Fig. 4.
Dual potentiator treatment increases the single-channel function of G551D-CFTR without substantially altering the phospho-occupancy. (a, b) The effect of 3μM VX-770 (n = 19) or VX-770 + 50μM apigenin (n = 18) on the function of protein kinase A–activated G551D-CFTR reconstituted into an artificial phospholipid bilayer. The Po of protein kinase A–activated WT-(n = 13) and G551D-CFTR were analyzed at 28–30°C. Significance between the VX-770 and VX-770+apigenin treated channel Po was calculated by two-sided Mann-Whitney U test (**P < 0.01, b). Representative traces (c - closed, o - open state) are shown in a. (c, d) In vivo phospho-occupancy of the PKA consensus sites in G551D-CFTR expressed in CFBE cells was determined by EThcD-MS/MS. Relative phosphorylation (%) of PKA consensus sites in G551D-CFTR upon DMSO or forskolin (20μM, 5 min) treatment (c), forskolin alone and VX-770 (3μM, 5 min) or VX-770 + genistein (Gen, 50μM, 5 min) in the presence of forskolin (d). Data are means ± SEM of at least three independent experiments. * P < 0.05, ** P < 0.01, *** P < 0.001 by unpaired, two-tailed Student’s t-test.