Cystic fibrosis transmembrane conductance regulator (CFTR) confers glibenclamide sensitivity to outwardly rectifying chloride channel (ORCC) in Hi-5 insect cells.
Increasing evidence is now accumulating for the involvement of the cystic fibrosis transmembrane conductance regulator (CFTR) in the control of the outwardly rectifying chloride channel (ORCC). We have examined the sensitivity of ORCC to the sulfonylurea drug glibenclamide in Hi-5 (Trichoplusia ni) insect cells infected with recombinant baculovirus expressing either wild-type CFTR, DeltaF508-CFTR or E. coli beta galactosidase cDNA and in control cells either infected with virus alone or uninfected. Iodide efflux and single channel patch-clamp experiments confirmed that forskolin and 1-methyl-3-isobutyl xanthine (IBMX) or 7-methyl-1,3 dipropyl xanthine (DPMX) activate CFTR channels (unitary conductance: 9.1 +/- 1.6 pS) only in cells expressing CFTR. In contrast, we identified 4-acetamido-4'-isothiocyanatostilbene-2,2'-disulfonic acid (SITS)-sensitive ORCC in excised membrane patches in any of the cells studied, with similar conductance (22 +/- 2.5 pS at -80 mV; 55 +/- 4.1 pS at +80 mV) and properties. In the presence of 500 microm SITS, channel open probability (Po) of ORCC was reversibly reduced to 0.05 +/- 0.01 in CFTR-cells, to 0.07 +/- 0.02 in non-CFTR expressing cells and to 0.05 +/- 0.02 in DeltaF508-cells. In Hi-5 cells that did not express CFTR, glibenclamide failed to inhibit ORCC activity even at high concentrations (100 microm), whereas 500 microm SITS reversibly inhibited ORCC. In contrast in cells expressing CFTR or DeltaF508, glibenclamide dose dependently (IC50 = 17 microm, Hill coefficient 1.2) and reversibly inhibited ORCC. Cytoplasmic application of 100 microm glibenclamide reversibly reduced Po from 0.88 +/- 0.03 to 0.09 +/- 0.02 (wash: Po = 0.85 +/- 0.1) in CFTR cells and from 0.89 +/- 0.05 to 0.08 +/- 0.05 (wash: Po = 0.87 +/- 0.1) in DeltaF508 cells. In non-CFTR expressing cells, glibenclamide (100 microm) was without effect on Po (control: Po = 0. 89 +/- 0.09, glib.: Po = 0.86 +/- 0.02; wash: Po = 0.87 +/- 0.05). These data strongly suggest that the expression of CFTR confers glibenclamide sensitivity to the ORCC in Hi-5 cells.
Julien
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Station de Recherche de Pathologie Comparée INRA-CNRS 30180 Saint-Christol-lez-Alès F-30380, France.
Verrier
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Cerutti
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Chappe
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Gola
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Devauchelle
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Becq
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D015056:1-Methyl-3-isobutylxanthine / Q000494:pharmacology; D012856:4-Acetamido-4'-isothiocyanatostilbene-2,2'-disulfonic Acid / Q000494:pharmacology; D018528:ATP-Binding Cassette Transporters / Q000037:antagonists & inhibitors; D000818:Animals; D015220:Calcium Channels / Q000187:drug effects*; D002478:Cells, Cultured; D002712:Chlorides / Q000378:metabolism*; D005576:Colforsin / Q000494:pharmacology; D019005:Cystic Fibrosis Transmembrane Conductance Regulator / Q000235:genetics / Q000502:physiology*; D005822:Genetic Vectors / Q000235:genetics; D005905:Glyburide / Q000494:pharmacology*; D006801:Humans; D015640:Ion Channel Gating / Q000187:drug effects*; D009036:Moths / Q000187:drug effects; D017924:Nucleopolyhedroviruses / Q000235:genetics; D018408:Patch-Clamp Techniques; D011993:Recombinant Fusion Proteins / Q000502:physiology; D018411:Spodoptera; D014970:Xanthines / Q000494:pharmacology; D001616:beta-Galactosidase / Q000235:genetics
D018528:ATP-Binding Cassette Transporters; C505032:CFTR protein, human; D015220:Calcium Channels; D002712:Chlorides; D011993:Recombinant Fusion Proteins; D014970:Xanthines; D019005:Cystic Fibrosis Transmembrane Conductance Regulator; D005576:Colforsin; D012856:4-Acetamido-4'-isothiocyanatostilbene-2,2'-disulfonic Acid; C059953:1,3-dipropyl-7-methylxanthine; D001616:beta-Galactosidase; D005905:Glyburide; D015056:1-Methyl-3-isobutylxanthine