Trafficking of GFP-tagged DeltaF508-CFTR to the plasma membrane in a polarized epithelial cell line.
The DeltaF508 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 DeltaF508-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 DeltaF508-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 DeltaF508-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 DeltaF508-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 DeltaF508-CFTR will allow investigation of DeltaF508-CFTR trafficking in living, polarized MDCK epithelial cells in real time.
Loffing-Cueni
D
D
Department of Physiology, Dartmouth Medical School, Hanover, New Hampshire 03755, USA.
Loffing
J
J
Shaw
C
C
Taplin
A M
AM
Govindan
M
M
Stanton
C R
CR
Stanton
B A
BA
D000818:Animals; D002087:Butyrates / Q000494:pharmacology; D002121:Calcium Channel Blockers / Q000494:pharmacology; D002460:Cell Line; D002462:Cell Membrane / Q000378:metabolism*; D016764:Cell Polarity; D000242:Cyclic AMP / Q000031:analogs & derivatives / Q000494:pharmacology; D019005:Cystic Fibrosis Transmembrane Conductance Regulator / Q000235:genetics / Q000378:metabolism*; D004594:Electrophysiology; D004791:Enzyme Inhibitors / Q000494:pharmacology; D004847:Epithelial Cells / Q000187:drug effects / Q000378:metabolism*; D019833:Genistein / Q000494:pharmacology; D049452:Green Fluorescent Proteins; D006801:Humans; D007202:Indicators and Reagents / Q000378:metabolism; D007476:Ionophores / Q000494:pharmacology; D008164:Luminescent Proteins / Q000235:genetics / Q000378:metabolism*; D018613:Microscopy, Confocal; D009761:Nystatin / Q000494:pharmacology; D021381:Protein Transport; D011993:Recombinant Fusion Proteins / Q000378:metabolism; D013696:Temperature; D019076:Transgenes; D062367:ortho-Aminobenzoates / Q000494:pharmacology
D002087:Butyrates; C505032:CFTR protein, human; D002121:Calcium Channel Blockers; D004791:Enzyme Inhibitors; D007202:Indicators and Reagents; D007476:Ionophores; D008164:Luminescent Proteins; D011993:Recombinant Fusion Proteins; D062367:ortho-Aminobenzoates; D019005:Cystic Fibrosis Transmembrane Conductance Regulator; D009761:Nystatin; D049452:Green Fluorescent Proteins; C001209:fenamic acid; D019833:Genistein; D000242:Cyclic AMP