CONICET | Buscador de Institutos y Recursos Humanos

Two differentmechanisms of PMCA inhibition by flavonoidsOntiveros, M; Rinaldi, D; Pantano S;Marder, M; Mangialavori, I, Rossi, RC, Rossi, JP; Ferreira-Gomes, M Research onflavonoids from plant sources has recently sparked increasing interest becauseof their beneficial health properties. Different studies have shown thatflavonoids change the intracellular Ca2+ homeostasis linked toalterations 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 extracellularmedium coupled to ATP hydrolysis, maintaining the cellular homeostasis. The aimof this study was to investigate the effect on purified preparations of PMCA ofquercetin and gossypin, two flavonoids that are very effective to inhibit PMCAactivity. Results showed thatquercetin and gossypin inhibit PMCA activity with Ki of 0.3 and 4.1 mM, respectively. Theinhibition of PMCA was dependent on the Mg2+ concentration and pH ina way that suggests that the main inhibitory species are Mg2+-flavonoidcomplexes, which show dissociation constants of around 1 mM at pH = 7.4. Themeasurement of partial reactions of phosphorylation and dephosphorylation ofPMCA under conditions that favor the formation of Mg2+-flavonoidscomplexes shows an increase in the concentration of phosphoenzymes, EP,particularly of that sensitive to ADP. These results suggest that both, the Mg2+-quercetinand Mg2+-gossypin complexes prevent the conformational changebetween E1P → E2P. Under experimental conditions not favoring theformation of the Mg2+-flavonoid complex, PMCA activity was inhibitedbut the phosphorylated intermediate decreased, suggesting that flavonoidscompete for the ATP-binding site. For a better comprehension of our results, weperformed docking assays of Mg2+-flavonoid complexes and PMCA.Structures of PMCA were obtained by homology modeling on Na,K-ATPasecrystallographic structure. Based on these simulations, we propose aninteraction model between the Mg2+-flavonoid complexes and the ATPbinding domain in the E1P intermediate.

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