Inhibition of the plasma membrane calcium ATPase by aurintricarboxylic acid

SOUTO, CECILIA; BRUNO, CAMILA; JUAN PABLO ROSSI; FERREIRA GOMES, MARIELA; MANGIALAVORI IRENE

Rosario

Congreso; L Reunión Anual de la Sociedad Argentina de Biofísica; 2022

Sociedad Argentina de Biofísica

Plasma membrane calcium pumps (PMCAs), are high affinity calcium pumps that extrudecalcium from the cytosol to the extracellular medium, using the energy of ATP hydrolysisand magnesium as a cofactor. There are 4 isoforms of PMCA, coded by 4 different genes.PMCA 1 and PMCA 4 are expressed in many tissues, while PMCA 2 and PMCA 3 are moretissue or cell specific.PMCA 4 is the most abundant isoform expressed in hematopoietic tissue. It has beendiscovered that it regulates in a negative way the angiogenesis, making the blockade ofthis isoform interesting for study. The aurintricarboxylic acid (ATA), has beendemonstrated to be an efficient inhibitor of the ATPase activity of PMCA4 at lowconcentrations, although its inhibition mechanism is unknown.The aim of this study is to continue the characterization of the inhibition of PMCA4 by ATA. The enzyme was obtained from human erythrocytes. We have previously demonstratedthat ATA inhibits the purified enzyme and its activity in native membranes. Furthermore, itis not a competitive inhibitor with calcium, magnesium or ATP. Our current results showthat:ATA binds to PMCA4 in presence of calcium and absence of ATP, where thepredominant conformation is E1Ca.ATA binds to PMCA4 in absence of calcium, where the equilibrium is displacedtowards conformation E2, and inhibits phosphatase activity of the enzyme.ATA does not interact with the ATP binding site of the pump but changes thefluorescence of the eosin probe when it is bound.The formation of a complex between ATA and magnesium is evidenced byfluorescence.ATA may act as a quencher of the fluorescence of aromatic aminoacids of PMCA.Inhibition by ATA of Ca2+-ATPase activity in membranes of erythrocytes is lesspotent than the observed in the purified enzyme, but it is incremented whenmembranes are diluted.Keywords: ATPases, calcium, aurintricarboxylic acid, inhibition mechanism.References:Nicholas Stafford, Claire Wilson, Delvac Oceandy, Ludwig Neyses, and Elizabeth J.Cartwright: The plasma membrane calcium ATPases and their role as major new players inhuman diseases. Physiol Rev 97: 1089 –1125, 2017, doi:10.1152.Conceição A. Minetti , David P. Remeta: Characterization of Aurintricarboxylic Acid (ATA)Interactions with Plasma Transporter Protein and SARS-CoV-2 Viral Targets: Correlation ofFunctional Activity and Binding Energetics. Life 2022, 12, 872. https://doi.org/10.3390/life12060872AcknowldegmentsUniversidad de Buenos Aires and Agencia Nacional de Promoción Científica y Tecnológica.