IQUIFIB   02644
INSTITUTO DE QUIMICA Y FISICOQUIMICA BIOLOGICAS "PROF. ALEJANDRO C. PALADINI"
Unidad Ejecutora - UE
congresos y reuniones científicas
Título:
PMCA DIFFERENTIAL EXPOSURE OF HYDROPHOBIC DOMAINS AFTER CALMODULIN AND PHOSPHATIDIC ACID ACTIVATION
Autor/es:
JUAN PABLO F. ROSSI ; IRENE MANGIALAVORI; ANA MARIA VILLAMIL GIRALDO; MARIA F. PIGNATARO; MARIELA S. FERREIRA GOMES. ; ARIEL J. CARIDE
Lugar:
Baltimore, Maryland
Reunión:
Congreso; 55th Annual Meeting of Biophysical Society.; 2011
Institución organizadora:
Biophysical Society.
Resumen:
The exposure of plasma membrane calcium pump to surrounding phospholipids was assessed by measuring the incorporation of the photoactivatable reagent [125I]TID-PC/16 into the membrane regions of this pump. In the absence of activators, Ca2+ increases the incorporation of [125I]TID-PC/16. On the contrary, in the presence of Ca2+ and either calmodulin or phosphatidic acid the incorporation of the labeled phospholipids is decreased. Proteolysis of PMCA with V8 protease results in 3 main fragments: fragment N (TM 1 and 2), fragment M (TM 3 and 4) and fragment C (TM 5 to 10). In the presence of Ca2+, 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, suggesting that the conformational changes induced by calmodulin or phosphatidic acid extend to the transmembrane domains. The result also indicates differences between the active conformations produced by calmodulin and acidic phospholipids. To verify this, we also measured FRET between PMCA labeled with eosin isothiocianate at the ATP binding site and Rho-PE included in PMCA-containing micelles. CaM decreased the efficiency of the energy transfer between these two probes while PA did not. The result indicates that activation by CaM increases the distance between the ATP binding site and the membrane, but acidic phospholipids do not. Moreover, the access of two proteases to their sites of cleavage was different in the presence of calmodulin or phosphatidic acid. The results indicate structural differences between the PMCA conformations induced by these activators.