“Measuring the dissociation constants of ligands from PMCA complexes by a photoactivatablephosphatidylcholine membrane domain probe

IRENE MANGIALAVORI, MARIELA FERREIRA GOMES, MARÍA F. PIGNATARO, EMANUEL E STREHLER AND JUAN PABLO F.C. ROSSI.

San Francisco

Congreso; 54rd Annual Meeting of Biophysical Society. San Francisco, California-USA.; 2010

Biophysical Society

MEASURING THE DISSOCIATION CONSTANTS OF LIGANDS FROM PMCA COMPLEXES BY A PHOTOACTIVATABLE PHOSPHATIDYLCHOLINE MEMBRANE DOMAIN PROBE Irene Mangialavori1, Mariela Ferreira Gomes 1, María F. Pignataro1, Ana Villamil1, Ariel Caride2 , Emanuel E. Strehler2 and Juan Pablo F.C. Rossi*1 1Instituto de Química y Fisicoquímica Biológicas, Facultad de Farmacia y Bioquímica, UBA-CONICET, Junín 956 (1113) Buenos Aires, Argentina and 2Department of Biochemistry and Molecular Biology, Mayo Clinic College of Medicine, 200 First Street South West, Rochester, MN 55905, USA The purpose of this work was to obtain structural information about conformational changes of the plasma membrane Ca2+ pump (PMCA) in the membrane region upon interaction with ATP, Ca2+, calmodulin and acidic phospholipids. To this end, we have quantified labeling of PMCA with the photoactivatable phosphatidylcholine analog [125I]TID-PC/16, measuring the shift of conformation E2 to the auto-inhibited conformation E1I and to the activated E1A state, titrating the effect of Ca2+ and ATP under different conditions. With this method we were able to measure apparent and equilibrium constants for the dissociation of Ca2+, ATP and calmodulin and other ligands from PMCA complexes through the change of transmembrane conformations of the pump. The results indicate that the PMCA possesses a high-affinity site for Ca2+ regardless of the presence or absence of activators. Modulation of pump activity is exerted through the C-terminal domain, which induces an apparent auto-inhibited conformation for Ca2+ transport but does not modify the affinity for Ca2+ at the transmembrane domain. The C-terminal domain is affected by calmodulin and calmodulin-like treatments driving the auto-inhibited conformation E1I to the activated E1A conformation and thus modulating the transport of Ca2+. The data further suggest that the hydrophobic transmembrane domain of the PMCA undergoes major rearrangements resulting in altered lipid accessibility upon Ca2+ binding and activation. With grants from ANPCYT, CONICET, UBACYT and NIH.