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Proceedings of the National Academy of Sciences of the United States of America logoLink to Proceedings of the National Academy of Sciences of the United States of America
. 2026 Jan 23;123(5):e2523950123. doi: 10.1073/pnas.2523950123

Chronic and acute modulator treatment restore wild-type-like activity and stability to the primary cystic fibrosis–causing CFTR variant

Mayuree Rodrat a,b, James N Charlick a, David N Sheppard a,1
PMCID: PMC12867690  PMID: 41576103

Significance

The genetic disease cystic fibrosis (CF) is caused by dysfunction of the anion channel cystic fibrosis transmembrane conductance regulator (CFTR). Treatment of the most common cause of CF, F508del-CFTR has been revolutionized by the combination therapy elexacaftor-tezacaftor-ivacaftor, which corrects defective channel assembly, allowing transport to the plasma membrane where channel activity is enhanced. By studying the behavior of individual F508del-CFTR channels, we investigated the molecular mechanism of action of elexacaftor-tezacaftor-ivacaftor. Chronic treatment of cells with elexacaftor-tezacaftor-ivacaftor stabilized the channel, preventing irreversible loss of activity and together with acute treatment with elexacaftor and ivacaftor restored to F508del-CFTR wild-type-like levels of channel activity. Thus, chronic and acute elexacaftor-tezacaftor-ivacaftor treatment suppresses the molecular defects of F508del-CFTR, providing an explanation for its clinical effectiveness.

Keywords: CFTR chloride ion channel, cystic fibrosis, F508del-CFTR, elexacaftor-tezacaftor-ivacaftor

Abstract

Elexacaftor-tezacaftor-ivacaftor has transformed the treatment of people with cystic fibrosis (CF) and the predominant cystic fibrosis transmembrane conductance regulator (CFTR) variant F508del by delivering the faulty channel to the plasma membrane and potentiating its activity. However, the action of elexacaftor-tezacaftor-ivacaftor on the molecular behavior of F508del-CFTR is unknown. Here, we investigated the impact of elexacaftor-tezacaftor-ivacaftor on individual human F508del-CFTR Cl− channels using the patch-clamp technique and cells heterologously expressing F508del-CFTR. Chronic treatment of F508del-CFTR-expressing cells with elexacaftor-tezacaftor-ivacaftor, but not pairs of CFTR modulators, stabilized F508del-CFTR, preventing channel deactivation in cell-free membrane patches and conferring upon it the current amplitude of wild-type CFTR with channel activity sustained at a level one-third that of wild-type CFTR. By incubating cells with different drug concentrations, we demonstrated that elexacaftor stabilizes the open-channel conformation of elexacaftor-tezacaftor-ivacaftor-rescued F508del-CFTR, while acute treatment of wild-type CFTR with elexacaftor increased current flow through the channel and potentiated channel gating. Of note, acute treatment of elexacaftor-tezacaftor-ivacaftor-rescued F508del-CFTR Cl− channels with elexacaftor and ivacaftor restored wild-type levels of channel activity to the CFTR variant, albeit its gating pattern was distinct from that of wild-type CFTR and characterized by infrequent prolonged bursts of channel openings. In conclusion, chronic treatment with elexacaftor-tezacaftor-ivacaftor plus acute treatment with elexacaftor and ivacaftor robustly corrects the processing, stability, and gating defects of F508del-CFTR. Our results reveal that elexacaftor is a triple-acting CFTR modulator with corrector, potentiator, and conduction activities, making it a valuable therapeutic to rescue CFTR variants with distinct molecular mechanisms of CFTR dysfunction.

With the approval of elexacaftor (ELX)-tezacaftor (TEZ)-ivacaftor (IVA) (Trikafta/Kaftrio; VX-445–VX-661–VX-770; Vertex Pharmaceuticals) (1, 2), people with cystic fibrosis (CF) and the predominant cystic fibrosis transmembrane conductance regulator (CFTR) variant F508del (p.Phe508del; c.1521_1523delCTT) have an orally bioavailable, highly effective therapy that targets the root cause of disease in all affected organs. F508del causes a temperature-sensitive processing defect, which prevents the correct assembly of CFTR, obstructing its delivery to the plasma membrane (3–7). What little F508del-CFTR protein that reaches the plasma membrane is further incapacitated by defective protein stability and channel regulation (8, 9). In consequence, F508del causes a profound loss of CFTR function as an ATP-gated anion channel with wide-ranging deleterious impacts on epithelial ion transport throughout the body and host defense mechanisms in the respiratory airways (10, 11).

