It has been difficult to separate/identify the roles of ClC-2 and CFTR in Cl- transport studies. The present study aimed to differentiate functionally between ClC-2 and CFTR Cl- channel currents using pharmacological agents. Effects of CFTRinhibitor172 (CFTRinh172), DASU-02 (N-(4-methylphenylsulfonyl)-N′-(4-trifluoromethylphenyl)urea) and methadone were examined by whole cell patch clamp on Cl- currents in recombinant human ClC-2/293EBNA and human CFTR/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 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-EBIO (1-ethyl-2-benzimidazolinone), 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 wherein ClC-2 or CFTR was knocked down (KD) using siRNAs were constructed. T84 ClC-2 KD cells had no response to lubiprostone, but responded to forskolin/IBMX in a methadone-insensitive, DASU-02-sensitive manner indicating CFTR function. T84 CFTR KD cells responded separately to both lubiprostone and forskolin/IBMX in a methadone-sensitive and DASU-02-insensitive manner indicating ClC-2 function. Low lubiprostone concentrations activated ClC-2, 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.
- Copyright © 2014, American Journal of Physiology - Cell Physiology