Fig. 4.

Role of domain–domain interactions in CFTR channel gating. (A) Inhibition of CFTR channel gating by cross-linking. Single-channel recording after exposure to 10 μM M1M from the cis side of the bilayer followed by 10 mM DTT. For Cys-less CFTR, the last 4 min from the total 20 min of M1M treatment is shown in the first half of the trace before the interruption. The last 4 min of the total 20 min of DTT treatment is shown in the second half. O, open state; c, closed state. (Top) Cys-less CFTR (n = 4). (Middle) Cys-less CFTR with 276C/Q1280C (n = 3). (Bottom) Cys-less CFTR with F508C/F1068C (n = 4). (Middle and Bottom) Change in functional state in the middle of a 4-min portion of the total 20-min recording. No effect of MTS reagents was observed in constructs containing single cysteines that contribute to cross-linkable pairs (SI Fig. 11). (B) NBD1 and CL4 participate early in the gating cycle. Brønsted plots for wild-type CFTR gated by 2 mM nucleotide ligands shown by each experimental point (Left) and substitutions of CL4 residues (Right). Both graphs are linear with the slope Φ values indicated. Points on both graphs are shown as mean values ± SEM of at least four different experiments. (Inset) Hypothetical free energy landscape of CFTR gating where troughs (stable states) are colored blue and crests (unstable states) are red. At the transition state (saddle point), NBD1 has proceeded 98% toward the open state, and CL4 has proceeded 86%.