Functional characterisation of mammalian epithelial sodium channels and the pathological consequences of sodium hyperabsorption in the airways

Abstract

The epithelial sodium channel (ENaC) plays a key role in salt homeostasis in mammals. Four ENaC subunits (α, β, γ, δ) form heterotrimeric αβγ- or δβγ-ENaCs. While the physiology of αβγ-ENaCs is well understood, the function of δβγ-ENaC is unknown. No standard laboratory animal model is available for studying δ-ENaC physiology as the sodium channel epithelial 1 subunit delta (SCNN1D) gene that encodes δ-ENaC, is absent in rats and mice. Cavia porcellus (guinea pig) has an intact SCNN1D gene. Functional characterisation of guinea pig αβγ- and δβγ-ENaC provides the first evidence of two functional isoforms in rodents. Guinea pig αβγ- and δβγ-ENaC were found to have similar biophysical and regulatory features to their respective human ENaC orthologues. Additionally, functional data suggested mammalian δβγ- ENaC can detect higher sodium concentrations and may fulfil a sensory function over canonical sodium absorption. Guinea pigs represent a commercially available rodent model that allows the investigation of δ-ENaC physiology in mammals. Through facilitating sodium absorption, αβγ-ENaC contributes to maintaining the airway surface liquid. Overexpression of the β-ENaC subunit in the airways of mice (β- ENaC-tg) promotes sodium hyperabsorption and airway surface liquid dehydration causing a pulmonary phenotype similar to chronic obstructive pulmonary disease and cystic fibrosis. Through the application of longitudinal micro-computed tomography, the pathogenesis of β-ENaC-tg was noninvasively recorded in vivo, defining a window for therapeutic intervention and the stages of pathogenesis. While future studies need to determine the cause of the change in phenotype severity, this research demonstrates that β-ENaC-tg mice represent a valuable model for studying diseases involving airway surface liquid dehydration. Airway surface liquid dehydration prevents proper mucociliary clearance. This causes the volume of mucus to increase, reducing the bioavailability of therapeutic drugs. ENaC is a valuable therapeutic target to rehydrate the airway surface liquid. The in vitro mucus penetration rate of the ENaC blocker amiloride was determined by coupling a simulated mucus plug with liquid chromatography-mass spectrometry detection and analysis. Establishing this assay and the mucus penetration rate of amiloride serves as a reference point for novel next generation amiloride-derived molecules designed to better permeate mucus. Additionally, a library of novel amiloride-derived molecules was shown to retain ENaC-inhibitory properties despite being modified in a moiety of the structure which is crucial to ENaC inhibition. This opens a new avenue for modifying the pharmacological properties of amiloride. Overall, this research demonstrates that the guinea pig represents the first viable mammalian model for the study of δ-ENaC physiology. Additionally, micro-computed tomography provides a method to measure the effect of increased ENaC-mediated sodium absorption on pulmonary physiology of β-ENaC-tg mice. Furthermore, this work provides an in vivo option for screening novel, improved ENaC-inhibitors developed in conjunction with the in vitro mucus penetration assay.