Highly Cooperative Dependence of Sarco/EndoplasmicReticulum Calcium ATPase (SERCA) 2a Pump Activity onCytosolic Calcium in Living Cells*□S

Received for publication, November 18, 2010, and in revised form, April 20, 2011 Published, JBC Papers in Press, April 22, 2011, DOI 10.1074/jbc.M110.204685

Kanayo Satoh‡§1, Toru Matsu-ura‡2, Masahiro Enomoto‡3, Hideki Nakamura‡4, Takayuki Michikawa‡¶5,6,and Katsuhiko Mikoshiba‡¶7

From the ‡Laboratory for Developmental Neurobiology, Brain Science Institute, RIKEN, 2-1 Hirosawa, Wako,Saitama 351-0198, Japan, the §Department of Cognitive Neuroscience, Graduate School of Medicine, The University ofTokyo, 7-3-1 Hongo, Bunkyo-ku, Tokyo 113-0033, Japan, and the ¶Japan Science and Technology Agency, InternationalCooperative Research Project and Solution-oriented Research for Science and Technology, Calcium Oscillation Project,4-1-8 Honcho, Kawaguchi, Saitama 332-0012, Japan

Sarco/endoplasmic reticulum (SR/ER) Ca2�-ATPase(SERCA) is an intracellular Ca2� pump localized on the SR/ERmembrane. The role of SERCA in refilling intracellular Ca2�

stores is pivotal formaintaining intracellular Ca2� homeostasis,and disturbed SERCA activity causes many disease phenotypes,including heart failure, diabetes, cancer, andAlzheimer disease.Although SERCA activity has been described using a simpleenzyme activity equation, the dynamics of SERCA activity inliving cells is still unknown. Tomonitor SERCAactivity in livingcells, we constructed an enhanced CFP (ECFP)- and FlAsH-tagged SERCA2a, designated F-L577, which retains the ATP-dependent Ca2� pump activity. The FRET efficiency betweenECFP and FlAsH of F-L577 is dependent on the conformationalstate of the molecule. ER luminal Ca2� imaging confirmed thatthe FRET signal changes directly reflect the Ca2� pump activity.Dual imaging of cytosolic Ca2� and the FRET signals of F-L577in intact COS7 cells revealed that SERCA2a activity is coinci-dent with the oscillatory cytosolic Ca2� concentration changesevoked by ATP stimulation. The Ca2� pump activity ofSERCA2a in intact cells can be expressed by the Hill equation

with an apparent affinity forCa2� of 0.41� 0.0095�Mand aHillcoefficient of 5.7 � 0.73. These results indicate that in the cel-lular environment the Ca2� dependence of ATPase activation ishighly cooperative and that SERCA2a acts as a rapid switch torefill Ca2� stores in living cells for shaping the intracellularCa2�

dynamics. F-L577 will be useful for future studies on Ca2� sig-naling involving SERCA2a activity.

Intracellular Ca2� plays a pivotal role in controlling numer-ous cellular processes such as exocytosis, gene transcription,cell proliferation, muscle contraction, and cell survival (1). Thelevel of intracellularCa2� is determined by the balance betweenthe influx that introduces Ca2� into the cytoplasm and theefflux that removes it from the cytoplasm. The key moleculesinvolved in the regulation of intracellular Ca2� such as chan-nels, pumps, and exchangers have been identified (2). Channelsin the plasma membrane and sarco/endoplasmic reticulum(SR/ER)8 membrane are responsible for the Ca2� influx,whereas pumps and exchangers carry out the Ca2� efflux.SR/ER Ca2�-ATPase (SERCA) is a Ca2� pump that transfersCa2� from the cytosol to the lumen of the SR/ER at the expenseof ATP hydrolysis (3). SERCA functions to determine the rest-ing level of intracellular Ca2� (4) and to control the spatiotem-poral profile ofCa2� transients and the frequency ofCa2� oscil-lations (5). Impairment of SERCA causes Ca2� homeostaticdysfunction, resulting in several important disease states suchas heart failure, hypertension, diabetes, and Alzheimer disease(6).The SERCA family consists of three isoforms and their splic-

ing variants (SERCA1a, -1b, -2a, -2b, -3a, -3b, and -3c). Themolecular masses of SERCA isoforms range from 105 to 115kDa. Each SERCA isoform consists of four distinct functionaldomains, namely the nucleotide-binding, phosphorylation,actuator, and transmembrane domains (7). The three-dimen-sional structures of different conformational states of SERCA1ahave been defined by x-ray crystallography (8), and structuralmodels for the catalytic cycle of SERCA are well established (9).

* This work was supported by Ministry of Education, Culture, Sports, Scienceand Technology of Japan Grants 20370054 (to T. M.) and 2022007 (to K. M.).

□S The on-line version of this article (available at http://www.jbc.org) containssupplemental “Experimental Procedures,” Table S1, Figs. S1–S5, and addi-tional references.

1 Present address: Tanz Centre for Research in Neurodegenerative Diseases,Faculty of Medicine, 6 Queen’s Park Crescent West, University of Toronto,Toronto, Ontario M5S 3H2, Canada.

2 Present address: Dept. of Pathology and Laboratory of Medicine, 231 AlbertSabin Way, University of Cincinnati, Cincinnati, OH 45267-0529.

3 Present address: Div. of Signaling Biology, Ontario Cancer Institute andDept. of Medical Biophysics, University of Toronto, Toronto, Ontario M5G1L7, Canada.

4 Present address: Dept. of Life Science and Medical Bioscience, School ofAdvanced Science and Engineering, Faculty of Science and Engineering,Waseda University, 2-2 Wakamatsu-cho, Shinjuku, Tokyo 162-8480, Japan.

5 Present address: Laboratory for Molecular Neurogenesis, RIKEN Brain Sci-ence Institute, 2-1 Hirosawa, Wako, Saitama 351-0198, Japan.

