Bile Acids Directly Bind ENaC and Allosterically Regulate its Activity

Abstract

The epithelial sodium channel (ENaC) mediates Na+ transport in several epithelia, including the aldosterone‐sensitive distal nephron, distal colon, and biliary epithelium. ENaC is a heterotrimer composed of α, β, and γ subunits. Numerous extracellular ligands regulate the activity of the channel. Bile acids are abundant in the biliary tree and intestinal tract, and can be elevated in the urine of patients with advanced liver disease. Our previous work showed that bile acids that regulate ENaC activity have specific chemical moieties, but are not necessarily membrane permeable. We hypothesized that bile acids directly bind ENaC. To test this, we used light‐sensitive bile acid probes derived from cholic acid and deoxycholic acid (p‐CA and p‐DCA). We incubated Fisher Rat Thyroid cells expressing V5‐tagged ENaC subunits with p‐CA and p‐DCA. After brief exposure to light, crosslinked proteins were conjugated to biotin, precipitated with streptavidin, subjected to SDS‐PAGE/immunoblotting with detection by anti‐V5. We found that both p‐DCA and p‐CA crosslinked to α, β, and γ ENaC subunits. In competition experiments with excess unlabeled bile acids, both DCA and CA decreased crosslinking by the corresponding bile acid. DCA competition vs. p‐DCA was more effective than CA competition vs. p‐CA. These data show that these bile acids directly and specifically bind ENaC subunits. Using Xenopus oocytes, we observed that taurocholic acid (t‐CA) activation of ENaC currents is voltage‐dependent. Modeling of the voltage‐dependent data suggests at least two binding sites, consistent with the crosslinking data. We also determined whether t‐CA regulated currents of αβγ, α‐only, αβ‐, and αγ‐channels. We found that α‐only channels were greatly activated by t‐CA, followed by αβγ and αβ channels. Our data suggest that the a subunit is sufficient for bile acid regulation. Our results suggest that bile acids regulate ENaC through direct ligand‐channel interactions.