{"status":"ok","message-type":"work","message-version":"1.0.0","message":{"indexed":{"date-parts":[[2024,7,1]],"date-time":"2024-07-01T14:37:15Z","timestamp":1719844635464},"reference-count":44,"publisher":"American Physiological Society","issue":"6","funder":[{"name":"Sucampo AG"}],"content-domain":{"domain":[],"crossmark-restriction":false},"short-container-title":["American Journal of Physiology-Cell Physiology"],"published-print":{"date-parts":[[2017,6,1]]},"abstract":"<jats:p> Fatty acids (including lubiprostone and cobiprostone) are human ClC-2 (hClC-2) Cl<jats:sup>\u2212<\/jats:sup> channel activators. Molecular and cellular mechanisms underlying this activation were examined. Role of a four-amino acid PKA activation site, RGET<jats:sub>691<\/jats:sub>, 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]<jats:sub>i<\/jats:sub>), EP<jats:sub>2<\/jats:sub>, or EP<jats:sub>4<\/jats:sub> receptor agonist activity. All fatty acids [lubiprostone, cobiprostone, eicosatetraynoic acid (ETYA), oleic acid, and elaidic acid] caused significant rightward shifts in concentration-dependent Cl<jats:sup>\u2212<\/jats:sup> current activation (increasing EC<jats:sub>50<\/jats:sub>s) 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 (RRAA<jats:sub>655<\/jats:sub>\/RGEA<jats:sub>691<\/jats:sub>) mutant. Cobiprostone did not activate human CFTR. Fatty acids did not increase [cAMP]<jats:sub>i<\/jats:sub> in hClC-2\/293EBNA or T84 cells. Using T84 CFTR knockdown cells, cobiprostone increased hClC-2 Cl<jats:sup>\u2212<\/jats:sup> currents without increasing [cAMP]<jats:sub>i,<\/jats:sub> while PGE<jats:sub>2<\/jats:sub> and forskolin-IBMX increased both. Fatty acids were not agonists of EP<jats:sub>2<\/jats:sub> or EP<jats:sub>4<\/jats:sub> receptors. L-161,982, a supposed EP<jats:sub>4<\/jats:sub>-selective inhibitor, had no effect on lubiprostone-activated hClC-2 Cl<jats:sup>\u2212<\/jats:sup> currents but significantly decreased T84 cell barrier function measured by transepithelial resistance and fluorescent dextran transepithelial movement. The present findings show that RGET<jats:sub>691<\/jats:sub> of hClC-2 (possible binding site) plays an important functional role in fatty acid activation of hClC-2. PKA, [cAMP]<jats:sub>i<\/jats:sub>, and EP<jats:sub>2<\/jats:sub> or EP<jats:sub>4<\/jats:sub> receptors are not involved. These studies provide the molecular basis for fatty acid regulation of hClC-2. <\/jats:p>","DOI":"10.1152\/ajpcell.00267.2016","type":"journal-article","created":{"date-parts":[[2017,4,20]],"date-time":"2017-04-20T01:00:29Z","timestamp":1492650029000},"page":"C707-C723","source":"Crossref","is-referenced-by-count":7,"title":["Identification of the fatty acid activation site on human ClC-2"],"prefix":"10.1152","volume":"312","author":[{"given":"John","family":"Cuppoletti","sequence":"first","affiliation":[{"name":"Department of Molecular and Cellular Physiology, University of Cincinnati, Cincinnati, Ohio"}],"role":[{"role":"author","vocabulary":"crossref"}]},{"given":"Kirti P.","family":"Tewari","sequence":"additional","affiliation":[{"name":"Department of Molecular and Cellular Physiology, University of Cincinnati, Cincinnati, Ohio"}],"role":[{"role":"author","vocabulary":"crossref"}]},{"given":"Jayati","family":"Chakrabarti","sequence":"additional","affiliation":[{"name":"Department 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