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

Abstract

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 μm 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.

Introduction

Intracellular Ca²⁺ plays a pivotal role in controlling numerous cellular processes such as exocytosis, gene transcription, cell proliferation, muscle contraction, and cell survival (1). The level of intracellular Ca²⁺ is determined by the balance between the influx that introduces Ca²⁺ into the cytoplasm and the efflux that removes it from the cytoplasm. The key molecules involved in the regulation of intracellular Ca²⁺ such as channels, pumps, and exchangers have been identified (2). Channels in 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 transfers Ca²⁺ from the cytosol to the lumen of the SR/ER at the expense of ATP hydrolysis (3). SERCA functions to determine the resting level of intracellular Ca²⁺ (4) and to control the spatiotemporal profile of Ca²⁺ transients and the frequency of Ca²⁺ oscillations (5). Impairment of SERCA causes Ca²⁺ homeostatic dysfunction, resulting in several important disease states such as heart failure, hypertension, diabetes, and Alzheimer disease (6).

The SERCA family consists of three isoforms and their splicing variants (SERCA1a, -1b, -2a, -2b, -3a, -3b, and -3c). The molecular masses of SERCA isoforms range from 105 to 115 kDa. Each SERCA isoform consists of four distinct functional domains, namely the nucleotide-binding, phosphorylation, actuator, and transmembrane domains (7). The three-dimensional structures of different conformational states of SERCA1a have been defined by x-ray crystallography (8), and structural models for the catalytic cycle of SERCA are well established (9). The sequential conformational changes of SERCA are accompanied by active transport of 2 mol of Ca²⁺ per 1 mol of bound ATP, and 1 mol of ATP is hydrolyzed during one reaction cycle (10). Thus, the conformational changes are directly linked with SERCA activity.

Cytosolic Ca²⁺ increases evoked by extracellular stimuli are often observed in the form of oscillating Ca²⁺ spikes (11). In most cell types, Ca²⁺ spikes are generated by inositol 1,4,5-trisphosphate (IP₃)-induced Ca²⁺ release from intracellular Ca²⁺ stores (12). The IP₃ receptor is an intracellular Ca²⁺ release channel, and its activity is controlled by not only IP₃ but also cytosolic Ca²⁺ (13). Because submicromolar Ca²⁺ concentrations activate IP₃ receptor, the upstroke of Ca²⁺ spikes may occur 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 the Ca²⁺ stores at a rate that is solely dependent on the intracellular Ca²⁺ concentration ([Ca²⁺]_i) (15). The pump activity of SERCA has been approximated by the Hill equation to have a Hill coefficient of 2 for Ca²⁺ binding (16) in theoretical studies of Ca²⁺ dynamics (15, 17). However, it remains to be elucidated whether the enzymatic activity of SERCA in living cells is identical to that measured in vitro.

The FRET technique is commonly used to study the conformational changes of functional proteins (18), and labeling with fluorescent proteins has been widely used for this purpose (19). However, as ectopic expression and/or dysfunction of the target protein can sometimes result from the labeling with these relatively large (∼27 kDa) proteins (20), techniques using small fluorescent molecules with less steric bulk have been greatly advanced (21). As an example, FlAsH-EDT₂ is a small membrane-permeable synthetic ligand with high affinity and specificity for a tetracysteine tag (CCPGCC; TC tag) (22). The binding of FlAsH-EDT₂ to a TC tag induces chromophore formation, giving rise to fluorescence (23).

Real-time visualization of SERCA pump activity in living cells should facilitate our understanding of the mechanism for the generation of intracellular Ca²⁺ dynamics. In this study, we successfully detected the pump activity of SERCA2a in living cells using the FRET technique with a combination of enhanced CFP (ECFP) and FlAsH. Dual imaging of intracellular Ca²⁺ and the FRET signals of our ECFP- and FlAsH-tagged SERCA2a, designated F-L577, provides significant insights into the regulatory mechanism of SERCA pump activity in living cells.

EXPERIMENTAL PROCEDURES

Plasmid Constructions

The details of these procedures are given in the supplemental data.

Cell Culture and Transfection

COS7 cells were cultured in DMEM (Invitrogen) supplemented with 10% FBS, 100 units/ml penicillin and 0.1 mg/ml streptomycin, and maintained in a humidified incubator with 5% CO₂ at 37 °C. The cells were grown on 35-mm poly-l-lysine-coated glass-bottomed dishes (Matsunami). F-L577 and mRFP-KDEL cDNAs were transfected into the cells using TransIT (Mirus). The cells were used for 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 and washed twice with BAL wash buffer (Invitrogen).

Western Blot Analysis

F-L577-expressing and non-transfected COS7 cells (1 × 10⁶) were solubilized with 100 μl of 1× SDS sample buffer (2% SDS, 10% (w/v) glycerol, 5% (v/v) 2-β-mercaptoethanol, 0.05% bromphenol blue, 0.0625 m Tris-HCl, pH 6.8). Aliquots (2 μl) of the cell lysates were analyzed by Western blot using the anti-SERCA2 antibody (BD Biosciences) at a dilution of 1:1000 and goat anti-mouse IgG, HRP-linked F(ab′)₂ fragment (GE Healthcare) as the secondary antibody at a dilution of 1:2000. Signals were detected with an ECL kit (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 confocal scanning microscope (CSU-XI; Yokogawa) attached to an IX81 inverted microscope (Olympus) with an EMCCD camera (CascadeII; Roper) and a 60× (oil; numerical aperture, 1.42) objective lens (Olympus). A 440-nm excitation filter, a 520-nm emission filter, and 450-nm dichroic mirrors were inserted into the light path. Data were analyzed using MetaMorph software (Molecular Devices).

