Potent stimulation and inhibition of the CFTR Cl- current by phloxine B

1 The effects of the fluoresceine derivative, phloxine B, on the Cl- current through the cystic fibrosis transmembrane conductance regulator (CFTR) were examined in Xenopus oocytes expressing human CFTR. 2 In whole oocytes, the CFTR Cl- current (I-CFTR) was activated by superfusion with isobutylmethylxanthine and forskolin. ICFTR was stable during activation and deactivated rapidly upon washout of the activation solution. Phloxine B slowed deactivation and, at high concentrations, inhibited ICFTR weakly. 3 In excised inside-out macroparches, I-CFTR was activated by the catalytic subunit of protein kinase 4 (cPKA) and MgATP. Phloxine B (0.01-3 mu M), applied after activation, increased ICFTR within 30 s followed by a slow decrease which became dominant at high concentrations. Slowing of deactivation of the CFTR was observed at all concentrations. 4 The effect of phloxine B after 30 s had a bell-shaped concentration-dependence with midpoints at 35 and 1600 nM for the stimulatory and the inhibitory limb, respectively; maximum stimulation was about 1.8 times. The slow inhibitory component, measured after 6 min, occurred with an IC50 value of similar to 1 mu M. 5 In the absence of cPKA, phloxine B did not stimulate I-CFTR. In the presence of cPKA and MgATP? the effects of phloxine B were more prominent at low (0.02 mM) than at high ATP (2 mM). 6 The data show that phloxine B modulates ICFTR by increasing channel activity and slowing channel deactivation; at high concentrations inhibition dominates. The effects may be mediated by direct interactions with CFTR from the inside of the cell.