Structural determinants for activation and block of CFTR-mediated chloride currents by apigenin

Apigenin (4',5,7-trihydroxyflavone) is an activator of cystic fibrosis transmembrane conductance regulator (CFTR)-mediated Cl^- currents across epithelia at low concentrations and a blocker at high concentrations. We determined the roles of structural components of apigenin for both stimulation and block of Cl^- currents across Calu-3 epithelia. The half-maximal binding affinity of apigenin for current stimulation (K(S)) was 9.1 ± 1.3 μM, and the rank-order of molecular structures was 7-hydroxyl > pyrone = 4'-hydroxyl > 5-hydroxyl. Both the 7-hydroxyl and the 4'-hydroxyl served as H-bond acceptors, whereas the 5-hydroxyl was an H-bond donor. The half-maximal binding affinity of apigenin during current block was 74 ± 11 μM. Blocked Cl^- currents were structurally determined by 7-hydroxyl = 4'-hydroxyl > pyrone > 5-hydroxyl. Prestimulation of tissues with forskolin significantly affected activation kinetics and binding characteristics. After forskolin stimulation, K(S) was 4.1 ± 0.9 μM, which was structurally determined by pyrone > all hydroxyls > single hydroxyls. In contrast, block of Cl^- current by apigenin was not affected by forskolin stimulation. We conclude that apigenin binds to a stimulatory and an inhibitory binding site, which are distinguished by their affinities and the molecular interactions during binding.