, E. M. Schwiebert, D. Loffing-Cueni, P. A. Halpin, K. H. Karlson, I. I. Ismailov, W. B. Guggino, G. M. Langford, and B. A. Stanton. “Membrane Trafficking of the Cystic Fibrosis Gene Product, Cystic Fibrosis Transmembrane Conductance Regulator, Tagged with Green Fluorescent Protein in Madin-Darby Canine Kidney Cells.” The Journal of Biological Chemistry 273, no. 34 (August 21, 1998): 21759–68.
Citation Information
Brian D Moyer, Johannes Loffing, Eric M Schwiebert, Dominique Loffing-Cueni, et al.. "Membrane trafficking of the cystic fibrosis gene product, cystic fibrosis transmembrane conductance regulator, tagged with green fluorescent protein in madin-darby canine kidney cells" The Journal of Biological Chemistry Vol. 279 Iss. 34 (1998)
Available at: http://works.bepress.com/george_langford/32/