Differential thermostability and response to cystic fibrosis transmembrane conductance regulator potentiators of human and mouse F508del-CFTR

Abstract

Cross-species comparative studies have highlighted differences between human and mouse cystic fibrosis transmembrane conductance regulator (CFTR), the epithelial Cl^- channel defective in cystic fibrosis (CF). Here, we compare the impact of the most common CF mutation F508del on the function of human and mouse CFTR heterologously expressed in mammalian cells and their response to CFTR modulators using the iodide efflux and patch-clamp techniques. Once delivered to the plasma membrane, human F508del-CFTR exhibited a severe gating defect characterized by infrequent channel openings and was thermally unstable, deactivating within minutes at 37°C. By contrast, the F508del mutation was without effect on the gating pattern of mouse CFTR, and channel activity demonstrated thermostability at 37°C. Strikingly, at all concentrations tested, the clinically approved CFTR potentiator ivacaftor was without effect on the mouse F508del-CFTR Cl^- channel. Moreover, eight CFTR potentiators, including ivacaftor, failed to generate CFTR-mediated iodide efflux from CHO cells expressing mouse F508del-CFTR. However, they all produced CFTR-mediated iodide efflux with human F508del-CFTR-expressing CHO cells, while fifteen CFTR correctors rescued the plasma membrane expression of both human and mouse F508del-CFTR. Interestingly, the CFTR potentiator genistein enhanced CFTR-mediated iodide efflux from CHO cells expressing either human or mouse F508del-CFTR, whereas it only potentiated human F508del-CFTR Cl^- channels in cell-free membrane patches, suggesting that its action on mouse F508del-CFTR is indirect. Thus, the F508del mutation has distinct effects on human and mouse CFTR Cl^- channels.

Keywords: CFTR chloride ion channel; CFTR potentiation; F508del-CFTR; cystic fibrosis; ivacaftor (VX-770).

Fig. 1.

The single-channel behavior of human and mouse wild-type (WT) and F508del-cystic fibrosis transmembrane conductance regulator (CFTR). A and B: representative recordings of human and mouse wild-type and F508del-CFTR Cl⁻ channels in excised inside-out membrane patches from NIH-3T3 and Chinese hamster ovary (CHO) cells heterologously expressing CFTR variants. Prior to study, the plasma membrane expression of human and mouse F508del-CFTR was rescued by low-temperature incubation. The recordings were acquired at 37°C in the presence of ATP (1 mM) and PKA (75 nM) in the intracellular solution. The closed-channel state (C), the subconductance state of mouse CFTR (O₁), and the full open state [human (O), mouse (O₂)] are indicated by dotted lines. Traces on the left were filtered at 500 Hz, whereas the 1-s portions indicated by the bars shown on an expanded time scale to the right were filtered at 50 Hz. In this and subsequent figures with single-channel data, a large Cl⁻ concentration gradient was imposed across excised membrane patches ([Cl⁻]_int, 147 mM; [Cl⁻]_ext, 10 mM), and membrane voltage was clamped at –50 mV. C and D: summary single-channel current amplitude (i) and open probability (P_o) data for the full open states of human and mouse CFTR determined from prolonged recordings (≥5 min) acquired from baby hamster kidney (BHK) and NIH-3T3 cells heterologously expressing human CFTR and CHO cells heterologously expressing mouse CFTR using the conditions described in A and B before channel deactivation (human F508del-CFTR). Dark gray and light gray circles represent individual values and columns means ± SE (human wild-type, n = 6; human F508del-CFTR, n = 6; mouse wild type, n = 6; mouse F508del-CFTR, n = 18); *P < 0.05 vs. human wild-type CFTR.

Fig. 7.

