The endoplasmic reticulum luminal Ca regulates cardiac Ca pump function.

The type 2a sarcoplasmic/endoplasmic reticulum Ca-ATPase (SERCA2a) plays a central role in Ca signaling of cardiomyocytes. The speed at which SERCA2a pumps Ca from the cytosol into the sarcoplasmic reticulum (SR) determines the diastolic relaxation rate. SERCA2a activity also sets SR Ca load, which determines the amplitude of SR Ca release and the systolic contraction strength. While SERCA2a controls the SR luminal [Ca] ([Ca]), less is known about how dynamic changes in [Ca] affect SERCA2a function. By measuring the endoplasmic reticulum [Ca] ([Ca]) with the Ca sensor R-CEPIA1er, we characterized the function of recombinant human and native mouse SERCA2a. We found that despite low endoplasmic reticulum (ER) Ca gradient, SERCA2a-mediated Ca transport was significantly slower at low [Ca] than at intermediate [Ca]. It appears that certain [Ca] is required for optimal SERCA2a Ca transport. We tested whether negatively charged amino acids within the luminal loop between transmembrane helices M7 and M8 contribute to SERCA2a regulation by [Ca]. We found that the triple mutation E877L/D878L/E883L in the M7-M8 loop reduces SERCA2a Ca transport particularly at intermediate [Ca]. Destabilizing the M7-M8 loop by breaking a disulfide bond between cysteines 875 and 887 abolished ER Ca transport. Complementary molecular dynamics simulations showed that the triple mutant E877L/D878L/E883L stabilizes a Ca-bound E2 state of the pump, slowing down release of Ca from the transport sites into the ER compared with the wild-type SERCA2a. These results revealed, for the first time, that SERCA2a Ca transport is regulated by the luminal Ca by interacting with the M7-M8 loop.