Two types of CFTR modulators are required to restore function to F508del-CFTR: correctors to traffic the CFTR variant to the plasma membrane and potentiators to augment its function (12). Elexacaftor-tezacaftor-ivacaftor consists of two correctors, elexacaftor and tezacaftor and a potentiator, ivacaftor (13–15). Elexacaftor and tezacaftor interact directly with F508del-CFTR at distinct sites to synergistically improve protein folding, domain assembly, and transport to the plasma membrane (7, 15–18). Once CFTR is phosphorylated by protein kinase A (PKA), ivacaftor increases the frequency and duration of channel openings by modifying channel gating (19, 20). Moreover, elexacaftor also enhances CFTR function (7, 21–23), demonstrating that it is a dual-acting molecule with both corrector and potentiator activities. However, understanding of the mechanism of action of elexacaftor-tezacaftor-ivacaftor remains incomplete because the impact of the combination therapy on the molecular behavior of F508del-CFTR is unknown.

Here, we investigated the impact of elexacaftor-tezacaftor-ivacaftor on the function of individual F508del-CFTR Cl− channels. To address this aim, we studied mammalian cells heterologously expressing CFTR with high-resolution single-channel recording. Chronic exposure of F508del-CFTR-expressing cells to elexacaftor-tezacaftor-ivacaftor stabilized the open-channel conformation of CFTR in cell-free membrane patches and, when F508del-CFTR was continuously exposed to elexacaftor and ivacaftor, restored wild-type-like levels of channel activity. Thus, chronic and acute treatment with CFTR modulators is required to rescue the plasma membrane expression, stability, and function of F508del-CFTR.

Results

Elexacaftor-Tezacaftor-Ivacaftor Stabilizes the F508del-CFTR Cl− Channel.

To investigate the action of elexacaftor-tezacaftor-ivacaftor at the molecular level, we studied CFTR Cl− channels in excised inside-out membrane patches from F508del-CFTR-expressing baby hamster kidney (BHK) cells, chronically treating cells with CFTR modulators [elexacaftor (2 μM), tezacaftor (3 μM), and ivacaftor (1 μM)] to resemble drug exposure in vivo. As controls, we studied wild-type CFTR and F508del-CFTR rescued either by low temperature incubation or pairs of CFTR modulators. We did not study the action of individual CFTR modulators because, by themselves, they lack clinical benefit for people with CF and the F508del-CFTR variant (24, 25). To analyze the single-channel behavior of CFTR, we acquired prolonged uninterrupted recordings at 37 °C in the continuous presence of ATP (1 mM) and protein kinase A (PKA; 75 nM), but absence of CFTR modulators, clamping membrane voltage at –50 mV and imposing a large Cl− concentration gradient [(Cl−)in, 147 mM; (Cl−)out, 10 mM] across membrane patches to magnify the size of channel openings. Under these conditions, wild-type CFTR demonstrates stability, whereas F508del-CFTR rescued by either low temperature or lumacaftor deactivates promptly (e.g., 26).

Fig. 1 demonstrates that the single-channel activity of F508del-CFTR rescued by either low temperature or pairs of CFTR modulators was unstable at 37 °C in cell-free membrane patches, whereas that of F508del-CFTR rescued by elexacaftor-tezacaftor-ivacaftor was sustained over 60-min recordings at 37 °C. Similarly, SI Appendix, Fig. S1 shows that only elexacaftor-tezacaftor-ivacaftor stabilized populations of F508del-CFTR Cl− channels in the apical membrane of Fischer rat thyroid (FRT) epithelia heterologously expressing the CFTR variant. To quantify single-channel behavior in cell-free membrane patches, we measured single-channel current amplitude (i) and open probability (Po) at the start (0 to 5 min), middle (25 to 30 min), and end (55 to 60 min) of recordings. Initially, i was equal to wild-type for F508del-CFTR rescued by either low temperature or pairs of CFTR modulators, but Po was greatly reduced except for channels rescued by tezacaftor-ivacaftor, where Po was equivalent to wild-type (Fig. 1). Consistent with previous results (26), at later time points, both the i and Po of F508del-CFTR rescued by either low temperature or pairs of CFTR modulators progressively declined to zero, except channels rescued by tezacaftor-ivacaftor, where channel deactivation was precipitous (Fig. 1). By contrast, the i and Po of elexacaftor-tezacaftor-ivacaftor-rescued F508del-CFTR remained unchanged over 60 min with i equivalent to that of wild-type CFTR, but Po sustained at a level around one-third that of wild-type CFTR (Fig. 1). Thus, chronic treatment with elexacaftor-tezacaftor-ivacaftor restores stability, but not wild-type-like channel activity to F508del-CFTR.

