The fluorescein derivative phloxine B is a potent modulator of the cystic fibrosis transmembrane conductance regulator (CFTR). 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 how fluorescein derivatives modulate the activity of CFTR, we studied eosin Y, an agent with a chemical structure closely related to phloxine B.

We used the patch-clamp technique 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. Like phloxine B, low micromolar concentrations of eosin Y (1-5 mM) stimulated CFTR Cl^– currents, whereas higher concentrations of the drug (20-100 mM) inhibited CFTR. For eosin Y, the drug concentration causing half-maximal inhibition of CFTR (K_i) was 48 mM (n = 4-16), whereas for phloxine B, K_i was 17 mM (n = 4-13). However, the effects of eosin Y were readily reversible, but those of phloxine B only partially reversible.

To learn how eosin Y stimulates and inhibits CFTR, we studied single CFTR Cl^– channels. Eosin Y (1 mM) increased the open probability (P_o; control, P_o = 0.38 ± 0.02; eosin Y, P_o = 0.43 ± 0.02; n = 8; means ± S.E.M.; P < 0.05; Student’s paired t test) without changing either single-channel current amplitude (i) or the number of active channels. Eosin Y (1 mM) increased P_o by prolonging mean burst duration (MBD; control, MBD = 133 ± 7 ms; eosin Y, MBD = 166 ± 11 ms; n = 4; P < 0.05) without altering the interburst interval (IBI; control, IBI = 194 ± 24 ms; eosin Y, IBI = 201 ± 13 ms; n = 4; P > 0.05). In contrast, inhibitory concentrations of eosin Y (100 mM) decreased both P_o and i (control, P_o = 0.38 ± 0.02 and i = -0.78 ± 0.04 pA; eosin Y, P_o = 0.23 ± 0.02 and i = -0.68 ± 0.04 pA; n = 7; P < 0.01 for P_o and i). Eosin Y (100 mM) decreased P_o both by shortening MBD and prolonging IBI (control, MBD = 139 ± 6 ms and IBI = 196 ± 23 ms; eosin Y, MBD = 75 ± 12 ms and IBI = 348 ± 49 ms; n = 3; P < 0.05 for MBD and IBI). These data indicate that eosin Y is a less potent modulator of CFTR than phloxine B. They also suggest that like phloxine B, eosin Y might interact directly with CFTR at multiple sites to modulate channel activity.

This work was supported by the CF Trust and NKRF.