Novel adenoviral vectors coding for GFP-tagged wtCFTR and ΔF508-CFTR:: characterization of expression and electrophysiological properties in A549 cells

E1/E3-deleted adenoviral vectors expressing an N-terminal green fluorescent protein (GFP) reporter gene fused to either wtCFTR (H5.040CMVEGFP-wtCFTR) or DeltaF508-CFTR (H5.040CMVEGFP-DeltaF508CFTR) were generated. To characterize the expression and activity, A549 cells were infected with vectors expressing GFP-tagged and non-tagged forms of CFTR and DeltaF508CFTR. CFTR activity was assayed in cell-attached and excised patches. For H5.040CMVEGFP-wtCFTR, forskolin-dependent outward current was observed in cell-attached patches from 56 of 67 GFP-positive cells. Single-channel conductances, open probability, mean open and mean closed time values for GFP-CFTR and CFTR were not significantly different. After excision, GFP-CFTR activity required ATP and exhibited a linear I-V relationship. For H5.040CMVEGFP-DeltaF508CFTR, media were supplemented with 5 mM butyrate 16 h after infection. Forskolin-dependent outward current was observed in cell-attached patches from 21 of 30 butyrate-treated GFP-positive cells and 0 of 8 GFP-positive cells without butyrate. Single-channel conductances, open probability, mean open and mean closed time values for GFP-DeltaF508CFTR and DeltaF508CFTR were not significantly different. However, the increase in open probability with genistein was significantly smaller for GFP-DeltaF508CFTR than for DeltaF508CFTR. In excised patches, GFP-DeltaF508CFTR activity required ATP and exhibited a linear I-V relationship. Despite the consistent detection of GFP-CFTR and GFP-DeltaF508CFTR channels in the plasma membrane by patch clamping, GFP fluorescence was observed only in intracellular regions and was not altered by butyrate. The data show that high levels of functional GFP-tagged CFTR channels can be expressed with these adenoviral vector constructs.