In:
American Journal of Physiology-Cell Physiology, American Physiological Society, Vol. 307, No. 5 ( 2014-09-01), p. C479-C492
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 (CFTR inh 172), 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 ( I sc ) measurements in T84 cells. Lubiprostone and forskolin-IBMX were used as activators. CFTR inh 172 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 I sc components were observed. CFTR inh 172 inhibited major portions of both components. DASU-02 had no effect on lubiprostone-stimulated I sc but partially inhibited FICA-stimulated I sc . 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.
Type of Medium:
Online Resource
ISSN:
0363-6143
,
1522-1563
DOI:
10.1152/ajpcell.00077.2014
Language:
English
Publisher:
American Physiological Society
Publication Date:
2014
detail.hit.zdb_id:
1477334-X
SSG:
12
