Interaction between recombinant SERCA hydrophilic domains and P-type ATPases: effects on the ATPase activity of PMCA and SERCA
Santos, D.F.1; Freire, M.M.1; Albernaz, F.P.1; Almeida, W.I.1; Costa, E.S.1; Verjovski-Almeida, S.2; Almeida, F.C.L.1; Valente, A.P.1; Carvalho-Alves, P.C.1 and Scofano, H.M.1
1 Instituto de Bioquímica Médica, Universidade Federal do Rio de Janeiro.
2 Instituto de Química, Universidade de São Paulo.
SERCA (sarco(endo)plasmic reticulum Ca2+-ATPase) and PMCA (plasma membrane Ca2+-ATPase) belong to the P-type family of membrane proteins that actively transport cations across biological membranes. In muscle, SERCA restores submicromolar cytosolic Ca2+ level resulting in relaxation. PMCA is the sole high affinity Ca2+ extrusion mechanism in the plasma membrane and is subject to various regulatory controls, being calmodulin (CaM) the most important physiological activator. The mechanism by which Ca2+ is translocated to the lumen of the endoplasmic reticulum involves major conformational rearrangements among the three cytoplasmic domains: Actuator (A), nucleotide-binding (N) and phosphorylation (P) domains, anchored by ten transmembrane segments (TM1-TM10). The A domain is believed to undergo movement during Ca2+ transport coupled to enzyme phosphorylation, interacting with the N and P domains.
In previous studies, we showed that the SERCA A domain recombinant (rDA-19 kDa protein), was able to stimulate the ATP hydrolysis activity of SERCA1 by uncoupling the Ca2+ transport, without changing the integrity of the membrane. To better understand this effect, we decided to extend these studies to either the PMCA, as the subject pump system, and recombinant SERCA P/N domains (large cytoplasmatic loop, rLCD-48 kDa), as the modifier agent. The rDA-19 kDa protein also stimulated PMCA ATPase activity three-fold, at saturating Ca2+ concentrations, in the absence, but not in the presence of CaM, which was a better activator. Curiously, the rLCD-48 kDa protein stimulated two-fold the SERCA1 ATP hydrolysis, in a dose-dependent manner with 50mM Ca2+, even in the presence of Ca2+ ionophore. This activation seems to be more important than that observed with the rDA-19 kDa protein, at any Ca2+ concentration tested.
These data showed that the recombinant domains are interesting tools for the understanding of the dynamic interplay of these three domains during the catalytic cycle of P-type ATPases.
Supporting: CNPq and FAPERJ
|