Fig. 1.

Recordings and graphs of C F T R C l super sub-channels with drug treatments over time. Single-channel current amplitude and open probability data.

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Chronic treatment with elexacaftor-tezacaftor-ivacaftor prolongs F508del-CFTR activity in cell-free membrane patches. (A) Representative prolonged recordings of CFTR Cl− channels in excised inside-out membrane patches from F508del-CFTR-expressing BHK cells incubated at 27 °C for 24 h or treated with the indicated combinations of elexacaftor (ELX; 2 μM), tezacaftor (TEZ; 3 μM), and ivacaftor (IVA; 1 μM) for 24 h at 37 °C. The recordings were made at 37 °C with ATP (1 mM) and PKA (75 nM) continuously present in the intracellular solution. Dotted lines indicate the closed channel state, arrowheads identify different open-channel current levels and downward deflections correspond to channel openings. In this and subsequent figures, membrane voltage was clamped at –50 mV and a large Cl− concentration gradient imposed across the membrane patch [(Cl−)int, 147 mM; (Cl−)ext, 10 mM]. (B and C) Summary single-channel current amplitude (i) and open probability (Po) data of F508del-CFTR Cl− channels for the experimental conditions described in A determined at the Start (0 to 5 min), Middle (25 to 30 min), and End (55 to 60 min) of 60-min time courses compared to wild-type CFTR analyzed over the first 9 to 15 min of recordings. Symbols represent individual values and columns are means ± SD (wild-type, n = 6; low temperature, n = 3; ELX/TEZ, n = 7; TEZ/IVA, n = 7; ELX/IVA, n = 6; ELX/TEZ/IVA, n = 7); *P < 0.05, **P < 0.01 and ***P < 0.001 vs. wild-type CFTR; one-way ANOVA with Dunn’s multiple comparison test.

Pairs of CFTR Modulators Have Distinct Effects On F508del-CFTR.

To assess the action of CFTR modulators on the molecular behavior of F508del-CFTR, we studied individual channels in excised inside-out membrane patches from cells chronically treated with the drugs over the initial 30 min at 37 °C following channel activation, but prior to channel deactivation. The single-channel behavior of wild-type CFTR is characterized by bursts of channel openings interrupted by brief, flickery closures, separated by longer closures between bursts, which are greatly prolonged in F508del-CFTR rescued by low temperature incubation (Fig. 2A). Fig. 2A demonstrates that all combinations of CFTR modulators altered channel gating with the frequency and duration of openings differing markedly from those of wild-type CFTR. Fig. 2 A and D reveals that the interburst interval (IBI, the average duration of the long closures separating open-channel bursts) was increased for all CFTR modulator combinations, albeit the increase was not statistically significant for elexacaftor-tezacaftor-ivacaftor. Although the differences were not statistically significant, mean burst duration (MBD, the average duration of open-channel bursts) was increased with all CFTR modulator combinations, except elexacaftor-tezacaftor, and particularly with tezacaftor-ivacaftor (Fig. 2 A and E). As a result, prior to channel deactivation, the Po of rescued F508del-CFTR varied widely, decreasing in the rank order: wild-type CFTR = tezacaftor-ivacaftor ≫ elexacaftor-tezacaftor-ivacaftor = elexacaftor-ivacaftor > elexacaftor-tezacaftor = low temperature-rescued F508del-CFTR (Fig. 2F). Of note, prior to channel deactivation the i of F508del-CFTR Cl− channels rescued by CFTR modulator combinations, which included elexacaftor was larger than those of wild-type and F508del-CFTR rescued by either low temperature or tezacaftor-ivacaftor, albeit the difference was only statistically significant for elexacaftor-ivacaftor-rescued F508del-CFTR (P = 0.022) (Fig. 2 A–C).

Fig. 2.

A six-part figure shows single-channel recordings and current amplitude histograms of wild-type and F 508 del-C F T R with C F T R modulators.

