Inhibition of the plasma membrane calcium pump by aurintricarboxylic acid

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

Rosario, Santa Fe

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

Plasma membrane calcium pumps (PMCAs), are high affinity calcium pumps that extrude calcium from the cytosol to the extracellular medium, using the energy of ATP hydrolysis and 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 more tissue or cell specific. PMCA 4 is the most abundant isoform expressed in hematopoietic tissue. It has been discovered that it regulates in a negative way the angiogenesis, making the blockade of this isoform interesting for study. The aurintricarboxylic acid (ATA), has been demonstrated to be an efficient inhibitor of the ATPase activity of PMCA4 at low concentrations, 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 demonstrated that ATA inhibits the purified enzyme and its activity in native membranes. Furthermore, it is not a competitive inhibitor with calcium, magnesium or ATP. Our current results show that:1)ATA binds to PMCA4 in presence of calcium and absence of ATP, where the predominant conformation is E1Ca.2)ATA binds to PMCA4 in absence of calcium, where the equilibrium is displaced towards conformation E2, and inhibits phosphatase activity of the enzyme.3)ATA does not interact with the ATP binding site of the pump but changes the fluorescence of the eosin probe when it is bound.4)The formation of a complex between ATA and magnesium is evidenced by fluorescence.5)ATA may act as a quencher of the fluorescence of aromatic amino acids of PMCA.6)Inhibition by ATA of Ca2+-ATPase activity in membranes of erythrocytes is less potent than the observed in the purified enzyme, but it is incremented when membranes are diluted. 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 in human 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 of Functional Activity and Binding Energetics. Life 2022, 12, 872. https://doi.org/10.3390/life12060872