A NEW CONFORMATION IN SERCA AND PMCA Ca2+ PUMPS REVEALED BY A PHOTOACTIVATABLE PHOSPHOLIPIDIC PROBE

IRENE MANGIALAVORI, ANA MARÍA VILLAMIL GIRALDO, CRISTINA MARINO BUSLJE, MARIELA GOMES FERREIRA, ARIEL J. CARIDE AND JUAN PABLO F.C. ROSSI

Buenos Aires. Argentina

Simposio; International Symposium of the International Master in Biomedical Sciences University of Buenos Aires (Argentina) and Albert Ludwigs University of Freiburg (Germany). School of Pharmacy and Biochemistry, UBA; 2008

University of Buenos Aires (Argentina) and Albert Ludwigs University of Freiburg (Germany)

The purpose of this work was to obtain structural information about conformational changes in PMCA membrane regions and their interaction with surrounding lipids. To this end, we have quantified labeling of the sarcoplasmic reticulum Ca2+ pump (SERCA) and the plasma membrane Ca2+ pump (PMCA) with the photoactivatable phosphatidylcholine analog [125I]TID-PC/16, under different conditions. This probe has been used previously to analyze lipid?protein interfaces. We determined that: (1) Incorporation of the photoactivatable reagent to SERCA decreases 25% when labeling is performed in the presence of Ca2+ as opposed to EGTA (2) The decrease in labeling matches qualitatively with the decrease in transmembrane surface exposed to the solvent calculated by the Lee-Richards method, when comparing the known SERCA structures 2ear (E2) (pdb. file) and 1su4 (E1Ca) (pdb. file). (3) Labeling of PMCA incubated with Ca2+ and calmodulin decreases by approximately the same amount as compared to EGTA. However incubation with Ca2+ alone (no calmodulin) increases labeling by 55%. This suggests that the conformation in which the enzyme is fully active (Ca2+ for SERCA and Ca2+-CaM for PMCA) exhibits a more compact transmembrane arrangement in both proteins. Addition of C28, a peptide containing the calmodulin binding region of PMCA, to SERCA in the presence of Ca2+ increases [125I]TID-PC/16 incorporation, confirming the suggestion made above. The results indicate that there is an autoinhibited conformation in these P-type ATPases that affects not only the cytoplasmic regions but also the transmembrane segments