TY - JOUR
T1 - Both CFTR and outwardly rectifying chloride channels contribute to cAMP- stimulated whole cell chloride currents
AU - Schwiebert, E. M.
AU - Flotte, T.
AU - Cutting, G. R.
AU - Guggino, W. B.
PY - 1994
Y1 - 1994
N2 - From whole cell patch-clamp recordings at 35°C utilizing either nystatin perforation or conventional methods with 5 mM MgATP in the pipette solution, it was demonstrated that both cystic fibrosis transmembrane conductance regulator (CFTR) chloride (Cl-) channels and outwardly rectifying Cl- channels (ORCC) contribute to adenosine 3',5'-cyclic monophosphate (cAMP)- activated whole cell Cl- currents in cultured human airway epithelial cells. These results were similar whether recordings were performed on two normal human cell lines or on two cystic fibrosis (CF) cell lines stably complemented with wild-type CF gene. These results were obtained by exploiting dissimilar biophysical properties of CFTR and ORCC currents such as the degree of rectification of the current-voltage relationship, the difference in sensitivity to Cl- channel-blocking drugs such as 4,4'- diisothiocyanostilbene-2,2'-disulfonic acid (DIDS), calixarenes, and diphenylamine carboxylic acid (DPC), and the opposing Cl- relative to I- permeabilities of the two channels. In normal cells or complemented CF cells, 8-(4-chlorophenylthio)adenosine 3',5'-cyclic monophosphate stimulated outwardly rectifying whole cell Cl- currents. Addition of DIDS in the presence of cAMP inhibited the outwardly rectifying portion of the cAMP- activated Cl- current. The remaining cAMP-activated, DIDS-insensitive, linear CFTR Cl- current was inhibited completely by DPC. Additional results showed that not only do ORCC and CFTR Cl- channels contribute to cAMP- activated Cl- currents in airway epithelial cells where wild-type CFTR is expressed but that both channels fail to respond to cAMP in ΔF508-CFTR- containing CF airway cells. We conclude that CFTR not only functions as a cAMP-regulated Cl- channel in airway epithelial cells but also controls the regulation of ORCC.
AB - From whole cell patch-clamp recordings at 35°C utilizing either nystatin perforation or conventional methods with 5 mM MgATP in the pipette solution, it was demonstrated that both cystic fibrosis transmembrane conductance regulator (CFTR) chloride (Cl-) channels and outwardly rectifying Cl- channels (ORCC) contribute to adenosine 3',5'-cyclic monophosphate (cAMP)- activated whole cell Cl- currents in cultured human airway epithelial cells. These results were similar whether recordings were performed on two normal human cell lines or on two cystic fibrosis (CF) cell lines stably complemented with wild-type CF gene. These results were obtained by exploiting dissimilar biophysical properties of CFTR and ORCC currents such as the degree of rectification of the current-voltage relationship, the difference in sensitivity to Cl- channel-blocking drugs such as 4,4'- diisothiocyanostilbene-2,2'-disulfonic acid (DIDS), calixarenes, and diphenylamine carboxylic acid (DPC), and the opposing Cl- relative to I- permeabilities of the two channels. In normal cells or complemented CF cells, 8-(4-chlorophenylthio)adenosine 3',5'-cyclic monophosphate stimulated outwardly rectifying whole cell Cl- currents. Addition of DIDS in the presence of cAMP inhibited the outwardly rectifying portion of the cAMP- activated Cl- current. The remaining cAMP-activated, DIDS-insensitive, linear CFTR Cl- current was inhibited completely by DPC. Additional results showed that not only do ORCC and CFTR Cl- channels contribute to cAMP- activated Cl- currents in airway epithelial cells where wild-type CFTR is expressed but that both channels fail to respond to cAMP in ΔF508-CFTR- containing CF airway cells. We conclude that CFTR not only functions as a cAMP-regulated Cl- channel in airway epithelial cells but also controls the regulation of ORCC.
KW - adeno-associated viral vectors
KW - adenosine 3',5'-cyclic monophosphate
KW - airway epithelia
KW - cell culture
KW - cystic fibrosis
KW - cystic fibrosis transmembrane conductance regulator
KW - gene transfection
KW - patch clamp
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U2 - 10.1152/ajpcell.1994.266.5.c1464
DO - 10.1152/ajpcell.1994.266.5.c1464
M3 - Article
C2 - 7515570
AN - SCOPUS:0027966771
SN - 0363-6143
VL - 266
SP - C1464-C1477
JO - American Journal of Physiology - Cell Physiology
JF - American Journal of Physiology - Cell Physiology
IS - 5 35-5
ER -