Abstract
The type 2a sarco/endoplasmic reticulum (ER) Ca(2+)-ATPase (SERCA2a) plays a key role in intracellular Ca(2+) regulation in the heart. We have previously shown evidence of stable homodimers of SERCA2a in heterologous cells and cardiomyocytes. However, the functional significance of the pump dimerization remains unclear. Here, we analyzed how SERCA2a dimerization affects ER Ca(2+) transport. Fluorescence resonance energy transfer experiments in HEK293 cells transfected with fluorescently labeled SERCA2a revealed increasing dimerization of Ca(2+) pumps with increasing expression level. This concentration-dependent dimerization provided means of comparison of the functional characteristics of monomeric and dimeric pumps. SERCA-mediated Ca(2+) uptake was measured with the ER-targeted Ca(2+) sensor R-CEPIA1er in cells cotransfected with SERCA2a and ryanodine receptor. For each individual cell, the maximal ER Ca(2+) uptake rate and the maximal Ca(2+) load, together with the pump expression level, were analyzed. This analysis revealed that the ER Ca(2+) uptake rate increased as a function of SERCA2a expression, with a particularly steep, nonlinear increase at high expression levels. Interestingly, the maximal ER Ca(2+) load also increased with an increase in the pump expression level, suggesting improved catalytic efficiency of the dimeric species. Reciprocally, thapsigargin inhibition of a fraction of the population of SERCA2a reduced not only the maximal ER Ca(2+) uptake rate but also the maximal Ca(2+) load. These data suggest that SERCA2a dimerization regulates Ca(2+) transport by improving both the SERCA2a turnover rate and catalytic efficacy. Analysis of ER Ca(2+) uptake in cells cotransfected with human wild-type SERCA2a (SERCA2a(WT)) and SERCA2a mutants with different catalytic activity revealed that an intact catalytic cycle in both protomers is required for enhancing the efficacy of Ca(2+) transport by a dimer. The data are consistent with the hypothesis of functional coupling of two SERCA2a protomers in a dimer that reduces the energy barrier of rate-limiting steps of the catalytic cycle of Ca(2+) transport.