Cystic fibrosis transmembrane conductance regulator (CFTR) potentiator VX-770 (ivacaftor) opens the defective channel gate of mutant CFTR in a phosphorylation-dependent but ATP-independent manner

Abstract

The cystic fibrosis transmembrane conductance regulator (CFTR) acts as a channel on the apical membrane of epithelia. Disease-causing mutations in the cystic fibrosis gene can lead to CFTR protein misfolding as in the case of the F508del mutation and/or channel dysfunction. Recently, a small molecule, VX-770 (ivacaftor), has shown efficacy in restoring lung function in patients bearing the G551D mutation, and this has been linked to repair of its channel gating defect. However, these studies did not reveal the mechanism of action of VX-770 in detail. Normally, CFTR channel activity is regulated by phosphorylation, ATP binding, and hydrolysis. Hence, it has been hypothesized that VX-770 modifies one or more of these metabolic events. In this study, we examined VX-770 activity using a reconstitution system for purified CFTR protein, a system that enables control of known regulatory factors. We studied the consequences of VX-770 interaction with CFTR incorporated in planar lipid bilayers and in proteoliposomes, using a novel flux-based assay. We found that purified and phosphorylated CFTR was potentiated in the presence of Mg-ATP, suggesting that VX-770 bound directly to the CFTR protein, rather than associated kinases or phosphatases. Interestingly, we also found that VX-770 enhanced the channel activity of purified and mutant CFTR in the nominal absence of Mg-ATP. These findings suggest that VX-770 can cause CFTR channel opening through a nonconventional ATP-independent mechanism. This work sets the stage for future studies of the structural properties that mediate CFTR gating using VX-770 as a probe.

FIGURE 1.

Purification and functional reconstitution of WT-CFTR. A, silver-stained gel lanes are as follows: lane A, prestained marker with the associated molecular mass indicated at left; lane B, 1/80th of the membrane fraction; lane C, 1% of the 10 mm imidazole wash fraction; lane D, 1/8th of the protein remaining associated with the beads after the 10 mm wash step; lane E, 0.4% of the 50 mm imidazole wash step; lane F, 1/8th of the protein remaining associated with the beads after the 50 mm wash step; lane G, 1/8th of the protein remaining associated with the beads after the 600 mm wash step; lane H, 1 μg of human CFTR purified, concentrated, and quantified as described under “Experimental Procedures”; lane I, Western blot for 0.1 μg of purified CFTR protein using the M3A7 anti-CFTR antibody. Gel and blot data are representative of three purifications. B, diagrammatic representation of the flux assay using CFTR-proteoliposomes. See under “Experimental Procedures” and “Results” for further details on the flux assay. C, sample flux assay traces. Purified CFTR was reconstituted into egg PC liposomes at a protein/lipid ratio of 1:1200 by mass. Control vesicles were prepared identically but in the absence of CFTR (empty or control; gray trace). Proteoliposomes were treated with 200 nm PKA and 1 mm Mg-ATP for 5 min, and flux was initiated by the addition of valinomycin at a concentration of 20 nm. Vesicles were lysed by addition of 0.5% Triton X-100 to verify that iodide was trapped (as indicated by the instantaneous rise in iodide near the end of the traces). D, initial iodide release (or flux) rates mediated by CFTR determined immediately after valinomycin addition. The efflux measured in the presence of PKA and Mg-ATP was significantly greater than that measured in the absence of PKA (also see Fig. 2A). The control trace (gray line) shows the lack of flux by liposomes lacking CFTR. CFTR_inh-172 (20 μm) prevented the flux by PKA- and ATP-treated CFTR. E, bars represent the mean of 5–7 trials ± S.E. * indicates treatment with CFTR_inh-172 causes significantly lower flux than in its absence (+PKA+ATP bar), p = 0.02. ** indicates efflux activity in proteoliposomes (−PKA+ATP) is significantly higher than control vesicles, p = 0.02, but significantly less than when treated with PKA, p = 0.005. *** indicates samples containing phosphorylated CFTR (+PKA+ATP) had efflux activity significantly higher than control vesicles, p < 0.0001.

FIGURE 2.