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The single-channel behavior of F508del-CFTR rescued by chronic treatment with CFTR modulators. (A) Representative single-channel recordings of wild-type and F508del-CFTR rescued either by low temperature incubation or chronic treatment with different combinations of CFTR modulators in excised inside-out membrane patches from CFTR-expressing BHK cells acquired during the first 30 min of 60-min recordings prior to channel deactivation. The recordings were made at 37 °C with ATP (1 mM) and PKA (75 nM) continuously present in the intracellular solution. Dotted lines indicate the closed channel state and downward deflections correspond to channel openings. (B) Single-channel current amplitude histograms for the 10-s recordings shown in A. The labels C and O denote the closed- and open-channel amplitudes, respectively. Small leak currents have shifted the histograms of F508del-CFTR relative to that of wild-type CFTR. (C–F) Summary single-channel current amplitude (i), interburst interval (IBI), mean burst duration (MBD), and open probability (Po) data of wild-type and F508del-CFTR for the experimental conditions described in A. Symbols represent individual values and columns means ± SD (wild-type, n = 6; low temperature, n = 3; ELX/TEZ, n = 7; TEZ/IVA, n = 6 to 7; ELX/IVA, n = 6; ELX/TEZ/IVA, n = 5 to 13); *P < 0.05, **P < 0.01 and ***P < 0.001 vs. wild-type CFTR; one-way ANOVA with Tukey’s post-hoc test.

Elexacaftor Increases Current Flow Through the CFTR Cl− Channel.

The action of chronic treatment with CFTR modulator combinations on F508del-CFTR suggests that elexacaftor might stabilize its open-channel conformation. To investigate this possibility, we tested different concentrations of elexacaftor in chronic treatments and analyzed the acute effects of the drug on the CFTR Cl− channel.

Fig. 3 A and B shows representative recordings and corresponding current amplitude histograms acquired at 37 °C prior to the deactivation of F508del-CFTR Cl− channels in excised inside-out membrane patches from F508del-CFTR-expressing BHK cells treated chronically with elexacaftor (0.1 to 10 μM) plus tezacaftor (3 μM) and ivacaftor (1 μM). Visual inspection of these single-channel records reveals two important points. First, as the elexacaftor concentration was raised, the i of F508del-CFTR increased (Fig. 3 A–C). At elexacaftor (2 μM), i was 28% greater than at elexacaftor (0.1 μM), while at elexacaftor (10 μM), i was 33% larger (Fig. 3C). Second, elexacaftor (10 μM), but not other drug concentrations, increased the Po of F508del-CFTR by prolonging the duration of channel openings with little effect on their frequency (Fig. 3 D–F). However, this potentiation of F508del-CFTR gating was transient and not sustained at later times in cell-free membrane patches (SI Appendix, Fig. S2). Only using elexacaftor (2 μM) were individual and populations of F508del-CFTR Cl− channels stabilized in excised inside-out membrane patches from BHK cells and the apical membrane of FRT epithelia, respectively; at either lower (0.1 and 0.3 μM) or higher (10 μM) concentrations, F508del-CFTR function declined with time (SI Appendix, Fig. S2 and S3).

Fig. 3.

Multi-part figure shows single-channel recordings of F 508 del-C F T R C l super minus channels with elexacaftor, tezacaftor, and ivacaftor.

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Elexacaftor enhances the single-channel current amplitude of F508del-CFTR. (A) Representative single-channel recordings of F508del-CFTR Cl− channels rescued by chronic treatment with the indicated concentrations of elexacaftor (ELX) and tezacaftor (TEZ; 3 μM) and ivacaftor (IVA; 1 μM) in excised inside-out membrane patches from CFTR-expressing BHK cells. The recordings were made at 37 °C with ATP (1 mM) and PKA (75 nM) continuously present in the intracellular solution. Dotted lines indicate the closed channel state and downward deflections correspond to channel openings. (B) Single-channel current amplitude histograms for the 10-s recordings shown in A. The labels C and O denote the closed- and open-channel amplitudes, respectively. Small leak currents have shifted the histograms of F508del-CFTR acquired at different concentrations of elexacaftor. (C–F) Summary single-channel current amplitude (i), interburst interval (IBI), mean burst duration (MBD), and open probability (Po) data of rescued F508del-CFTR for the experimental conditions described in A determined from time interval 0 to 30 min of 60-min time courses prior to channel deactivation. Symbols represent individual values and columns means ± SD (ELX 0.1 μM, n = 3 to 4; ELX 0.3 μM, n = 5; ELX 2 μM, n = 5 to 9; ELX 10 μM, n = 4 to 5); **P < 0.01 vs. 0.1 μM elexacaftor; one-way ANOVA with Tukey’s post-hoc test. Data for F508del-CFTR rescued with elexacaftor (2 μM)-tezacaftor-ivacaftor are from Fig. 2.

