ACTIVATION OF THE PLASMA MEMBRANE Ca2+ PUMP AS REVEALED BY FRET BETWEEN BFP AND GFP FUSED TO THE N AND C TERMINUS.

CORRADI GERARDO RAUL; ADAMO HUGO PEDRO

Pinamar, Pcia. Bs. As.

Congreso; XLI Reunion de la Sociedad Argentina de Investigacion en Bioquimica y Biologia Molecular (SAIB); 2005

Sociedad Argentina de Investigacion en Bioquimica y Biologia Molecular (SAIB)

 Activation of the Plasma Membrane Ca2+ Pump as revealed by FRET between BFP and GFP fused to the N and C terminus.  Gerardo R. Corradi and Hugo P. Adamo, IQUIFIB-Facultad de Farmacia y Bioquímica (UBA-CONICET), Buenos Aires. gcorradi@qb.ffyb.uba.ar   The autoinhibition of the plasma membrane Ca2+ pump (PMCA) is believed to involve the interaction of the C-terminal domain with the catalytic region which contains the domains A and N (Bredeston and Adamo. 2004. J. Biol. Chem. 279:41619–41625). Calmodulin binds to the inhibitory sequence and promotes an activated state characterized by high affinity for Ca2+ and high transport activity. The BFP was inserted after Thr2 of the human PMCA isoform 4xb while the GFP was located at the COOH-terminal. Excitation of the BFP-PMCA-GFP at 387 nm resulted in a differential GFP emission by energy transfer (FRET) at 509 nm. The calculated average distance between chromophores (r) in the BFP-PMCA-GFP was 45 Å. The activation of the PMCA by the addition of 10 µM Ca2+ and 200 nM calmodulin increased the value of r to 50 Å.  A similar value of r was obtained when the PMCA was activated by phosphoinositides in the presence of   0.5 mM EGTA. Under these conditions the addition of Ca2+ calmodulin caused no further change. According to these results, the NH2 terminus and the COOH terminus of the PMCA would re-orient or slightly separate during activation. [Supported by grants of CONICET and UBA.]