title: Highly cooperative dependence of sarco/endoplasmic reticulum calcium ATPase SERCA2a pump activity on cytosolic calcium in living cells.
abstract: Sarco/endoplasmic reticulum (SR/ER) Ca(2+)-ATPase (SERCA) is an intracellular Ca(2+) pump localized on the SR/ER membrane. The role of SERCA in refilling intracellular Ca(2+) stores is pivotal for maintaining intracellular Ca(2+) 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 Ca(2+) pump activity. The FRET efficiency between ECFP and FlAsH of F-L577 is dependent on the conformational state of the molecule. ER luminal Ca(2+) imaging confirmed that the FRET signal changes directly reflect the Ca(2+) pump activity. Dual imaging of cytosolic Ca(2+) and the FRET signals of F-L577 in intact COS7 cells revealed that SERCA2a activity is coincident with the oscillatory cytosolic Ca(2+) concentration changes evoked by ATP stimulation. The Ca(2+) pump activity of SERCA2a in intact cells can be expressed by the Hill equation with an apparent affinity for Ca(2+) of 0.41 ± 0.0095 μm and a Hill coefficient of 5.7 ± 0.73. These results indicate that in the cellular environment the Ca(2+) dependence of ATPase activation is highly cooperative and that SERCA2a acts as a rapid switch to refill Ca(2+) stores in living cells for shaping the intracellular Ca(2+) dynamics. F-L577 will be useful for future studies on Ca(2+) signaling involving SERCA2a activity.
authors.affiliation: Laboratory for Developmental Neurobiology, Brain Science Institute, RIKEN, 2-1 Hirosawa, Wako, Saitama 351-0198, Japan.
authors.forename: Kanayo
authors.initials: K
authors.lastname: Satoh
authors.forename: Toru
authors.initials: T
authors.lastname: Matsu-Ura
authors.forename: Masahiro
authors.initials: M
authors.lastname: Enomoto
authors.forename: Hideki
authors.initials: H
authors.lastname: Nakamura
authors.forename: Takayuki
authors.initials: T
authors.lastname: Michikawa
authors.forename: Katsuhiko
authors.initials: K
authors.lastname: Mikoshiba
mesh_terms: D000818:Animals; D019556:COS Cells; D002118:Calcium / Q000378:metabolism*; D020013:Calcium Signaling / Q000502:physiology*; D002522:Chlorocebus aethiops; D003600:Cytosol / Q000378:metabolism*; D049452:Green Fluorescent Proteins / Q000235:genetics / Q000378:metabolism; D006801:Humans; D011993:Recombinant Fusion Proteins / Q000235:genetics / Q000378:metabolism; D053498:Sarcoplasmic Reticulum Calcium-Transporting ATPases / Q000235:genetics / Q000378:metabolism*; D018411:Spodoptera
chemical_list: D011993:Recombinant Fusion Proteins; C485184:enhanced green fluorescent protein; D049452:Green Fluorescent Proteins; D053498:Sarcoplasmic Reticulum Calcium-Transporting ATPases; C506946:ATP2A2 protein, human; D002118:Calcium