Exploring the molecular mechanism of the effect of puerarin on P2X₃

P2X₃ is a ligand-gated nonselective cation channel and permeable to Na⁺, K⁺ and Ca²⁺. Adenosine triphosphate (ATP) activation of the P2X₃ on primary sensory ganglion neurons is involved in nociceptive transmission. Puerarin is a major active ingredient extracted from the traditional Chinese medicine Ge-gen. Puerarin inhibits nociceptive signal transmission by inhibiting the P2X₃ in the dorsal root ganglia (DRG) and sympathetic ganglia, but its molecular mechanism is unclear. The aim of this study was to explore the molecular mechanism of puerarin on the P2X₃. Here, molecular docking results revealed that puerarin binds well to the human P2X₃ protein in the vicinity of the ATP binding pocket. Protein-ligand docking showed that the V64A mutation reduced the effect of puerarin but had little effect on ATP. V64A site-directed mutagenesis of P2X₃ was performed using an overlap extension PCR technique. The wild-type and V64A mutant pEGFP-C1-P2X₃ recombinant plasmids were transfected into HEK 293 cells. The electrophysiology results demonstrated that puerarin exerted an obvious inhibitory effect on ATP-activated currents in HEK 293 cells transfected with the wild-type P2X₃, while little inhibition was observed in HEK 293 cells transfected with the mutant P2X₃. These studies suggest that puerarin inhibits the P2X₃ by binding to V64A.