6 To whom correspondence may be addressed: Laboratory for MolecularNeurogenesis, RIKEN Brain Science Institute, 2-1 Hirosawa, Wako, Saitama351-0198, Japan. Tel.: 81-48-467-5906; Fax: 81-48-467-6079; E-mail:t-michikawa@brain.riken.jp.

7 To whom correspondence may be addressed: Laboratory for Developmen-tal Neurobiology, RIKEN Brain Science Institute, 2-1 Hirosawa, Wako,Saitama 351-0198, Japan. Tel.: 81-48-467-9745; Fax: 81-48-467-9744;E-mail: mikosiba@brain.riken.jp.

8 The abbreviations used are: SR/ER, sarco/endoplasmic reticulum; SERCA,sarco/endoplasmic reticulum calcium ATPase; ECFP, enhanced CFP; IP3,inositol 1,4,5-trisphosphate; TC, tetracysteine.

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The sequential conformational changes of SERCA are accom-panied by active transport of 2 mol of Ca2� per 1 mol of boundATP, and 1mol of ATP is hydrolyzed during one reaction cycle(10). Thus, the conformational changes are directly linked withSERCA activity.Cytosolic Ca2� increases evoked by extracellular stimuli are

often observed in the form of oscillating Ca2� spikes (11). Inmost cell types, Ca2� spikes are generated by inositol 1,4,5-trisphosphate (IP3)-induced Ca2� release from intracellularCa2� stores (12). The IP3 receptor is an intracellular Ca2�

release channel, and its activity is controlled by not only IP3 butalso cytosolic Ca2� (13). Because submicromolar Ca2� concen-trations activate IP3 receptor, the upstroke of Ca2� spikes mayoccur by regenerative Ca2� release through IP3 receptor (14).On the other hand, the role of SERCA in the generation of Ca2�

spikes is more passive. SERCA transports Ca2� back to theCa2� stores at a rate that is solely dependent on the intracellularCa2� concentration ([Ca2�]i) (15). The pump activity ofSERCA has been approximated by the Hill equation to have aHill coefficient of 2 for Ca2� binding (16) in theoretical studiesof Ca2� dynamics (15, 17). However, it remains to be elucidatedwhether the enzymatic activity of SERCA in living cells is iden-tical to that measured in vitro.The FRET technique is commonly used to study the confor-

mational changes of functional proteins (18), and labeling withfluorescent proteins has been widely used for this purpose (19).However, as ectopic expression and/or dysfunction of the tar-get protein can sometimes result from the labeling with theserelatively large (�27 kDa) proteins (20), techniques using smallfluorescent molecules with less steric bulk have been greatlyadvanced (21). As an example, FlAsH-EDT2 is a small mem-brane-permeable synthetic ligand with high affinity and speci-ficity for a tetracysteine tag (CCPGCC; TC tag) (22). The bind-ing of FlAsH-EDT2 to a TC tag induces chromophoreformation, giving rise to fluorescence (23).Real-time visualization of SERCA pump activity in living

cells should facilitate our understanding of the mechanism forthe generation of intracellular Ca2� dynamics. In this study, wesuccessfully detected the pump activity of SERCA2a in livingcells using the FRET techniquewith a combination of enhancedCFP (ECFP) and FlAsH. Dual imaging of intracellular Ca2� andthe FRET signals of our ECFP- and FlAsH-tagged SERCA2a,designated F-L577, provides significant insights into the regu-latory mechanism of SERCA pump activity in living cells.

EXPERIMENTAL PROCEDURES

Plasmid Constructions—The details of these procedures aregiven in the supplemental data.Cell Culture and Transfection—COS7 cells were cultured in

DMEM (Invitrogen) supplemented with 10% FBS, 100 units/mlpenicillin and 0.1 mg/ml streptomycin, and maintained in ahumidified incubator with 5% CO2 at 37 °C. The cells weregrown on 35-mm poly-L-lysine-coated glass-bottomed dishes(Matsunami). F-L577 and mRFP-KDEL cDNAs were trans-fected into the cells using TransIT (Mirus). The cells were usedfor experiments at 2 days after transfection. For FRET imaging,F-L577-expressing COS7 cells were loaded with FlAsH-EDT2

reagent (Invitrogen) for 60–90 min at room temperature andwashed twice with BAL wash buffer (Invitrogen).Western Blot Analysis—F-L577-expressing and non-trans-

fected COS7 cells (1 � 106) were solubilized with 100 �l of 1�SDS sample buffer (2% SDS, 10% (w/v) glycerol, 5% (v/v) 2-�-mercaptoethanol, 0.05% bromphenol blue, 0.0625 M Tris-HCl,pH 6.8). Aliquots (2 �l) of the cell lysates were analyzed byWestern blot using the anti-SERCA2 antibody (BD Biosci-ences) at a dilution of 1:1000 and goat anti-mouse IgG, HRP-linked F(ab�)2 fragment (GE Healthcare) as the secondary anti-body at a dilution of 1:2000. Signals were detected with an ECLkit (GE Healthcare) and an LAS-4000 Image Reader (FujiFilm).Subcellular Localization of F-L577—Fluorescence images of

F-L577 expressing COS7 cells were taken under a confocalscanningmicroscope (CSU-XI; Yokogawa) attached to an IX81invertedmicroscope (Olympus) with an EMCCD camera (Cas-cadeII; Roper) and a 60� (oil; numerical aperture, 1.42) objec-tive lens (Olympus). A 440-nm excitation filter, a 520-nm emis-sion filter, and 450-nm dichroic mirrors were inserted into thelight path. Data were analyzed using MetaMorph software(Molecular Devices).Acceptor Photobleaching—Acceptor (FlAsH) photobleach-

ing was performed using a confocal microscope (A1; Nikon).Repeated scans (120 s at maximum rates) with an unattenuated514-nm illumination from a multi-argon laser were applied(total, 30 milliwatt maximum output). ECFP and FlAsH emis-sion signals were acquired with a 515-nm dichroicmirror and apair of 460–500-nm and 520–620-nm band pass filters,respectively. Data were analyzed using NIS elements (Nikon)and MetaMorph software (Molecular Devices).Generation of Recombinant Baculoviruses and Expression—

