A7410

Fully differentiated HBECs on Snapwell filters were mounted in Ussing chambers (Easymount chamber; Physiologic Instruments) connected to a VCC MC6 voltage clamp unit (Physiologic Instruments, San Diego, CA, USA). Solutions were maintained at 37 °C by heated water jackets and bubbled with air. For BK activity, basolateral membranes were permeabilized for 30 min with 20 µM amphotericin B, 10 µM nigericin and 10 µM valinomycin because whole cell short circuit current recordings cannot distinguish K⁺ efflux (it measures net current, a combination of K⁺ and Cl⁻ efflux)^{10 (link)}. Furthermore, cells were exposed to a basolateral (140 mM) to apical (5 mM) K⁺ gradient, in the presence of apically applied 10 µM amiloride (Sigma-Aldrich #A7410, St. Louis, MO, USA) and 10 µM ATP (Sigma-aldrich #A1852). CFTR activity was measured with apical 5 mM Cl⁻ in the presence of apically applied 10 µM amiloride, 10 µM forskolin (Sigma-Aldrich #F3917) followed by 10 µM CFTR_inh172 (Sigma-Aldrich #C2992)^{8 (link)}.

Cells grown on 6.5 mm-inserts were mounted into the EasyMount Ussing Chamber Systems (VCC MC8 Physiologic Instrument) and bathed in bilateral HCO₃⁻KRB continuously gassed with 95% O₂₋ 5% CO₂ and maintained at 37 °C. Monolayers were voltage-clamped to 0 mV and cells were left to equilibrate for at least 20 min before ion transport agonists and inhibitors were added. These were added in the following sequence: amiloride (10 µM, apical; A7410, Sigma-Aldrich), forskolin (Fsk, 10 µM, bilateral; 1099, Tocris Bioscience), potentiator 5, P5 (dF508act-02, 5 µM, apical; CFF Therapeutics, CFTR Compound Program), and CFTR_inh172 (172, 20 µM, apical; 3430, Tocris Bioscience). Changes in short-circuit current (ΔIsc) were monitored using Ag/AgCl reference electrodes. The transepithelial short-circuit current (Isc) and the transepithelial electrical resistance (TEER) were recorded using Ag-AgCl electrodes in 3 M KCl agar bridges and the Acquire & Analyze software (Physiologic Instruments, San Diego, CA, USA), as previously described [52 (link)], and results normalized to an area of 1 cm² and expressed as Isc (µAmp.cm⁻²).