Highly Cooperative Dependence of Sarco/Endoplasmic Reticulum Calcium ATPase (SERCA) 2a Pump Activity on Cytosolic Calcium in Living Cells

Sarco/endoplasmic reticulum (SR/ER) Ca2+-ATPase (SERCA) is an intracellular Ca2+ pump localized on the SR/ER membrane. The role of SERCA in refilling intracellular Ca2+ stores is pivotal for maintaining intracellular Ca2+ homeostasis, and disturbed SERCA activity causes many disease phenotypes, including heart failure, diabetes, cancer, and Alzheimer disease. Although SERCA activity has been described using a simple enzyme activity equation, the dynamics of SERCA activity in living cells is still unknown. To monitor SERCA activity in living cells, we constructed an enhanced CFP (ECFP)- and FlAsH-tagged SERCA2a, designated F-L577, which retains the ATP-dependent Ca2+ pump activity. The FRET efficiency between ECFP and FlAsH of F-L577 is dependent on the conformational state of the molecule. ER luminal Ca2+ imaging confirmed that the FRET signal changes directly reflect the Ca2+ pump activity. Dual imaging of cytosolic Ca2+ and the FRET signals of F-L577 in intact COS7 cells revealed that SERCA2a activity is coincident with the oscillatory cytosolic Ca2+ concentration changes evoked by ATP stimulation. The Ca2+ pump activity of SERCA2a in intact cells can be expressed by the Hill equation with an apparent affinity for Ca2+ of 0.41 +/- 0.0095 +/- mu M and a Hill coefficient of 5.7 +/- 0.73. These results indicate that in the cellular environment the Ca2+ dependence of ATPase activation is highly cooperative and that SERCA2a acts as a rapid switch to refill Ca2+ stores in living cells for shaping the intracellular Ca2+ dynamics. F-L577 will be useful for future studies on Ca2+ signaling involving SERCA2a activity.