Forskolin-induced apical membrane insertion of virally expressed, epitope-tagged CFTR in polarized MDCK cells.
Channel gating of the cystic fibrosis transmembrane conductance regulator (CFTR) is activated in response to cAMP stimulation. In addition, CFTR activation may also involve rapid insertion of a subapical pool of CFTR into the plasma membrane (PM). However, this issue has been controversial, in part because of the difficulty in distinguishing cell surface vs. intracellular CFTR. Recently, a fully functional, epitope-tagged form of CFTR (M2-901/CFTR) that can be detected immunologically in nonpermeabilized cells was characterized (Howard M, Duvall MD, Devor DC, Dong J-Y, Henze K, and Frizzell RA. Am J Physiol Cell Physiol 269: C1565-C1576, 1995; and Schultz BD, Takahashi A, Liu C, Frizzell RA, and Howard M. Am J Physiol Cell Physiol 273: C2080-C2089, 1997). We have developed replication-defective recombinant adenoviruses that express M2-901/CFTR and used them to probe cell surface CFTR in forskolin (FSK)-stimulated polarized Madin-Darby canine kidney (MDCK) cells. Virally expressed M2-901/CFTR was functional and was readily detected on the apical surface of FSK-stimulated polarized MDCK cells. Interestingly, at low multiplicity of infection, we observed FSK-stimulated insertion of M2901/CFTR into the apical PM, whereas at higher M2-901/CFTR expression levels, no increase in surface expression was detected using indirect immunofluorescence. Immunoelectron microscopy of unstimulated and FSK-stimulated cells confirmed the M2-901/CFTR redistribution to the PM upon FSK stimulation and demonstrates that the apically inserted M2-901/CFTR originates from a population of subapical vesicles. Our observations may reconcile previous conflicting reports regarding the effect of cAMP stimulation on CFTR trafficking.
Laboratory of Epithelial Cell Biology, Renal-Electrolyte Division, University of Pittsburgh, Pennsylvania 15213-2500, USA.
M
M
Howard
X
X
Jiang
D B
DB
Stolz
W G
WG
Hill
J A
JA
Johnson
S C
SC
Watkins
R A
RA
Frizzell
C M
CM
Bruton
P D
PD
Robbins
O A
OA
Weisz
D000256:Adenoviridae / Q000378:metabolism; D000818:Animals; D002478:Cells, Cultured / Q000821:virology; D018118:Chloride Channels / Q000187:drug effects / Q000378:metabolism; D005576:Colforsin / Q000494:pharmacology; D019005:Cystic Fibrosis Transmembrane Conductance Regulator / Q000187:drug effects / Q000378:metabolism*; D004285:Dogs; D000939:Epitopes; D007668:Kidney / Q000166:cytology / Q000187:drug effects / Q000378:metabolism*
D018118:Chloride Channels; D000939:Epitopes; D019005:Cystic Fibrosis Transmembrane Conductance Regulator; D005576:Colforsin