Spodoptera frugiperda (Sf9) cells (Invitrogen) were cultured at27 °C with stirring in Sf-900II SFM medium (Invitrogen) con-taining 10% FBS. P1 viral stocks of recombinant baculovirusesfor F-L577 and WT-SERCA2a were generated using a Bac-to-Bac Baculovirus Expression System (Invitrogen) according tothe manufacturer’s instructions. Amplification of P2 viralstocks and recombinant protein expression of F-L577 andWT-SERCA2awere performed using Sf9 cells as described pre-viously (24, 25).In Vitro Kinetic Assay of ATP-induced Ca2� Uptake by

SERCA Pump—Microsomal vesicles were prepared from Sf9cells as described previously (26). The measurements and anal-ysis of the ATP-inducedCa2� accumulationwere conducted asdescribed elsewhere.9 Briefly, the volume of the microsomalvesicles used in each measurement was adjusted using theabsorbance at 600nmacquired by aDU640 spectrophotometer(Beckman Coulter). The microsomal vesicles were suspendedin 500 �l of cytosol-like medium (110 mM KCl, 10 mM NaCl, 5mMKH2PO4, 1mMDTT, 50mMHEPES-KOH, pH 7.2, at roomtemperature), supplemented with 1 �g/ml oligomycin (Sigma),2 �M MgCl2, 25 �g/ml creatine kinase (Roche Diagnostics), 10mM phosphocreatine (Sigma), and 2 �M fura-2 (Dojindo). Thesuspension was continuously stirred at 30 °C during measure-ments. The fluorescent signals of fura-2 were monitored with a

9 M. Enomoto, T. Michikawa, and K. Mikoshiba, unpublished data.

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CAF-110 spectrofluorometer (Jasco) and a PowerLab dataacquisition system (ADInstruments). Fluorescence signalswere recorded every 0.01 s at an emissionwavelength of 510 nmwith alternate excitation at 340 and 380 nm. The 1 mM ATP-induced Ca2� uptake into microsomal vesicles by the SERCApumpwas initiated andmonitored for 300 s. Free Ca2� concen-trations were calculated from acquired fluorescent signals asdescribed previously (27). To estimate the rate of Ca2� uptake,curve fitting of three-exponential function was conductedusing Igor Pro 6.02 software (WaveMetrics) and t1⁄2 (half-decaytime) was calculated.Cell Permeabilization and State Fixation of F-L577—FlAsH-

EDT2-loaded F-L577-expressing COS7 cells were permeabi-lized with 60�M �-escin (Sigma) in an internal solution (19mM

NaCl, 125mMKCl, 10mMHEPES-KOH, pH7.4) supplementedwith 5 mM EGTA for 3–5 min. The permeabilized cells weregently washed with the internal solution containing 5 mM

EGTA and then used for FRETmeasurements. Reagents for thefixation of F-L577 in the four major conformational stateswere prepared according to methods described previously inthe crystallization experiments of SERCA1a (28–31). Cellswere perfused with an internal solution containing 30�M thap-sigargin and 5 mM EGTA for the E2 state; 100 �M CaCl2 for theE1-Ca2� state; 1 mM ADP, 0.33 mM AlCl3, 5 mM NaF, 1 mM

MgCl2, and 100 �MCaCl2 for the E1-ATP state; or 2 mM BeCl2,8mMNaF, 1mMMgCl2, and 5mMEGTA for the E2P state. Thefree Ca2� concentration in the internal solutions was adjustedwith K2HEDTA and CaHEDTA at 37 °C as described previ-ously (32). Fluorescent signals were acquired at 0.25 Hz with anIX71 inverted fluorescence microscope (Olympus) (see below).ER Ca2� Imaging—ER luminal Ca2� imaging was performed

with 10 �M Mag-Indo-1 AM (Molecular Probes), according tothe method described previously (33, 34) with some modifica-tions. The free Ca2� concentrations in the internal solutionswere carefully adjusted with a Ca2� buffer and confirmed usinga Ca2�-sensitive electrode (Metrohm) prior to use (41). ForFRET and Ca2� imaging, the permeabilized COS7 cellsexpressing F-L577 were perfused with the internal solutioncontaining 1 �M free Ca2�, and then 0.01, 0.1, or 1 mMMgATP(Sigma) was applied in the internal solution containing 1 �M

free Ca2�. Fluorescent signals were acquired at 0.25 Hz with anIX71 inverted microscope (Olympus) (see below).Live Cell Imaging—For imaging of intact cells, F-L577-ex-

pressing COS7 cells were loaded with 10 �M Indo-5F AM(Dojindo) and FlAsH-EDT2 reagent. Images were obtainedunder a constant flow (2 ml/min) of a balanced salt solutioncontaining 115 mM NaCl, 5.4 mM KCl, 1 mM MgCl2, 2 mM

CaCl2, 10mM glucose, and 20mMHEPES-NaOH, pH 7.4. Fluo-rescent signals were acquired at 2 Hz with an IX71 invertedfluorescence microscope (see below). For [Ca2�]i calibration,fluorescence imaging of F-L577 and Indo-5F was performedusing the IX81 inverted microscope. [Ca2�]i was calculatedusing the formula of Grynkiewicz et al. (27). After agonist stim-ulation (1 �M ATP for 2 min), the minimum and maximumfluorescence ratio of Indo-5F was obtained by treatment of thecells with 2 �M ionomycin (Br-A23187) and 5 mM EGTA fol-lowed by 2 �M ionomycin and 10 mM CaCl2.

