Varying selection pressure for a Na+ sensing site in epithelial Na+ channel subunits reflect divergent roles in Na+ homeostasis

The epithelial Na+ channel (ENaC) emerged early in vertebrates and has played a role in Na+ and fluid homeostasis throughout vertebrate evolution. We previously showed that proteolytic activation of the channel evolved at the water to land transition of vertebrates. Sensitivity to extracellular Na+, known as Na+ self-inhibition, reduces ENaC function when Na+ concentrations are high and is a distinctive feature of the channel. A fourth ENaC subunit, {delta}, emerged in jawed fishes from an subunit gene duplication. Here, we analyzed 846 and {delta} subunit sequences and found that a key Asp in a postulated Na+ binding site was nearly always present in the subunit, but frequently lost in the {delta} subunit (e.g., human). Analysis of site evolution and codon substitution rates provide evidence that the ancestral subunit had the site and that purifying selection for the site relaxed in the {delta} subunit after its divergence from the subunit, coinciding with a loss of {delta} subunit expression in renal tissues. We also provide evidence that the proposed Na+ binding site in the subunit is a bona fide site by conferring novel function to channels comprising human {delta} subunits. Together, our findings provide evidence that ENaC Na+ self-inhibition improves fitness through its role in Na+ homeostasis in vertebrates.