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 (I(CFTR)) was activated by superfusion with isobutylmethylxanthine and forskolin. I(CFTR) was stable during activation and deactivated rapidly upon washout of the activation solution. Phloxine B slowed deactivation and, at high concentrations, inhibited I(CFTR) weakly. In excised inside-out macropatches, I(CFTR) was activated by the catalytic subunit of protein kinase A (cPKA) and MgATP. Phloxine B (0.01 - 3 microM), applied after activation, increased I(CFTR) 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 IC(50) value of approximately 1 microM. 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). The data show that phloxine B modulates I(CFTR) 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.
Bachmann
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A
Department of Pharmacology, University of Tübingen, Wilhelmstr. 56, D-72074 Tübingen, Germany.
Russ
U
U
Waldegger
S
S
Quast
U
U
D018528:ATP-Binding Cassette Transporters; D000818:Animals; D019005:Cystic Fibrosis Transmembrane Conductance Regulator / Q000037:antagonists & inhibitors / Q000378:metabolism* / Q000502:physiology; D004594:Electrophysiology; D004791:Enzyme Inhibitors / Q000494:pharmacology; D010697:Eosine I Bluish / Q000494:pharmacology*; D005456:Fluorescent Dyes / Q000494:pharmacology; D019833:Genistein / Q000494:pharmacology; D006801:Humans; D009865:Oocytes / Q000502:physiology; D018408:Patch-Clamp Techniques; D015221:Potassium Channels / Q000378:metabolism / Q000502:physiology; D024661:Potassium Channels, Inwardly Rectifying; D011955:Receptors, Drug / Q000378:metabolism / Q000502:physiology; D064233:Sulfonylurea Receptors; D014982:Xenopus laevis
D018528:ATP-Binding Cassette Transporters; C505032:CFTR protein, human; D004791:Enzyme Inhibitors; D005456:Fluorescent Dyes; D015221:Potassium Channels; D024661:Potassium Channels, Inwardly Rectifying; D011955:Receptors, Drug; D064233:Sulfonylurea Receptors; D019005:Cystic Fibrosis Transmembrane Conductance Regulator; D019833:Genistein; D010697:Eosine I Bluish