PMCA differential exposure hydrophobic domains after Calmodulin and phosphatidic acid activation

IRENE MANGIALAVORI; ANA MARÍA VILLAMIL GIRALDO; MARÍA FLORENCIA PIGNATARO; MARIELA FERREIRA GOMES; ARIEL CARIDE; JUAN PABLO ROSSI

JOURNAL OF BIOLOGICAL CHEMISTRY

AMER SOC BIOCHEMISTRY MOLECULAR BIOLOGY INC

Año: 2011 vol. 286 p. 18397 - 18404

0021-9258

The exposure of plasma membrane calcium pump (PMCA) to surrounding phospholipids was assessed by measuring the incorporation of the photoactivatable phosphatidylcholine analogue [125I]TID-PC/16 to the protein. In the presence of Ca2+ both calmodulin (CaM) and phosphatidic acid (PA) greatly decreased the incorporation of [125I]TID-PC/16 to PMCA. Proteolysis of PMCA with V8 protease results in 3 main fragments: N which includes transmembrane segments M1 and M2, M which includes M3 and M4 and C which includes M5 to M10. CaM decreased the level of incorporation of [125I]TID-PC/16 to fragments M and C, while phosphatidic acid decreased the incorporation of [125I]TID-PC/16 to fragments N and M. This suggests that the conformational changes induced by binding of CaM or PA extend to the adjacent transmembrane domains. Interestingly, this result also denotes differences between the active conformations produced by CaM and PA. To verify this point, we measured RET between PMCA labeled with eosin-isothiocyanate at the ATP-binding site and the phospholipid Rho-PE included in PMCA micelles. CaM decreased the efficiency of the energy transfer between these two probes while PA did not. This result indicates that activation by CaM increases the distance between the ATP-binding site and the membrane, but PA does not affect this distance. Our results disclose main differences between PMCA conformations induced by CaM or PA and show that those differences involve transmembrane regions.