Cystic Fibrosis Transmembrane Conductance Regulator (CFTR) Confers Glibenclamide Sensitivity to Outwardly Rectifying Chloride Channel (ORCC) in Hi-5 Insect Cells |
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| Authors: | M Julien B Verrier M Cerutti V Chappe M Gola G Devauchelle F Becq |
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| Institution: | (1) Station de Recherche de Pathologie Comparée INRA-CNRS 30180 Saint-Christol-lez-Alès F-30380, France, FR;(2) Laboratoire de Neurobiologie UPR-9024 CNRS 31 ch. J. Aiguier F-13402 Marseille Cedex, 20, France, FR |
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| Abstract: | 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, ΔF508-CFTR or E. coliβ 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 μm SITS, channel open probability (P
o
) 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 ΔF508-cells. In Hi-5 cells that did not express CFTR, glibenclamide failed to inhibit ORCC activity even at high concentrations
(100 μm), whereas 500 μm SITS reversibly inhibited ORCC. In contrast in cells expressing CFTR or ΔF508, glibenclamide dose dependently (IC50= 17 μm, Hill coefficient 1.2) and reversibly inhibited ORCC. Cytoplasmic application of 100 μm glibenclamide reversibly reduced P
o
from 0.88 ± 0.03 to 0.09 ± 0.02 (wash: P
o
= 0.85 ± 0.1) in CFTR cells and from 0.89 ± 0.05 to 0.08 ± 0.05 (wash: P
o
= 0.87 ± 0.1) in ΔF508 cells. In non-CFTR expressing cells, glibenclamide (100 μm) was without effect on P
o
(control: P
o
= 0.89 ± 0.09, glib.: P
o
= 0.86 ± 0.02; wash: P
o
= 0.87 ± 0.05). These data strongly suggest that the expression of CFTR confers glibenclamide sensitivity to the ORCC in Hi-5
cells.
Received: 23 October 1998/Revised: 29 December 1998 |
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| Keywords: | :Trichoplusia ni insect cells (Hi-5) — Baculovirus — Cystic fibrosis transmembrane conductance regulator (CFTR) chloride channel — Glibenclamide — Outwardly rectifying chloride channel (ORCC) |
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