Dimerization of SERCA2a Enhances Transport Rate and Improves Energetic Efficiency in Living Cells

The type 2a sarco/endoplasmic reticulum (ER) Ca²⁺-ATPase (SERCA2a) plays a key role in intracellular Ca²⁺ 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 Ca²⁺ transport. Fluorescence resonance energy transfer experiments in HEK293 cells transfected with fluorescently labeled SERCA2a revealed increasing dimerization of Ca²⁺ pumps with increasing expression level. This concentration-dependent dimerization provided means of comparison of the functional characteristics of monomeric and dimeric pumps. SERCA-mediated Ca²⁺ uptake was measured with the ER-targeted Ca²⁺ sensor R-CEPIA1er in cells cotransfected with SERCA2a and ryanodine receptor. For each individual cell, the maximal ER Ca²⁺ uptake rate and the maximal Ca²⁺ load, together with the pump expression level, were analyzed. This analysis revealed that the ER Ca²⁺ uptake rate increased as a function of SERCA2a expression, with a particularly steep, nonlinear increase at high expression levels. Interestingly, the maximal ER Ca²⁺ 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 Ca²⁺ uptake rate but also the maximal Ca²⁺ load. These data suggest that SERCA2a dimerization regulates Ca²⁺ transport by improving both the SERCA2a turnover rate and catalytic efficacy. Analysis of ER Ca²⁺ 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 Ca²⁺ 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 Ca²⁺ transport.