To test the acute effects of elexacaftor on the single-channel behavior of CFTR, we studied wild-type CFTR in excised inside-out membrane patches. Fig. 4 A and B shows representative recordings and corresponding current amplitude histograms of a single wild-type CFTR Cl− channel in the absence and presence of elexacaftor (2 μM) in the intracellular solution, while Fig. 4 C–F quantifies the drug’s acute effects. Elexacaftor (2 μM) strongly potentiated CFTR gating by enhancing the frequency and duration of channel openings with the result that Po increased by 50% (Fig. 4 D–F). Moreover, although the effect was small (5%), elexacaftor (2 μM) caused a statistically significant increase in the i of wild-type CFTR (P = 0.026) (Fig. 4C and SI Appendix, Fig. S4). Taken together, the data indicate that elexacaftor-tezacaftor-ivacaftor stabilizes F508del-CFTR at the plasma membrane in part by elexacaftor enhancing current flow through open channels.

Fig. 4.

Multi-part figure shows single-channel recordings and current amplitude histograms of wild-type and F 508 del-C F T R with drug treatments.

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Acute treatment of elexacaftor-tezacaftor-ivacaftor-rescued F508del-CFTR with elexacaftor and ivacaftor confers wild-type-like activity on the variant. (A) Representative single-channel recordings of wild-type and F508del-CFTR rescued by chronic treatment with elexacaftor-tezacaftor-ivacaftor in excised inside-out membrane patches from BHK cells stably expressing either wild-type or F508del-CFTR. The wild-type CFTR recordings were made from a single membrane patch in the absence and presence of elexacaftor (ELX; 2 μM) in the intracellular solution, whereas the F508del-CFTR recordings were from two different membrane patches in the absence and presence of elexacaftor (2 μM) and ivacaftor (100 nM) in the intracellular solution. All recordings were made 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. (B) Single-channel current amplitude histograms for the 10-s recordings shown in A. The labels C and O denote the closed- and open-channel amplitude, respectively, and the horizontal dotted lines the peak values of the closed- and open-channel current amplitudes. (C–F) Summary single-channel current amplitude (i), interburst interval (IBI), mean burst duration (MBD), and open probability (Po) data of wild-type and elexacaftor-tezacaftor-ivacaftor-rescued F508del-CFTR for the experimental conditions described in A determined from recordings ≥ 10 min in durations. Symbols connected by lines indicate individual datasets and columns means ± SD (wild-type: i and Po, n = 6; MBD and IBI, n = 4; F508del: i and Po, n = 8 to 13; MBD and IBI, n = 5 to 6). [Wild-type (C, E, and F): *P < 0.05 and **P < 0.01 vs. control; Student’s paired t test; normality test (Shapiro–Wilk), P > 0.05 (passed); F508del-CFTR (C): **P < 0.01 vs. elexacaftor-tezacaftor-ivacaftor-rescued F508del-CFTR; Student’s unpaired t test; normality test (Shapiro–Wilk), P < 0.05 (failed); Mann–Whitney rank sum test, P < 0.01; F508del-CFTR (E and F): ***P < 0.001 vs. elexacaftor-tezacaftor-ivacaftor-rescued F508del-CFTR; Student’s unpaired t test; normality test (Shapiro–Wilk), P > 0.05 (passed)]. Data for F508del-CFTR rescued with elexacaftor (2 μM)-tezacaftor-ivacaftor are from Fig. 2.

Copotentiation With Elexacaftor and Ivacaftor Restores Wild-Type-Like Channel Activity to Elexacaftor-Tezacaftor-Ivacaftor-Rescued F508del-CFTR.

