Identification of the fatty acid activation site on human ClC-2
| Autor: | Jayati Chakrabarti, Danuta H. Malinowska, Kirti P. Tewari, John Cuppoletti |
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| Rok vydání: | 2017 |
| Předmět: |
0301 basic medicine
Physiology Stereochemistry Amino Acid Motifs Cystic Fibrosis Transmembrane Conductance Regulator Oleic Acids Arachidonic Acids Thiophenes 03 medical and health sciences Lubiprostone 0302 clinical medicine Chlorides Chloride Channels 1-Methyl-3-isobutylxanthine Cyclic AMP medicine Humans Protein Kinase Inhibitors chemistry.chemical_classification Binding Sites Ion Transport Chemistry Cobiprostone Colforsin Fatty acid Cell Biology Receptors Prostaglandin E EP2 Subtype Triazoles Cyclic AMP-Dependent Protein Kinases CLC-2 Chloride Channels Kinetics HEK293 Cells 030104 developmental biology Biochemistry Prostaglandins Identification (biology) Channel (broadcasting) Receptors Prostaglandin E EP4 Subtype Methadone 030217 neurology & neurosurgery Oleic Acid Protein Binding medicine.drug |
| Zdroj: | American Journal of Physiology-Cell Physiology. 312:C707-C723 |
| ISSN: | 1522-1563 0363-6143 |
| Popis: | 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. |
| Databáze: | OpenAIRE |
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