CONICET | Buscador de Institutos y Recursos Humanos

Research on flavonoids from plant sources has recently sparked increasing interest because of their beneficial health properties. Different studies have shown that flavonoids change the intracellular Ca2+ homeostasis linked to alterations in the function of mitochondria, Ca2+ channels and Ca2+ pumps. These findings hint at plasma membrane Ca2+-ATPase (PMCA) involvement, as it transports Ca2+ actively to the extracellular medium coupled to ATP hydrolysis, maintaining the cellular homeostasis. The aim of this study was to investigate the effect on purified preparations of PMCA of quercetin and gossypin, two flavonoids that are very effective to inhibit PMCA activity. Results showed that quercetin and gossypin inhibit PMCA activity with Ki of 0.3 and 4.1 M, respectively. The inhibition of PMCA was dependent on the Mg2+ concentration and pH in a way that suggests that the main inhibitory species are Mg2+-flavonoid complexes, which show dissociation constants of around 1 mM at pH = 7.4. The measurement of partial reactions of phosphorylation and dephosphorylation of PMCA under conditions that favor the formation of Mg2+-flavonoids complexes shows an increase in the concentration of phosphoenzymes, EP, particularly of that sensitive to ADP. These results suggest that both, the Mg2+-quercetin and Mg2+-gossypin complexes prevent the conformational change between E1P → E2P. Under experimental conditions not favoring the formation of the Mg2+-flavonoid complex, PMCA activity was inhibited but the phosphorylated intermediate decreased, suggesting that flavonoids compete for the ATP-binding site. For a better comprehension of our results, we performed docking assays of Mg2+-flavonoid complexes and PMCA. Structures of PMCA were obtained by homology modeling on Na,K-ATPase crystallographic structure. Based on these simulations, we propose an interaction model between the Mg2+-flavonoid complexes and the ATP binding domain in the E1P intermediate.