FLAG EPITOPE POSITIONED IN AN EXTERNAL LOOP PRESERVES NORMAL BIOPHYSICAL PROPERTIES OF CFTR

We asked whether inclusion of the FLAG epitope in the fourth extracell ular loop of the cystic fibrosis transmembrane conductance regulator ( M2-901/CFTR), which permits-detection of cell surface expression, affe cted CFTR's biophysical properties or channel regulation by kinases, p hosphatases, and nucleotides. Channel activity of M2-901/CFTR was eval uated in numerous cell types and expression systems to characterize it s gating and regulation. Our results show that M2-901/CFTR required ad enosine 3',5'-cyclic monophosphate-dependent protein kinase phosphoryl ation to initiate channel activity. Subsequently, ATP alone was suffic ient to support channel gating, and ADP inhibited channel opening. Cur rent fluctuation analysis indicated that the nucleotide-dependent gati ng rates were indistinguishable from those of wild-type (wt) cystic fi brosis transmembrane conductance regulator (CFTR). Channel conductance in symmetric Cl- (11.2 pS), anion permeability ratio (1.66), and bloc k by gluconate indicate that the anion conduction pathway is indisting uishable from wtCFTR. Sulfonylureas (glibenclamide and LY-295501) inhi bited M2-901/CFTR channel activity by an identical mechanism to that d escribed for wtCFTR. Finally, CFTR-dependent insertion and retrieval o f cell membrane was unaffected by the presence of the FLAG epitope. Th ese results indicate that this structural alteration does not affect t he control mechanisms for channel gating and suggest that the fourth e xtracellular loop of CFTR does not contribute to the ion pore. Detecti on 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 cell ular location.