Based on the potentiation of wild-type CFTR by elexacaftor (Fig. 4) and the copotentiation of F508del-CFTR Cl− currents by elexacaftor and ivacaftor (22, 23), we tested the acute effects of elexacaftor and ivacaftor on elexacaftor-tezacaftor-ivacaftor-rescued F508del-CFTR Cl− channels. By increasing the duration of channel openings 426% without altering their frequency (Fig. 4 A, D and E), copotentiation with elexacaftor (2 μM) and ivacaftor (100 nM) augmented strongly the gating behavior of F508del-CFTR rescued by chronic treatment with elexacaftor-tezacaftor-ivacaftor. As a result, the Po of copotentiated elexacaftor-tezacaftor-ivacaftor-rescued F508del-CFTR was 232% greater than that of F508del-CFTR rescued by elexacaftor-tezacaftor-ivacaftor and 52% greater than that of wild-type CFTR in the absence of elexacaftor, while i was equivalent to that of wild-type CFTR and channel activity was sustained in cell-free membrane patches (Fig. 4 C and F and SI Appendix, Fig. S4 and S5).

Chronic treatment of F508del-CFTR-expressing epithelia with elexacaftor-tezacaftor-ivacaftor leads to robust CFTR-mediated Cl− currents prior to CFTR activation with forskolin (16, 27). These data suggest that elexacaftor-tezacaftor-ivacaftor might cause the constitutive activation of F508del-CFTR Cl− channels. To investigate this possibility, we assessed the PKA dependence of elexacaftor-tezacaftor-ivacaftor-rescued F508del-CFTR. Following the excision of inside-out membrane patches, F508del-CFTR Cl− channels opened infrequently and Po was extremely low (SI Appendix, Fig. S6). By contrast, following PKA addition to the intracellular solution, the frequency and duration of channel openings increased markedly and Po approached wild-type levels (Fig. 4 and SI Appendix, Fig. S6). Thus, by enhancing current flow, channel gating, and plasma membrane stability, elexacaftor-ivacaftor copotentiation of elexacaftor-tezacaftor-ivacaftor-rescued F508del-CFTR restores wild-type-like function to the CFTR variant.

Discussion

This study investigated the rescue of individual F508del-CFTR Cl− channels by elexacaftor-tezacaftor-ivacaftor, the therapy that has transformed the treatment of most people with CF. Chronic treatment of cells with elexacaftor-tezacaftor-ivacaftor greatly stabilized F508del-CFTR function in cell-free membrane patches and the apical membrane of epithelia. Of note, with copotentiation by elexacaftor and ivacaftor, the combination therapy restored wild-type-like levels of channel activity to F508del-CFTR by sustaining current flow through the channel and prolonging greatly the duration of channel openings.

A striking finding of this study was the marked stabilization of F508del-CFTR Cl− channels in cell-free membrane patches by chronic treatment of F508del-CFTR-expressing cells with elexacaftor-tezacaftor-ivacaftor, but not pairs of CFTR modulators. This effect, which depended on the elexacaftor concentration, was characterized by stability of both i and Po during prolonged recordings lasting one hour. This result contrasts with previous studies, which revealed that micromolar concentrations of ivacaftor destabilize F508del-CFTR both by perturbing its structure (28, 29) and disrupting the integrity of the lipid bilayer (30). The marked stabilization of elexacaftor-tezacaftor-ivacaftor-rescued F508del-CFTR is reminiscent of the action of revertant (second site) mutations in cis with F508del, which abrogate the deactivation of low temperature-rescued F508del-CFTR (31–34). These and other data (7) argue that the thermostability of elexacaftor-tezacaftor-ivacaftor-rescued F508del-CFTR is a consequence of stabilization of CFTR structure through the correct assembly of different domains. They also suggest that treating cells with ivacaftor (1 μM) in the presence of fetal bovine serum-containing culture media might prevent the deleterious actions of solutions supersaturated with micromolar concentrations of ivacaftor (35). Such solutions lead to the accumulation of ivacaftor in the plasma membrane of cells, which compromises the physicochemical properties of cell membranes and disrupts CFTR function [(28–30, 35); for discussion see (36)].

CFTR modulators restore function to CFTR variants by either rescuing their plasma membrane expression or potentiating channel gating (12). No CFTR modulators have been discovered that enhance current flow through the CFTR pore, even class I potentiators (e.g., ivacaftor and GLPG1837), which augment channel gating by binding near the pore constriction in a pocket involving the unstructured region of transmembrane segment 8, which gates the CFTR pore (37–39). By contrast, many small molecules inhibit current flow through the CFTR pore by either open-channel block or allosteric inhibition (40). Under certain circumstances, some inhibitors enhance CFTR function, but they do so by modifying channel gating, not current flow (41–43).

