Identification of the fatty acid activation site on human ClC-2

John Cuppoletti, Kirti P. Tewari, Jayati Chakrabarti, Danuta H. Malinowska
2017 American Journal of Physiology - Cell Physiology  
Fatty acids (including lubiprostone and cobiprostone) are human ClC-2 (hClC-2) Cl Ϫ channel activators. Molecular and cellular mechanisms underlying this activation were examined. Role of a four-amino acid PKA activation site, RGET691, of hClC-2 was investigated using wild-type (WT) and mutant (AGET, RGEA, and AGAA) hClC-2 expressed in 293EBNA cells as well as involvement of PKA, intracellular cAMP concentration ([cAMP]i), EP2, or EP4 receptor agonist activity. All fatty acids [lubiprostone,
more » ... s [lubiprostone, cobiprostone, eicosatetraynoic acid (ETYA), oleic acid, and elaidic acid] caused significant rightward shifts in concentrationdependent Cl Ϫ current activation (increasing EC50s) with mutant compared with WT hClC-2 channels, without changing time and voltage dependence, current-voltage rectification, or methadone inhibition of the channel. As with lubiprostone, cobiprostone activation of hClC-2 occurred with PKA inhibitor (myristoylated protein kinase inhibitor) present or when using double PKA activation site (RRAA655/RGEA691) mutant. Cobiprostone did not activate human CFTR. Fatty acids did not increase [cAMP]i in hClC-2/293EBNA or T84 cells. Using T84 CFTR knockdown cells, cobiprostone increased hClC-2 Cl Ϫ currents without increasing [cAMP]i, while PGE2 and forskolin-IBMX increased both. Fatty acids were not agonists of EP2 or EP4 receptors. L-161,982, a supposed EP4-selective inhibitor, had no effect on lubiprostone-activated hClC-2 Cl Ϫ currents but significantly decreased T84 cell barrier function measured by transepithelial resistance and fluorescent dextran transepithelial movement. The present findings show that RGET691 of hClC-2 (possible binding site) plays an important functional role in fatty acid activation of hClC-2. PKA, [cAMP]i, and EP2 or EP4 receptors are not involved. These studies provide the molecular basis for fatty acid regulation of hClC-2. RESULTS Putative fatty acid activation site on hClC-2. To investigate the possibility of direct interaction of fatty acids with the hClC-2 Cl Ϫ channel, a series of mutant hClC-2 channels was used. In preliminary studies a four amino acid sequence, C709 FATTY ACID ACTIVATION SITE ON HUMAN ClC-2 AJP-Cell Physiol •
doi:10.1152/ajpcell.00267.2016 pmid:28424169 fatcat:znzjsohwbbcolgcvpixncqf54i