The fluorescein derivative phloxine B (4,5,6,7-tetrachloro-2′,4′,5′,7′-tetrabromofluorescein) is a potent modulator of the cystic fibrosis transmembrane conductance regulator (CFTR) Cl^– channel. Low micromolar concentrations of phloxine B stimulate CFTR Cl^– currents, whereas higher concentrations of the drug inhibit CFTR (Bachmann et al. 2000; Cai & Sheppard, 2002). To understand better the structure-activity relationship of fluorescein derivatives, we studied Bengal Rose B (4,5,6,7-tetrachloro-2′,4′,5′,7′-tetraiodofluorescein), an agent with a chemical structure closely related to that of phloxine B.

The patch-clamp technique was used to investigate CFTR Cl^– channels in excised inside-out membrane patches from C127 cells stably expressing wild-type human CFTR (Cai & Sheppard, 2002). The pipette (external) solution contained 10 mM Cl^–, whereas the bath (internal) solution contained 147 mM Cl^–, PKA (75 nM) and ATP (0.3 mM) at 37 °C; voltage was -50 mV. Following the activation of CFTR Cl^– currents by cAMP-dependent phosphorylation, drugs were added to the intracellular solution. Nanomolar concentrations of Bengal Rose B (0.1-1 µM) stimulated CFTR Cl^– currents, whereas low micromolar concentrations of the drug (2-10 µM) inhibited CFTR. Bengal Rose B (0.1 µM) increased open probability (P_o) from 0.31 ± 0.06 to 0.40 ± 0.08 (means ± S.E.M., n = 5, P < 0.05, Student’s paired t test), but caused a small, but significant decrease in current amplitude (i; P < 0.05). To determine how Bengal Rose B increased P_o, we investigated the drug’s effects on gating kinetics using the QuB software suite (Cai & Sheppard, 2002). Bengal Rose B (0.1 µM) increased P_o by greatly prolonging mean burst duration (MBD) (control MBD = 116 ± 8 ms; Bengal Rose B MBD = 232 ± 30 ms; n = 4; P < 0.05) without significantly altering the inter-burst interval (IBI) (control IBI = 219 ± 19 ms; Bengal Rose B IBI = 307 ± 37 ms; n = 4; P > 0.05). However, the characteristics of channel block by Bengal Rose B differed from those of phloxine B. Inhibition of CFTR by phloxine is time independent. In contrast, during constant exposure to the drug, Bengal Rose B (2-10 µM) caused a progressive decrease of i until channel activity completely disappeared (n = 10).

We interpret these data to suggest that (i) Bengal Rose B interacts directly with CFTR at multiple sites to modulate channel activity; (ii) Bengal Rose B might stimulate CFTR by a similar mechanism to that of phloxine B; (iii) by modifying the residues at positions 2′,4′,5′,7′ of fluorescein, potent modulators of CFTR might be developed.

This work was supported by the CF Trust and NKRF.