Regulation of TMEM16A by a novel mechanism involving PKC in biliary epithelium

TMEM16A is a Ca 2+ ‐activated Cl − channel in the apical membrane of biliary epithelial cells (BECs), and contributes to biliary secretion and bile formation [Dutta et al. JBC, 2010]. Extracellular nucleotides activate TMEM16A via binding P2 receptors and increasing [Ca 2+ ] i . While PKC contributes to ATP‐mediated secretion in BECs, the role in TMEM16A regulation is unknown. Aim To determine if PKC contributes to Cl − transport in BECs through regulation of TMEM16A. Methods Studies were performed in human biliary Mz‐cha‐1 cells. Cl − currents were measured by whole‐cell patch clamp. Results TMEM16A Cl − currents were activated by exposure to extracellular ATP or increasing Ca 2+ in the patch pipette. Currents had a reversal potential of 0 mV, time‐dependent activation and outward rectification. Currents were significantly inhibited by anti‐TMEM16A siRNA. In the presence of the PKC inhibitors Chelerithrine or Gö6976 Cl − currents were significantly inhibited. Conversely, activation by PMA resulted in large Cl − currents. Intracellular dialysis with recombinant PKCα activated Cl − currents with identical biophysical properties to TMEM16A. Conclusion PKCα couples ATP‐mediated signaling to TMEM16A activation in BECs. Modulation of kinase signaling pathways may represent a target for increasing biliary secretion and bile formation. [Studies are supported by AASLD/ALF Liver Scholar Award 2010, Children's Medical Center Foundation grant (Dutta), and NIH NIDDK DK078587]