Figure 11.
Figure 11. D110C forms a spontaneous disulfide bond with K892C when channels are in the closed state. (A) Representative macropatch currents of WT- and D110C/K892C-CFTR were recorded in inside-out mode with symmetrical 150 mM Cl− solution. Pipette was backfilled with 1 mM DTT solution. A voltage protocol that held at 0 mV then stepped to 100 mV for 30 ms followed by a ramp to −100 mV over 300-ms duration was applied every 5 s. a, control, before ATP + PKA; b, peak current (Imax), with cytoplasmic ATP + PKA; c, current after DTT perfused fully to the tip of pipette, with cytoplasmic ATP + PKA; d, current with DTT perfused to the tip of pipette, wash out of cytoplasmic ATP + PKA. I-V plots of currents at times a–c for both WT- and D110C/K892C-CFTR are shown in the middle panel. Summary data are shown in the right panel. Imax, maximum current activated by ATP and PKA, likely before DTT perfused fully to the tip of pipette; I, current activated by ATP and PKA with DTT perfused to the tip of pipette. **, P < 0.01 indicates a significant difference in the pre- and post-DTT conditions; #, P < 0.001 indicates a significant difference in the pre- and post-DTT conditions. (B) 1 mM DTT further activated D110C/K892C-CFTR current but not WT-CFTR current in TEVC recording condition. Representative traces (left) and summary data (right) for macroscopic currents measured from WT- and D110C/K892C-CFTR with addition of 1 mM DTT in the presence of ISO. ND96, control bath solution. VM = −60 mV. ISO1, 10 µM ISO alone; ISO2 + DTT, 1 mM DTT in 10 µM ISO solution. Current levels in the summary data are given relative to control conditions before first exposure to ISO and normalized to maximal current in response to ISO1 (Imax). **, P < 0.01 compared with ISO1 in n = 4 for WT-CFTR and n = 5 for D110C/K892C-CFTR experiments. (C) ISO plus DTT failed to further activate D110C/K892C-CFTR current in oocytes pretreated with DTT in TEVC recording condition. Representative trace (left) and summary data (right) for macroscopic currents measured from D110C/K892C-CFTR with prior addition of 1 mM DTT for 3 min in ND96 solution. Current levels in the summary data are given relative to control conditions before first exposure to ISO and normalized to maximal current in response to ISO1 (Imax). #, P < 0.001 compared with ISO1 in n = 4 experiments. Mean ± SEM is shown.

D110C forms a spontaneous disulfide bond with K892C when channels are in the closed state. (A) Representative macropatch currents of WT- and D110C/K892C-CFTR were recorded in inside-out mode with symmetrical 150 mM Cl− solution. Pipette was backfilled with 1 mM DTT solution. A voltage protocol that held at 0 mV then stepped to 100 mV for 30 ms followed by a ramp to −100 mV over 300-ms duration was applied every 5 s. a, control, before ATP + PKA; b, peak current (Imax), with cytoplasmic ATP + PKA; c, current after DTT perfused fully to the tip of pipette, with cytoplasmic ATP + PKA; d, current with DTT perfused to the tip of pipette, wash out of cytoplasmic ATP + PKA. I-V plots of currents at times a–c for both WT- and D110C/K892C-CFTR are shown in the middle panel. Summary data are shown in the right panel. Imax, maximum current activated by ATP and PKA, likely before DTT perfused fully to the tip of pipette; I, current activated by ATP and PKA with DTT perfused to the tip of pipette. **, P < 0.01 indicates a significant difference in the pre- and post-DTT conditions; #, P < 0.001 indicates a significant difference in the pre- and post-DTT conditions. (B) 1 mM DTT further activated D110C/K892C-CFTR current but not WT-CFTR current in TEVC recording condition. Representative traces (left) and summary data (right) for macroscopic currents measured from WT- and D110C/K892C-CFTR with addition of 1 mM DTT in the presence of ISO. ND96, control bath solution. VM = −60 mV. ISO1, 10 µM ISO alone; ISO2 + DTT, 1 mM DTT in 10 µM ISO solution. Current levels in the summary data are given relative to control conditions before first exposure to ISO and normalized to maximal current in response to ISO1 (Imax). **, P < 0.01 compared with ISO1 in n = 4 for WT-CFTR and n = 5 for D110C/K892C-CFTR experiments. (C) ISO plus DTT failed to further activate D110C/K892C-CFTR current in oocytes pretreated with DTT in TEVC recording condition. Representative trace (left) and summary data (right) for macroscopic currents measured from D110C/K892C-CFTR with prior addition of 1 mM DTT for 3 min in ND96 solution. Current levels in the summary data are given relative to control conditions before first exposure to ISO and normalized to maximal current in response to ISO1 (Imax). #, P < 0.001 compared with ISO1 in n = 4 experiments. Mean ± SEM is shown.

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