Cf inhibitor 172

FRT cells (obtained from Dr. M. Welsh, University of Iowa, Iowa City, IA) stably transduced with F508del CFTR were grown on permeable supports (3378; Costar) in Coon’s modified media containing 11.5g/l of F12 Ham nutrient mixture (Sigma-Aldrich, St. Louis, MO), 2.68g/l sodium bicarbonate, and 5% FBS. Transepithelial conductance of the cells was measured using a 24 channel current clamp (EP-Devices, Bertum, Belgium) equipped robot (PrecisePlace 2,300 Robot; Precise Automation Inc, La Jolla, CA) [22 (link), 23 (link)]. Cells were bathed in FBS and sodium bicarbonate-free media and treated first with 10 μM amiloride followed by an agonist (20 μM forskolin), and then with a CF inhibitor 172 (10 μM; Sigma-Aldrich) to block CFTR dependent conductance.

CFBE41o-cells expressing WT or F508del CFTR were seeded onto permeable supports (Costar) after coating with fibronectin [24 (link)]. Cells were grown to confluence and transferred to an air—liquid interface, after which they were mounted in modified Ussing chambers. Monolayers were initially bathed on both sides with identical Ringers solution containing (in mM): 115 NaCl, 25 NaHCO₃, 2.4 KH₂PO₄, 1.24 K₂HPO₄, 1.2 CaCl₂, 1.2 MgCl₂, 10 D-glucose (pH 7.4) and vigorously stirred and gassed with 95%O₂: 5% CO₂ at 37°C. Short-circuit current (Isc) was obtained using an epithelial voltage clamp. The mucosal bathing solution was changed to a low Cl^- solution containing (in mM): 1.2 NaCl, 115 Na gluconate, plus 100 μM amiloride followed by addition of agonists (20 μM forskolin, 50 μM genistein, and/or 10 μM ivacaftor) to the mucosal surface. CF inhibitor 172 (10 μM; Sigma-Aldrich, St. Louis, MO) was added to the bathing solution at the conclusion of each experiment to block CFTR-dependent Isc.