Exploring Cardiac Calcium ATPase Dimerization

Abstract

Previous studies have suggested that the cardiac calcium ATPase (SERCA) can form homo-dimers. To investigate this in heterologous cells, we fused Cerulean (Cer) or yellow fluorescent protein (YFP) to the N-terminus of canine SERCA2a. We measured 18% maximum fluorescence resonance energy transfer (FRET) from Cer-SERCA to YFP-SERCA, indicating an interaction between SERCA protomers. To determine whether the measured FRET was specific, we co-transfected cells with unlabeled SERCA to compete for binding with Cer-SERCA and YFP-SERCA. FRET progressively decreased with increasing molar ratios of unlabeled competitor SERCA to a minimum of 5%. Competition with increasing molar ratios of unlabeled phospholamban (PLB) did not reduce the measured FRET or the apparent affinity of the SERCA dimer. In order to determine whether PLB phosphorylation status altered SERCA dimerization, we enhanced phosphorylation of PLB at serine 16 by foskolin or using a phosphomimetic PLB (S16E). This did not alter the maximum FRET or the apparent affinity of the SERCA protomers for each other. Additionally, the apparent affinity was unchanged with various concentrations of cytosolic calcium or ATP, or in the presence of the SERCA inhibitor, thapsigargin. SERCA dimerization was also suggested by co-immunoprecipitation experiments in cells co-transfected with YFP-SERCA and myc-tagged SERCA. Immunoprecipitation with an anti-mycTag antibody co-immunoprecipitated YFP-SERCA, as detected by western blot. To analyze SERCA dimerization in cardiac myocytes, we used benzophenone-4-maleimide crosslinking. Rabbit left ventricular myocytes were isolated and permeabilized in the presence of crosslinker. Western blot analysis revealed two bands: the first at approximately 110 kD, consistent with monomeric SERCA, and the second at approximately 200 kD, a value compatible with the predicted molecular weight of a homo-dimer. Our data suggest that SERCA2a forms obligate homo-dimers in live cells, including cardiac myocytes. We have not determined a regulatory component for this interaction.