1. Determination of the dissociation constants for Ca2+ and calmodulin from the plasma membrane Ca2+ Pump by a lipid probe that senses membrane domain changes.

MANGIALAVORI IRENE; FERREIRA GOMES MARIELA; PIGNATARO, MARÍA FLORENCIA; STREHLER, EMANUEL E; ROSSI, J.P.F.C.

JOURNAL OF BIOLOGICAL CHEMISTRY

AMER SOC BIOCHEMISTRY MOLECULAR BIOLOGY INC

Año: 2009 vol. 285

0021-9258

The purpose of this work was to obtain information aboutconformational changes of the plasma membrane Ca2
-pump(PMCA) in the membrane region upon interaction with Ca2
,calmodulin (CaM) and acidic phospholipids. To this end, wehave quantified labeling of PMCA with the photoactivatablephosphatidylcholine analog [125I]TID-PC/16, measuring theshift of conformation E2 to the auto-inhibited conformation E1Iand to the activated E1A state, titrating the effect of Ca2
underdifferent conditions. Using a similar approach, we also determinedthe CaM-PMCA dissociation constant. The results indicatethat the PMCA possesses a high affinity site for Ca2
regardless of the presence or absence of activators. Modulationof pump activity is exerted through the C-terminal domain,which induces an apparent auto-inhibited conformation forCa2
transport but does not modify the affinity for Ca2
at thetransmembrane domain. The C-terminal domain is affected byCaM and CaM-like treatments driving the auto-inhibited conformationE1I to the activated E1A conformation and thus modulatingthe transport of Ca2
. This is reflected in the differentapparent constants for Ca2
in the absence of CaM (calculatedby Ca2
-ATPase activity) that sharply contrast with the lack ofvariation of the affinity for the Ca2
site at equilibrium. This isthe first time that equilibrium constants for the dissociation ofCa2
and CaM ligands from PMCA complexes are measuredthrough the change of transmembrane conformations of thepump.Thedatafurthersuggestthatthetransmembranedomainofthe PMCA undergoes major rearrangements resulting in alteredlipid accessibility upon Ca2
binding and activation.