VX-770 acted as a potentiator, not an activator, of G551D- and F508del CFTR in recombinant cells. (A) Structure of VX-770 (N-(2,4-di-tert-butyl-5-hydroxyphenyl)-4-oxo-1,4-dihydroquinoline-3-carboxamide)). (B) Representative I_T recording from G551D-FRT showing the response to the sequential application of 10 μM forskolin (FSK), 10 μM VX-770, and 20 μM CFTR_inh-172. (C) Concentration–response curve of the net increase in forskolin-stimulated I_T after sequential application of VX-770 at the concentrations indicated in G551D-FRT (filled circles; n = 12) and temperature-corrected F508del-FRT (open circles; n = 5). (D) Mean (± SEM) of the I_T response in G551D- (filled bars; n = 6) and F508del- (open bars; n = 5) FRT under the conditions indicated in B. Single asterisk indicates significant difference relative to unstimulated; double asterisk indicates significant difference relative to forskolin and unstimulated (P < 0.05; one-way ANOVA followed by Tukey's multiple comparison test). (E) Representative I_T recording from G551D-FRT showing the response to the sequential application of 10 μM VX-770, 10 μM FSK, and 20 μM CFTR_inh-172. (F) Total cAMP (cellular and secreted) was measured after 30 min incubation of FRT cells with 0.5 μM FSK plus DMSO, VX-770, and the nonspecific PDE inhibitor, IBMX (open bars) or with 20 μM FSK alone (filled bar).

VX-770 potentiated the gating activity of CFTR. Representative patch-clamp recording of the single channel current resulting from activation of G551D CFTR in FRT cells (A) and F508del CFTR in NIH 3T3 cells (B) by 1 mM ATP and 75 nM PKA before and during VX-770 application. The maximum effective concentration of VX-770 based on Fig. 1C was used for each CFTR form. All modulators were added to the cytoplasmic surface. Dotted lines indicate closed state. (C) Mean P_o of F508del-, G551D-, and wild-type CFTR in the presence of PKA and ATP alone (open bars) and with VX-770 (filled bars). For G551D CFTR, 10 μM VX-770 was added, whereas 1 μM VX-770 was added to F508del- and wild-type CFTR. F508del-NIH 3T3 cells were incubated at 27 °C for 24 h before recording. Asterisks indicate significant difference (P < 0.05; t test; n = 5–6).

VX-770 potentiated CFTR-mediated Cl⁻ secretion in primary cultures of G551D/F508del HBE and F508del HBE. Ussing chamber techniques were used to record the I_T resulting from CFTR-mediated Cl⁻ secretion. To isolate the CFTR-mediated I_T, a basolateral to apical Cl⁻ gradient was established, 30 μM amiloride was added to block ENaC, and 10 μM (EC₉₉) forskolin (FSK) was applied to activate CFTR. The addition CFTR_inh-172 (20 μM) to the apical surface was used to confirm that the forskolin-stimulated I_T was caused by CFTR. (A) Representative I_T tracing from G551D/F508del HBE. (B) The concentration–response curve for VX-770 in the presence of FSK is shown for G551D/F508del HBE isolated from the bronchi of a single individual (filled circles; n = 16) and F508del HBE isolated from the bronchi of the three individuals that responded to VX-770 (open circles; n = 7–24). Left y axis shows I_T responses; right y axis shows I_T normalized to the 10 μM FSK-stimulated I_T in non-CF HBE (mean ± SEM). Note that the error bars for the F508del HBE were smaller than the symbol. (C) The I_T before FSK addition (unstimulated) and during the sequential addition of 10 μM FSK followed by 10 μM VX-770 in F508del HBE isolated from the bronchi of six F508del-homozygous individuals (n = 4–24). (D) Representative I_T tracing from F508del HBE isolated from the bronchi represented by the open circles in C.

VX-770 increased CFTR-mediated Cl⁻ secretion and reduced ENaC-mediated Na⁺ absorption in G551D/F508del HBE. Representative recording of the PD in non-CF HBE (A) and G551D/F508del HBE (B) obtained with the Ussing chamber technique (open circuit mode) using equimolar Cl⁻ on the basolateral and apical sides. In G551D/F508del HBE, DMSO (vehicle; solid lines) or 10 μM VX-770 (dashed lines) was added to the apical surface before sequential addition of amiloride, low Cl⁻ (5 mM) and 10 μM forskolin (FSK) to the apical surface. (C) Concentration–response curve (mean ± SEM, n = 6) of the PD response to VX-770 addition in the presence of DMSO (filled circles), 10 μM FSK (open circles) or 20 μM CFTR_inh-172 (open squares). (D) Net change in PD (ΔPD) after the addition of 30 μM amiloride in the presence of the treatments indicated. (E) Representative PD recording in G551D/F508del HBE showing the response to DMSO (solid line) or 10 μM VX-770 without (dashed line) or with 10 μM FSK (dotted line) followed by sequential addition of 30 μM amiloride and 20 μM CFTR_inh-172. (F) The contribution of ENaC and CFTR to the PD in non-CF HBE and G551D/F508del HBE (bar) was determined by measuring the PD change in response to amiloride (open bars) and CFTR_inh-172 (filled bars), respectively. All data are from G551D/F508del HBE isolated from the bronchi of a single individual. Asterisks indicate significant difference compared with vehicle-treated controls (ANOVA followed by Tukey's test; mean ± SEM, n = 6).

Potentiation of CFTR by VX-770 partially restored fluid regulation and cilia beating in G551D/F508del HBE. (A) The ASL volume in G551D/F508del HBE and wild-type HBE after up to 72 hours' incubation at 37 °C in the presence of 30 nM VIP with or without VX-770. All modulators were added to the basolateral surface. (B) Concentration–response curve (n = 3–9) of the change in ASL volume in G551D/F508del HBE after VX-770 addition at the indicated concentrations in the absence (open circles) or presence (filled circles) of 30 nM VIP. All data were normalized to ASL volume in the vehicle (DMSO)–treated controls and expressed as percent control. (C) Mean (n = 3–9) ASL volume in the absence (open bars) or presence (filled bars) of 10 μM VX-770 and in the presence of 30 nM VIP and/or 20 μM CFTR_inh-172. (D) Representative tracings of the light intensity (y axis in relative units) derived from a single region of interest in G551D/F508del HBE monitored 5 days after adding 100 μl of fluid to the apical surface. (E) Mean (± SEM; n = 6) CBF wild-type HBE (filled bars) or G551D/F508del HBE (open bars) after a 5-day treatment with DMSO, 30 nM VIP, 10 μM VX-770, or 30 nM VIP with 10 μM VX-770. Single asterisk indicates significantly different (P < 0.05) from vehicle control in G551D/F508del HBE; double asterisk indicates significantly different (P < 0.05) from vehicle control and VX-770 alone. All data are from G551D/F508del HBE isolated from the bronchi of a single individual.