CFTR-Mediated Ion Transport Measurements

CFBE or HBE cells expressing WT- or ΔF508-CFTR were grown to confluence on permeable filters at an air-liquid interface and mounted in modified Ussing chambers. I_sc measurements were obtained under voltage clamp conditions using MC8 equipment and P2300 Ussing chambers (Physiologic Instruments) as previously described [51 (link),52 (link),103 (link)]. Cells were equilibrated for 5–10 min in regular Ringer solution (115 mM NaCl, 25 mM NaHCO₃, 2.4 mM KH₂PO₄, 1.24 mM K₂HPO₄, 1.2 mM CaCl₂, 1.2 mM MgCl₂, 10 mM D-glucose, pH 7.4). In some measurements, this was followed by the exchange of low chloride Ringer (115 mM NaCl reduced to 1.2 mM NaCl and 10 mM D-glucose replaced with 115 mM Na-gluconate) to the apical surface. After addition of the sodium channel inhibitor amiloride (100 μM), the CFTR agonists Frk (10 μM) and gen (50 μM) were sequentially added, followed by inhibitor₁₇₂ (10 μM) at the conclusion of each experiment, in order to specifically inhibit CFTR activity. Changes in CFTR-mediated ion transport were calculated using the highest current value for each sample after achievement of a stable plateau for several minutes.