Two mechanisms of genistein inhibition of cystic fibrosis transmembrane conductance regulator Cl⁻ channels expressed in murine cell line

Abstract

The isoflavone genistein may either stimulate or inhibit cystic fibrosis transmembrane conductance regulator (CFTR) Cl− channels. To investigate how genistein inhibits CFTR, we studied CFTR Cl− channels in excised inside‐out membrane patches from cells expressing wild‐type human CFTR. Addition of genistein (100 μM) to the intracellular solution caused a small decrease in single‐channel current amplitude (i), but a large reduction in open probability (Po). Single‐channel analysis of channel block suggested that genistein (100 μM) may inhibit CFTR by two mechanisms: first, it may slow the rate of channel opening and second, it may block open channels. Acidification of the intracellular solution relieved channel block, suggesting that the anionic form of genistein may inhibit CFTR. Genistein inhibition of CFTR Cl− currents was weakly voltage dependent and unaffected by changes in the extracellular Cl− concentration. Channel block was relieved by pyrophosphate (5 mM) and ATP (5 mM), two agents that interact with the nucleotide‐binding domains (NBDs) of CFTR to greatly stimulate channel activity. ATP (5 mM) prevented the genistein‐induced decrease in Po, but was without effect on the genistein‐induced decrease in i. The genistein‐induced decrease in i was voltage dependent, whereas the genistein‐induced decrease in Po was voltage independent. The data suggest that genistein may inhibit CFTR by two mechanisms. First, it may interact with NBD1 to potently inhibit channel opening. Second, it may bind within the CFTR pore to weakly block Cl− permeation.

Phloxine B Interacts with the Cystic Fibrosis Transmembrane Conductance Regulator at Multiple Sites to Modulate Channel Activity

“…To investigate whether phloxine B inhibition of CFTR was voltagedependent and enhanced when the external Cl Ϫ concentration was reduced, we used voltage ramp protocols to acquire macroscopic current-voltage (I-V) relationships as previously described (31). Basal currents recorded before the addition of PKA and ATP were subtracted from those recorded in the absence and presence of phloxine B to determine the effect of phloxine B on CFTR Cl Ϫ currents.…”

“…For open probability (P o ) and burst analyses, lists of open-and closed-times were created and analyzed as previously described (31). Burst analysis was performed as described by Carson et al (33), using a t c (the time that separates interburst closures from intraburst closures) of 15 ms.…”

“…Elevated concentrations of the CFTR activator genistein inhibit channel activity by slowing greatly the rate of channel opening (13,31) (31), under control conditions, CFTR Cl Ϫ currents exhibited weak inward rectification (Fig. 8A).…”

Direct Sensing of Intracellular pH by the Cystic Fibrosis Transmembrane Conductance Regulator (CFTR) Cl− Channel

“…Mean burst duration (MBD or T MBD ) and open probability within a burst (P o(burst) ) were determined directly from experimental data using pCLAMP software. P o was calculated from open and closed times as described (23). However, for G551D and G1349D, because the number of active channels in a membrane patch was unknown, we measured NP o instead of P o .…”

The Therapeutic Potential of Small-molecule Modulators of the Cystic Fibrosis Transmembrane Conductance Regulator (CFTR) Cl− Channel

“…However, its mechanism of action is distinct from other allosteric inhibitors (e.g. genistein) 113,114 that interfere with the control of CFTR gating by ATP-driven NBD dimerisation by competing with ATP for binding to ATP-binding site 2. First, Kopeikin et al 107 demonstrated that CFTR inhibition by CFTR inh -172 occurs from either the open or closed state and does not involve disassembly of the NBD dimer, but instead is reminiscent of the inactivation mechanism of voltage-gated cation channels.…”