title: Analysis of ClC-2 channels as an alternative pathway for chloride conduction in cystic fibrosis airway cells.
abstract: 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. Compounds that exploit the pH mechanism of activating endogenous ClC-2 channels may provide a pharmacologic option for increasing chloride conductance in the airways of CF patients.
authors.affiliation: Department of Physiology, Johns Hopkins University School of Medicine, Baltimore, MD 21205, USA.
authors.forename: E M
authors.initials: EM
authors.lastname: Schwiebert
authors.forename: L P
authors.initials: LP
authors.lastname: Cid-Soto
authors.forename: D
authors.initials: D
authors.lastname: Stafford
authors.forename: M
authors.initials: M
authors.lastname: Carter
authors.forename: C J
authors.initials: CJ
authors.lastname: Blaisdell
authors.forename: P L
authors.initials: PL
authors.lastname: Zeitlin
authors.forename: W B
authors.initials: WB
authors.lastname: Guggino
authors.forename: G R
authors.initials: GR
authors.lastname: Cutting
mesh_terms: D002460:Cell Line; D018118:Chloride Channels / Q000378:metabolism*; D002712:Chlorides / Q000378:metabolism*; D003550:Cystic Fibrosis / Q000378:metabolism* / Q000473:pathology; D004847:Epithelial Cells / Q000378:metabolism* / Q000473:pathology; D006801:Humans; D017136:Ion Transport; D018408:Patch-Clamp Techniques; D012137:Respiratory System / Q000378:metabolism* / Q000473:pathology; D015398:Signal Transduction
chemical_list: D018118:Chloride Channels; D002712:Chlorides