The temperature dependence of mouse F508del-cystic fibrosis transmembrane conductance regulator (CFTR) treated with nanomolar concentrations of ivacaftor. A: representative recordings of mouse F508del-CFTR Cl⁻ channels in an excised inside-out membrane patch from a Chinese hamster ovary (CHO) cell heterologously expressing CFTR to show the effects of acute addition of ivacaftor (VX-770; 100 nM) to the intracellular solution. The recordings were acquired at the indicated temperatures in the continuous presence of ATP (1 mM) and PKA (75 nM) in the intracellular solution. Prior to study, the plasma membrane expression of mouse F508del-CFTR was rescued by low-temperature incubation. Traces on the left were filtered at 500 Hz, whereas the 2-s portions indicated by the bars shown on an expanded time scale on the right were filtered at 50 Hz. The closed-channel state (C), the subconductance state of mouse CFTR (O₁), and the full open state (O₂) are indicated by dotted lines. B and C: summary data show the change in single-channel current amplitude (i) and open probability (P_o) between 23 and 37°C for the full open state of mouse F508del-CFTR in membrane patches excised from CHO cells heterologously expressing mouse F508del-CFTR. Data are means ± SE (control, n = 10–12; VX-770, n = 5). In B, the continuous lines are the fit of first-order regression functions to mean data, whereas in C, they are the fit of second-order regression functions to mean data. In B and C, control data are the same as the mouse F508del-CFTR data in Fig. 6.

Fig. 2.

Thermostability of mouse F508del-cystic fibrosis transmembrane conductance regulator (CFTR) in excised inside-out membrane patches A: representative recordings of human and mouse F508del-CFTR in excised inside-out membrane patches from baby hamster kidney (BHK) and Chinese hamster ovary (CHO) cells heterologously expressing CFTR variants made in the continuous presence of ATP (1 mM) and PKA (75 nM) once channel activation was complete. Membrane patches were excised, and channels were activated at 27°C to delay temperature-dependent channel deactivation. Only after channels were fully activated was temperature increased to 37°C and thermostability evaluated. Prior to study, the plasma membrane expression of human and mouse F508del-CFTR was rescued by low-temperature incubation. Arrows denote the closed-channel state, and downward deflections correspond to channel openings. B and C: time courses of open probability (P_o) for human and mouse F508del-CFTR using the conditions described in A. P_o values were calculated for each 30-s interval. Data are means ± SE (human F508del-CFTR heterologously expressed in BHK cells, n = 10; mouse F508del-CFTR heterologously expressed in CHO cells, n = 7). The human F508del-CFTR data in A and C were originally published in Meng et al. (51).

Fig. 3.

The effects of ivacaftor on the single-channel behavior of mouse wild-type cystic fibrosis transmembrane conductance regulator (CFTR). A: representative recordings of mouse wild-type CFTR in an excised inside-out membrane patch from a Chinese hamster ovary (CHO) cell heterologously expressing CFTR in the absence and presence of the indicated concentrations of ivacaftor added acutely to the intracellular solution. The recordings were acquired at 37°C in the continuous presence of ATP (1 mM) and PKA (75 nM) in the intracellular solution. Dotted lines indicate the closed-channel state, and downward deflections correspond to channel openings; the subconductance state of mouse CFTR is not readily apparent in these recordings. B: ivacaftor concentration-response relationship for human and mouse wild-type CFTR. Data are means ± SE (human wild-type CFTR heterologously expressed in NIH-3T3 cells, n = 3; mouse wild-type CFTR heterologously expressed in CHO cells, n = 5); *P < 0.05 vs. control.

Fig. 4.