Channel activity of purified and reconstituted phosphorylated WT-CFTR is directly modified by VX-770. A, PKA phosphorylation increased the initial rate of flux mediated by purified and reconstituted WT-CFTR in the presence of Mg-ATP (1 mm; white bars, *, p = 0.027). The flux rate exhibited by unphosphorylated CFTR (in the presence of Mg-ATP and normalized to the flux by PKA and Mg-ATP protein, i.e. control) is unchanged in the absence and presence of VX-770 (black bar, 10 μm). However, the flux rate exhibited by phosphorylated CFTR (in the presence of Mg-ATP) is significantly increased by 10 μm VX-770 addition (black bar, *, p = 0.039). Bars represent the mean of three (unphosphorylated) or four (phosphorylated) trials ± S.E. B, proteoliposomes containing purified and phosphorylated WT-CFTR were fused to planar lipid bilayers. Single channel current steps were recorded at −50 mV in the presence of 1 mm Mg-ATP following fusion to a planar lipid bilayer containing 300 and 50 mm KCl on the cis- and trans-compartments of the apparatus, respectively. c and o indicate closed channel and open channel current levels, respectively. The current-voltage relationships yielded a unitary conductance of ∼10 pS. Open probability measurements were possible by combining channel events lists from different trials. The trace shown in B is representative of five trials (or five different protein preparations with 874 events analyzed to yield a P_o of 0.47). C, single channel current steps measured as described in B but in the presence of 2 μm VX-770 and 1 mm Mg-ATP. These open probability measurements were possible by combining channel events lists from seven different trials (or seven different protein preparation with 855 events analyzed to yield a P_o of 0.79).

FIGURE 3.

ATP-independent opening of phosphorylated CFTR by VX-770. A, VX-770 (10 μm) stimulates the initial rate of iodide flux in reconstituted pre-phosphorylated WT-CFTR in the nominal absence of ATP. B, VX-770 dependence of the ATP-independent flux mediated by pre-phosphorylated WT-CFTR. Data points shown represent the mean of five independent dose-response trials and are expressed relative to a sample treated with DMSO alone. Data were fitted to a hyperbolic single site binding equation, yielding an EC₅₀ of ∼1 μm (see “Results”). C, planar lipid bilayer studies of purified and PKA-phosphorylated WT-CFTR in the nominal absence of ATP. As expected, despite multiple proteoliposome fusion events (nystatin spikes indicated with stars), there were no single channel events detected in the absence of ATP (representative of eight studies). At the point indicated with the arrow, the bilayer was treated with 2 μm VX-770, which induced multiple channel current steps ∼10–20 s after the addition artifact. In this experiment, ∼4 CFTR channels were activated. D, in studies (n = 2 protein preparations) showing single channel activity after VX-770 addition, the unitary current steps corresponded to the low single channel conductance expected for WT-CFTR (10 pS). Single channel current steps were recorded at −50 mV in a lipid bilayer chamber containing 300 and 50 mm KCl on the cis- and trans-compartments of the apparatus, respectively. P_o of 0.42 was determined from a combined events list of 2625 events.

FIGURE 4.

Additive effect of VX-770 and ATP in regulation of phosphorylated WT CFTR. A, representative iodide efflux traces mediated by proteoliposomes containing pre-phosphorylated WT-CFTR in the absence or presence of 1 mm Mg-ATP and the absence or presence of 10 μm VX-770 as indicated, also see summary bar graphs in Fig. 6C. Data in gray indicate the sum of the efflux rates +VX-770 and +ATP traces. B, representative iodide efflux traces mediated by proteoliposomes containing pre-phosphorylated WT-CFTR in the absence (gray) or presence (black) of 2 μm VX-770 and in the presence of 0.1 or 2 mm ATP. C, no effect of VX-770 on ATP dependence of WT-CFTR flux activity in the presence or absence of VX-770 (2 μm). Data points represent initial rates mediated by PKA-phosphorylated WT-CFTR proteoliposomes pretreated in the presence of 2 μm VX-770 (+VX-770; closed circles) or in its absence but in the presence of DMSO vehicle (−VX-770; open circles) at a range of Mg-ATP concentrations. Although treatment with VX-770 significantly increased flux activity (p < 0.0001), to 1.4-fold the value in its absence, data were normalized to 1 in the absence of ATP in both cases to illustrate similarity of the ATP dependence. Data represent the mean of three traces ± S.E.

FIGURE 5.

