FLAG epitope positioned in an external loop preserves normal biophysical properties of CFTR.
We asked whether inclusion of the FLAG epitope in the fourth extracellular loop of the cystic fibrosis transmembrane conductance regulator (M2-901/CFTR), which permits detection of cell surface expression, affected CFTR's biophysical properties or channel regulation by kinases, phosphatases, and nucleotides. Channel activity of M2-901/CFTR was evaluated in numerous cell types and expression systems to characterize its gating and regulation. Our results show that M2-901/CFTR required adenosine 3',5'-cyclic monophosphate-dependent protein kinase phosphorylation to initiate channel activity. Subsequently, ATP alone was sufficient to support channel gating, and ADP inhibited channel opening. Current fluctuation analysis indicated that the nucleotide-dependent gating rates were indistinguishable from those of wild-type (wt) cystic fibrosis transmembrane conductance regulator (CFTR). Channel conductance in symmetric Cl- (11.2 pS), anion permeability ratio (1.66), and block by gluconate indicate that the anion conduction pathway is indistinguishable from wtCFTR. Sulfonylureas (glibenclamide and LY-295501) inhibited M2-901/ CFTR channel activity by an identical mechanism to that described for wtCFTR. Finally, CFTR-dependent insertion and retrieval of cell membrane was unaffected by the presence of the FLAG epitope. These results indicate that this structural alteration does not affect the control mechanisms for channel gating and suggest that the fourth extracellular loop of CFTR does not contribute to the ion pore. Detection of M2-901/CFTR by a commercially available monoclonal antibody (M2), together with presentation of normal functional properties, makes M2-901/CFTR a valuable tool to evaluate CFTR protein expression and cellular location.
Schultz
B D
BD
Department of Cell Biology and Physiology, University of Pittsburgh, Pennsylvania 15261, USA.
Takahashi
A
A
Liu
C
C
Frizzell
R A
RA
Howard
M
M
D000818:Animals; D001572:Benzofurans / Q000494:pharmacology; D002460:Cell Line; D002462:Cell Membrane / Q000187:drug effects / Q000502:physiology; D002712:Chlorides / Q000378:metabolism; D000242:Cyclic AMP / Q000031:analogs & derivatives / Q000494:pharmacology; D017868:Cyclic AMP-Dependent Protein Kinases / Q000378:metabolism; D019005:Cystic Fibrosis Transmembrane Conductance Regulator / Q000096:biosynthesis / Q000737:chemistry* / Q000502:physiology*; D004285:Dogs; D004553:Electric Conductivity; D000939:Epitopes; D005260:Female; D005942:Gluconates / Q000494:pharmacology; D005905:Glyburide / Q000494:pharmacology; D006367:HeLa Cells; D006801:Humans; D015640:Ion Channel Gating; D007668:Kidney; D008564:Membrane Potentials / Q000187:drug effects / Q000502:physiology; D009842:Oligopeptides; D009865:Oocytes / Q000187:drug effects / Q000502:physiology; D010455:Peptides; D010671:Phenylurea Compounds / Q000494:pharmacology; D010766:Phosphorylation; D017433:Protein Structure, Secondary; D011994:Recombinant Proteins / Q000096:biosynthesis / Q000737:chemistry / Q000187:drug effects; D013873:Thionucleotides / Q000494:pharmacology; D014162:Transfection; D014982:Xenopus laevis
D001572:Benzofurans; C505032:CFTR protein, human; D002712:Chlorides; D000939:Epitopes; D005942:Gluconates; D009842:Oligopeptides; D010455:Peptides; D010671:Phenylurea Compounds; D011994:Recombinant Proteins; D013873:Thionucleotides; D019005:Cystic Fibrosis Transmembrane Conductance Regulator; C094382:N-(5-(2,3-dihydrobenzofuryl)sulfonyl)-N'-(3,4-dichlorophenyl)urea; C015929:8-((4-chlorophenyl)thio)cyclic-3',5'-AMP; C085706:FLAG peptide; D000242:Cyclic AMP; D017868:Cyclic AMP-Dependent Protein Kinases; C030691:gluconic acid; D005905:Glyburide