Regulation of mechanosensitive biliary epithelial transport by the epithelial Na⁺ channel

Intrahepatic biliary epithelial cells (BECs), also known as cholangiocytes, modulate the volume and composition of bile through the regulation of secretion and absorption. While mechanosensitive Cl^- efflux has been identified as an important secretory pathway, the counterabsorptive pathways have not been identified. In other epithelial cells, the epithelial Na⁺ channel (ENaC) has been identified as an important contributor to fluid absorption; however, its expression and function in BECs have not been previously studied. Our studies revealed the presence of α, β, and γ ENaC subunits in human BECs and α and γ subunits in mouse BECs. In studies of confluent mouse BEC monolayers, the ENaC contributes to the volume of surface fluid at the apical membrane during constitutive conditions. Further, functional studies using whole-cell patch clamp of single BECs demonstrated small constitutive Na⁺ currents, which increased significantly in response to fluid-flow or shear. The magnitude of Na⁺ currents was proportional to the shear force, displayed inward rectification and a reversal potential of +40 mV (E_Na+=+60 mV), and were abolished with removal of extracellular Na⁺ (N-methyl-d-glucamine) or in the presence of amiloride. Transfection with ENaCα small interfering RNA significantly inhibited flow-stimulated Na⁺ currents, while overexpression of the α subunit significantly increased currents. ENaC-mediated currents were positively regulated by proteases and negatively regulated by extracellular adenosine triphosphate. Conclusion: These studies represent the initial characterization of mechanosensitive Na⁺ currents activated by flow in biliary epithelium; understanding the role of mechanosensitive transport pathways may provide strategies to modulate the volume and composition of bile during cholestatic conditions.