Imaging Equipment—Imaging in intact and permeabilizedcells was performed at 37 °C using an IX71 or IX81 invertedmicroscope (Olympus) with a cooled CCD camera (ORCA-ER;Hamamatsu Photonics), a 40� (numerical aperture, 1.35)objective lens (Olympus) and a xenon lamp (Ushio). For fluo-rescent images of F-L577, Indo-5F (Dojindo), and Mag-Indo-1(Molecular Probes), an emission splitter (W-view; HamamatsuPhotonics) was used with a light source exchanger (DG-4; Sut-ter Instrument Co.) on the IX71 inverted microscope. Sequen-tial excitations of F-L577 and Indo-5F (or Mag-Indo-1) wereperformed using a 450-nm dichroic mirror and two excitationfilters (425–445 nm for F-L577, 360 nm for Indo-5F and Mag-Indo-1). Emissions were split with a 460–510-nm filter (forF-L577, Indo-5F, andMag-Indo-1), a long path 520-nm barrierfilter (for F-L577) and two 505-nm dichroic mirrors (for allfluorophores) equipped in W-view. Images were acquired at0.25 or 2 Hz, with an exposure time of 100 or 150 ms. For[Ca2�]i calibration, fluorescence imaging of F-L577 andIndo-5F was performed using the IX81 inverted microscopeequipped with two excitation filters (425–445 nm for F-L577and 333–348nm for Indo-5F), twodichroicmirrors (450nm forF-L577 and 400 nm for Indo-5F), two emission filters (460–510nm for F-L577 and Indo-5F, 515–565 nm for F-L577). The fil-ters were controlled by Lambda 10–2 (Sutter Instrument Co.)and IX2-RFACA (Olympus). Image acquisition was performedwith theMetaFluor software (Molecular Devices). Offline anal-yses were performed with the MetaFluor Analyst software(Molecular Devices) and Igor Pro software (WaveMetrics).

RESULTS AND DISCUSSION

Construction and Characterization of Fluorescently LabeledSERCA2a—Fig. 1A shows a schematic representation of theF-L577 protein constructed in this study to investigate the con-formational changes linked with SERCA2a activity in livingcells. Because the N-terminal fusion of ECFP to SERCA1a hasno notable effects on itsATPase activity (35), ECFPwas fused totheN terminus of the A-domain of SERCA2a (Fig. 1A). The siteof the TC tag insertion was determined based on the followingcriteria: 1) the relative distance between the donor and acceptorshould change dramatically during the reaction cycle ofSERCA, and 2) the site should be located within a surface loopconnecting secondary structures, such as an �-helix and a�-sheet. Fig. 1B shows a schematic representation of the con-formational changes during the reaction cycle of SERCA1a(28–31, 36). Because the relative orientation of theN-domain ismarkedly changed during the cycle, the penultimate loopbetween Leu-577 and Glu-578 in the N-domain was chosen asthe TC tag insertion site (Fig. 1, A and B).Fig. 2A shows aWestern blot analysis of F-L577 expressed in

COS7 cells. The signal for F-L577 (lane 2) was detectedwith theexpected size. The subcellular localization of F-L577 in the ERwas confirmed by confocal microscopy (Fig. 2B). The ER waslabeled by cotransfection with mRFP-KDEL (an ER-targetedmonomeric red fluorescent protein) (34). The fluorescent sig-nals of ECFP and FlAsH colocalized well with the mRFP-KDELsignals, indicating that the ECFP fusion and TC tag insertiondid not disrupt the ER localization of F-L577.

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To confirm that FRET occurred in living COS7 cells, we per-formed acceptor (FlAsH) photobleaching of F-L577. After thephotobleachingofFlAsH, theECFP(donor) fluorescence intensitywas increasedto130.1�10.1%(mean�S.D.,n�6) (Fig.2C).Thisfinding indicates that FRET occurred between the N-terminalECFP and the FlAsH label in the N-domain in resting COS7 cells.Wemeasured the Ca2� pump activity of F-L577 in vitro (Fig.

2D). Recombinant WT-SERCA2a and F-L577 were expressedin Sf9 insect cells. ATP-induced Ca2� accumulation in themicrosomal vesicles containing WT-SERCA2a or F-L577 wasmonitored by spectrofluorometry. A representative trace ofCa2� uptake is shown in supplemental Fig. S1. The half-decaytime (t1⁄2) of the ATP-induced Ca2� accumulation in themicro-somal vesicles prepared from cells expressing WT-SERCA2awas significantly smaller than the t1⁄2 of Ca2� accumulation inthe microsomal vesicles prepared from non-transfected cells(Fig. 2D). These findings indicate that the activity of the recom-binant WT-SERCA2a was successfully detected in this experi-mental system. Overexpression of F-L577 also significantlydecreased the t1⁄2 (Fig. 2D). Based on these findings, we con-

cluded that the ECFP fusion and TC tag insertion did not abol-ish the SERCA2 pump activity.FRET Signals of F-L577 in FourMajor Conformational States

of SERCA—To examine the ability of F-L577 to reflect the var-ious conformational changes linked to SERCA2 pump activity,wemeasured its FRET signals in the fourmajor conformationalstates (Fig. 3A). To isolate these states, we performed state fix-ation of F-L577 expressed in COS7 cells. Briefly, permeabilizedCOS7 cells expressing F-L577 were initially perfused with aninternal solution containing 5 mM EGTA to provide an arbi-trary F-L577 reference state. The cells were then perfused withan internal solution containing 30 �M thapsigargin and 5 mM

EGTA (E2 state) (29); 100 �M CaCl2 (E1-Ca2� state) (28);1mMADP, 0.33mMAlCl3, 5mMNaF, 1mMMgCl2, and 100�M

CaCl2 (E1-ATP state) (30); or 2 mM BeCl2, 8 mM NaF, 1 mM

MgCl2, and 5 mM EGTA (E2P state) (31). AlF4� and BeF3� arephosphate analogues that can be used to fix SERCA1a in theE1-ATP and E2P states, respectively (30, 36). Supplemental Fig.S2 shows the relationship between the FRET signals of F-L577and the concentration of thapsigargin, as a SERCA-specificinhibitor, in permeabilizedCOS7 cells. The EC50 valuewas�20�M, which was higher than that measured in vitro (37). There-fore, a saturating concentration (30 �M) of thapsigargin wasused for the state fixation experiments. Fig. 3B shows repre-sentative traces of the FRET signal changes from the referencestate to the four different states. The average values of the FRETsignal changes (�R/Rbase) for the four different states are shownin Fig. 3C. When the cells were fixed in the E2 state (thapsi-gargin-bound state), F-L577 showed a 7.3� 1.6% (mean� S.D.)

