A common mechanism for cystic fibrosis transmembrane conductance regulator protein activation by genistein and benzimidazolone analogs

Abstract

We have investigated the mechanism of action of two benzimidazolone analogs (NS004 and NS1619) on ΔF508-CFTR using both whole-cell and cell-attached patch-clamp techniques and compared their effects with those of genistein. We conclude that benzimidazolone analogs and genistein act through a common mechanism, based on the following evidence: 1) both act only on phosphorylated CFTR, 2) the maximal ΔF508-CFTR current activated by benzimidazolone analogs is identical to that induced by genistein, 3) benzimidazolone analogs increase the open probability of the forskolin-dependent ΔF508-CFTR channel activity through an increase of the channel open time and a decrease of the channel closed time (effects indistinct from those reported for genistein), and 4) the prolonged K1250A-CFTR channel open time (in the presence of 10 μM forskolin) is unaffected by benzimidazolone analogs or genistein, supporting the hypothesis that these compounds stabilize the open state by inhibiting ATP hydrolysis at nucleotide binding domain 2 (NBD2). In addition, we demonstrate that NS004 and NS1619 are more potent CFTR activators than genistein (EC50 values are 87 ± 14 nM, 472 ± 88 nM, and 4.4 ± 0.5 μM, respectively). From our studies with the double mutant ΔF508/K1250A-CFTR, we conclude that benzimidazolone analogs and genistein rectify the ΔF508-CFTR prolonged closed time independent of their effects on channel open time, since these agonists enhance ΔF508/K1250A-CFTR activity by shortening the channel closed time. These studies should pave the way toward understanding the agonist binding sites at a molecular level.