Vx 770

HEK cells were seeded in 96-well plates (black, flat bottom; Greiner). After 24 h the cells were transfected with either M1101K-, G85E-or N1303K-CFTR constructs, and 18 h post-transfection were treated with DMSO, 3 µM VX-809, 0.5 µM AC1, 3 µM AC2-1, 3 µM AC2-2, 0.5 µM AC1+ 3 µM AC2-1 and 0.5 µM AC1+ 3 µM AC2-2 for 24 h at 37 °C. Cells were then loaded with blue membrane potential dye for dissolved in chloride free buffer (136 mM sodium gluconate, 3 mM potassium, gluconate, 10 mM glucose, 20 mM HEPES, pH 7.35, 300 mOsm, at a concentration of 0.5 mg/mL, Molecular Devices), for 45 min at 37 °C. The plate was then read in a fluorescence plate reader (SpectraMax i3; Molecular Devices) at 37 °C, and after reading the baseline fluorescence (excitation: 530 nm, emission: 560 nm) for 5 min; CFTR was stimulated using the forskolin (10 µM; Sigma) and the potentiators VX-770 (1 µM) or AP2 (1.5 µM). CFTR-mediated depolarization of the plasma membrane was detected as an increase in fluorescence. Then, CFTR inhibitor (CFTRinh-172, 10 µM) was added to inactivated CFTR. The peak changes in fluorescence to CFTR agonists were normalized relative to fluorescence immediately before agonist (forskolin) addition [ 20 Laselva

Primary nasal epithelial cells were grown on transwells and studied in a nonperfused Ussing chamber (Physiologic Instruments, San Diego, CA, USA). Cells expressing mutant CFTR were transduced as described earlier. Where indicated, cells were treated with either 0.1% dimethyl sulfoxide (DMSO) or the CFTR modulator 3 µM VX-809, 48 h before the experiments at 37°C. The buffer solution (126 mM NaCl, 24 mM NaHCO 3 , 2.13 mM K 2 HPO 4 , 0.38 mM KH 2 PO 4 , 1 mM MgSO 4 , 1 mM CaCl 2 and 10 mM glucose) was maintained at pH 7.4 and 37°C and continuously gassed with a 5% CO 2 /95% O 2 mixture. The transepithelial potential was recorded in open-circuit mode and the baseline resistance was measured following repeated, brief short-circuit current pulses (1 µA every 30 s). The results are presented as equivalent transepithelial current (I eq ), which was calculated using Ohm's law. CFTR function was determined after inhibition of the epithelial sodium channel (ENaC) with amiloride (30 µM, Spectrum Chemical, Gardena, CA, USA) and following cAMP activation with forskolin (10 µM, Sigma-Aldrich, St Louis, MO, USA) and when indicated 1 µM VX-770. CFTR activity was confirmed as I eq difference following CFTR inhibition with CFTR inhibitor 172 (CFTR Inh172 ) (10 µM, EMD Millipore Corp., Billerica, MA, USA) [28, 29] .