VX-770 acts directly to potentiate phosphorylated F508del-CFTR and phosphorylated G551D-CFTR in the absence of Mg-ATP. A, SDS-PAGE analysis and silver stain of purified F508del-CFTR and purified G551D-CFTR protein isolated using the PFO detergent extraction method. In this method, the mutant CFTR proteins are solubilized in PFO and applied to a Ni-NTA affinity column. The full-length mutant protein is eluted by the application of a continuous pH gradient (FPLC). The PFO method requires 3 days and yields functional protein as published previously (, –, –49). The full-length PFO-purified proteins run as expected as broad 150-kDa bands in overloaded gels as expected for Sf9-expressed proteins (21). Data are representative of 10 separate purifications. However, full-length (150 kDa) mutant proteins plus other proteins are evident in silver-stained gels after the fos-choline 14-based methods (a method wherein CFTR protein solubilized in fos-choline is eluted from a Ni-NTA affinity column in a batchwise method described for the first time in this paper and requiring half a day). Immunoblots using a CFTR-selective antibody shows that other bands likely correspond to CFTR fragments. Hence, the fast batchwise method employing fos-choline purifies the full-length mutants plus degradation products. Data are representative of three separate purifications. B, initial iodide efflux rates mediated by phosphorylated F508del-CFTR or phosphorylated G551D-CFTR are potentiated by VX-770 (10 μm) in the presence of 1 mm ATP, regardless of the purification method used (PFO or fos-choline). These data show that the activity mediated by reconstituted fos-choline-extracted and Ni-NTA-purified mutant CFTR is conferred by the full-length mutant protein. An inactive analog of VX-770 (V-09-1188, labeled In-VX, provided by Vertex Pharmaceuticals) fails to mediate potentiation of F508del-CFTR, whereas P1 (or VRT-532) is effective in potentiating G551D-CFTR. Means ± S.E. of five and four studies for the PFO purification and three and five studies for the fos-choline method for F508del-CFTR and G551D-CFTR, respectively, are shown. **, p = 0.0034 for PFO-purified F508del-CFTR in the presence versus absence of VX-770; p = 0.001 for fos-choline purified F508del-CFTR in the presence of In-VX versus sample treated with VX-770; and p = 0.0019 for fos-choline purified G551D in the presence of VX-770 versus its absence. ***, p ≤ 0.0001, when compared with the associated control sample in the absence of VX-770. PKA-phosphorylated F508del-CFTR (C) or PKA phosphorylated G551D-CFTR (D) purified using either method exhibits potentiation by VX-770 in the nominal absence of Mg-ATP. We show traces representative (n = 3) for mutant proteins extracted either using fos-choline 14 or PFO detergent (B and C). Samples were phosphorylated with 200 nm and 1 mm ATP and treated with 10 μm VX-770, In-VX, or VRT-532 where indicated. The similarity of these responses support the idea that the activities shown for fos-choline-extracted mutant proteins report the intrinsic activities of the full-length mutants and support the utility of this rapid process for studying mutant CFTR proteins in a cell-free reconstitution system that enables excellent control of ligand concentrations.

FIGURE 6.

Additive effect of VX-770 and ATP in regulation of phosphorylated F508del-CFTR and phosphorylated G551D-CFTR. Representative iodide efflux traces are from proteoliposomes containing F508del-CFTR (A) or G551D-CFTR (B). Additive effect of ATP (1 mm) and VX-770 (10 μm) on F508del-CFTR. In contrast to F508del-CFTR (and WT-CFTR), G551D-CFTR proteoliposomes are not activated Mg-ATP but do show robust response to VX-770 alone. C, bar graph shows summary data for initial flux rates by the WT-CFTR and mutant CFTR proteins. Data are means of three replicates ± S.E., except for WT +ATP and WT +ATP VX-770, which are means of 10 ± S.E., and the G551D-CFTR data, which are means of 5–7, respectively, ± S.E. Samples were normalized to their respective untreated sample (−ATP-VX-770). Asterisks indicate samples that are significantly different from values for WT-CFTR treated similarly; * 0.05>p > 0.01; ** 0.01>p > 0.001; ***, p < 0.001. Arrows to the right indicate the mathematical addition of VX-770 alone flux and ATP alone flux values for upper WT-, middle F508del-, and lower G551D-CFTR. D, VX-770 modestly increases the ATPase activity of G551D-CFTR. Significantly more ATPase activity was measured for PKA-phosphorylated G551D-CFTR samples treated with VX-770 versus control (***, p < 0.0001) or the inactive analog (In-VX, p = 0.0027). Bars represent the mean of 3 (WT-CFTR), 22 (−VX-770), 17 (+VX-770), or 7 (In-VX) trials ± S.E.

FIGURE 7.

Schematic showing potential mechanism of action of VX-770. According to this model, the phosphorylated form of the CFTR channel (enclosed in the orange rectangle), but not the unphosphorylated form (gray), is capable of ATP and/or VX-770-mediated opening. The closed state of the CFTR channel is shown with the gate (thin blue bar) in the membrane domain in the horizontal position. We speculate in this model that the gate is closed when the canonical catalytic site between the NBDs (half-moons) is not occupied by ATP (yellow sphere), and the NBDs are separated at this site. Normally, ATP binding to the catalytic site induces a tight NBD dimer which in turn leads to conformational changes along the loop regions (dark teal rectangles) to the membrane domains (cyan rectangles) inducing their conformational change and opening of the channel gate. Our results suggest that in the absence of ATP the interaction of VX-770 (at an unknown site) also induces conformational changes effective in opening the channel gate. Together, the consequences of VX-770 binding and ATP binding induce an additive effect to stabilize the open conformation of the channel gate and enhance open probability.