Flavonoids and calcium transport through biological membranes

MALLKU Q ONTIVEROS; DEBORA RINALDI; MARIEL MARDER; ESPELT, M.V.; MAXIMILIANO VIGIL; IRENE MANGIALAVORI; ROLANDO ROSSI; JUAN PABLO ROSSI; MARIELA FERREIRA-GOMES

Congreso; XLVII Reunión Annual de la Sociedad de Biofísica Argentina.; 2018

Flavonoids are polyphenolic compounds synthesized by plants that are widely distributed in fruits and vegetables. These compounds have been reported to reach a micromolar concentration in human blood plasma. Flavonoids have a broad bioactivity, mainly antioxidant, modulate the catalytic activity of enzymes like P-type ATPase and trigger the apoptosis of malignant cells through the Ca2+-dependent mitochondrial pathway. Some flavonoids, like quercetin, inhibit Ca2+ removal systems, as the sarcoplasmic/endoplasmic reticulum (SERCA) and the plasma membrane (PMCA) calcium ATPases. However, the molecular mechanism of this effect is not yet defined. PMCA is a P-type ATPase regulated by calmodulin that maintains the homeostasis of intracellular Ca2+ in eukaryotic cells, coupling the transport of Ca2+ with ATP hydrolysis. The present study seeks to investigate the effect of several natural flavonoids on hPMCA4 (human Plasma Membrane Calcium Pump isoform 4) both in the purified system and in living cells, and to establish the relationship between the flavonoid structure and its inhibitory activity. Results obtained with purified PMCA show that some flavonoids inhibit the PMCA activity and that the increase in the number of -OH in the B ring enhances the potency of the inhibition. The best inhibitors were quercetin, a flavonol aglycone, and gossypin, a flavonol glycoside, with Ki of 0.3 and 4.1 M, respectively. Magnesium competition assays indicate that the mechanism of inhibition of these flavonoids is dependent on the concentration of Mg2+, suggesting that the inhibitor is a flavonoid-Mg2+ complex. On the other hand, when phosphorylated intermediates (EP) were measured, quercetin led to the increase whereas gossypin induced a decrease of EP. The dephosphorylation of EP formed in the presence of quercetin was sensitive to ADP indicating that this flavonoid could stabilize an E1P intermediate. These results suggest that gossypin could affect the binding of ATP while quercetin could prevent the conformational change E1P → E2P. Altogether, these flavonoids inhibit PMCA through different mechanisms.In order to assess whether the effects of quercetin and gossypin on the purified hPMCA4 could occur in living cells, we evaluated the effect of these flavonoids monitoring changes in the cytoplasmic calcium in HeK293T cells that overexpress hPMCA4. Results show that quercetin and gossypin inhibited the PMCA activity in HeK293T cells. Fluorescence microscopy images indicate that both flavonoids distributed widely in the cytoplasm of HEK293T cells, suggesting that it is possible a direct interaction between these flavonoids and the cytoplasmic domains of PMCA.