Trafficking of GFP-tagged ΔF508-CFTR to the plasma membrane in a polarized epithelial cell line
American Journal of Physiology-Cell Physiology, 2001•journals.physiology.org
The ΔF508 mutation reduces the amount of cystic fibrosis transmembrane conductance
regulator (CFTR) expressed in the plasma membrane of epithelial cells. However, a reduced
temperature, butyrate compounds, and “chemical chaperones” allow ΔF508-CFTR to traffic
to the plasma membrane and increase Cl− permeability in heterologous and nonpolarized
cells. Because trafficking is affected by the polarized state of epithelial cells and is cell-type
dependent, our goal was to determine whether these maneuvers induce ΔF508-CFTR …
regulator (CFTR) expressed in the plasma membrane of epithelial cells. However, a reduced
temperature, butyrate compounds, and “chemical chaperones” allow ΔF508-CFTR to traffic
to the plasma membrane and increase Cl− permeability in heterologous and nonpolarized
cells. Because trafficking is affected by the polarized state of epithelial cells and is cell-type
dependent, our goal was to determine whether these maneuvers induce ΔF508-CFTR …
The ΔF508 mutation reduces the amount of cystic fibrosis transmembrane conductance regulator (CFTR) expressed in the plasma membrane of epithelial cells. However, a reduced temperature, butyrate compounds, and “chemical chaperones” allow ΔF508-CFTR to traffic to the plasma membrane and increase Cl− permeability in heterologous and nonpolarized cells. Because trafficking is affected by the polarized state of epithelial cells and is cell-type dependent, our goal was to determine whether these maneuvers induce ΔF508-CFTR trafficking to the apical plasma membrane in polarized epithelial cells. To this end, we generated and characterized a line of polarized Madin-Darby canine kidney (MDCK) cells stably expressing ΔF508-CFTR tagged with green fluorescent protein (GFP). A reduced temperature, glycerol, butyrate, or DMSO had no effect on 8-(4-chlorophenylthio)-cAMP (CPT-cAMP)-stimulated transepithelial Cl− secretion across polarized monolayers. However, when the basolateral membrane was permeabilized, butyrate, but not the other experimental maneuvers, increased the CPT-cAMP-stimulated Cl− current across the apical plasma membrane. Thus butyrate increased the amount of functional ΔF508-CFTR in the apical plasma membrane. Butyrate failed to stimulate transepithelial Cl− secretion because of inhibitory effects on Cl− uptake across the basolateral membrane. These observations suggest that studies on heterologous and nonpolarized cells should be interpreted cautiously. The GFP tag on ΔF508-CFTR will allow investigation of ΔF508-CFTR trafficking in living, polarized MDCK epithelial cells in real time.
American Physiological Society