Phospholamban Interaction with SR Ca-ATPase Investigated by Pre-Steady State Charge Measurements

Abstract

Sarcoplasmic reticulum (SR) Ca-ATPase (SERCA) promotes muscle relaxation by pumping Ca2+ ions from the cytoplasm into the SR lumen. The activity of SERCA is modulated by Phospholamban (PLN), a membrane protein present in the SR of cardiomyocytes (1). The molecular mechanism of PLN regulation is not yet fully understood.Proteoliposomes incorporating SERCA only or SERCA and PLN were adsorbed on a solid supported membrane (SSM) and then activated by an ATP concentration jump. The current transient observed following an ATP jump is related to Ca2+ translocation through the ATPase upon ATP phosphorylation during the first enzymatic cycle (2).We found that when PLN is co-reconstituted with SERCA into proteoliposomes, the ATP-dependent current signal is significantly reduced with respect to the control measurement (proteoliposomes containing SERCA only) at 1mM MgCl2. On the other hand, no effect of PLN on the ATP-induced current transient was observed at high Mg2+ (5mM).ATP jumps were performed on proteoliposomes incorporating SERCA and PLN in the presence of increasing Ca2+ concentrations yielding a K0.5 of 0.7 µM and a cooperativity coefficient of 1.37. Interestingly, we observed higher values of K0.5 (0.84 µM) and cooperativity coefficient (1.67) if simultaneous ATP and Ca2+ jumps were carried out on co-reconstituted SERCA/PLN proteoliposomes. The higher K0.5 and cooperativity values suggest that PLN may affect the SERCA conformational change that follows binding of the first calcium ion, as proposed in previous studies (3,4).Supported by the Ente Cassa di Risparmio di Firenze, POR CRO FSE 2007-2013 project and SIBPA.1) Kranias E. G., Hajjar R. J. 2012. Circ. Res.110, 1646–1660.2) Tadini-Buoninsegni F. et al. 2008. Arch. Biochem. Biophys. 476:75-86.3) Trieber C.A. et al. 2009. Biochemistry. 48(39):9287-96.4) Cantilina T. et al. 1993. J Biol Chem. 268(23):17018-25.