マツウラ　トオル   MATSUURA TORU   松浦　徹    所属   関西医科大学  病理学講座    職種   講師
論文種別	原著(症例報告除く)
言語種別	英語
査読の有無	査読あり
表題	Highly Cooperative Dependence of Sarco/Endoplasmic Reticulum Calcium ATPase (SERCA) 2a Pump Activity on Cytosolic Calcium in Living Cells
掲載誌名	正式名：Journal of Biological Chemistry 略　 称：J Biol Chem ISSNコード：00219258
掲載区分	国外
巻・号・頁	286(23),pp.20591-20599
著者・共著者	Kanayo Satoh, Toru Matsu-ura, Masahiro Enomoto, Takayuki Michikawa, Katsuhiko Mikoshiba
担当区分	2nd著者
発行年月	2011/06
概要	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.