Inhibition of the plasma membrane calcium pump by aurintricarboxylic acid

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

Congreso; XLIX Reunión Anual de la Sociedad Argentina de Biofísica; 2021

The plasma membrane calcium pump (PMCA), is a high-affinity calcium pump that ejects calcium from the cell into the extracellular environment, using the hydrolysis of ATP. There are four isoforms of PMCA, encoded by four different genes. PMCA1 and PMCA4 are expressed in many tissues, while PMCA2 and PMCA3 are more tissue or cell-specific. PMCA4 is the most common isoform expressed in red blood cells. It has been found to negatively regulate angiogenesis, which makes this PMCA blockade of interest for research1. In living cells, aurintricarboxylic acid (ATA) has been shown to be a specific inhibitor of PMCA4 at low concentrations2, although its inhibitory mechanism remains unknown. This study aims to characterize the PMCA inhibition of ATA. For this purpose, PMCA was purified from human erythrocytes, containing 80% PMCA4 and 20% PMCA1, and the inhibition of PMCA by ATA was studied under different conditions. Our current results show that: (1) ATA inhibits PMCA Ca2+ATPase activity with an apparent affinity (Ki) of 0.25 µM; (2) The inhibition and activity of PMCA Ca2+ATPase were completely restored after dilution of ATA in the medium (3) The apparent affinity for ATA increased with Mg2+ concentration, but did not modify with ATP or Ca2+; (4 ) In the absence of Ca2+ and ATP, ATA also inhibited PMCA phosphatase activity with a Ki of 0.1 µM; (5) When increased concentrations of ATA were added to the purified PMCA, the fluorescence of ATA increased suggesting that ATA binds to the pump. Our results suggest that ATA does not bind to the ATP binding site of PMCA and that Mg2+ is required for complete inhibition of activity. Moreover, the variation in fluorescence of the binding can make it possible to obtain information on the interaction between ATA and different structural states of PMCA.