(614.9) Bile acids directly bind and allosterically regulate the epithelial sodium channel

Poster Board Number: E584

Xueping Wang (University of Pittsburgh), Victor Tomilin (University of Texas Health Science Center), Merve Ertem (University of Pittsburgh), Abigail McKernan (University of Pittsburgh), Xiaoguang Lei (Peking University), Oleh Pochynyuk (University of Texas Health Science Center), Ossama Kashlan (University of Pittsburgh)

Bile acids are critical for the digestion and absorption of dietary fats and have important roles as signaling molecules. Bile acids are normally restricted to the enterohepatic circulation, but can be elevated in the blood and urine of patients with advanced liver diseases. The epithelial sodium channel (ENaC) mediates Na+ transport in the biliary epithelium, where it regulates bile viscosity, and in the aldosterone-sensitive distal nephron, where it regulates extracellular fluid volume. We previously showed that bile acid regulation of ENaC depends on the position and stereochemistry of specific functional groups, but not on their chemico-physical properties. To determine whether bile acids regulate ENaC in ex vivo native tissues, we performed patch clamp experiments on isolated split open renal tubules from mice, using recording pipettes backfilled with taurocholic acid (t-CA) or taurohyodeoxycholic acid (t-HDCA). Consistent with in vitro experiments, t-CA increased ENaC open probability (PO) by 2-fold while t-HDCA decreased ENaC PO by nearly two-thirds. To determine whether bile acids regulate ENaC through direct binding, we performed photoaffinity labeling experiments. We employed a bile acid derivative (p-DCA) with diazirine and terminal alkyne groups that facilitate uv-crosslinking and affinity purification, respectively. Using cultured Fisher Rat Thyroid cells transfected with ENaC, we found that the deoxycholic acid derivative (p-DCA) crosslinked to the α, β, and γ subunits. To determine whether crosslinking was specific, we performed experiments in the presence of competing bile acids. Crosslinking to the β subunit was significantly reduced in the presence of deoxycholic acid (DCA). Crosslinking to the γ subunit was significantly reduced by taurocholic acid (t-CA). These data suggest direct binding and multiple sites. To determine the effect of subunit composition on regulation, we measured the effect of t-CA on Li+ currents in Xenopus oocytes expressing different functional ENaC subunit combinations. Channels composed of α subunits alone were strongly activated by t-CA, followed by αβγ- and αβ- channels, suggesting that the α subunit is sufficient for ENaC sensitivity to t-CA. Our data are consistent with bile acids directly binding all three ENaC subunits at multiple sites. Bile acid moieties provide for selective preferences for each of the sites, resulting in distinct effects for each bile acid.

This work was supported by grants from NIDDK, National Institutes of Health: R01 DK125439 to OBK, the Pittsburgh Liver Research Center was supported by P30 DK120531, and the Pittsburgh Center for Kidney Research was supported by P30 DK079307.