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

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. In whole oocytes, the CFTR Cl− current (ICFTR) 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. In excised inside-out macropatches, ICFTR was activated by the catalytic subunit of protein kinase A (cPKA) and MgATP. Phloxine B (0.01–3 μM), 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. The effect of phloxine B after 30 s had a bell-shaped concentration-dependence with midpoints at 45 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 ∼1 μM. In the absence of cPKA, phloxine B did not stimulate ICFTR. 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). 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. British Journal of Pharmacology (2000) 131, 433–440; doi:10.1038/sj.bjp.0703600