Data for 2023 PNAS publication on Airway Surface Liquid (ASL) pH in Cystic Fibrosis (CF) human airway cells

In Cystic Fibrosis (CF), defects in the CF transmembrane conductance regulator (CFTR) channel leads to an acidic airway surface liquid (ASL), which compromises innate defence mechanisms, predisposing to pulmonary failure. Restoring ASL pH is a potential therapy for people with CF, particularly for those who cannot benefit from current highly effective modulator therapy (HEMT). However, we lack a comprehensive understanding of the complex mechanisms underlying ASL pH regulation. The calcium-activated chloride channel, TMEM16A, and the anion exchanger, SLC26A4, have been proposed as targets for restoring ASL pH, but current results are contradictory and often utilise non-physiological conditions. To provide better evidence for a role of these two proteins in ASL pH homeostasis we developed an efficient CRISPR-Cas9-based approach to knock-out (KO) relevant transporters in primary airway basal cells lacking CFTR, and then measured dynamic changes in ASL pH under thin-film conditions in fully differentiated 2D airway cultures, which better simulate the in vivo situation. Unexpectantly, we found that both proteins regulated steady-state as well as agonist-stimulated ASL pH, but only under inflammatory conditions. Furthermore, we identified two FDA-approved drugs which raised ASL pH by activating SLC26A4. While we identified a role for SLC26A4 in fluid absorption, KO had no effect on cAMP-stimulated fluid secretion in 3D airway organoids. Overall, we have identified a novel role of TMEM16A in ASL pH homeostasis and shown that both TMEM16A and SLC26A4 could be important alternative targets for ASL pH therapy in CF, particularly for those people who do not produce any functional CFTR.