Quercetin-Magnesium (II) Complex Inhibits the Plasma Membrane Calcium Pump.

LUCILA GENTILE; MARIEL MARDER; MALLKU QHAPAJ ONTIVEROS; IRENE MANGIALAVORI; MARIELA FERREIRA-GOMES; DEBORA RINALDI; JUAN PABLO ROSSI

Buenos Aires

Congreso; Reunión Conjunta de Sociedades de Biociencias; 2017

Abstract: Flavonoids are commonly found in fruit and vegetables and have been reported to reach micromolar concentration in the human blood plasma. These compounds have antioxidant capacity and are related to cancer chemoprotective properties, by triggering apoptosis of malignant cells through the Ca2+?dependent mitochondrial pathway. Some flavonoids, like quercetin inhibit the activity of sarcoplasmic/endoplasmic reticulum Ca2+-ATPase and the plasma membrane Ca2+-ATPase (PMCA), however the mechanism of its effect is not yet defined. PMCA is a calmodulin regulated P-type ATPase that maintains the homeostasis of intracellular Ca2+ in eukaryotic cells, coupling the transport of Ca2+ with ATP hydrolysis. The aim of this work is to characterize the inhibition of PMCA by quercetin. We measured the Ca2+-ATPase activity in purified PMCA from human erythrocytes and the [Ca2+]c transport in HEK293T cells culture. Results using purified PMCA show that quercetin inhibits PMCA activity. Quercetin absorption spectra with added magnesium Mg2+ show the formation of a quercetin-Mg2+ complex. Competitive kinetics with Mg2+ has shown that the mechanism of inhibition of quercetin is totally dependent on the concentration of Mg2+. Assays with [γ-32P] ATP indicate that inhibition of the ATPase activity by quercetin led to the increase of phosphoenzyme level (EP), implying that quercetin blocks the dephosphorylation of the pump. The EP formed is sensitive to dephosphorylation by ADP, suggesting a stabilization of the E1P intermediate. To characterize the effect of quercetin at the cellular level we studied its transport in HEK293T cell culture by measuring the intrinsic quercetin fluorescence. On the other hand, PMCA activity in HEK293T cells that overexpress PMCA4 is inhibited on a concentration-dependent behavior, in a similar way that in purified PMCA. Altogether, results show that the quercetin- Mg2+ complex is the true inhibitor of PMCA both in purified enzyme and in cultured cells.