Several lines of evidence from the present study suggest that elexacaftor enhances current flow through the CFTR pore. First, chronic treatment with combinations of CFTR modulators that contained elexacaftor achieved higher F508del-CFTR i values than those without (Fig. 2). Second, raising the elexacaftor concentration from 0.1 to 10 μM increased the i of F508del-CFTR rescued by coincubation with tezacaftor and ivacaftor by 33% (Fig. 3). Third, acute treatment of wild-type CFTR with elexacaftor increased i and potentiated channel gating (Fig. 4). Taken together, the data suggest that elexacaftor increases current flow through the CFTR pore and that when used chronically with tezacaftor and ivacaftor, the drug stabilizes the open-channel conformation of F508del-CFTR.

Only by acutely treating elexacaftor-tezacaftor-ivacaftor-rescued F508del-CFTR Cl− channels with elexacaftor and ivacaftor were wild-type-like levels of channel activity (as measured by Po) conferred upon the CFTR variant. In contrast to previous studies (44, 45), this result identifies a drug combination that copotentiates F508del-CFTR. However, the gating pattern of F508del-CFTR Cl− channels chronically and acutely treated with CFTR modulators was distinct from that of wild-type CFTR and characterized by less frequent prolonged bursts of channel openings. The reduced frequency of channel openings suggests that subtle structural defects persist even though elexacaftor-tezacaftor-ivacaftor fully corrected the F508del domain assembly defects (7). These subtle structural defects might hinder dimerization of the NBDs or communication between the NBD1:NBD2 dimer and the channel pore (46–48). By contrast, the prolonged duration of channel openings suggests that copotentiation with elexacaftor and ivacaftor might stabilize the NBD1:NBD2 dimer by tighter ATP binding or slower ATP hydrolysis at ATP-binding site 2, where ATP hydrolysis promptly closes the channel (46, 49). Alternatively, the prolonged openings might reflect the decoupling of the gating cycle from the ATP hydrolysis cycle (50, 51).

The elexacaftor binding site on phosphorylated ATP-bound human F508del-CFTR is a shallow groove at the MSD–lipid interface, principally composed of residues from transmembrane segment 11 (7). The location of this binding site suggests that elexacaftor acts allosterically to augment current flow and channel gating like its action on NBD1 stability, and hence, CFTR processing (18, 52). However, two lines of evidence suggest that elexacaftor might act at a distinct site to mediate these effects. First, the half-maximal effective concentration for elexacaftor potentiation of wild-type and G551D-CFTR is > 100-fold less than that required to correct the F508del-CFTR processing defect (correction, EC50 ~0.28 μM; potentiation, EC50 ~1 nM) (16, 22, 23). Second, using molecular docking and molecular dynamics simulations, Baatallah et al. (52) identified two binding sites for elexacaftor distinct from the site identified by Fiedorczuk and Chen (7) using cryoelectron microscopy, one in MSD1 and a second in NBD1. Interestingly, the MSD1 site located at the CFTR–lipid interface lies in a position where it might influence current flow through the CFTR pore and channel gating (37, 39, 52, 53). This suggests that the mechanism by which elexacaftor augments current flow and channel gating in CFTR is distinct from the master key mechanism of negatively charged activators, which potentiate different K+ channels (54). Binding of these small molecules close to the selectivity filter, enhances K+ occupancy, dilating the pore and opening the selectivity gate thereby, augmenting channel gating (54).

This study is not without limitations. First, all the studies undertaken were performed with heterologous cells overexpressing CFTR variants because of the technical difficulty of studying native CFTR Cl− channels in polarized epithelial cells (55). Although cellular background influenced the rescue of G1244E-CFTR by elexacaftor-tezacaftor-ivacaftor (56), the differential effects of CFTR modulators on F508del-CFTR-expressing human bronchial epithelia argue that elexacaftor-tezacaftor-ivacaftor enhances both the processing and function of F508del-CFTR in these cells (15). Second, we studied individual F508del-CFTR Cl− channels using cell-free membrane patches because of the superior quality of single-channel recordings when compared with cell-attached membrane patches. Using excised inside-out membrane patches, CFTR function is studied directly. However, in intact cells and epithelia, CFTR function is influenced by the activity of ion channels, transporters, and interacting proteins that establish and/or modify the electrochemical gradient for transmembrane ion movements (57). Third, we only assessed the stability of individual channels in cell-free membrane patches for 1 h and populations of F508del-CFTR in the apical membrane of epithelia for 6 h. Thus, although elexacaftor-tezacaftor-ivacaftor greatly stabilized F508del-CFTR, we do not know whether the stability of wild-type CFTR [t½ ~12 h; (9)] was completely restored to F508del-CFTR. Indeed, Graeber et al. (58) found that elexacaftor-tezacaftor-ivacaftor incompletely restored CFTR function to nasal and intestinal epithelia of people with CF and one or two copies of the F508del variant.

