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, δ, emerged in jawed fishes from an α subunit gene duplication. Here, we analyzed 846 α and δ subunit sequences and found that a key Asp in a postulated Na⁺binding site was nearly always present in the α subunit, but frequently lost in the δ subunit (e.g., human). Analysis of site evolution and codon substitution rates provide evidence that the ancestral α subunit had the site and that purifying selection for the site relaxed in the δ subunit after its divergence from the α subunit, coinciding with a loss of δ subunit expression in renal tissues. We also provide evidence that the proposed Na⁺binding site in the α subunit is a bona fide site by conferring novel function to channels comprising human δ subunits. Together, our findings provide evidence that ENaC Na⁺self-inhibition improves fitness through its role in Na⁺homeostasis in vertebrates.