Novel flavonoid-magnesium complexes as inhibitors of plasma membrane calcium ATPase

Plasma membrane calcium ATPases (PMCAs) are essential for regulating intracellular calcium (Ca²⁺) levels by extruding it from the cytosol. Improper regulation of these transporters is associated with numerous diseases, including neurological, cardiovascular, oncological, and metabolic problems, rendering them interesting targets for therapeutic intervention. However, there is a scarcity of specific tools to adjust PMCA activity. Flavonoids, a varied group of polyphenolic compounds with numerous biological effects, have been demonstrated to affect the function of several ATPases, including PMCAs. In this study, we investigated the inhibitory mechanism of quercetin on the human PMCA4 isoform (hPMCA4). Using UV-visible spectroscopy and ATPase activity assay, we identified a high-affinity inhibition mediated by a quercetin‑magnesium (Mg²⁺) complex with a Ki of 49.7 ± 1.5 nM. Functional and phosphorylation studies at different pHs suggest that quercetin affects PMCA activity through two inhibitory mechanisms: a high-affinity one mediated by the quercetin-Mg²⁺ complex and a low-affinity one mediated by the free flavonoid. Analysis of the structure-activity relationship revealed that hydroxyl groups at positions 3', 4', and 3 are critical for complex formation and inhibitory potency. Specifically, the 3' and 4' hydroxyls are required to form the PMCA inhibitory complex. These findings demonstrate a novel mechanism of PMCA activity modulation involving flavonoid-Mg²⁺ complexes, which emerge as selective molecular tools capable of regulating Ca²⁺ transport. This knowledge provides new insights into designing PMCA inhibitors and exploring therapeutic strategies targeting diseases linked to calcium signalling dysfunction.