title: Determination of the dissociation constants for Ca2+ and calmodulin from the plasma membrane Ca2+ pump by a lipid probe that senses membrane domain changes.
abstract: The purpose of this work was to obtain information about conformational changes of the plasma membrane Ca(2+)-pump (PMCA) in the membrane region upon interaction with Ca(2+), calmodulin (CaM) and acidic phospholipids. To this end, we have quantified labeling of PMCA with the photoactivatable phosphatidylcholine analog [(125)I]TID-PC/16, measuring the shift of conformation E(2) to the auto-inhibited conformation E(1)I and to the activated E(1)A state, titrating the effect of Ca(2+) under different conditions. Using a similar approach, we also determined the CaM-PMCA dissociation constant. The results indicate that the PMCA possesses a high affinity site for Ca(2+) 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 Ca(2+) transport but does not modify the affinity for Ca(2+) at the transmembrane domain. The C-terminal domain is affected by CaM and CaM-like treatments driving the auto-inhibited conformation E(1)I to the activated E(1)A conformation and thus modulating the transport of Ca(2+). This is reflected in the different apparent constants for Ca(2+) in the absence of CaM (calculated by Ca(2+)-ATPase activity) that sharply contrast with the lack of variation of the affinity for the Ca(2+) site at equilibrium. This is the first time that equilibrium constants for the dissociation of Ca(2+) and CaM ligands from PMCA complexes are measured through the change of transmembrane conformations of the pump. The data further suggest that the transmembrane domain of the PMCA undergoes major rearrangements resulting in altered lipid accessibility upon Ca(2+) binding and activation.
authors.affiliation: Facultad de Farmacia y Bioquímica, Instituto de Química y Fisicoquímica Biológicas, Universidad de Buenos Aires, Consejo Nacional de Investigaciones Científicas y Técnicas, Junín 956, 1113 Buenos Aires, Argentina.
authors.forename: Irene
authors.initials: I
authors.lastname: Mangialavori
authors.forename: Mariela
authors.initials: M
authors.lastname: Ferreira-Gomes
authors.forename: María F
authors.initials: MF
authors.lastname: Pignataro
authors.forename: Emanuel E
authors.initials: EE
authors.lastname: Strehler
authors.forename: Juan Pablo F C
authors.initials: JP
authors.lastname: Rossi
mesh_terms: D001389:Azirines / Q000378:metabolism*; D002118:Calcium / Q000378:metabolism*; D002147:Calmodulin / Q000378:metabolism*; D002462:Cell Membrane / Q000187:drug effects / Q000201:enzymology*; D002918:Chymotrypsin / Q000494:pharmacology; D004789:Enzyme Activation / Q000187:drug effects; D006801:Humans; D007700:Kinetics; D015335:Molecular Probes / Q000378:metabolism*; D019301:Oleic Acid / Q000494:pharmacology; D010712:Phosphatidic Acids / Q000494:pharmacology; D010713:Phosphatidylcholines / Q000378:metabolism*; D053499:Plasma Membrane Calcium-Transporting ATPases / Q000737:chemistry / Q000378:metabolism*; D011499:Protein Processing, Post-Translational / Q000187:drug effects; D017434:Protein Structure, Tertiary; D017384:Sequence Deletion / Q000187:drug effects; D017186:Titrimetry; D014107:Tosyllysine Chloromethyl Ketone / Q000494:pharmacology
chemical_list: D001389:Azirines; D002147:Calmodulin; D015335:Molecular Probes; D010712:Phosphatidic Acids; D010713:Phosphatidylcholines; C088423:1-palmitoyl-2-(decanedioyl mono-(2-iodo-4-(3-trifluoromethyl-3H-diazirin-3-yl)benzyl)ester)glycero-3-phosphocholine; D014107:Tosyllysine Chloromethyl Ketone; D019301:Oleic Acid; D002918:Chymotrypsin; D053499:Plasma Membrane Calcium-Transporting ATPases; D002118:Calcium