FIGURE 1. The schematic structure of F-L577. A, the basic design of F-L577.ECFP was fused at the N terminus of SERCA2a with a linker sequence (GSL),and a TC tag (-CCPGCC-) was inserted after the Leu-577 site of SERCA2a. B, aschematic representation of the three-dimensional structures of SERCA in itsfour different conformational states during the reaction cycle. The four dis-tinct functional domains, namely the nucleotide-binding (N), phosphoryla-tion (P), actuator (A), and transmembrane (TM) domains, are highlighted. Thesequential conformational changes of SERCA are accompanied by activetransport of 2 mol of Ca2� per 1 mol of bound ATP, and 1 mol of ATP ishydrolyzed during one reaction cycle. Ca2� binding stabilizes the conforma-tion for binding of ATP (E1-Ca2� state). The conformational change inducedby phosphorylation decreases the Ca2� affinity (E1-ATP state), resulting in aCa2� shift from the cytosolic side of the membrane to the luminal side. Duringthis process, 1 mol of ATP is hydrolyzed. The Ca2� affinity then decreases, andCa2� is mobilized into the ER lumen (E2P state). This Ca2� mobilization pro-cess promotes the dephosphorylation of the Asp-351 phosphorylated resi-due, thereby making the Ca2�-binding site accessible to cytosolic Ca2� again(E2 state).

FIGURE 2. The characterization of F-L577. A, Western blot analysis of endog-enous SERCA2 and F-L577 with an anti-SERCA2 antibody. Cell lysates (2 �leach) prepared from non-transfected (lane 1) and F-L577-transfected (lane 2)COS7 cells were applied to the gel. Molecular mass markers (in kDa) are shownon the left. B, the subcellular distribution of F-L577 in COS7 cells. Confocalimages of COS7 cells expressing F-L577 and mRFP-KDEL are shown. C, accep-tor photobleaching of F-L577-expressing COS7 cells. Representative emissionintensities of ECFP (continuous line, donor) and FlAsH (broken line, acceptor) areshown. D, half-decay times (t1⁄2) of ATP-induced Ca2� uptake of microsomal ves-icles prepared from non-infected Sf9 cells (NC) and Sf9 cells expressing recombi-nant WT-SERCA2a or F-L577 were calculated. The error bars represent the S.D. Thenumbers of measurements are shown in parentheses. **, p 0.01; n.s., not signif-icant by Tukey-Kramer test. a.u., arbitrary units.

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decrease in the FRET signal relative to the baseline signal in thereference state (Fig. 3, B and C). The FRET signals in theE1-Ca2� state (Ca2�-bound state) and E1-ATP state (ATP-

bound state) showed 11.9 � 3.2% and 6.2 � 1.9% decreasesrelative to the baseline signal, respectively (Fig. 3, B and C). Incontrast, the FRET signal showed a 2.4 � 0.86% increase fol-lowing the transition from the reference state to the E2P state(Fig. 3, B and C). The overall dynamic range of the FRET signalchanges of F-L577 was �15%.The distances between the N terminus and Leu-577 of

SERCA1a in the four major conformational states were calcu-lated using the molecular modeling software PyMOL. The cal-culated distances were 39.19 Å in the E2 state (Protein DataBank code 1IWO) (29), 69.79 Å in the E1-Ca2� state (ProteinData Bank code 1SU4) (28), 36.83 Å in the E1-ATP state (Pro-tein Data Bank code 1T5T) (30), and 33.32 Å in the E2P state(ProteinData Bank code 3B9B) (36). Therefore, the order of thedistance between the N terminus and Leu-577 was E1-Ca2� E2 E1-ATP E2P. This order was consistent with the orderof the relative amounts of the FRET signal changes of SERCA2a(Fig. 3C), suggesting that the FRET signal changes containinformation about the distance between the N terminus andLeu-577. Themeasured FRET signal changeswere predictive ofthe defined structural changes during the cycling of SERCA,strongly suggesting that F-L577 is capable of detecting the con-formational changes of SERCA2a.FRET Signal Changes of F-L577 Directly Reflect Ca2� Pump

Activity—To confirm that the FRET signal changes of F-L577reflect the Ca2� pump activity in living cells, we performedtime-lapse dual imaging of F-L577 and the ER luminal Ca2�

concentration during Ca2� pumping in permeabilized cells(Fig. 4A). For ER luminal Ca2� imaging, COS7 cells were loadedwith a low affinity fluorescent Ca2� indicator, Mag-Indo-1 AM(Kd � �35 �M) using amethod described previously (33, 34). Fortime-lapse imaging during Ca2� pumping, a baseline was initiallyestablished by perfusionwith an internal solution containing 1�M

Ca2� without MgATP. Under these ATP-free conditions, the E2and E1-Ca2� states should dominate (Fig. 3A). To initiate Ca2�

accumulation in theER, 1mMMgATPwas added to theperfusate.Following the application of MgATP, the FRET signal of F-L577increased by 3–6%, accompanied by an increase in the ER luminalCa2� concentration (Fig. 4A). The increase in the FRET signalsuggests that the fractions of the E1-ATP and E2P states, whichshowed higher FRET signals than the E2 and E1-Ca2� states (Fig.3C), are increased when SERCA2a is activated.To determine the timing of the FRET changes relative to Ca2�

uptake,wecompared the firstderivativesof the tworesponses (Fig.4B). The peak responses were detected at the same samplingpoints, indicating that the timingof the conformational changesofF-L577 was coincident with Ca2� uptake into the ER.

TheCa2� pump activity of SERCA2 is dependent on theATPconcentration (38). To measure the ATP dependence of theFRET signals of F-L577, we repeated the above experiments inthe presence of 0.01, 0.1, or 1 mM ATP (Fig. 4C). The resultsdemonstrated that the FRET signals of F-L577 showed ATPconcentration-dependent changes (Fig. 4C, left) and that theATP dependence of the FRET signals paralleled the accumula-tion of Ca2� into the ER lumen (Fig. 4C, right).

