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

SAFFIOTI, NICOLÁS A.; RIESCO, ANA; DE SAUTU, MARILINA; ROSSI ROLANDO C; BERLIN, JOSHUA; FERREIRA GOMES, MARIELA; ROSSI JUAN PABLO F. C.; MANGIALAVORI IRENE

La Plata

Congreso; XLVII Reunión Anual de la Sociedad Argentina de Biofísica; 2018

Sociedad Argentina de Biofísica

The Plasma Membrane Ca2+ Pump (PMCA) is one of the most importantparticipants in cytoplasmic Ca2+ regulation, and belongs to the P-type ATPasesfamily (P-ATPases). The Albers and Post model postulates that P-ATPases exist intwo main conformations E1 and E2 that can be phosphorylated forming E1-P andE2-P. Recently, the first structural model of PMCA was obtained by crioelectronmicroscopy1. In the context of progresses in the study of PMCA structure,new methods to stabilize this protein in different conformations are necessary inorder to investigate the relationship between its structure and function.In this work we studied the effects of fluoride complexes of aluminium (AlFx),beryllium (BeFx) and magnesium (MgFx) on PMCA by means of measurement ofenzyme activity, and employing fluorescent or hydrophobic photoactivatableprobes. These complexes stabilize different states of the phosphorylatedintermediates in other P-ATPases, but they have never been tested on PMCA. Ourresults show that the three complexes behaved as slow reversible inhibitors ofCa2+-ATPase and phosphatase activities by preventing phosphorylation from ATP.The inhibition was not competitive with Ca2+ in the case of AlF x and MgF x, incontrast with results observed in other P-ATPases. The affinities for AlFx, BeFx andMgFx increased slightly when PMCA was activated by calmodulin. On the otherhand, the water content in the nucleotide binding site increased as a result of theconformational change produced by fluoride complexes binding. This effectoccurred also when PMCA was phosphorylated from ATP, which allowed tomeasure the concentration of the phosphorylated intermediate in real time. Insummary, our results show that these fluoride complexes are a useful to revealthe properties of different phosphorylated intermediates that are involved in themechanism of hydrolysis of ATP by the PMCA.Refrences1) Gong D., Chi X., Ren K., Huang G., Zhou G., Yan N., Lei J., Zhou Q. (2018) Nat.Commun. 3623, 9 1AcknowldegmentsThis work was supported by ANPCyT PICT 2014 0065, CONICET PIP 11220150100250CO, and UBA Cienciay Técnica grant 2014-2017: 20020130100254B.