Vx-770 potentiates CFTR function by promoting decoupling between the gating cycle and ATP hydrolysis cycle

Abstract

Vx-770 (Ivacaftor), a Food and Drug Administration (FDA)-approved drug for clinical application to patients with cystic fibrosis (CF), shifts the paradigm from conventional symptomatic treatments to therapeutics directly tackling the root of the disease: functional defects of the cystic fibrosis transmembrane conductance regulator (CFTR) chloride channel caused by pathogenic mutations. The underlying mechanism for the action of Vx-770 remains elusive partly because this compound not only increases the activity of wild-type (WT) channels whose gating is primarily controlled by ATP binding/hydrolysis, but also improves the function of G551D-CFTR, a disease-associated mutation that abolishes CFTR's responsiveness to ATP. Here we provide a unified theory to account for this dual effect of Vx-770. We found that Vx-770 enhances spontaneous, ATP-independent activity of WT-CFTR to a similar magnitude as its effects on G551D channels, a result essentially explaining Vx-770's effect on G551D-CFTR. Furthermore, Vx-770 increases the open time of WT-CFTR in an [ATP]-dependent manner. This distinct kinetic effect is accountable with a newly proposed CFTR gating model depicting an [ATP]-dependent "reentry" mechanism that allows CFTR shuffling among different open states by undergoing multiple rounds of ATP hydrolysis. We further examined the effect of Vx-770 on R352C-CFTR, a unique mutant that allows direct observation of hydrolysis-triggered gating events. Our data corroborate that Vx-770 increases the open time of WT-CFTR by stabilizing a posthydrolytic open state and thereby fosters decoupling between the gating cycle and ATP hydrolysis cycle. The current study also suggests that this unique mechanism of drug action can be further exploited to develop strategies that enhance the function of CFTR.

Fig. 1.

Energetic coupling model of CFTR gating and the structure of Vx-770. (A) Energetic coupling model for CFTR gating (modified from ref. 24). The red box marks the reentry pathway. The rate of C2 → C1 transition is extremely slow for WT-CFTR, therefore in the continuous presence of ATP, the channel rarely visits the C1 state. C1, C2, C2 ATP, and C2 ATP dimerized (C2 AD) represent closed states and O1, O2, and O2 ATP represent open states with different NBD configurations as illustrated in the cartoon. Semiquantitative analysis suggested that the O1 state is extremely stable (see ref. for detail). As a result, for WT-CFTR, closure of most opening events is the consequence of ATP hydrolysis, which effectively creates a shortcut for the channel to escape from the otherwise very stable O1 state. (B) Chemical structure of Vx-770. A was modified from figure 6 in ref. .

Fig. 2.

Effects of Vx-770 on ATP-independent activity of WT-CFTR. (A and B) Representative traces for ATP-induced macroscopic current of WT-CFTR channels in the absence (A) or presence (B) of Vx-770. Patches were perfused with ATP until the current reaches the steady state to measure ATP-induced current. ATP-independent activity was measured after ATP washout (expanded in the red and blue boxes). Traces shown in A and B were chosen because they exhibit similar amplitude of ATP-independent current, whereas the ATP-induced current is much greater in A, indicating a higher ATP-independent activity in the presence of Vx-770. (C) Effects of Vx-770 on the ratio of ATP-independent currents to ATP-induced currents. (D) Estimated Po for ATP-independent activity in the presence or absence of Vx-770. Bars above each trace mark applications of the indicated ligand(s) to the patch (same in all figures). *P < 0.05.

Fig. 3.

Vx-770 prolongs the open time of WT-CFTR in an ATP-dependent manner. (A) Representative single-channel traces for WT-CFTR in conditions depicted above each trace. (B) Summary of the mean open time at different [ATP] in the presence (black markers) or absence (blue markers) of 200 nM Vx-770. Mean Po for conditions denoted in A–C are 0.38 ± 0.03, n = 13; 0.71 ± 0.02, n = 25 and 0.45 ± 0.02, n = 17, respectively. (C) Summary of the opening rate of WT-CFTR at different [ATP] in the presence (black markers) or absence (blue markers) of 200 nM Vx-770. (D) In the presence of 200 nM Vx-770, the relaxation time constants of macroscopic CFTR current after removing specified concentrations of ATP. *P < 0.05 compared with 10 mM ATP. ^#P < 0.05 compared with the same [ATP] but without Vx-770.

Fig. 4.

Effects of Vx-770 on R352C-CFTR. (A and B) Representative single-channel traces for R352C-CFTR in the absence (A) or presence (B) of 200 nM Vx-770. When treated by Vx-770, the percentage of opening events contain more than one O1 → O2 transition is increased (summarized in Table 1) and the mean open time is prolonged (Fig. S3).

Fig. 5.

W401F mutation and Vx-770 work additively on the reentry pathway. Representative single-channel traces for R352C/W401F-CFTR treated with 2.75 mM ATP in the absence (A) or presence (B) of 200 nM Vx-770. Frequency of opening bursts containing multiple rounds of O1 → O2 transition (summarized in Table 1) is increased and the overall open time is prolonged (Fig. S3D) compared with R352C-CFTR recorded in the same condition.