Calcium Occlusion in the Phosphorylated Intermediate of Plasma Membrane Ca2+-ATPase

MARIELA FERREIRA-GOMES, RODOLFO M GONZÁLEZ-LEBRERO, MARÍA CANDELARIA DE LA FUENTE, ROLANDO ROSSI AND JUAN PABLO ROSSI

Salta

Congreso; SAB 2010 Workshop CeBEM-Protein Society Meeting; 2010

SAB-Protein Society

“Calcium Occlusion in the Phosphorylated Intermediate of Plasma Membrane Ca2+-ATPase”. Mariela Ferreira-Gomes, Rodolfo M González-Lebrero, María Candelaria de la Fuente, Rolando Rossi andJuan Pablo Rossi. The Plasma Membrane Calcium ATPase (PMCA) is a calmodulin-regulated P-type ATPase responsible for the maintenance of low intracellular concentrations of Ca2+ in most eukaryotic cells. It couples the transport of Ca2+ out of the cells with the hydrolysis of ATP into ADP and inorganic phosphate. The current kinetic model proposes that the enzyme exists in two main conformations, E1 and E2. After binding of intracellular Ca2+ to high-affinity sites, E1 can be phosphorylated by ATP with formation of the intermediate E1P, which would result in occlusion of bound Ca2+. After a conformational transition to E2P, Ca2+ would be released to the extracellular medium from low-affinity sites, the phosphoenzyme is hydrolyzed and the resulting E2 intermediate state undergoes a new conformational transition to E1 (fig. 1). The principal aim of this work was to identify the calcium occlusion intermediate and to assess whether the occlusion of calcium is concomitant with the phosphorylation of the enzyme by ATP. For this, PMCA was produced by Baculovirus–mediated expression in Sf9 insect cells and isolated as microsomal membranes. Calcium occlusion was detected in this microsomal preparation of PMCA using the method of Rossi et al (1999) with minor modifications. In pre-steady-state conditions, the time course of  retained calcium showed a biphasic behavior, an initial rapid phase and a second slow one. Results show that: (1) The addition of alamethicin allowed a more efficient removal of Ca2+ ions from the intravesicular medium, without affecting the enzyme activity. With this treatment, the slow phase disappeared without affecting the fast phase (2) The Ca2+-ATPase activity and the amount of occluded calcium showed the same dependence on [Ca2+], with similar values of K0.5. This is an evidence that the intermediate containing occluded calcium is an intermediate of the reaction cycle of  PMCA (3) The ratio CaOcc/E1P was compatible with the stoichiometry of transport, where one Ca2+ is transported per ATP hydrolyzed. Calcium is occluded in E1P (4) The occlusion of calcium is concomitant with the phosphorylation of the enzyme and probably occurs in the E1P intermediate.  With grants of ANPCYT, CONICET and UBA.