Effects of mutations at positions 460 and 1375 on nonhydrolytic gating in the K1250R background. (A) Representative normalized decay time courses of macroscopic currents for H1375A/K1250R and T460S/H1375A/K1250R CFTR after the removal of 2 mM ATP. Solid blue and green lines are fitted bi-exponentials. Fitted parameters were τ₁ = 2.8 s, τ₂ = 11 s, A₁ = 0.77, and A₂ = 0.23 for the H1375A/K1250R trace, and τ₁ = 2.8 s, τ₂ = 15 s, A₁ = 0.82, and A₂ = 0.18 for the T460S/H1375A/K1250R trace. Average steady-state burst durations (τ*; inset) were estimated from the two fitted time constants (τ₁ and τ₂) and their fractional amplitudes (A₁ and A₂) as τ* = (A₁+A₂)τ₁τ₂/(A₁τ₂+A₂τ₁). (B) Thermodynamic mutant cycle for target pair T460-H1375 built on average nonhydrolytic closing rates (1/τ*). The top two corners of the mutant cycle were taken from Fig. 5 B. (C) Noise analysis for estimation of P_o for H1375A (blue symbols) and T460S/H1375A (green symbols); each symbol represents one patch. (D; left) Mean ± SEM P_o for H1375A (blue bar) and T460S/H1375A (green bar). (Right) Thermodynamic mutant cycle for target pair T460-H1375 built on K_eq = P_o/(1−P_o) values under nonhydrolytic conditions. The top two corners of the mutant cycle were taken from Fig. 5 D.