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, Delta F508-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 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, glibenclamide failed to inhibit ORCC activity even at high concentrations (100 mu M), whereas 500 mu M SITS reversibly inhibited ORCC. In contrast in cells expressing CFTR or Delta F508, glibenclamide dose dependently (IC50 = 17 mu M, Hill coefficient 1.2) and reversibly inhibited ORCC. Cytoplasmic application 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 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.