Actions of Genistein on Cystic Fibrosis Transmembrane Conductance Regulator Channel Gating : Evidence for Two Binding Sites with Opposite Effects

Previous studies have shown that genistein increased cystic fibrosis transmembrane conductance regulator (CFTR) channel activity in the presence of saturating concentrations of forskolin and calyculin A in intact cells. Possible molecular mechanisms for genistein's action include inhibition of tyrosine kinases, inhibition of serine/threonine protein phosphatases, or direct binding of genistein to CFTR. Since genistein inhibits several enzymes that hydrolyze ATP, and ATP hydrolysis is an intrinsic property of CFTR, we examined the effect of genistein on CFTR gating in excised inside-out patches from Hi-5 insect cells and NIH3T3 cells expressing recombinant CFTR. Genistein (50 μM) did not open phosphorylated CFTR channels by itself, but increased the ATP- induced CFTR channel current by approximately twofold. A similar magnitude of enhancement was observed when genistein was applied with PKI, a specific inhibitor of protein kinase A, or vanadate, a tyrosine phosphatase inhibitor, suggesting that inhibition of protein phosphatases or tyrosine kinases does not account for genistein's effects. The enhancement of channel current increased with increasing concentrations of genistein and reached a maximum at 35 μM genistein. At higher concentrations of genistein concentration, CFTR channel current decreased, resulting in a bell-shaped dose–response relationship. In the absence of genistein, both open- and closed-time histograms could be fitted with a single exponential function, yielding a mean open time (τ_O) of 0.302 ± 0.002 s, and a mean closed time (τ_C) of 0.406 ± 0.003 s. In the presence of 50 μM genistein, the open time histogram could be fitted with a double exponential function with τ_O1 = 0.429 ± 0.003 s and τ_O2 = 2.033 ± 0.173 s. Thus, genistein induced a prolonged open state, an effect that mimics that of nonhydrolyzable ATP analogs. Closed time analysis showed that 50 μM genistein caused a prolonged closed state with a time constant of 2.410 ± 0.035 s. We thus conclude that (a) the effects of genistein are likely caused by a direct binding of the drug to the CFTR protein, and (b) at least two binding sites are required to explain the effects of genistein: a high affinity site that decreases the closing rate and a low affinity site that reduces the opening rate.