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 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.

Cirrhosis of the liver often leads to edema and increased circulating and urinary levels of bile acids and bilirubin conjugates. We hypothesized that ENaC activation by increased concentrations of biliary metabolites in renal tubular ultrafiltrate could contribute to renal Na+ retention and edema in cirrhosis of the liver. The epithelial Na+ channel (ENaC) plays a key role in regulating Na+ reabsorption in the kidney, and is regulated by an array of factors, including extracellular ligands (e.g. H+, Na+, Cl−) and post‐translational modifications (e.g. cleavage and palmitoylation). To begin testing this, we expressed wild type and mutant ENaCs in Xenopus oocytes and measured effects on channel current by two‐electrode voltage clamp. We found that specific bile acids and conjugated‐bilirubin activate ENaC in a dose‐dependent manner, with deoxycholic acid having the strongest stimulatory effect. Notably, deoxycholic acid stimulated both wild type channels and ‘near silent’ channels in which the furin cleavage sites were mutated. However, fully activating channels by trypsin abolished deoxycholic acid stimulation in both wild type channels and channels lacking furin cleavage sites, consistent with an effect on open probability. We hypothesized that bile acids, conjugated bilirubin and Cys‐palmitoylation (palmitate addition to specific intracellular cysteines on the β and γ subunits) regulate ENaC currents through a common mechanism: by increasing the membrane association of specific intracellular structures, driving conformation changes in the channel's pore. To begin testing our hypothesis, we determined whether mutation of all 4 palmitoylation sites in the β and γ subunits (βC43, βC557, γC33 and γC41) to alanine would affect activation by deoxycholic acid. We found that deoxycholic acid stimulated currents of ENaC lacking of all 4 palmitoylation site cysteines by 7‐fold over wild type ENaC. When we tested the effect of mutating individual sites, we found that removal of the N‐terminal palmitoylation site cysteines resulted in the strongest stimulatory effects. We also found that deoxycholic acid had a stronger stimulatory effect when we removed both sites from the γ subunit as compared to the β subunit. Our results demonstrate that ENaC can be activated by biliary metabolites, including bile acids and bilirubin, and that these metabolites may activate ENaC through a mechanism in common with ENaC activation by palmitoylation.Support or Funding InformationDK098204 to O.B.K. and DK110332 to E.C.R.This abstract is from the Experimental Biology 2018 Meeting. There is no full text article associated with this abstract published in The FASEB Journal.