Figure 3.

Figure 3. Strong coupling between positions 576 and 1371 of hCFTR E1371Q in the bursting state.

(A) Macroscopic current relaxations following ATP removal for indicated human CFTR channel mutants (color coded). Inside-out patch currents were activated by exposure of pre-phosphorylated channels to saturating ATP. Current amplitudes are shown normalized by their steady-state values in ATP (i.e., just before ATP removal). V_m was −20 to −80 mV. (B) Relaxation time constants of the currents in (A), obtained by fits to single exponentials. Data are shown as mean ± standard error of the mean (SEM) from seven to nine experiments using a logarithmic ordinate. (C) Thermodynamic mutant cycle showing mutation-induced changes in the height of the free enthalpy barrier for the B₁ → IB₁ transition (ΔΔG⁰_T^‡−B1, numbers on arrows; k, Boltzmann’s constant; T, absolute temperature). Each corner is represented by the mutations introduced into positions Q1371 and G576 of E1371Q-hCFTR. ΔΔG_int(B₁ → T^‡) (purple number) is obtained as the difference between ΔΔG⁰_T^‡−B1 values along two parallel sides of the cycle. (D) Currents of last open channels after ATP removal for the indicated human CFTR constructs (color coded). Recordings were done as in panel (A), but on patches with smaller numbers of channels. Membrane potential was −80 mV. Black lines indicate the analyzed segments. (E) Intraburst equilibrium constants obtained by dwell-time analysis for the four human CFTR constructs, plotted on a logarithmic scale. Data are shown as mean ± standard error of the mean (SEM) from five to six experiments. (F) Thermodynamic mutant cycle showing mutation-induced changes in the stability of the O state relative to the C_f state (ΔΔG⁰_O-CF, numbers on arrows; k, Boltzmann’s constant; T, absolute temperature). Each corner of the cycle is represented by the mutations introduced into positions Q1371 and G576 of E1371Q-hCFTR. ΔΔG_int(C_f → O) (purple number) is obtained as the difference between ΔΔG⁰_O-CF values along two parallel sides of the cycle.