Cross-linking of TMs 1 and 6 by the oxidizing agent CuPhe. (A–C) Example leak-subtracted I-V relationships for K95C/I344C (A), Q98C/I344C (B), and Q98C/M348C (C) after channel activation with 20 nM PKA and 1 mM ATP. In A and B, current amplitude is decreased by the subsequent addition of CuPhe to the intracellular solution, whereas in C, CuPhe is without effect. In both CuPhe-sensitive channel constructs, the inhibitory effects of CuPhe were not reversed by washing CuPhe from the bath (top panels in both A and B), but were reversed by the addition of 5 mM DTT to the intracellular solution (bottom panels in both A and B). (D) Mean effect of internal CuPhe on macroscopic current amplitude under these conditions, measured at membrane potentials of −80 mV (white bars) and +80 mV (black bars). Note that cys-less CFTR, the single mutants K95C, Q98C, or I344C, and the double mutant Q98C/M348C were all insensitive to CuPhe under these conditions. Also note that CuPhe had a stronger inhibitory effect on currents carried by K95C/I344C when measured at +80 mV compared with −80 mV; this same apparent voltage dependence was previously reported for K95C/S1141C under similar experimental conditions (Zhou et al., 2010). In contrast, the inhibitory effects of CuPhe on Q98C/I344C were similar when measured at −80 mV or +80 mV. Asterisks indicate a significant difference from control: *, P < 0.005; **, P < 0.00005. (E) Mean effects of CuPhe (black bars), CuPhe followed by washing with normal bath solution (white bars), and CuPhe followed by DTT (gray bars) on macroscopic current amplitude in K95C/I344C (left) and Q98C/I344C (right) at +80 mV. Daggers indicate a significant difference from CuPhe alone (P < 0.005); washing alone (white bars) had no significant effect compared with CuPhe alone (P > 0.6). Mean of data from three to seven patches is shown in D and E.