The Functional Significance of Cardiac SERCA Dimerization

Abstract

It has been shown that the sarcoplasmic/endoplasmic reticulum Ca-ATPase (SERCA) can form homodimers. However, the functional significance of SERCA dimerization remains unclear. FRET measurements in HEK293 cells transfected with fluorescently labeled cardiac SERCA2a revealed that SERCA forms homodimers in a dose-dependent manner. In parallel experiments, properties of endoplasmic reticulum (ER) Ca uptake were studied at different levels of SERCA2a expression. Intra-ER [Ca] ([Ca]ER) was measured in HEK293 cells transfected with SERCA2a (tagged with cerulean), cardiac ryanodine receptor (RyR2 tagged with GFP) and the ER-targeted Ca sensor Cepia-1er. The plasma membrane was permeabilized with saponin to control cytosolic milieu. Activation of RyR2 with caffeine was used to completely deplete [Ca]ER. Once caffeine was removed, RyR2 inhibitors were applied to measure the rate of [Ca]ER recovery. At the end of each experiment, RyR2-independent Ca leak was measured during SERCA inhibition with thapsigargin. For each individual cell, ER Ca uptake and leak as a function of [Ca]ER were analyzed to determine maximum ER Ca uptake rate and maximum ER Ca load. SERCA2a expression level was estimated from the cerulean fluorescence. This analysis revealed that ER Ca uptake increased as a function of [SERCA], with a particularly steep increase at high SERCA expression level. We also found that maximum ER Ca load (or SERCA thermodynamic limit) increased with an increase of SERCA expression. Partial inhibition of SERCA with thapsigargin reduced not only ER Ca uptake rate but also maximum ER Ca load. These results suggest that SERCA dimerization can facilitate ER Ca uptake and improve the efficacy of coupling between ATP hydrolysis and Ca transport. Malfunction of this mechanism (due to SERCA downregulation) can contribute to reduced SR Ca uptake in the failing heart.