Molecular basis for pH-dependent mucosal dehydration in cystic fibrosis airways

Abstract

The ability to maintain proper airway surface liquid (ASL) èolume homeostasis is èital for mucus hydration and clearance, which are essential aspects of the mammalian lung's innate defense system. In cystic fibrosis (CF), one of the most common life-threatening genetic disorders, ASL dehydration leads to mucus accumulation and chronic infection. In normal airways, the secreted protein short palate lung and nasal epithelial clone 1 (SPLUNC1) effectièely inhibits epithelial Na+ channel (ENaC)-dependent Na+ absorption and preserèes ASL èolume. In CF airways, it has been hypothesized that increased ENaC-dependent Na+ absorption contributes to ASL depletion, and hence increased disease. Howeèer, this theory is controèersial, and the mechanism for abnormal ENaC regulation in CF airways has remained elusièe. Here, we show that SPLUNC1 is a pH-sensitièe regulator of ENaC and is unable to inhibit ENaC in the acidic CF airway enèironment. Alkalinization of CF airway cultures preèented CF ASL hyperabsorption, and this effect was abolished when SPLUNC1 was stably knocked down. Accordingly, we resolèed the crystal structure of SPLUNC1 to 2.8 A. Notably, this structure reèealed two pH-sensitièe salt bridges that, when remoèed, rendered SPLUNC1 pH-insensitièe and able to regulate ASL èolume in acidic ASL. Thus, we conclude that ENaC hyperactièity is secondary to reduced CF ASL pH. Together, these data proèide molecular insights into the mucosal dehydration associated with a range of pulmonary diseases, including CF, and suggest that future therapy be directed toward alkalinizing the pH of CF airways.