Metal fluoride effects on the Plasma Membrane Ca2+- ATPase: Characterization of the fluoride-stabilized phosphoenzyme analogues

NICOLAS ANDRÉS SAFFIOTI; ANA RIESCO; MARILINA DE SAUTU; ROLANDO ROSSI; MARIELA FERREIRA-GOMES; JUAN PABLO ROSSI; IRENE MANGIALAVORI

Congreso; XLVII Reunión Annual de la Sociedad de Biofísica Argentina.; 2018

The Plasma Membrane Ca2+ Pump (PMCA) is one of the most important participants in cytoplasmic Ca2+ regulation, and belongs to the P-type ATPases family (P-ATPases). The reaction cycle of this group is explained by the Albers and Post model, which postulates that these proteins exist in two main conformations E1 and E2 that can be phosphorylated forming the intermediates E1-P and E2-P. Recently, the first structural model of PMCA was obtained by crio-electronmicroscopy1. In the context of progresses in the study of PMCA structure, new methods to stabilize this protein in different conformations are necessary in order to investigate the relationship between its structure and mechanism of function and regulation.In this work we studied the effects of fluoride complexes of aluminium (AlFx), beryllium (BeFx) and magnesium (MgFx) on PMCA by means of measurement of enzyme activity, and employing fluorescent or hydrophobic photoactivatable probes. These complexes stabilize different states of the phosphorylated intermediates in other P-ATPases, but they have never been tested on PMCA. Our results show that the three complexes behaved as slow reversible inhibitors of Ca2+-ATPase and phosphatase activities by preventing phosphorylation from ATP. The inhibition was not competitive with Ca2+ in contrast with results observed in other P-ATPases. The affinities for AlFx, BeFx and MgFx increased slightly when PMCA was activated by calmodulin. On the other hand, the water content in the nucleotide binding site increased as a result of the conformational change produced by fluoride complexes binding. This effect occurred also when PMCA is phosphorylated from ATP, which allowed the measurement of the concentration of the phosphorylated intermediate in real time. In summary, our results show that these fluoride complexes are a useful to reveal the properties of different phosphorylated intermediates that are involved in the mechanism of hydrolysis of ATP by the PMCA.