Exploring the molecular mechanism of the effect of puerarin on P2X₃

P2X(3) is a ligand-gated nonselective cation channel and permeable to Na+, K+ and Ca2+. Adenosine triphosphate (ATP) activation of the P2X(3) 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 P2X(3) 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 P2X(3). Here, molecular docking results revealed that puerarin binds well to the human P2X(3) 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 P2X(3) was performed using an overlap extension PCR technique. The wildtype and V64A mutant pEGFP-C1-P2X(3) 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 P2X(3), while little inhibition was observed in HEK 293 cells transfected with the mutant P2X(3). These studies suggest that puerarin inhibits the P2X(3) by binding to V64A. (C) 2019 Elsevier B.V. All rights reserved.