CONICET | Buscador de Institutos y Recursos Humanos

The plasma membrane calcium pump (PMCA) is widely distributed along the eukaryotic cells. This pump transports Ca2+ actively from cytoplasm to extracellular medium coupled to the hydrolysis of ATP maintaining a low intracellular concentration of this cation. PMCA is mainly regulated by the Ca2+-calmodulin complex (Ca2+-CaM). In the absence of CaM the pump is auto-inhibited; while binding of CaM to the C-terminal domain produces the activation of the pump1. The aim of this work is to study the transport of several divalent cations (C2+) by PMCA in both auto-inhibited and activated state. Purified PMCA preparations and inside-out vesicles (IOVS) were obtained from human erythrocytes membranes. We measured the C2+-ATPase activity as function of Ca2+, Sr2+, Ba2+ and Pb2+, in the presence and absence of CaM as well as, with the enzyme previously activated by removing the C terminal domain with chymotrypsin2. Additionally, we study the active transport of divalent cations into IOVS by measuring light scattering changes of the vesicles. Our results show that: (a) PMCA is able to transport divalent cations other than Ca2+; (b) These cations are transported with different affinities and velocities; (c) PMCA activated state presents an increased affinity for the studied cations; (d) Both C2+-ATPase and transport of C2+ measured by light scattering show a similar behavior revealing that both processes are coupled. These results suggest that the mechanism of expulsion of Ca2+ by PMCA would also transport other divalent cations. This fact indicates that PMCA may contribute to detoxification process under the eventual access of other divalent cations into the cell. [1] Filomatori, C. V. and Rega, A. F. (2003) J Biol Chem. 278, 22265-22271 [2] Enyedi, A., Flura, M., Sarkadi, B., Gardos, G., Carafoli, E. (1987) J. Biol. Chem. 262,6425-6430 With grants of ANPCYT, CONICET y UBACYT