PMCA differential exposure of hydrophobic domains after calmodulin and phos-phatidic acid activation

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

JOURNAL OF BIOLOGICAL CHEMISTRY

AMER SOC BIOCHEMISTRY MOLECULAR BIOLOGY INC

Año: 2011 vol. 286 p. 18397 - 18404

0021-9258

The exposure of plasma membranecalcium pump (PMCA) to surroundingphospholipids was assessed bymeasuring the incorporation of thephotoactivatable phosphatidylcholineanalogue [125I]TID-PC/16 to theprotein. In the presence of Ca2+ bothcalmodulin (CaM) and phosphatidicacid (PA) greatly decreased theincorporation of [125I]TID-PC/16 toPMCA. Proteolysis of PMCA with V8protease results in 3 main fragments: Nwhich includes transmembranesegments M1 and M2, M whichincludes M3 and M4 and C whichincludes M5 to M10.CaM decreased the level ofincorporation of [125I]TID-PC/16 tofragments M and C, while phosphatidicacid decreased the incorporation of[125I]TID-PC/16 to fragments N and M.This suggests that the conformationalchanges induced by binding of CaM orPA extend to the adjacenttransmembrane domains. Interestingly,this result also denotes differencesbetween the active conformationsproduced by CaM and PA. To verifythis point, we measured RET betweenPMCA labeled with eosinisothiocyanateat the ATP-binding siteand the phospholipid Rho-PE includedin PMCA micelles. CaM decreased theefficiency of the energy transferbetween these two probes while PA didnot. This result indicates that activationby CaM increases the distance betweenthe ATP-binding site and themembrane, but PA does not affect thisdistance. Our results disclose maindifferences between PMCAconformations induced by CaM or PAand show that those differences involvetransmembrane regions.