We further analyzed the correlation between the F-L577FRET and Mag-Indo-1 responses (Fig. 4D). Interestingly, theF-L577 (�R/Rbase) and Mag-Indo-1 (�F/Fbase) responses were

FIGURE 3. FRET signals of F-L577 in the four major conformational states.A, a schematic representation of the SERCA reaction cycle using the four-statemodel. The components of the solutions required for state fixation are shownin parentheses. B, representative traces of the FRET signal changes of F-L577(�R/Rbase) in permeabilized COS7 cells. Fixation of F-L577 in the four stateswas performed by perfusion of solutions containing thapsigargin (E2), Ca2�

(E1-Ca2�), Ca2� with Mg2�-ADP and AlF4� (E1-ATP) and BeF3

� in the absenceof Ca2� (E2P) (horizontal black bars) after perfusion of an internal solutioncontaining 5 mM EGTA. Each image was acquired at 0.25 Hz. R, ratio of FlAsHfluorescence to ECFP fluorescence; Rbase, basal level of R; �R/Rbase, (R � Rbase)/Rbase. C, the average values of the FRET signal changes of F-L577 in the fourmajor conformational states. The error bars represent the S.D. The numbers ofmeasurements are shown in parentheses.

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significantly correlated (r � 0.767, n � 61). We also exploredthe effect of the cytosolic Ca2� concentration on the F-L577response (supplemental Fig. S3). The FRET signal changes ofF-L577 and the Mag-Indo-1 signal changes were significantlycorrelated even when the cytosolic Ca2� concentration variedfrom0.13–1.0�M (r� 0.651,n� 38). These results suggest thatthe positive FRET signal changes of F-L577 are directly corre-lated with the instantaneous Ca2� pump activity of SERCA2a.We compared the Mag-Indo-1 signal changes of non-trans-

fected cells and F-L577-expressing cells under three differentconditions (Fig. 4E). When permeabilized COS7 cells werestimulated by 0.1 or 1 mM MgATP in an internal solution con-taining 1 �M Ca2�, the Mag-Indo-1 signal change in F-L577-expressing cells was significantly larger than that in non-trans-fected cells. These results indicate that F-L577 itself has Ca2�

pump activity in living cells.The data described thus far demonstrate that 1) F-L577

shows different FRET signals depending on the conformationalstate, (2) the conformational change of F-L577 is coincidentwith Ca2� uptake, (3) the ATP and Ca2� concentration depen-dences of the F-L577 FRET signal are comparable with those ofCa2� uptake, (4) the FRET signal change of F-L577 is correlatedwith Ca2� uptake in permeabilized cells, and (5) F-L577 itself hasCa2� pump activity in living cells. In summary, the FRET signal

changes of F-L577 directly reflect the Ca2� pump activity linkedwith its conformational changes in the SERCA reaction cycle.Visualization of Ca2� Pump Activities of SERCA During

Ca2� Oscillations in COS7 Cells—To investigate the dynamicsof SERCAactivity in living cells duringATP-evokedCa2� oscil-lations, we performed dual imaging of intracellular Ca2� andthe FRET signal changes of F-L577 in COS7 cells. Fig. 5A showsrepresentative FRET signal changes of F-L577 (upper panel)and cytoplasmic Ca2� concentration changes (lower panel)observed in COS7 cells stimulated with 1 �M ATP. The FRETsignals of F-L577 showed oscillatory dynamics during Ca2�

oscillations. Although the oscillatory SERCA activity has beenpredictable, these findings comprise the first experimental evi-dence for the detection of oscillatory SERCA activity in livingcells. The representative traces of the FRET signal changes ofF-L577 (continuous line) and Ca2� changes (broken line) arealso shown on an enlarged time scale (Fig. 5B). There was nodetectable delay between the F-L577 signals and the Indo-5Fsignals during either the rising or falling phases of the Ca2�

spikes. These findings indicate that the Ca2� pump activity ofSERCA2a is synchronized with cytosolic Ca2� concentrationchanges without any detectable delay.Fig. 5C shows the relationship between the FRET signal

changes of F-L577 and [Ca2�]i during the period of Ca2� oscil-

FIGURE 4. FRET signal changes of F-L577 reflect the Ca2� uptake activity. A, representative traces of the FRET signal changes of F-L577 (�R/Rbase) and thefluorescent signal changes of Mag-Indo-1 loaded in the ER (�F/Fbase) induced by MgATP. An increase in �F/Fbase represents Ca2� uptake from the cytosol tothe ER lumen. F-L577-expressing permeabilized COS7 cells were stimulated with 0.01, 0.1, or 1 mM MgATP (black horizontal bars). White horizontal bars indicatethe presence of 1 �M Ca2�. Each image was acquired at 0.25 Hz. R, ratio of FlAsH fluorescence to ECFP fluorescence; Rbase, basal level of R; �R/Rbase, (R �Rbase)/Rbase; F, fluorescence of Mag-Indo-1 at 450 – 495 nm; Fbase, basal level of F; �F/Fbase, (F � Fbase)/Fbase. B, superimposition of the first derivatives of �R/Rbase(d(�R/Rbase)/dt; continuous lines) and �F/Fbase (d(�F/Fbase)/dt; broken lines). The lower panel shows magnified versions of parts of the traces in the upper panel(1–2 min). C, ATP dependences of �R/Rbase and �F/Fbase in the presence of 1 �M Ca2�. The data represent means � S.D. The numbers of measurements areshown in parentheses. D, the correlation between �R/Rbase and �F/Fbase. The data were analyzed by the Pearson correlation coefficient test (r � 0.767, n � 61, p 0.001). The linear regression equation is y � 3.9702 x � 0.0233. E, Ca2� uptake (�F/Fbase) of F-L577-expressing (�) and non-expressing (�) cells under the threeconditions shown on the right. The error bars represent the S.D. *, p 0.05, ***, p 0.001, by Student’s t test. The numbers of measurements are shown in parentheses.

