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.

ClC-2 regulation of intestinal barrier function: Translation of basic science to therapeutic target

“…In addition, ClC-2 knockdown T84 cells did not respond to lubiprostone whereas CFTR knockdown T84 cells had significantly increased Cl ¡ current in response to lubiprostone ( Table 2). 9,98,102,111,112,[114][115][116][117][118][119][120][121][122][123][124][125][126] Collectively, these findings indicate that lubiprostone selectively stimulates ClC-2 Cl ¡ currents in intestinal epithelial cells at low doses. However, there are several alternate mechanisms of action of lubiprostone revealed by recent studies including ion transporter trafficking, mucus release, and smooth muscle contraction.…”

“…[111][112][113] However, a recent study has shown that CFTR ihn 172 also inhibits ClC-2 Cl ¡ currents. 114 Other laboratories have detected dual activation of CFTR and ClC-2 in a dose-dependent manner. However, this may relate to dosedependent effects of lubiprostone, which when used at dosages 10-fold higher that those required to activate ClC-2 can stimulate CFTR Cl ¡ currents.…”

Opioid-induced constipation: advances and clinical guidance

“…Drugs approved for OIC (Table 1) Lubiprostone Lubiprostone is a bicyclic fatty acid derived from prostaglandin E1 (PGE1) metabolite which increases fluid secretion in the gastrointestinal tract [Cuppoletti et al 2004[Cuppoletti et al , 2014 by stimulating the cystic fibrosis transmembrane regulator and type 2 chloride channels (ClC2) in the apical membrane to secrete chloride and water into the lumen [Bijvelds et al 2009;Schiffhauer et al 2013]. This results in increased peristalsis, laxation, and acceleration of small intestinal and colonic transit [Camilleri et al 2006].…”

Research and progress on ClC-2

“…Previous studies have shown that ClC-2 is also regulated by hormones (89–91), drugs (92–100), proteins (101–107), kinases (108–114), transcription factors (115,116), inhibitors (117–120), scorpion venom (121), α1-adrenoceptor (122), Plasmodium berghei (123), adenosine triphosphate (124,125), ClH 3 (40,126–137), permeant anions (138,139), membrane cholesterol (140) and the tyrosine endocytosis motif (141). …”