The endoplasmic reticulum luminal Ca²⁺ regulates cardiac Ca²⁺ pump function

The type 2a sarcoplasmic/endoplasmic reticulum Ca²⁺-ATPase (SERCA2a) plays a central role in Ca²⁺ signaling of cardiomyocytes. The speed at which SERCA2a pumps Ca²⁺ from the cytosol into the sarcoplasmic reticulum (SR) determines the diastolic relaxation rate. SERCA2a activity also sets SR Ca²⁺ load, which determines the amplitude of SR Ca²⁺ release and the systolic contraction strength. While SERCA2a controls the SR luminal [Ca²⁺] ([Ca²⁺]_SR), less is known about how dynamic changes in [Ca²⁺]_SR affect SERCA2a function. By measuring the endoplasmic reticulum [Ca²⁺] ([Ca²⁺]_ER) with the Ca²⁺ sensor R-CEPIA1er, we characterized the function of recombinant human and native mouse SERCA2a. We found that despite low endoplasmic reticulum (ER) Ca²⁺ gradient, SERCA2a-mediated Ca²⁺ transport was significantly slower at low [Ca²⁺]_ER than at intermediate [Ca²⁺]_ER. It appears that certain [Ca²⁺]_ER is required for optimal SERCA2a Ca²⁺ transport. We tested whether negatively charged amino acids within the luminal loop between transmembrane helices M7 and M8 contribute to SERCA2a regulation by [Ca²⁺]_ER. We found that the triple mutation E877L/D878L/E883L in the M7-M8 loop reduces SERCA2a Ca²⁺ transport particularly at intermediate [Ca²⁺]_ER. Destabilizing the M7-M8 loop by breaking a disulfide bond between cysteines 875 and 887 abolished ER Ca²⁺ transport. Complementary molecular dynamics simulations showed that the triple mutant E877L/D878L/E883L stabilizes a Ca²⁺-bound E2 state of the pump, slowing down release of Ca²⁺ from the transport sites into the ER compared with the wild-type SERCA2a. These results revealed, for the first time, that SERCA2a Ca²⁺ transport is regulated by the luminal Ca²⁺ by interacting with the M7-M8 loop.