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lations evoked with 1 �M ATP.We found a good fit between thedata and the Hill equation with an apparent affinity for Ca2� of0.41� 0.0095�Mand aHill coefficient of 5.7� 0.73 (Fig. 5C). TheapparentCa2� sensitivityofF-L577wasconsistentwithpreviously

reported values (0.34� 0.45�M) for native SERCA2ameasured invitro (15, 39, 40). In contrast, the high degree of cooperativity dif-fered from that measured in vitro (16).To determine whether the high degree of cooperativity

shown in this in vivo experiment is due to overexpression of theSERCApumpor fluorescent labeling, we investigated the FRETsignal change of F-L577 under the steady-state condition in[Ca2�]i (Fig. 6). Fig. 6 shows the relationship between [Ca2�]i inthe solutions applied to permeabilized cells and the FRET signalchange of F-L577.We found a good fit between the data and theHill equation with an apparent affinity for Ca2� of 0.46 � 0.26�M and a Hill coefficient of 0.94� 0.41 (Fig. 6). Both the appar-ent Ca2� sensitivity of F-L577 and the Hill coefficient wereconsistentwith thatmeasured in vitro (15, 39, 40). These resultsconfirm that the highly cooperative dependence of the FRETsignal changes of F-L577 on cytosolic Ca2� does not arise fromartifacts caused by the overexpression and/or fluorescent label-ing. These findings indicate that the Ca2� pump activity ofSERCA2a shows a high degree of cooperativity in the physio-logical condition of living cells.We assumed that the FRET signal of F-L577 was propor-

tional to the instantaneous SERCA pump activity based on thelinear relationship between the FRET signal of F-L577 and theMag-Indo-1 signal reflecting the ER luminal Ca2� concentra-tion ([Ca2�]ER) (Fig. 4, B and D). To investigate the possibilitythat the high degree of cooperativity is caused artificially by anonlinear relationship between the Mag-Indo-1 signal and[Ca2�]ER (Fig. 7A), we evaluated the effect of the nonlinearityon the Hill coefficient as described below. Because [Ca2�]ER isdifficult to calibrate, we could not estimate the range of Ca2�

concentrations observed in the ER Ca2� imaging experimentsprecisely. Therefore, we assumed several possible ranges inwhich the Mag-Indo-1 signal maximally changed by 30% rela-tive to the basal level (Fig. 4D and supplemental Fig. S3). Fig. 7Bshows the relationship between the Mag-Indo-1 signal change

FIGURE 5. FRET signal changes of F-L577 during Ca2� oscillations. A, FRETsignal changes of F-L577 (�R/Rbase) and fluorescence changes of Indo-5F (�F/Fbase) evoked by 1 �M ATP (horizontal bar) in intact F-L577-expressing COS7cells. B, the superimposition of representative traces of the F-L577 signal (con-tinuous line) and Ca2� concentration (broken line) on an enlarged time scale.Each image was acquired at 2 Hz. C, Ca2� sensitivity of the FRET signalchanges of F-L577 (�R/Rbase) in intact COS7 cells. The values for �R/Rbase dur-ing Ca2� oscillations were plotted against the calibrated Ca2� concentration.Filled circles, samples in the rising phase of Ca2� spikes; open circles, samples inthe falling phase of Ca2� spikes; daggers, samples at the peak of Ca2� spikes.The estimated values for the Kd and Hill coefficient by non-linear regressionwith the Hill equation are 0.41 � 0.0095 �M and 5.7 � 0.73, respectively(means � S.D.). The thin line shows the Hill equation with the same Kd valuebut a Hill coefficient of 2. The data were collected from six independent mea-surements with a sampling rate of 0.25 Hz.

FIGURE 6. FRET signal changes of F-L577 under the steady-state condi-tion. Ca2� sensitivity of the FRET signal changes of F-L577 (�R/Rbase) in per-meabilized F-L577-expressing COS7 cells is shown. FRET signal changes ofF-L577 were induced by 1 mM MgATP in the presence of 0.13, 0.4, 1.0, 10, and30 �M Ca2� as shown in Fig. 4. The values for �R/Rbase under the equilibriumcondition were plotted against the Ca2� concentration of each internal solu-tion. The estimated values for the Kd and Hill coefficient by non-linear regres-sion with the Hill equation are 0.46 � 0.26 �M and 0.94 � 0.41, respectively(means � S.D.). The numbers of measurements are shown in parentheses.

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and [Ca2�]ER change when the basal Mag-Indo-1 signal wasvaried from 0.01 to 0.7 of the maximum. On the basis of thelinear relationship between the Mag-Indo-1 signal and theFRET signal of F-L577 (Fig. 4D), we obtained the relationshipbetween [Ca2�]ER change and the FRET signal change ofF-L577 (Fig. 7C). By using this relationship, we estimated thecytosolic Ca2� dependence of the SERCA pump activity, whichwas directly correlated with [Ca2�]ER change (Fig. 7D). Wefound that theHill coefficient of the cytosolicCa2�dependence ofthe SERCA pump activity was almost constant regardless of thevalueof thebasalMag-Indo-1 signal (Fig. 7E). These results clearlydemonstrate that the high cooperativity was not caused by a non-linear relationship between theMag-Indo-1 signal and [Ca2�]ER.