The effects of ivacaftor on the single-channel behavior of mouse F508del-cystic fibrosis transmembrane conductance regulator (CFTR) rescued by low temperature or lumacaftor. A and D: representative recordings of mouse F508del-CFTR in excised inside-out membrane patches from Chinese hamster ovary (CHO) cells heterologously expressing CFTR in the absence and presence of the indicated concentrations of ivacaftor added acutely to the intracellular solution. Prior to study, the plasma membrane expression of mouse F508del-CFTR was rescued by either low-temperature incubation (A) or treatment with lumacaftor (VX-809; 3 μM for 24 h at 37°C; D). The recordings were acquired at 37°C in the continuous presence of ATP (1 mM) and PKA (75 nM) in the intracellular solution. Dotted lines indicate the closed-channel state, and downward deflections correspond to channel openings; the subconductance state of mouse CFTR is not readily apparent in these recordings. B, C, E, and F: summary single-channel current amplitude (i; B and E) and open probability (P_o; C and F) for the full open states of human and mouse F508del-CFTR determined from prolonged recordings (≥5 min) acquired from baby hamster kidney (BHK) and NIH-3T3 cells heterologously expressing human F508del-CFTR and CHO cells heterologously expressing mouse F508del-CFTR using the conditions described in A and D before channel deactivation (human F508del-CFTR). Dark gray and light gray circles represent individual values and columns means ± SE (B and C: human F508del-CFTR, n = 4–6; mouse F508del-CFTR, n = 8–10; E and F: human F508del-CFTR, n = 3–4; mouse F508del-CFTR, n = 5); *P < 0.05 vs. control.

Fig. 5.

The temperature dependence of mouse F508del-cystic fibrosis transmembrane conductance regulator (CFTR) single-channel activity. A: representative recordings of mouse F508del-CFTR Cl⁻ channels in an excised inside-out membrane patch from a Chinese hamster ovary (CHO) cell heterologously expressing CFTR. Prior to study, the plasma membrane expression of mouse F508del-CFTR was rescued by low-temperature incubation. The recordings were acquired at the indicated temperatures in the presence of ATP (1 mM) and PKA (75 nM) in the intracellular solution. Traces on the left were filtered at 500 Hz, whereas the 2-s portions indicated by the bars shown on an expanded time scale to the right were filtered at 50 Hz. The closed-channel state (C), the subconductance state of mouse CFTR (O₁), and the full open state (O₂) are indicated by dotted lines. B: single-channel current amplitude histograms for the 10-s traces shown in A, filtered at 50 Hz, which removes transient events. Arrows denote C, O₁, and O₂.

Fig. 6.

Analysis of the temperature dependence of human and mouse wild-type and F508del-cystic fibrosis transmembrane conductance regulator (CFTR) Cl⁻ channels. A and B: summary data show the change in single-channel current amplitude (i) and open probability (P_o) between 23 and 37°C for the full open states of human and mouse wild-type and F508del-CFTR. Data are from inside-out membrane patches excised from baby hamster kidney (BHK) cells heterologously expressing human CFTR and Chinese hamster ovary (CHO) cells heterologously expressing mouse CFTR. Prior to study, human and mouse F508del-CFTR were rescued by low-temperature incubation. Data are means ± SE (human wild-type CFTR, n = 6–8; human F508del-CFTR, n = 4–8; mouse wild-type CFTR, n = 4; mouse F508del-CFTR, n = 10–12). In A, the continuous lines are the fit of first-order regression functions to mean data, whereas in B, they are the fit of second-order regression functions to mean data. Note the break in the ordinate scale in B. The human wild-type and F508del-CFTR data were originally published in Wang et al. (90).

Fig. 8.

Genistein and VRT-532 potentiate iodide efflux by human F508del-cystic fibrosis transmembrane conductance regulator (CFTR) rescued by low temperature or the CFTR corrector suberoylanilide hydroxamic acid (SAHA). A and B: time courses of iodide efflux from Chinese hamster ovary (CHO) cells heterologously expressing human F508del-CFTR. In A, CHO cells were pretreated with DMSO (0.1% vol/vol) or SAHA (3 μM) for 26 h at 37°C before study. In B, CHO cells were cultured at 26°C for 26 h before study. During the periods indicated by the black bars, forskolin (10 μM) and CFTR potentiators [A: genistein (50 μM); B: DMSO (0.1% vol/vol), genistein (50 μM), or VRT-532 (10 μM)] were added to the extracellular solution. Data are means ± SE (n = 3).

Fig. 9.

