Figure 3. Cross-linking of cysteines... | Rockefeller University Press

Figure 3.

$Figure 3. Cross-linking of cysteines substituted for K95 and S1141. (A) Example leak-subtracted macroscopic I-V relationships for the different CFTR variants named, using symmetrical high (154-mM) Cl− concentrations, after maximal channel activation with 5–10 nM PKA, 1 mM ATP, and 2 mM PPi. Currents were recorded before (control) and after the addition of CuPhe to the intracellular (bath) solution. (B) Mean fractional current remaining after the addition of CuPhe as a function of voltage in wild type (•), K95C (□), S1141C (○), and K95C/S1141C (■). Data values for K95C/S1141C were significantly different from wild type, K95C, or S1141C (P < 0.05 in each case) at all voltages examined. (C) Mean fractional current remaining after the addition of CuPhe at +80 mV for different channel variants as indicated, and for K95C/S1141C after washing with normal bath solution (wash) or with bath solution supplemented with 5 mM DTT (wash + DTT). Mean of data from three to nine patches in B and C. (D) Example leak-subtracted macroscopic I-V relationships for cys-less K95C/S1141C-CFTR recorded under the same conditions as in A. Both panels are from the same inside-out membrane patch before (control) and after the addition of 400 µM CuPhe to the intracellular (bath) solution (left), and after the removal of CuPhe and reapplication of ATP, PKA, and PPi (wash) and subsequent application of 5 mM DTT (right, +DTT). Representative example of five patches.$

Cross-linking of cysteines substituted for K95 and S1141. (A) Example leak-subtracted macroscopic I-V relationships for the different CFTR variants named, using symmetrical high (154-mM) Cl⁻ concentrations, after maximal channel activation with 5–10 nM PKA, 1 mM ATP, and 2 mM PPi. Currents were recorded before (control) and after the addition of CuPhe to the intracellular (bath) solution. (B) Mean fractional current remaining after the addition of CuPhe as a function of voltage in wild type (•), K95C (□), S1141C (○), and K95C/S1141C (■). Data values for K95C/S1141C were significantly different from wild type, K95C, or S1141C (P < 0.05 in each case) at all voltages examined. (C) Mean fractional current remaining after the addition of CuPhe at +80 mV for different channel variants as indicated, and for K95C/S1141C after washing with normal bath solution (wash) or with bath solution supplemented with 5 mM DTT (wash + DTT). Mean of data from three to nine patches in B and C. (D) Example leak-subtracted macroscopic I-V relationships for cys-less K95C/S1141C-CFTR recorded under the same conditions as in A. Both panels are from the same inside-out membrane patch before (control) and after the addition of 400 µM CuPhe to the intracellular (bath) solution (left), and after the removal of CuPhe and reapplication of ATP, PKA, and PPi (wash) and subsequent application of 5 mM DTT (right, +DTT). Representative example of five patches.

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