Lipid-protein interactions in Plasma Membrane Calcium Pump A fluorescence and photolabeling study

MANGIALAVORI IRENE CECILIA, VILLAMIL GIRALDO ANA MARÍA, ROSSI JUAN PABLO F.C.

Montevideo-Uruguay, 27-31 de Agosto de 2007

Congreso; • 6th International Conferences of Biological Physics. ICBP 2007; 2007

Plasma membrane calcium pump (PMCA) is a 134 KDa integral membrane protein that actively transports Ca2+ towards the extra cellular milieu, maintaining low intracellular Ca2+ concentrations [1]. ATP binding site is located in a large citoplasmatic loop between transmembrane 4 and 5. The principal regulatory mechanism of enzymatic activity is a binding of calmodulin (CaM) to a domain located near the C-terminus of PMCA. 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+. We have previously employed a photoactivatable phosphatidilcholine analogue [125I] TID-PC/16 to study PMCA-lipids interactions. The experimental design includes reconstitution of PMCA form erythrocytes membranes, in detergent or mixed detergent-phosphatidilcholine micelles, photolabeling and further electrophoretic isolation of the labeled protein. Which 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. In addition, we employed study of fluorescence that include the labeling the intracellular loop (Lys 495) of the pump which both eosin isothiocianate (EITC) and fluorecein isothiocianate (FITC) this procedure not interfere which the CaM binding and this methodology can permit see conformationals changes sensing the environment and realize FÖrster Resonance Energy Transfer (FRET) study between EITC-PMCA - rhodamin phosphatiyletanolamine (RhoPE), and FITC-PMCA - BODIPY 530/550 C5-HPC for have information to lipids-protein interactions of the pump which different phospholipids in presence or absence of CaM.    Results shown 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 observe as a decrease in the amount  of reagent covalently bound to PMCA, both in the absence or in the presence of CaM.. This suggests that regardless micelle composition, PMCA exposure to neighboring phospholipids is higher in basal auto inhibited conditions. In the other hands, the transfer between donor-acceptor pair not change in presence of CaM, but can see displacement which different amphiphiles, suggested that the citoplasmatic loop not sense change, almost in the employed conditions.