Analysis of ClC-2 channels as an alternative pathway for chloride conduction in cystic fibrosis airway cells

作者：Erik M. Schwiebert, L. Pablo Cid, Diane M. Stafford, Mark Carter, Carol J. Blaisdell, Pamela Leslie Zeitlin, William B. Guggino, Garry R. Cutting · 发表于：Proceedings of the National Academy of Sciences · 年份：1998 · DOI：10.1073/pnas.95.7.3879 · 被引用次数：144 · 研究领域：Cystic Fibrosis Research Advances、Ion channel regulation and function、Asthma and respiratory diseases

Cystic fibrosis (CF) is a lethal inherited disease that results from abnormal chloride conduction in epithelial tissues. ClC-2 chloride channels are expressed in epithelia affected by CF and may provide a key "alternative" target for pharmacotherapy of this disease. To explore this possibility, the expression level of ClC-2 channels was genetically manipulated in airway epithelial cells derived from a cystic fibrosis patient (IB3-1). Whole-cell patch-clamp analysis of cells overexpressing ClC-2 identified hyperpolarization-activated Cl- currents (HACCs) that displayed time- and voltage-dependent activation, and an inwardly rectifying steady-state current-voltage relationship. Reduction of extracellular pH to 5.0 caused significant increases in HACCs in overexpressing cells, and the appearance of robust currents in parental IB3-1 cells. IB3-1 cells stably transfected with the antisense ClC-2 cDNA showed reduced expression of ClC-2 compared with parental cells by Western blotting, and a significant reduction in the magnitude of pH-dependent HACCs. To determine whether changes in extracellular pH alone could initiate chloride transport via ClC-2 channels, we performed 36Cl- efflux studies on overexpressing cells and cells with endogenous expression of ClC-2. Acidic extracellular pH increased 36Cl- efflux rates in both cell types, although the ClC-2 overexpressing cells had significantly greater chloride conduction and a longer duration of efflux than the parental cells. Compound...