In conclusion, elexacaftor-tezacaftor-ivacaftor has transformed CF from a life-threatening illness to a treatable condition for most people with the predominant CFTR variant F508del (1, 2, 59). By studying individual F508del-CFTR Cl− channels, we revealed that chronic treatment with elexacaftor-tezacaftor-ivacaftor greatly stabilizes F508del-CFTR at the plasma membrane and plus acute treatment with elexacaftor and tezacaftor restored wild-type-like levels of channel activity to the CFTR variant. Our results provide molecular insight into the clinical efficacy of the combination therapy, demonstrating that the therapeutic addresses each of the F508del-CFTR defects, processing, gating, and stability (4, 8, 9), which together cause a profound loss of CFTR function. They also reveal that elexacaftor is a triple-acting CFTR modulator with corrector, potentiator, and conduction activities. This suggests that elexacaftor might be particularly beneficial to CFTR variants, which perturb CFTR conduction, some of which are unresponsive to ivacaftor (60, 61).

Materials and Methods

Cells and CFTR Expression.

For single-channel studies of human CFTR, we used BHK cells (RRID:CVCL_1915) stably expressing human wild-type and F508del-CFTR from MD Amaral (University of Lisboa) (62) (SI Appendix, Materials and Methods). To promote the plasma membrane expression of F508del-CFTR, cells expressing the variant were either incubated at 27 °C for 24 h or treated with combinations of CFTR modulators in media containing 5% v·v-1 fetal bovine serum for 24 h at 37 °C (5, 13–15). Prior to study, F508del-CFTR-expressing cells were washed in drug-free solutions to remove CFTR modulators. However, the maximum period cells were left in drug-free solution before study did not exceed 30 min.

Patch-Clamp Experiments.

The single-channel activity of wild-type and F508del-CFTR was studied using excised inside-out membrane patches (36). For further information, see the SI Appendix, Materials and Methods.

Reagents.

For the source of reagents and their preparation and use, see the SI Appendix, Materials and Methods.

Statistics.

Results are expressed as means ± SD of n observations. To test for differences between two groups of data acquired within the same experiment, we used Student’s paired t test, whereas to test for differences between multiple groups of data, we used one-way repeated measures ANOVA followed by Tukey’s multiple comparison test when a statistically significant difference was observed. Differences were considered statistically significant when P < 0.05 (SI Appendix, Materials and Methods).

Supplementary Material

Appendix 01 (PDF)

pnas.2523950123.sapp.pdf (989.3KB, pdf)

Acknowledgments

We thank M.D. Amaral and L.J.V. Galietta for CFTR-expressing cells and A.K. Singh and laboratory colleagues for valuable discussions and assistance. This work was supported by the Cystic Fibrosis Trust [Grant no. SRC 021] to D.N.S. J.N.C. is a Cystic Fibrosis Trust-supported PhD student.

Author contributions

M.R., J.N.C., and D.N.S. designed research; M.R. and J.N.C. performed research; M.R., J.N.C., and D.N.S. analyzed data; and M.R., J.N.C., and D.N.S. wrote the paper.

Competing interests

The authors declare no competing interest.

Footnotes

This article is a PNAS Direct Submission.

Data, Materials, and Software Availability

Data are available at the University of Bristol data repository, data.bris, at https://doi.org/10.5523/bris.md1upto2lx8x2u1iu64x2k9sz (63). Study data are included in the article and/or SI Appendix.

Supporting Information

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Associated Data

This section collects any data citations, data availability statements, or supplementary materials included in this article.

Supplementary Materials

Appendix 01 (PDF)

pnas.2523950123.sapp.pdf (989.3KB, pdf)

Data Availability Statement

Data are available at the University of Bristol data repository, data.bris, at https://doi.org/10.5523/bris.md1upto2lx8x2u1iu64x2k9sz (63). Study data are included in the article and/or SI Appendix.

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