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 f ibrosis transmembrane conductance regulator (CFTR) in the control of the ou twardly rectifying chloride channel (ORCC). We have examined the sensitivit y of ORCC to the sulfonylurea drug glibenclamide in Hi-5 (Trichoplusia ni) insect cells infected with recombinant baculovirus expressing either wild-t ype CFTR, Delta F508-CFTR or E. coli beta galactosidase cDNA and in control cells either infected with virus alone or uninfected. Iodide efflux and si ngle channel patch-clamp experiments confirmed that forskolin and 1-methyl- 3-isobutyl xanthine (IBMX) or 7-methyl-1,3 dipropyl xanthine (DPMX) activat e CFTR channels (unitary conductance: 9.1 +/- 1.6 pS) only in cells express ing CFTR. In contrast, we identified 4-acetamido-4'-isothiocyanatostilbene- 2,2'-disulfonic acid (SITS)-sensitive ORCC in excised membrane patches in a ny 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 mu M 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 Delta F508-cells. In Hi-5 cells that did not express CFTR, g libenclamide failed to inhibit ORCC activity even at high concentrations (1 00 mu M), whereas 500 mu M SITS reversibly inhibited ORCC. In contrast in c ells expressing CFTR or Delta F508, glibenclamide dose dependently (IC50 = 17 mu M, Hill coefficient 1.2) and reversibly inhibited ORCC. Cytoplasmic a pplication of 100 mu M 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 Delta F508 cells. In non-CFTR expressing cells, glibenclamide (100 mu M) was without effect o n 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 c onfers glibenclamide sensitivity to the ORCC in Hi-5 cells.