The Asn879 of the human plasma membrane Ca2+-ATPase (SERCA Asn796): more than a Ca2+-binding residue.

DÉBORA E. RINALDI; HUGO P. ADAMO

Ahrus, Dinamarca

Congreso; 12th International ATPase Conference.Na,K-ATPase and Related Transport ATPases of P-type: Structures, Mechanisms, and Roles in Health and Disease.; 2008

University of Aarhus

Similarly to Asn796 from SERCA, the M6 residue Asn879 has been suggested coordinate Ca2+ through its carboxylate in the Ca2+ transport site of the plasma membrane Ca2+-ATPase (PMCA human isoform 4xb).   Consistently with this idea mutation of Asn879 to Ala abolish the transport of Ca2+ by the PMCA. This result however does not account for the conservation of an Asn residue in the Ca2+-ATPases since its replacement for example by Asp would still provide the lateral chain required for Ca2+ binding. We have expressed in Saccharomyces cerevisiae a mutant of the PMCA in which Asn879 was replaced by Asp. Yeast membranes were solubilized by the detergent C12E10, the PMCA was purified by calmodulin chromatography and reconstituted in liposomes. The Ca-ATPase activity of the Asn879Asp mutant was between 12% and 40 % of that of the wild type enzyme. Most strikingly the mutant exhibited a Ca2+ dependency similar to the wild type and reacted with ATP to form proportionate amounts of phosphoenzyme. Furthermore the mutation reduced the ability of the enzyme to hydrolyze pNPP in the absence of Ca2+ to a similar degree as the Ca2+-ATPase. These results suggest that the role of Asn879 in the PMCA are more complex than expected from its Ca2+ binding function and  involve alterations in the catalytic activity  associated with the E2 forms of the enzyme.   This work was supported in part by the University of Buenos Aires (UBA, Grant B029), by the Consejo Nacional de Investigaciones Científicas y Técnicas (CONICET, Grant PIP 6168), and by Agencia Nacional de Promoción Científica y Tecnológica (ANPCyT, Grant BID 1728 OC-AR PICT 15-25965).