Epitope tagging permits cell surface detection of functional CFTR.
The cystic fibrosis transmembrane conductance regulator (CFTR) is a phosphorylation-activated Cl channel responsible for adenosine 3',5'-cyclic monophosphate (cAMP)-induced Cl secretion across the apical membranes of epithelial cells. To optimize its detection for membrane localization studies, we tagged CFTR with epitope sequences at the carboxy terminus or in the fourth external loop. When epitopes were added to the fourth external loop, the N-linked glycosylation sites in that loop were either preserved or they were mutated to produce a deglycosylated CFTR (dgCFTR). Tagged CFTRs were expressed in HeLa cells, and their cAMP-sensitive Cl permeability was assayed using the halide-sensitive fluorophore SPQ. CFTRs containing the M2 epitope showed halide permeability responses to cAMP, whereas cells expressing CFTR with the hemagglutinin (HA) tag showed little or no cAMP response. Xenopus oocytes expressing dgCFTR, with or without the M2 epitope, showed Cl conductance responses that were 20% of the wild-type response, whereas M2-tagged constructs retaining the glycosylation sites responded like wild-type CFTR. External M2-tagged CFTR was detected in the surface membrane of nonpermeabilized cells. The surface expression of the mutant M2-tagged CFTRs correlated with processing of these mutants (Gregory et al. Mol. Cell. Biol. 11:3886-3893, 1991). M2-901/CFTR is a useful reporter for the trafficking of wild-type and mutant CFTRs to the cell surface.
Howard
M
M
Department of Physiology and Biophysics, University of Alabama at Birmingham 35294-0005, USA.
DuVall
M D
MD
Devor
D C
DC
Dong
J Y
JY
Henze
K
K
Frizzell
R A
RA
D000818:Animals; D001483:Base Sequence; D002462:Cell Membrane / Q000378:metabolism; D003550:Cystic Fibrosis / Q000378:metabolism; D019005:Cystic Fibrosis Transmembrane Conductance Regulator / Q000235:genetics / Q000378:metabolism*; D000939:Epitopes / Q000235:genetics*; D006031:Glycosylation; D006367:HeLa Cells; D006801:Humans; D008856:Microscopy, Fluorescence; D015335:Molecular Probes / Q000235:genetics; D008969:Molecular Sequence Data; D009154:Mutation; D009865:Oocytes / Q000378:metabolism; D016324:Sequence Tagged Sites; D014982:Xenopus laevis
C505032:CFTR protein, human; D000939:Epitopes; D015335:Molecular Probes; D019005:Cystic Fibrosis Transmembrane Conductance Regulator