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The purpose of this work was to obtain structural information about conformational changes of the plasma membrane calcium pump (PMCA) related to the activation by calmodulin (CaM) and phosphatidic acid (PA). The transmembrane region was studied by measuring the specific incorporation of photoactivatable phosphatidylcholine analog, [125I]TID-PC/161. 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 2, 3. The cytoplasmic region was studied by the access of two proteases to their sites of cleavage and FRET between the labeled pump with eosin isothiocyanate (EITC-PMCA) and fluorescent phosphatidylethanolamine analog (RhoPE). Our results suggest that: (1) The three hydrophobic domains (DH1-3) conforming PMCA transmembrane region were differentially affected by CaM and PA: CaM decreased the specific incorporation of [125I]TID-PC/16 in DH2 and DH3 and PA affected mainly DH2 and DH1. (2) Conformational changes associated with activation by CaM or PA did not expose additional cleavage sites but the proteolysis rate depends on the modulator. (3) FRET between PMCA-EITC and RhoPE was affected by CaM but not by PA indicating that activation by CaM modifies the distance between ATP binding site and the membrane. In conclusion, this data show the different mechanism of activation of PMCA by CaM and acidic phospholipids that involve not only cytoplasmic but also transmembrane regions of PMCA.