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.