The cooperative dependence of SERCA on cytosolic Ca2� isan important parameter for the generation of complex Ca2�

signals such as Ca2� oscillations because small changes in thisparameter had a large impact on the behavior of the Ca2�

dynamics. Similar to the action potential, which is generated bythe combination of opposite membrane potential changes pro-

moted by fast voltage-gated sodium channels and slowly acti-vated voltage-gated potassium channels (41), cytosolic Ca2�

spikes may be generated by the ingenious balance of Ca2�

influx and Ca2� efflux promoted by Ca2� release channels andCa2� pumps, respectively. Therefore, our findings provide uswith evidence to reconsider not only the SERCA pump activitybut also the Ca2� release activity in living cells to gain a betterunderstanding of the mechanism underlying the generation ofCa2� dynamics. In addition, Ca2� is one of the most importantcytosolic signals in living cells, but a prolonged elevation of thecytosolic Ca2� concentration results in irreversible damage, asobserved during cardiac or cerebral ischemia (42). The highcooperativity of SERCA should work to rapidly take up intra-cellular Ca2� to reduce its cytotoxicity.Currently, we do not know the exact reason for the discrep-

ancy between the Hill coefficients measured in intact cells(nH � 5.7 � 0.73) (Fig. 5C) and in vitro assays (nH � 2) (16). Toexplore the mechanism for the highly cooperative dependenceon cytosolic Ca2�, we carried out a simulation analysis based on

FIGURE 7. Effect of the nonlinearity of the Mag-Indo-1 signal on the ER luminal Ca2� concentration for the Hill coefficient estimation. A, the normalizedCa2� titration curve of Mag-Indo-1 fluorescence intensity (F). The fluorescence intensity of Mag-Indo-1 was calculated by assuming a 1:1 interaction with a Kdvalue of 35 �M. B, the relationship between the [Ca2�]ER change (�[Ca2�]ER) and the normalized Mag-Indo-1 signal relative to the baseline level. Six differentbaseline levels (Fbase � 0.01, 0.1, 0.3, 0.5, 0.6, and 0.7 of the maximum) were used. C, the relationship between �[Ca2�]ER and the normalized FRET signal changeof F-L577. The linear relationship between the Mag-Indo-1 signal and the FRET signal of F-L577 (y � 3.9702 x � 0.0233) (Fig. 4D) was used to convert �F/Fbaseto �R/Rbase. The shaded area shows the range of the FRET signals of F-L577 measured during Ca2� oscillations in living cells (Fig. 5C). D, the cytosolic Ca2�

dependence on the SERCA2a pump activity was assessed by �[Ca2�]ER when Fbase � 0.5. The measured values of �R/Rbase of F-L577 shown in Fig. 5C wereconverted into �[Ca2�]ER by the relationship shown in Fig. 7C. The Hill coefficient of the best-fit equation is 5.7. E, the Hill coefficient estimated from thecytosolic Ca2� dependence of the SERCA2a pump activity assessed by �[Ca2�]ER was plotted against the assumed Fbase value. The dashed line representsthe average value of 5.7. Similar results were obtained when the linear relationship between the Mag-Indo-1 signal and the FRET signal of F-L577 in thepresence of different [Ca2�]i (y � 2.5996, x � 0.0881) (supplemental Fig. S3) was used to convert �F/Fbase to �R/Rbase. The error bars represent the S.D.

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the six-state SERCA model described by Yano et al. (43) (sup-plemental Fig. S4A). The model includes the regulation ofSERCA pump activity by both [Ca2�]i and [Ca2�]ER. TheATPase activity of the SERCA pump was calculated for a cycleof cytosolic Ca2� oscillation with a peak value of 1 �M and thebaseline concentration of 150 nM with a sinusoidal decrease in[Ca2�]ER (44). [Ca2�]ER was set as 100 �M at the resting state,with the minimal [Ca2�]ER set as 50 �M during the cycle. Wefound that nonlinear modulation of the rate of the transitionfrom the E2 state to E1 state by cytosolic Ca2� can produce thehighly cooperative dependence of SERCA activity on cytosolicCa2� (supplemental Fig. S4B). We measured the intracellulardistribution of F-L577 in COS7 cells before and after ATP stim-ulation (supplemental Fig. S5A) and found that the distributionof F-L577 was not largely changed after ATP stimulation, eventhough the FRET signal was changed significantly (supplemen-tal Fig. S5C). These results suggest that the fluorescent signalchanges of F-L577 are caused mainly by intramolecular FRET,rather than intermolecular FRET. Therefore, the high cooper-ativity obtained in this study is not derived from oligomericinteraction of SERCApumps. These results suggest that factorssuch as Ca2�-dependent SERCA binding proteins, which canbe excluded by the permeabilization treatment, are involved inthemodulation of the transition rate from the E2 state to the E1state for the generation of the high cooperativity in living cells.In fact, there is a report that an interacting modulator, such asphospholamban, alters the cooperativity of Ca2� binding toSERCA (45). The mechanism of the highly cooperative depen-dence of SERCA2a pump activity on cytosolic Ca2� is an inter-esting issue to be clarified in future studies.In conclusion, we succeeded in visualizing the dynamics of

SERCA2a in living cells by constructing F-L577, whose FRETsignal changes reflect the instantaneous Ca2� pump activity.We found that the Ca2� pump activity of SERCA2a is synchro-nized with cytosolic Ca2� concentration changes without delayduring Ca2� oscillations observed in COS7 cells stimulatedwith ATP. The Ca2� pump activity of SERCA2a in intact cellscan be expressed by the Hill equation with an apparent affinityforCa2� of 0.41� 0.0095�Mand aHill coefficient of 5.7� 0.73.The marked Ca2� dependence allows SERCA2a to act as aswitch to refill the Ca2� stores efficiently. The F-L577 proteinconstructed in this study will be useful for future studies onCa2� signaling in normal and abnormal cellular processes thatinvolve SERCA pump activity.

Acknowledgments—We thank Drs. Hiroko Bannai, HarukaYamazaki, Mark W. Sherwood, Yoshiyuki Yamada, and AkitoshiMiyamoto for critical reading of the manuscript. We thank AkioSuzuki for technical help with the adjustment of the free Ca2� con-centrations in the internal solutions.

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Highly Cooperative Ca2� Dependence of SERCA in Living Cells

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Michikawa and Katsuhiko MikoshibaKanayo Satoh, Toru Matsu-ura, Masahiro Enomoto, Hideki Nakamura, Takayuki

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

doi: 10.1074/jbc.M110.204685 originally published online April 22, 20112011, 286:20591-20599.J. Biol. Chem. 

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