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Cleavage of ENaC α and γ Subunits Evolved with the Terrestrial Migration

The epithelial Na+ channel (ENaC) is critical to extracellular fluid volume regulation through its activity in several epithelia, including the aldosterone‐sensitive distal nephron and colon, and the airway and alveoli. Human ENaC subunits are subject to activating proteolysis. During processing in the trans‐Golgi network, furin cleaves the α subunit twice. This event liberates an imbedded 26‐mer inhibitory tract, moderately activates the channel, and leaves 30 kDa and 65 kDa fragments as parts of the channel complex. Furin cleaves the γ subunit once. This event has no effect on channel activity, but leaves 18 kDa and 75 kDa fragments as parts of the channel complex. Once at the cell surface, one of a number of proteases may cleave the γ subunit in a region ~35–50 residues distal to the furin cleavage site. Some of these proteases are hormonally regulated (e.g. prostasin), while others may be present in disease states (e.g. plasmin). This second cleavage event of the γ subunit also releases an imbedded inhibitory tract, 43‐residues when prostasin is the second protease, and greatly activates the channel. To examine the evolution of ENaC cleavage sites, we generated a phylogenetic tree of ENaC and ENaC‐like subunits to identify key branch points in the evolution of ENaC subunits. The four ENaC subunits arose from three gene duplication events. First, a single gene diverged to an α/δ precursor and a β/γ precursor. Shortly after, the β/γ precursor diverged to β and γ subunits. These two events occurred in time for the emergence of jawless fishes, as lampreys have α, β, and γ subunits. Later, but prior to the terrestrial migration, α and δ diverged from the α/δ precursor; the lobe‐finned coelacanth is the first known species to have all four subunits. We observed that the activating cleavage sites in the α and γ subunits appeared around the transition to terrestrial life. Among the fishes, only the Australian lungfish γ subunit had two cleavage sites, and all tetrapod α and γ subunits had two apparent cleavage sites. We ran nested likelihood models in BayesTraits and found a significant relationship between terrestrial status and having two cleavage sites in the α and γ subunits. Both sites showed a statistically significant coevolutionary pattern with the terrestrial state: proximal site, P = 0.00325; distal site, P = 0.0486. This patterns contrasts with the C‐terminal PY motif, which is important for aldosterone regulation through Sgk1/Nedd4‐2, and was present in ENaC α, β and γ subunits from all species, but not in the δ subunit of any species. The vertebrate terrestrial migration is associated with many changes, including accessibility to dietary Na+, differences in osmotic stress, and the development of lungs. One or a combination of these factors may have provided the selection pressure to develop this form of channel regulation.