The endoplasmic reticulum luminal Ca(2+) regulates cardiac Ca(2+) pump function.

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