Genistein, but not VRT-532, potentiates iodide efflux by mouse F508del-cystic fibrosis transmembrane conductance regulator (CFTR) rescued by low temperature or the CFTR corrector suberoylanilide hydroxamic acid (SAHA). A and B: time courses of iodide efflux from Chinese hamster ovary (CHO) cells heterologously expressing mouse F508del-CFTR. In A, CHO cells were pretreated with DMSO (0.1% vol/vol) or SAHA (3 μM) for 26 h at 37°C before study. In B, CHO cells were cultured at 26°C for 26 h before study. During the periods indicated by the black bars, forskolin (10 μM) and CFTR potentiators [A: genistein (50 μM); B: DMSO (0.1% vol/vol), genistein (50 μM), or VRT-532 (1 or 10 μM)] were added to the extracellular solution. Data are means ± SE (n = 3).

Fig. 10.

Comparison of the change in human and mouse F508del-cystic fibrosis transmembrane conductance regulator (CFTR)-mediated iodide efflux elicited by panels of CFTR correctors and potentiators. A and B: magnitude of CFTR-mediated iodide efflux in Chinese hamster ovary (CHO) cells heterologously expressing human and mouse F508del-CFTR. CFTR-mediated iodide efflux was quantified as magnitude of the initial increase in fractional ¹²⁵I⁻ efflux at 90 s, expressed as %/min. In A, CHO cells were pretreated with DMSO (0.1% vol/vol) or the indicated CFTR correctors for 26 h at 37°C before study, and CFTR-mediated iodide efflux was activated by forskolin (10 μM) and potentiated by genistein (50 μM). In B, CHO cells were cultured at 26°C for 26 h before study and CFTR-mediated iodide efflux was activated by forskolin (10 μM) and potentiated by the indicated CFTR potentiators. The concentration of test CFTR correctors and potentiators used was optimized with concentration-response relationships over a 100-fold concentration range chosen around the most effective concentrations reported in the literature for correction and potentiation of human F508del-CFTR. Data represent mean values of two identical experiments, each performed in triplicate and expressed as fold increase in CFTR activity relative to DMSO (0.1% vol/vol) alone. For further details, see the text.

Fig. 11.

Differential responses of human and mouse F508del-cystic fibrosis transmembrane conductance regulator (CFTR)-mediated iodide efflux to CFTR potentiators, but not correctors. A and B: relationship between CFTR-mediated iodide efflux for human and mouse F508del-CFTR heterologously expressed in Chinese hamster ovary (CHO) cells and rescued by either CFTR correctors or CFTR potentiators. Data are the fold increase in CFTR activity relative to DMSO (0.1% vol/vol) alone from Fig. 10. In A, the continuous line is the fit of a first-order regression function to the data (r² = 0.88). For further details, see the text.

Fig. 12.

Genistein potentiates the single-channel activity of human, but not mouse, F508del-cystic fibrosis transmembrane conductance regulator (CFTR) in excised inside-out membrane patches. A and B: representative recordings of human and mouse F508del-CFTR in excised inside-out membrane patches from baby hamster kidney (BHK) and Chinese hamster ovary (CHO) cells heterologously expressing CFTR in the absence and presence of genistein (20 μM) added acutely to the intracellular solution. Prior to study, the plasma membrane expression of F508del-CFTR was rescued by low-temperature incubation. The recordings were acquired at the indicated temperatures in the continuous presence of ATP (1 mM) and PKA (75 nM) in the intracellular solution. Dotted lines indicate the closed-channel state, and downward deflections correspond to channel openings; the subconductance state of mouse CFTR is not readily apparent in these recordings. C and D: summary single-channel current amplitude (i) and open probability (P_o) for the full open states of human and mouse CFTR determined from prolonged recordings (≥5 min) acquired from BHK cells heterologously expressing human F508del-CFTR and CHO cells heterologously expressing mouse F508del-CFTR using the conditions described in A and B before channel deactivation (human F508del-CFTR). Dark gray and light gray circles represent individual values and columns means ± SE (human F508del-CFTR, n = 7–8; mouse F508del-CFTR, n = 6–7); *P < 0.05 vs. human F508del-CFTR control.