“Activation of the plasma membrane Ca2+ pump as revealed by fret between BFP and GFP fused to the N and C terminus

CORRADI G. R; ADAMO H.P

Isla Margarita, Venezuela

Workshop; “International Workshop Membrane Transport in Health and Disease”; 2006

Physiological Society, el Centro Latinoamericano de Ciencias Biológicas (CLAB-UNESCO)

The autoinhibition of the plasma membrane Ca2+ pump is believed to involve the interaction of the COOHterminal domain with regions of 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. With the aim of studying the inhibition–activation process, we succeeded in creating an active PMCA fused to two fluorescent proteins. The BFP was inserted after Thr2 of the human isoform 4xb while the GFP was located at the COOH-terminal end 100 residues downstream of the sequence corresponding to the calmodulin binding peptide C28. The recombinant BFP-PMCA-GFP was obtained by expression in Saccharomyces cerevisiae and was purified by affinity chromatography. Excitation of the BFP-PMCAGFP at 387 nm resulted in a differential GFP emission by energy transfer (FRET) at 509 nm. The efficiency of transfer was estimated by comparing the emission of BFP at 450 nm in the BFP-PMCA-GFP with that of the BFPPMCA protein. 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.