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Fatty acids (including lubiprostone and cobiprostone) are human ClC-2 (hClC-2) Cl−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 EP4receptor agonist activity. All fatty acids [lubiprostone, cobiprostone, eicosatetraynoic acid (ETYA), oleic acid, and elaidic acid] caused significant rightward shifts in concentration-dependent Cl−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]iin hClC-2/293EBNA or T84 cells. Using T84 CFTR knockdown cells, cobiprostone increased hClC-2 Cl−currents without increasing [cAMP]i,while PGE2and forskolin-IBMX increased both. Fatty acids were not agonists of EP2or EP4receptors. L-161,982, a supposed EP4-selective inhibitor, had no effect on lubiprostone-activated hClC-2 Cl−currents but significantly decreased T84 cell barrier function measured by transepithelial resistance and fluorescent dextran transepithelial movement. The present findings show that RGET691of hClC-2 (possible binding site) plays an important functional role in fatty acid activation of hClC-2. PKA, [cAMP]i, and EP2or EP4receptors are not involved. These studies provide the molecular basis for fatty acid regulation of hClC-2.

Objective: HBE cells contain both ClC-2 and CFTR Cl- channels. The objective was to identify the Cl- channel in HBE cells that is activated by the prostone, cobiprostone (cobi) using knockdowns and...

G A A b st ra ct s 1.01 (95% CI, 0.83-1.23) for women who used 0.5 to 1.5 standard tablets/week, 1.25 (95% CI, 1.02-1.53) for 2 to 5 tablets/week, 1.71 (95% CI, 1.38-2.13) for 6-14 tablets/week, and 2.11 (95% CI, 1.53-2.91) for >14 tablets/week (P 5 years; P