A notch-regulated cell type in human airway epithelia performs active CFTR-mediated chloride secretion but lacks a passive CFTR-dependent pathway for apical-to-basolateral chloride flow

Cystic fibrosis (CF) results from mutations in the CFTR gene, which encodes an anion channel critical for airway physiology. Airway disease is the major cause of morbidity in CF, yet the cellular basis of cystic fibrosis transmembrane conductance regulator (CFTR)-mediated Cl– transport remains incompletely understood. Recent single cell transcriptomic studies revealed that CFTR mRNA expression is concentrated in rare ionocytes. Historically, CFTR in airway epithelia was thought to drive Cl– secretion; however, our previous reports indicate that ionocytes mediate Cl– absorption rather than secretion. The finding raises a fundamental question: which cells drive Cl– secretion in human airways? We hypothesized that CFTR in secretory cells—named for their secretory vesicles—perform Cl– secretion. To test this hypothesis, we engineered n = 6 primary human airway epithelial cultures to selectively deplete ionocytes or secretory cells by disrupting cell signaling essential for their cell type specification. We eliminated ionocytes by CRISPR-mediated disruption of FOXI1 and reduced the number of secretory cells by inhibiting notch signaling with the γ-secretase inhibitor N-N-(3,5-Difluorophenacetyl)-L-alanyl-S-phenylglycine t-butyl ester (DAPT). Cell populations were quantified by flow cytometry, and CFTR-dependent currents were measured using the transepithelial voltage-clamp technique under conditions favoring apical-to-basolateral Cl– flow (i.e., an apical-to-basolateral Cl– gradient, Vt = 0 mV) or active secretion (i.e., symmetrical Cl–, Vt = 0 mV). Approrate multivariant statistics were applied to each dataset and adjusted for multiple comparisons. FOXI1 disruption (gFOXI1) eliminated ionocytes without affecting other cell types and abolished apical-to-basolateral Cl– flow without affecting active secretion Igradient (µA*cm–2) control 1.21 ± 0.69 vs. gFOXI1 -0.53 ± 0.83, p = 0.03 and Isc (µA*cm–2) control 12.28 ± 6.53 vs. gFOXI1 12.36 ± 5.56, p = 0.99. This finding is consistent with our previous reports that ionocytes perform Cl– absorption and inefficiently perform active Cl– secretion. In contrast, DAPT-treated epithelia retained apical-to-basolateral Cl– flow but exhibited markedly reduced active secretion Igradient (µA*cm–2) control 1.21 ± 0.69 vs. DAPT 1.41 ± 1.18, p = 0.99 and Isc (µA*cm–2), control 12.28 ± 6.53 vs. DAPT 2.82 ± 2.32, p = 0.03. Flow cytometry revealed DAPT did not decrease ionocytes or the larger population of CEACAM6+ secretory cells but eliminated a subset of MUC5AC+ secretory cells raising the possibility that another rare CFTR-containing cell may oppose CFTR-mediated absorption by ionocytes ionocytes (% of epithelial cells) control 0.27 ± 0.17 vs. DAPT 0.14 ± 0.10, p = 0.12; secretory cells (% of epithelial cells) control 37.29 ± 21.97 vs. DAPT 32.70 ± 18.95, p = 0.40; and MUC5AC+ (% of epithelial cells), control 0.97 ± 1.88 vs. DAPT 0.49 ± 0.99, p = 0.04.These findings identify secretory cells as a driver of Cl– secretion, while ionocytes mediate absorption. Together, these results suggest a division of labor among airway epithelial cells with CFTR function partitioned between two specialized cell types. The proposed model provides a framework for understanding CF pathogenesis and informing strategies for targeted therapies. 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.