Apparent association and dissociation rates of VX-770 in site II_N mutants. (A and B) Macroscopic current traces of Y304A-CFTR (A) and F312A-CFTR (B) in response to application and removal of 200 nM VX-770. The time course of current rise (blue curve, τ_on) and current decline (red curve, τ_off) were fitted with a single-exponential function. The experiment with F312A-CFTR was performed at 30 µM ATP to adjust its P_o to the level comparable to WT-CFTR (see Fig. 7 for details). CFTR_Inh-172 (Inh) was applied to attain the baseline. (C) Summary of the time constants for the apparent association and dissociation of VX-770. While only Y304A slows down the current rise in response to VX-770, the relaxation time constants for the dissociation of VX-770 are decreased by F236A, Y304A, and F312A mutations. τ_on: 6.7 ± 1.2 s (WT); 6.2 ± 1.3 s (F229A); 19 ± 8 s (F236A); 49 ± 11 s (Y304A); 15 ± 1 s (F312A). τ_off: 361 ± 39 s (WT); 338 ± 49 s (F229A); 173 ± 45 s (F236A); 18 ± 6 s (Y304A); 38 ± 5 s (F312A). n = 3 for WT, F229A, and F312A. n = 4 for F236A and Y304A. *, P < 0.05 (ANOVA followed by Dunnett test). Error bars represent SEM.