Lipid-protein Interactions in Plasma Membrana Calcium Pump.

MANGIALAVORI IRENE; VILLAMIL GIRALDO A.M; ROSSI, J.P.F.C.

ROSARIO, SANTA FE

Congreso; XXXVI Reunión Annual de la Sociedad Argentina de Biofísica; 2006

SOCIEDAD ARGENTINA DE BIOFISICA

Transporters, receptors and channels -          Lipid-protein Interactions in Plasma Membrane Calcium Pump Mangialavori Irene Cecilia, Villamil Giraldo Ana María, Rossi Juan Pablo F.C. IQUIFIB. Departamento  de Química biológica.  Universidad de Buenos Aires. irenem@qb.ffyb.uba.ar   Plasma membrane calcium pump (PMCA) is an integral membrane protein that actively transports Ca2+ towards the extra cellular milieu, maintaining low intracellular Ca2+ concentrations which are necessary for efficient signaling [1]. The energy necessary for this transport is provided by ATP hydrolysis. PMCA is a 134 kDa protein with 10 putative transmembrane segments. ATP binding site and the aspartate residue which is phosphorylated during the catalytic cycle are located in a large intracellular loop between transmembrane segments 4 and 5. PMCA is sensitive to its phospholipidic environment: neutral phospholipids are required to preserve the enzyme in a basal functional state from which it can be further activated while acidic phospholipids have been well characterized as PMCA activity modulators. The principal regulatory mechanism of enzymatic activity is the binding of calmodulin (CaM) to a domain located near the C-terminus of PMCA. This region has been suggested to function as an auto regulatory domain that inhibits enzyme activity by binding to a region closer to its catalytic site. Binding of CaM leads to dissociation of this domain causing a large conformational change which results in activation of PMCA by increasing its affinity to Ca2+ and the maximum velocity of the pump. We have previously employed a photoactivatable phosphatidylcholine analogue [125I] TID-PC/16 to study PMCA-lipid interactions. The experimental design includes reconstitution of PMCA from erythrocyte membranes, in detergent or mixed detergent-phosphatidylcholine micelles, Photolabeling and further electrophoretic isolation of the labeled protein. With the aim of evaluating if the conformational change produce by binding of CaM to PMCA modifies the enzyme exposure to surrounding phospholipids; the same methodological approach was applied. Results show that maximum interaction between PMCA and amphiphiles occurs when the protein is devoid of CaM. In the presence of PC a displacement of [125I] TID-PC/16 is observed as a decrease in the amount of reagent covalently bound to PMCA, both in the absence or in the presence of CaM. However, the extent of labeling of PMCA reconstituted either in detergent or in mixed detergent-PC micelles is higher in the absence of calmodulin. This suggests that regardless micelle composition, PMCA exposure to neighboring phospholipids is higher in basal -auto inhibited- conditions. With grants of ANPCYT, CONICET and UBACYT. References [1] Carafoli, E. Intracellular calcium homeostasis. 1987. Annu. Rev. Biochem. 56:395-433 [2]  Filomatori CV, Rega AF  On mechanism of activation of the plasma membrane Ca2+-ATPase by ATP and acidic phospholipids. 2003. J Biol. Chem. 278:22265-71  [3].Enyedi, A. T Vortherr , P. James, D. J. Mc Cormick, A. G. Filoteo, E. Carafoli, and J. T. Penniston. The calmodulin binding domain of the plasma membrane Ca2+ pump interacts both with calmodulin and with another part of the pump. 1989.  J Biol. Chem. 264:12313-12321 [4] Villamil Giraldo AM, Castello PR, Gonzalez Flecha FL, Delfino JM, Rossi JP. Phospholipids distribution around the plasma membrane calcium pump: a hydrophobic photolabeling study. 2006. Cell Biochem Biophys. 44:431-7