Conformational changes of the transmembrane domain of the Plasma Membrane Calcium Pump by calmodulin and acidic phospholipids

M. FLORENCIA PIGNATARO, ANA VILLAMIL GIRALDO, MARIELA FERREIRA-GOMES, JUAN PABLO ROSSI AND IRENE MANGIALAVORI

Salta

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

SAB-Protein Society

“Conformational changes of the transmembrane domain of the Plasma Membrane Calcium Pump by calmodulin and acidic phospholipids”. M. Florencia Pignataro, Ana Villamil Giraldo, Mariela Ferreira-Gomes, Juan Pablo Rossi and Irene Mangialavori. The Plasma Membrane Calcium Pump (PMCA) is an integral membrane protein, from the family of the P-ATPases. The reaction cycle of these proteins is characterized for a phosphorylated intermediary. Its ATPase activity is enhanced by several modulators among which calmodulin (CaM) and acidic phospholipids appear to be the most effective ones. Whenever Ca2+ intracelular concentration rises, calmodulin binds to the C-terminal cytoplasmatic domain, generating a conformational change that releases the protein from its previous state of auto-inhibition. The acidic phospholipids binding site could be located in the cytoplasmatic loop that connects the transmembrane segments 2 and 3 (TM2 y TM3). Some author, suggest the existence of a second site in the C-terminal domain near to the CaM binding site, that would prevent CaM mediated activation when acidic phospholipids are present. The purpose of this work was to obtain structural information about conformational changes of PMCA related to the activation by calmodulin (CaM) and phosphatidic acid (PA). The transmembrane region was studied by measuring the specific incorporation of the photoactivatable phosphatidylcholine analog, [125I]TID-PC/16. Previously, this strategy allowed us to demonstrate the existence of a transmembrane inhibited conformation in the presence of Ca2+ (E1I) and an active conformation (E1As) in the presence of activators that expose a different surface to surrounding phospholipids [1, 2]. The cytoplasmatic region was studied by the access of two proteases to their sites of cleavage and measuring FRET between the labeled pump with eosin isothiocyanate (EITC-PMCA) and fluorescent phosphatidylethanolamine analog (RhoPE). 1) FRET experiments would be suggesting that in the conformation E1I (obtained with Ca2+ alone) the cytoplasmatic domain containing the ATP binding site (where PMCA is labeled with EITC) is closer to the membrane. Addition of CaM, to form E1 activated (E1A), results in this domain located in a position further away from the membrane. Regarding the experiment in which we measured FRET efficiency between EITC-PMCA and RhoPE with increasing amounts of PA, it will be said that, as expected, increasing the amount of lipids decreased the fluorescence because of RhoPE dilution. However, the decrease in FRET was the same regardless of the nature of the phospholipid added (phosphatidylcholine, phosphatidylethanolamine, or phosphatidic acid). This indicates that during the activation by acidic lipids, the cytoplasmic domain which contains the ATP binding site does not move significantly farther away from the membrane. This strongly differs from the results obtained with the enzyme activated by CaM. With grants of ANPCYT, CONICET and UBA.