title: Differentiation between human ClC-2 and CFTR Cl- channels with pharmacological agents.
abstract: It has been difficult to separate/identify the roles of ClC-2 and CFTR in Cl(-) transport studies. Using pharmacological agents, we aimed to differentiate functionally between ClC-2 and CFTR Cl(-) channel currents. Effects of CFTR inhibitor 172 (CFTRinh172), N-(4-methylphenylsulfonyl)-N'-(4-trifluoromethylphenyl)urea (DASU-02), and methadone were examined by whole cell patch clamp on Cl(-) currents in recombinant human ClC-2/human embryonic kidney 293 (ClC-2/HEK293) cells stably transformed with Epstein-Barr nuclear antigen 1 (hClC-2/293EBNA) and human CFTR/HEK293 (hCFTR/HEK293) cells and by short-circuit current (Isc) measurements in T84 cells. Lubiprostone and forskolin-IBMX were used as activators. CFTRinh172 inhibited forskolin-IBMX-stimulated recombinant human CFTR (hCFTR) and lubiprostone-stimulated recombinant human ClC-2 (hClC-2) Cl(-) currents in a concentration-dependent manner equipotently. DASU-02 inhibited forskolin-IBMX-stimulated Cl(-) currents in hCFTR/HEK293 cells, but not lubiprostone-stimulated Cl(-) currents in hClC-2/293EBNA cells. In T84 cells with basolateral nystatin or 1-ethyl-2-benzimidazolinone (1-EBIO), lubiprostone-stimulated and forskolin-IBMX-cyclosporin A (FICA)-stimulated Isc components were observed. CFTRinh172 inhibited major portions of both components. DASU-02 had no effect on lubiprostone-stimulated Isc but partially inhibited FICA-stimulated Isc. T84 cells in which ClC-2 or CFTR was knocked down using siRNAs were constructed. T84 ClC-2 knockdown cells did not respond to lubiprostone but did respond to forskolin-IBMX in a methadone-insensitive, DASU-02-sensitive manner, indicating CFTR function. T84 CFTR knockdown cells responded separately to lubiprostone and forskolin-IBMX in a methadone-sensitive and DASU-02-insensitive manner, indicating ClC-2 function. Low lubiprostone concentrations activated ClC-2, but not CFTR, and both channels were activated by forskolin-IBMX but have different inhibitor sensitivities. Methadone, but not DASU-02, inhibited ClC-2. DASU-02, but not methadone, inhibited CFTR. In T84 cells, both ClC-2 and CFTR are present and likely play roles in Cl(-) secretion.
authors.affiliation: Department of Molecular and Cellular Physiology, University of Cincinnati College of Medicine, Cincinnati, Ohio John.Cuppoletti@uc.edu.
authors.forename: John
authors.initials: J
authors.lastname: Cuppoletti
authors.affiliation: Department of Molecular and Cellular Physiology, University of Cincinnati College of Medicine, Cincinnati, Ohio.
authors.forename: Jayati
authors.initials: J
authors.lastname: Chakrabarti
authors.affiliation: Department of Molecular and Cellular Physiology, University of Cincinnati College of Medicine, Cincinnati, Ohio.
authors.forename: Kirti P
authors.initials: KP
authors.lastname: Tewari
authors.affiliation: Department of Molecular and Cellular Physiology, University of Cincinnati College of Medicine, Cincinnati, Ohio.
authors.forename: Danuta H
authors.initials: DH
authors.lastname: Malinowska
mesh_terms: D015056:1-Methyl-3-isobutylxanthine / Q000494:pharmacology; D000090007:CLC-2 Chloride Channels; D002460:Cell Line; D018118:Chloride Channels / Q000037:antagonists & inhibitors* / Q000502:physiology*; D019005:Cystic Fibrosis Transmembrane Conductance Regulator / Q000037:antagonists & inhibitors* / Q000502:physiology*; D004305:Dose-Response Relationship, Drug; D004847:Epithelial Cells / Q000187:drug effects; D057809:HEK293 Cells; D006801:Humans; D008691:Methadone / Q000494:pharmacology; D014508:Urea / Q000031:analogs & derivatives / Q000494:pharmacology
chemical_list: C505032:CFTR protein, human; D000090007:CLC-2 Chloride Channels; D018118:Chloride Channels; C416188:N-(4-methylphenylsulfonyl)-N'-(4-trifluoromethylphenyl)urea; D019005:Cystic Fibrosis Transmembrane Conductance Regulator; D014508:Urea; D015056:1-Methyl-3-isobutylxanthine; D008691:Methadone
keywords: CFTR inhibitor 172; DASU-02; forskolin; lubiprostone; methadone