Delta F508-CFTR channels: kinetics, activation by forskolin, and potentiation by xanthines.
Trafficking, activation, and kinetics of delta F508-cystic fibrosis transmembrane conductance regulator (CFTR) and CFTR were compared in stably transduced C127I mouse mammary epithelial cells. Western blots detected a small amount of fully glycosylated delta F508-CFTR Efflux of 125I was stimulated by forskolin with the same mean effective concentration (EC50; approximately 0.5 microM) for CFTR and delta F508-CFTR cells, but the maximum response was reduced more than fivefold and its latency increased approximately threefold in delta F508-CFTR cells. In delta F508-CFTR cells, 3-isobutyl-1-methylxanthine (IBMX; EC50 = 1.45 microM) and 8-cyclopentyl-1,3-dipropylxanthine (CPX; EC50 = 58 microM) increased the peak forskolin-stimulated efflux rate approximately 2.5-fold and decreased the time to peak. A sevenfold increase in intracellular adenosine 3',5'-cyclic monophosphate (cAMP) levels accompanied potentiation of forskolin-induced 125I efflux by IBMX but not by CPX. Elevation of intracellular cAMP increased linear voltage-independent whole cell currents 30-fold in CFTR and 4-fold in delta F508-CFTR cells; the response rate in delta F508-CFTR cells was much slower. Single-channel currents were detected in 57 of 68 cell-attached patches from forskolin-prestimulated CFTR cells vs. 6 of 35 patches in delta F508-CFTR cells. Mean number of active channels per patch was 4.1 for CFTR [open probability (Po) = 0.34] and 0.2 for delta F508-CFTR (Po = 0.11). The lower Po of delta F508-CFTR resulted from an approximately threefold longer mean interburst interval. We estimate that forskolin-stimulated chloride conductance of delta F508-CFTR C127I cells is < 5% of CFTR cells. CPX is approximately 25-fold more potent than IBMX in potentiating delta F508-CFTR and may operate by a mechanism other than elevation of cAMP.
Haws
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CM
Johns Hopkins University School of Medicine, Baltimore, Maryland 21205, USA.
Nepomuceno
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IB
Krouse
M E
ME
Wakelee
H
H
Law
T
T
Xia
Y
Y
Nguyen
H
H
Wine
J J
JJ
D015056:1-Methyl-3-isobutylxanthine / Q000494:pharmacology; D000818:Animals; D002460:Cell Line; D005576:Colforsin / Q000494:pharmacology*; D000242:Cyclic AMP / Q000378:metabolism; D019005:Cystic Fibrosis Transmembrane Conductance Regulator / Q000378:metabolism*; D004594:Electrophysiology; D006031:Glycosylation; D007455:Iodine / Q000493:pharmacokinetics; D051379:Mice; D009154:Mutation; D013997:Time Factors; D014970:Xanthines / Q000494:pharmacology*
D014970:Xanthines; C093349:cystic fibrosis transmembrane conductance regulator delta F508; D019005:Cystic Fibrosis Transmembrane Conductance Regulator; D005576:Colforsin; D007455:Iodine; C051360:1,3-dipropyl-8-cyclopentylxanthine; D000242:Cyclic AMP; D015056:1-Methyl-3-isobutylxanthine