The epithelial sodium channel (ENaC) mediates Na ؉ transport in several epithelia, including the aldosterone-sensitive distal nephron, distal colon, and biliary epithelium. Numerous factors regulate ENaC activity, including extracellular ligands, post-translational modifications, and membrane-resident lipids. However, ENaC regulation by bile acids and conjugated bilirubin, metabolites that are abundant in the biliary tree and intestinal tract and are sometimes elevated in the urine of individuals with advanced liver disease, remains poorly understood. Here, using a Xenopus oocyte-based system to express and functionally study ENaC, we found that, depending on the bile acid used, bile acids both activate and inhibit mouse ENaC. Whether bile acids were activating or inhibiting was contingent on the position and orientation of specific bile acid moieties. For example, a hydroxyl group at the 12-position and facing the hydrophilic side (12␣-OH) was activating. Taurine-conjugated bile acids, which have reduced membrane permeability, affected ENaC activity more strongly than did their more membranepermeant unconjugated counterparts, suggesting that bile acids regulate ENaC extracellularly. Bile acid-dependent activation was enhanced by amino acid substitutions in ENaC that depress open probability and was precluded by proteolytic cleavage that increases open probability, consistent with an effect of bile acids on ENaC open probability. Bile acids also regulated ENaC in a cortical collecting duct cell line, mirroring the results in Xenopus oocytes. We also show that bilirubin conjugates activate ENaC. These results indicate that ENaC responds to compounds abundant in bile and that their ability to regulate this channel depends on the presence of specific functional groups. Epithelial Na ϩ channel (ENaC) 2-mediated Na ϩ transport is rate-limiting for Na ϩ reabsorption in principal cells in the aldo

The epithelial Na + channel (ENaC) is a member of the ENaC/Degenerin family of ion channels. In the structure of a related family member, the 'thumb' domain's base interacts with the pore, and its tip interacts with the divergent 'finger' domain. Between the base and tip, the thumb domain is characterized by a conserved 5-rung disulfide ladder holding together two anti-parallel a helices. The ENaC a and g subunits' finger domains harbor autoinhibitory tracts that can be proteolytically liberated to activate the channel, and also host an ENaC-specific pair of cysteines. Using a crosslinking approach, we show that one of the finger domain cysteines in the a subunit (aC263) and both of the finger domain cysteines in the g subunit (gC213 and gC220) lie near the dynamic finger-thumb domain interface. Our data suggest that the aC256/aC263 pair is not disulfide bonded. In contrast, we found that the gC213/gC220 pair is disulfide bonded. Our data also suggest the g subunit lacks the terminal rung in the thumb domain disulfide ladder, suggesting asymmetry between the subunits. We also observed functional asymmetry between the a and g subunit finger-thumb domain interfaces: crosslinks bridging the a subunit fingerthumb interface only inhibited ENaC currents, while crosslinks bridging the g subunit fingerthumb interface activated or inhibited currents dependent on the length of the crosslinker. Our data suggest that reactive cysteines lie at the dynamic finger-thumb interfaces of the a and g subunits, and may play a yet undefined role in channel regulation.

The epithelial sodium channel (ENaC) mediates Na+ transport in several epithelia, including the aldosterone‐sensitive distal nephron, distal colon, and biliary epithelium. Numerous factors regulate the activity of the channel, including extracellular ligands, post‐translational modifications, and membrane‐resident lipids. Bile acids are abundant in the biliary tree and intestinal tract, and can be elevated in the urine of patients with advanced liver disease. Using Xenopus oocytes, we found that bile acids both activated and inhibited mouse ENaC, dependent on the bile acid. Whether bile acids were activating or inhibiting depended on the position and stereochemistry of specific moieties. Taurine conjugated bile acids had stronger effects than their more membrane permeant unconjugated counterparts, suggesting that bile acids regulate ENaC extracellularly. Bile acids that increased ENaC currents had a hydroxyl group at position 12, facing the hydrophilic side. Bile acids that decreased ENaC currents had a hydroxyl group at position 6. Bile acid dependent activation of ENaC currents was mildly voltage‐dependent, suggesting that regulation occurs in the outer leaflet of the membrane. Bile acids also regulated ENaC in a cortical collecting duct cell line, mirroring results in Xenopus oocytes. These results suggest that bile acids interact directly with ENaC near the interface between the outer leaflet and the extracellular solution.Support or Funding InformationThis work was supported by NIDDK, National Institutes of Health, Grant R01 DK098204 (to O.B.K), and a grant from the Pittsburgh Liver Research Center. The Pittsburgh Center for Kidney Research was supported by Grant DK P30 DK079307 from NIDDK, National Institutes of Health.This abstract is from the Experimental Biology 2019 Meeting. There is no full text article associated with this abstract published in The FASEB Journal.

Vertebrates evolved mechanisms for sodium conservation and gas exchange in conjunction with migration from aquatic to terrestrial habitats. Epithelial Na+ channel (ENaC) function is critical to systems responsible for extracellular fluid homeostasis and gas exchange. ENaC is activated by cleavage at multiple specific extracellular polybasic sites, releasing inhibitory tracts from the channel’s α and γ subunits. Here we investigated the evolution of ENaC regulatory mechanisms to determine which features coevolved with the marine‐terrestrial transition. We consistently found both activating cleavage sites in the ENaC α and γ subunits of terrestrial vertebrates, while they appeared only sporadically in fishes. We confirmed that cleavage occurred at sites found in the γ subunit from Australian lungfish, leading to channel activation. Phylogenetic analysis and likelihood ratio tests showed that proximal and distal polybasic tracts in ENaC subunits coevolved, consistent with the dual cleavage requirement for activation. They also showed a coevolutionary dependence of tandem polybasic tracts with terrestrial status and with lungs, coincident with the ENaC activator aldosterone. Amplification of transcripts by RT‐PCR in ray‐finned Polypteriformes and previously in lobe‐finned lungfish suggest that transcripts for ENaC subunits with cleavage sites are readily detected at important sites of ion exchange (gills and kidney), but are difficult to detect in lungs. Analysis of ancestral reconstructions strongly suggests that the polybasic tracts appeared independently in the α and γ subunits of ENaC. Similar analyses of the PY motif, required for Nedd4‐2 dependent regulation, showed no coevolutionary pattern and that the PY motif first arose in an ancient ancestral ENaC subunit. Our data suggest that changes associated with adaptation to terrestrial life provided selective pressure for the development of ENaC activation by proteolytic cleavage.