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F508-CFTR to the plasma membrane
in a polarized epithelial cell line
Department of Physiology, Dartmouth Medical School, Hanover, New Hampshire 03755
The 
F508 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 F508-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 F508-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 F508-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 F508-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
F508-CFTR will allow investigation of F508-CFTR trafficking in
living, polarized MDCK epithelial cells in real time.
cystic fibrosis; Madin-Darby canine kidney; butyrate; glycerol; dimethyl sulfoxide; green fluorescent protein; cystic fibrosis transmembrane conductance regulator
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