Natural flavonoids and calcium transport through biological membranes: molecular target and mechanisms of action

ONTIVEROS, MALLKU; RINALDI, DEBORA; MARDER, MARIEL; ESPELT, MARÍA V.; VIGIL MAXIMILIANO ANGEL; MANGIALAVORI IRENE; ROLANDO C ROSSI; ROSSI JUAN PABLO; FERREIRA GOMES, MARIELA

Lima

Congreso; Revisiting the Central Dogma of Molecular Biology at the Single-Molecule Level; 2019

Recently, there has been increasing interest in research on flavonoids from plant sources because of their beneficial properties for health. Several studies have attributed them anti-oxidative, anti-inflammatory, anti-mutagenic, and anti-carcinogenic properties coupled with their capacity to modulate key cellular enzyme functions. Flavonoids exert their effect in different ways, in particular, there are flavonoids related with changes of intracellular calcium concentration suggesting that they could disturb the calcium transport, including the sarcoplasmic/endoplasmic reticulum Ca2+-ATPase and the plasma membrane Ca2+-ATPase (PMCA). However, the molecular mechanisms underlying this effect are not yet clear. The present study seeks to investigate the effect of several natural flavonoids on hPMCA4 (human plasma membrane Ca2+-ATPase isoform 4), a P-type ATPase essential for the intracellular Ca2+ control in eukaryotic cells.Results obtained with purified PMCA show that some flavonoids inhibited the PMCA activity related to the increase in the number of -OH in the B ring which enhances the inhibition potency. The most potent inhibitors were quercetin and gossypin with a Ki of 0.3 and 4.1 µM, respectively. The mechanism of inhibition of these flavonoids was dependent on the Mg2+ concentration, suggesting that the real inhibitor is a flavonoid-Mg2+ complex. When phosphorylated intermediates (EP) were measured, quercetin led to the increase of EP, which was sensitive to the ADP concentration in the medium, whereas gossypin induced a decrease. These results suggest that gossypin could affect the ATP binding site, whereas quercetin could prevent the change between E1P → E2P, the two main phosphorylated conformations belonging to the mechanism of ATP hydrolysis.To assess whether the effects of quercetin and gossypin on the purified hPMCA4 could affect living cells, we evaluated the effect of these flavonoids monitoring the changes in the cytoplasmic Ca2+ in HeK293T cells that overexpress hPMCA4. Fluorescence microscopy images indicate that both flavonoids distributed widely in the nucleus and the cytoplasm of cells. Similar results were obtained in preliminary experiments in Caco-2 cells.Results reveal that quercetin and gossypin are incorporated into the cells, showing that the cytoplasmic Ca2+ dynamics is affected, probably because PMCA activity is inhibited when quercetin or gossypin pass into the cells. These findings suggest a direct interaction between PMCA cytoplasmic domains and these flavonoids. In conclusion, our results show that PMCA could be involved in the molecular mechanism underlying the flavonoids effects on biological systems.Acknowledgement, with grants of CONICET, ANPCYT and UBACYT