Interaction of ATP, Calcium and Vanadate with the plasma membrane calcium pump: The regulatory effect of ATP

MANGIALAVORI IRENE C.; FERREIRA GOMES MARIELA; GONZÁLEZ-LEBRERO RODOLFO; ROSSI ROLANDO ; ROSSI JUAN PABLO F.C.

Tucuman

Congreso; XLI Reunión Anual de la Sociedad Argentina de Biofísica; 2012

Sociedad Argentina de Biofísica

The plasma membrane calcium pump (PMCA) is a membrane-bound P-type ATPase that actively transports Ca2+ from the cytosol to the extracellular medium. The membrane-buried region of PMCA, comprise 10 membrane helices connected to a large cytoplasmic domain which contains the nucleotide binding and the phosphorylation sites. Ca2+ transport is achieved by means of a reaction cycle involving extensive protein conformational changes between four major states, E1, E1P, E2P, and E2. ATP is the substrate for phosphorylation of E1 state and also enhances the rate of the step involved in phosphoenzyme turnover. However, these regulatory effects of ATP remain largely unresolved for the PMCA. The aim of this work was to obtain information about conformational changes of a purified preparation of PMCA when it interacts with ATP, (VO4)3- and Ca2+. To this end, we used a photoactivatable phosphatidylcholine probe tagged with 125iodine ([125I]TID-PC/16) and an ATP fluorescent analogue (TNP-ATP) to study the transmembrane and the cytoplasmic domains, respectively. Using a rapid-mixing technique we have measured the amount of Ca2+ tightly bound to the E2(VO4)3- complex of the PMCA. In addition, we measured the effect of ATP on the conformation of the pump when it is stabilized in E2, E2P and Ca2+E2P analogue conformations. Our results show that: (1) ATP binds to the E2 and E2P conformations of the PMCA inducing a conformational change (2) ATP binding affinity is different for E2 and E2P (3) Ca2+ binds with a non-competitive behavior to the E2(VO4)3- complex both in the presence and in the absence of ATP generating a large conformational change. With grants of ANPCYT, CONICET, UBACYT y NIH.