Cobiprostone Activates Human Bronchial Epithelial (HBE) Cell ClC‐2, but not CFTR

Abstract

Objective HBE cells contain both ClC-2 and CFTR Cl- channels. The objective was to identify the Cl- channel in HBE cells that is activated by the prostone, cobiprostone (cobi) using knockdowns and differential inhibition using methadone (meth) and DASU-02. (Cuppoletti et al AJP Cell Physiol 307: C479, 2014). Methods: Cl- currents were measured by whole cell patch clamp of hClC-2/293EBNA cells, hCFTR/HEK293 cells as well as HBE cells with either CFTR or ClC-2 knocked down (HBE CFTR KD & HBE ClC-2 KD cells). Effects of cobi or forskolin/IBMX (F/I) followed by the inhibitors meth (1 µM) and DASU-02 (100 µM) were examined. Western blot confirmed absence of ClC-2 in HBE ClC-2 KD cells. Results: ClC-2 Cl- currents in hClC-2/293EBNA and HBE CFTR KD cells were inwardly rectified, activated by cobi (EC50's37.8± 3.0 and 25.6 ± 5.6 nM respectively) or F/I and inhibited by meth but not DASU-02. The cobi response was PKA independent since 0.4 µM myristoylated PKI (PKA inhibitor) had no effect. CFTR Cl- currents in hCFTR/HEK293 and HBE ClC-2 KD cells did not respond to cobi but were activated by F/I with a linear I/V and inhibited by DASU-02 but not meth. Conclusions In hClC-2/293 EBNA and HBE CFTR KD cells cobi or F/I potently activated ClC-2 Cl- currents in a meth-sensitive, DASU-02- and PKA-insensitive manner. In hCFTR/HEK293 and HBE ClC-2 KD cells CFTR was unaffected by cobi, but activated by F/I in a DASU-02-sensitive, meth-insensitive manner. These responses distinguish between ClC-2 and CFTR in epithelial (or other) cells that contain both Cl- channels. It is essential to understand that F/I activates both hClC-2 and hCFTR. Cobi activates recombinant and HBE cell ClC-2, but not CFTR. Cobi activation of hClC-2 may be useful in a variety of diseases including cystic fibrosis.