Two different mechanisms of PMCA inhibition by flavonoids

ONTIVEROS, M; RINALDI, D.E; PANTANO, S; MARDER, M; MANGIALAVORI, I.; ROSSI, R.C; ROSSI, J.P.; FERREIRA GOMES, M

San Luis

Congreso; XLVIII Reunión Anual de la SAB; 2019

Research on flavonoids from plant sources has recently sparked increasing interestbecause of their beneficial health properties. Different studies have shown thatflavonoids change the intracellular Ca2+ homeostasis linked to alterations in the functionof mitochondria, Ca2+ channels and Ca 2+ pumps. These findings hint at plasmamembrane Ca2+-ATPase (PMCA) involvement, as it transports Ca2+ actively to theextracellular medium coupled to ATP hydrolysis, maintaining the cellular homeostasis.The aim of this study was to investigate the effect on purified preparations of PMCA ofquercetin 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 4.1mM, respectively. The inhibition of PMCA was dependent on the Mg2+ concentration andpH in a 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 of PMCAunder conditions that favor the formation of Mg2+-flavonoids complexes shows anincrease in the concentration of phosphoenzymes, EP, particularly of that sensitive toADP. These results suggest that both, the Mg2+-quercetin and Mg2+-gossypin complexesprevent the conformational change between E1P → E2P. Under experimental conditionsnot favoring the formation of the Mg2+-flavonoid complex, PMCA activity was inhibitedbut the phosphorylated intermediate decreased, suggesting that flavonoids compete forthe ATP-binding site. For a better comprehension of our results, we performed dockingassays of Mg2+-flavonoid complexes and PMCA. Structures of PMCA were obtained byhomology modeling on Na,K-ATPase crystallographic structure. Based on thesesimulations, we propose an interaction model between the Mg2+-flavonoid complexesand the ATP binding domain in the E1P intermediate.