The cAMP-dependent anion channel activity of the cystic fibrosis transmembrane conductance regulator (CFTR) on the plasma membrane of intact cells can be detected by observing the iodide efflux using an iodide-selective electrode after stimulation with forskolin (Long and Walsh, 1997, Methods in Cell Science, 19:207-212). However, sensitive and reliable quantification of CFTR channel activity using this approach has been a challenge, limiting its wide use. Rigorous optimization was performed on the electrode-based real-time iodide efflux assay using cystic fibrosis bronchial epithelial cells (CFBE41o-) stably expressing wild-type CFTR (CFBE-WT) or F508del CFTR (CFBE-DF). Iodide-loaded cells were washed with iodide-free buffer and extracellular iodide concentrations were recorded at 37°C under baseline conditions and following forskolin stimulation. Relative efflux rate was calculated by normalizing the maximal forskolin-induced efflux rate to the baseline rate. At least two washes are required to reliably detect cAMP-dependent iodide efflux in CFBE-WT relative to CFBE-DF cells. Although baseline efflux rate decreases with additional washes, the relative efflux rate after 12 washes is comparable to that after 2 washes in CFBE-WT cells. Baseline efflux rate in CFBE-DF cells is 43% lower than in CFBE-WT cells after three washes. The state of cell growth significantly affects assay sensitivity: at 24 h post-seeding, relative efflux rates do not differ between CFBE-WT and CFBE-DF cells, whereas clear differences emerge at 48 h and 72 h, despite equivalent confluency across conditions. The impact of dimethyl sulfoxide (DMSO), a common solvent for CFTR modulators, was further assessed. Concentrations up to 1% are generally tolerated, whereas 5% DMSO abolishes cAMP-dependent iodide efflux in CFBE-WT cells. Finally, assay validity was confirmed by the immediate elimination of forskolin-stimulated iodide efflux in CFBE-WT cells and in CFBE-DF cells rescued with 2 µM Elexacaftor in the presence of 50 µM Genistein upon addition of CFTR channel inhibitor CFTRinh-172. The inclusion of CFTRinh-172 does not have a substantial effect on baseline efflux rate. Together, these findings define assay conditions that provide a robust dynamic range for quantifying functional rescue of F508del CFTR by modulators in airway epithelial cells. The electrode-based iodide efflux assay offers a physiologically relevant, real-time measure of CFTR anion channel activity in intact cells and serves as a valuable complement to Ussing chamber and patch-clamp approaches. This work was supported by NIH (5P30DK072482), a Samford University McWhorter Faculty Research Grant (to X.R.W.), and McWhorter School of Pharmacy Pharmaceutical Summer Research Internship (to P.N. and J.P.). Phi Nguyen and Justin Porter contributed equally to this work. This abstract was presented at the American Physiology Summit 2026 and is only available in HTML format. There is no downloadable file or PDF version. The Physiology editorial board was not involved in the peer review process.
Porter et al. (Fri,) studied this question.