Szollosi A , Muallem DR , Csanády L , Vergani P .

Medical Research Council , Wellcome Trust , G0501200 Medical Research Council , MRC_G0501200 Medical Research Council

	Figure 1. Spatial arrangement of target residues on opposite sides of CFTR’s composite site 1 in homology models. In a homology model of CFTR’s NBD dimer (Mornon et al., 2008), T460 (red) in the Walker A motif of NBD1 is close to NBD2 residues H1348 (gold; in the signature sequence), L1353 (blue; at the end of the signature sequence), and H1375 (magenta; in the D-loop).
	Figure 2. Effect of mutations at positions 460 and 1353 on channel closing rates. (A) Representative traces of multichannel recordings of prephosphorylated WT and mutant CFTR channels, used to determine burst duration. Downward deflection indicates inward current. (B) Closing rates of WT and mutant CFTR channels, defined as the inverse of the mean burst duration (see Materials and methods). (C) Thermodynamic mutant cycle for target pair T460-L1353 built on the closing rates from B; each corner is represented by the side chains at positions 460 and 1353, respectively. ΔΔG0 values (mean ± SEM) on arrows show mutation-induced changes in the stability of the transition state for closure with respect to the open ground state and were used to calculate (see Materials and methods) the coupling energy for the 460–1353 interaction (ΔΔG‡int (closing)).
	Figure 3. Effect of mutations at positions 460 and 1353 on channel opening rates. (A) The same records used for determining burst duration (see Fig. 2) were used for noise analysis. Each data point represents one recording. (B) Mean ± SEM of Po values estimated for each patch from the corresponding data point in A (see Materials and methods). (C) Mean ± SEM of opening rates calculated for each patch using the estimate for Po (see B) and the closing rate (see Fig. 2 B). (D) Thermodynamic mutant cycle for target pair T460-L1353 built on opening rates (from C); notation as in Fig. 2 C.
	Figure 4. Mutations at positions 460 and 1353 do not affect apparent affinity for ATP. (A) Representative traces showing macroscopic current response for WT and T460S to a test [ATP] of 50 µM, bracketed with applications of 2 mM ATP. Different [ATP] were tested several times within one recording. (B) [ATP] dose–response relationships for WT and mutant CFTR channels; currents in test [ATP] were normalized to the average of the currents observed in bracketing segments in the presence of 2 mM ATP. Solid lines show fits of the Michaelis-Menten equation; KPo values are plotted in the panel. Between 5 and 14 measurements were made for each concentration tested. (C) Estimates of KrCO for each construct, calculated (see Results) using KPo from B and Po;max from Fig. 3 B. (D) Thermodynamic mutant cycle for target pair T460-L1353 built on KrCO values.
	Figure 5. The T460S mutation destabilizes the open state of CFTR in the nonhydrolytic K1250R background. (A) Representative normalized decay time courses of WT and mutant CFTR macroscopic currents after the removal of 2 mM ATP (gray). Solid colored lines are fitted exponentials; mean ± SEM relaxation time constants (τrelaxation) are shown in the inset. (B) Thermodynamic mutant cycle for target pair T460-L1353 built on nonhydrolytic closing rates (1/τrelaxation). (C) Noise analysis was performed on 2–3-min records from patches containing <100 channels. Each point represents one patch. Po was calculated for each patch; mean ± SEM Po values are shown in the inset. (D) Thermodynamic mutant cycle for target pair T460-L1353 built on Keq = Po/(1−Po) values under nonhydrolytic conditions.
	Figure 6. Effects of mutations at positions 460 and 1348 on normal hydrolytic channel gating. (A) Representative single-channel current traces from prephosphorylated H1348A and T460S/H1348A CFTR channels gating in 2 mM ATP. Downward deflection indicates inward current. (B; left) Closing rates of H1348A (blue bar) and T460S/H1348A (green bar), defined as the inverse of the mean burst duration (see Materials and methods). (Right) Thermodynamic mutant cycle for target pair T460-H1348 built on closing rates. The top two corners of the mutant cycle (representing WT and T460S) were taken from Fig. 2 C. Because the bottom two corners (representing H1348A and T460S/H1348A) were evaluated in separate sets of experiments, the absolute ΔΔG values are not printed for the vertical sides of the cycle. (C) Noise analysis was used to estimate Po for H1348A (blue bar) and T460S/H1348A (green bar). (D; left) Opening rates of H1348A (blue bar) and T460S/H1348A (green bar), obtained using the estimate for Po (see C) and the closing rate (see B). (Right) Thermodynamic mutant cycle for target pair T460-H1348 built on opening rates. The top two corners of the mutant cycle were taken from Fig. 3 D.
	Figure 7. The H1348A mutation stabilizes the open state of CFTR in the nonhydrolytic K1250R background. (A) Representative normalized decay time courses of macroscopic currents for H1348A/K1250R and T460S/H1348A/K1250R CFTR after the removal of 2 mM ATP (gray). Solid blue and green lines are fitted exponentials; mean ± SEM relaxation time constants (τrelaxation) are shown in the inset. (B) Thermodynamic mutant cycle for target pair T460-H1348 built on nonhydrolytic closing rates (1/τrelaxation). The top two corners of the mutant cycle were taken from Fig. 5 B. (C) Noise analysis for estimation of Po for H1348A (blue symbols) and T460S/H1348A (green symbols); each symbol represents one patch. (D; left) Mean ± SEM Po for H1348A (blue bar) and T460S/H1348A (green bar). (Right) Thermodynamic mutant cycle for target pair T460-H1348 built on Keq = Po/(1−Po) values under nonhydrolytic conditions. The top two corners of the mutant cycle were taken from Fig. 5 D.
	Figure 8. Effects of mutations at positions 460 and 1375 on normal hydrolytic channel gating. (A) Representative single-channel current traces from prephosphorylated H1375A and T460S/H1375A CFTR channels gating in 2 mM ATP. Downward deflection indicates inward current. (B; left) Closing rates of H1375A (blue bar) and T460S/H1375A (green bar), defined as the inverse of the mean burst duration (see Materials and methods). (Right) Thermodynamic mutant cycle for target pair T460-H1375 built on closing rates. The top two corners of the mutant cycle (representing WT and T460S) were taken from Fig. 2 C. (C) Noise analysis was used to estimate Po for H1375A (blue bar) and T460S/H1375A (green bar). (D; left) Opening rates of H1375A (blue bar) and T460S/H1348A (green bar), obtained using the estimate for Po (see C) and closing rate (see B). (Right) Thermodynamic mutant cycle for target pair T460-H1375 built on opening rates. The top two corners of the mutant cycle were taken from Fig. 3 D.
	Figure 9. 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 Po for H1375A (blue symbols) and T460S/H1375A (green symbols); each symbol represents one patch. (D; left) Mean ± SEM Po for H1375A (blue bar) and T460S/H1375A (green bar). (Right) Thermodynamic mutant cycle for target pair T460-H1375 built on Keq = Po/(1−Po) values under nonhydrolytic conditions. The top two corners of the mutant cycle were taken from Fig. 5 D.

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