Analysis of ClC-2 channels as an alternative pathway for chloride conduction in cystic fibrosis airway cells.
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.
Schwiebert
E M
EM
Department of Physiology, Johns Hopkins University School of Medicine, Baltimore, MD 21205, USA.
Cid-Soto
L P
LP
Stafford
D
D
Carter
M
M
Blaisdell
C J
CJ
Zeitlin
P L
PL
Guggino
W B
WB
Cutting
G R
GR
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
D018118:Chloride Channels; D002712:Chlorides