Acceptor Photobleaching

Acceptor (FlAsH) photobleaching was performed using a confocal microscope (A1; Nikon). Repeated scans (120 s at maximum rates) with an unattenuated 514-nm illumination from a multi-argon laser were applied (total, 30 milliwatt maximum output). ECFP and FlAsH emission signals were acquired with a 515-nm dichroic mirror and a pair 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 at 27 °C with stirring in Sf-900II SFM medium (Invitrogen) containing 10% FBS. P1 viral stocks of recombinant baculoviruses for F-L577 and WT-SERCA2a were generated using a Bac-to-Bac Baculovirus Expression System (Invitrogen) according to the manufacturer's instructions. Amplification of P2 viral stocks and recombinant protein expression of F-L577 and WT-SERCA2a were performed using Sf9 cells as described previously (24, 25).

In Vitro Kinetic Assay of ATP-induced Ca²⁺ Uptake by SERCA Pump

Microsomal vesicles were prepared from Sf9 cells as described previously (26). The measurements and analysis of the ATP-induced Ca²⁺ accumulation were conducted as described elsewhere.⁹ Briefly, the volume of the microsomal vesicles used in each measurement was adjusted using the absorbance at 600 nm acquired by a DU 640 spectrophotometer (Beckman Coulter). The microsomal vesicles were suspended in 500 μl of cytosol-like medium (110 mm KCl, 10 mm NaCl, 5 mm KH₂PO₄, 1 mm DTT, 50 mm HEPES-KOH, pH 7.2, at room temperature), supplemented with 1 μg/ml oligomycin (Sigma), 2 μm MgCl₂, 25 μg/ml creatine kinase (Roche Diagnostics), 10 mm phosphocreatine (Sigma), and 2 μm fura-2 (Dojindo). The suspension was continuously stirred at 30 °C during measurements. The fluorescent signals of fura-2 were monitored with a CAF-110 spectrofluorometer (Jasco) and a PowerLab data acquisition system (ADInstruments). Fluorescence signals were recorded every 0.01 s at an emission wavelength of 510 nm with alternate excitation at 340 and 380 nm. The 1 mm ATP-induced Ca²⁺ uptake into microsomal vesicles by the SERCA pump was initiated and monitored for 300 s. Free Ca²⁺ concentrations were calculated from acquired fluorescent signals as described previously (27). To estimate the rate of Ca²⁺ uptake, curve fitting of three-exponential function was conducted using Igor Pro 6.02 software (WaveMetrics) and t½ (half-decay time) was calculated.

Cell Permeabilization and State Fixation of F-L577

FlAsH-EDT₂-loaded F-L577-expressing COS7 cells were permeabilized with 60 μm β-escin (Sigma) in an internal solution (19 mm NaCl, 125 mm KCl, 10 mm HEPES-KOH, pH 7.4) supplemented with 5 mm EGTA for 3–5 min. The permeabilized cells were gently washed with the internal solution containing 5 mm EGTA and then used for FRET measurements. Reagents for the fixation of F-L577 in the four major conformational states were prepared according to methods described previously in the crystallization experiments of SERCA1a (28–31). Cells were perfused with an internal solution containing 30 μm thapsigargin and 5 mm EGTA for the E2 state; 100 μm CaCl₂ for the E1-Ca²⁺ state; 1 mm ADP, 0.33 mm AlCl₃, 5 mm NaF, 1 mm MgCl₂, and 100 μm CaCl₂ for the E1-ATP state; or 2 mm BeCl₂, 8 mm NaF, 1 mm MgCl₂, and 5 mm EGTA for the E2P state. The free Ca²⁺ concentration in the internal solutions was adjusted with K₂HEDTA and CaHEDTA at 37 °C as described previously (32). Fluorescent signals were acquired at 0.25 Hz with an IX71 inverted fluorescence microscope (Olympus) (see below).

ER Ca²⁺ Imaging

ER luminal Ca²⁺ imaging was performed with 10 μm Mag-Indo-1 AM (Molecular Probes), according to the method described previously (33, 34) with some modifications. The free Ca²⁺ concentrations in the internal solutions were carefully adjusted with a Ca²⁺ buffer and confirmed using a Ca²⁺-sensitive electrode (Metrohm) prior to use (41). For FRET and Ca²⁺ imaging, the permeabilized COS7 cells expressing F-L577 were perfused with the internal solution containing 1 μm free Ca²⁺, and then 0.01, 0.1, or 1 mm MgATP (Sigma) was applied in the internal solution containing 1 μm free Ca²⁺. Fluorescent signals were acquired at 0.25 Hz with an IX71 inverted microscope (Olympus) (see below).

Live Cell Imaging

For imaging of intact cells, F-L577-expressing COS7 cells were loaded with 10 μm Indo-5F AM (Dojindo) and FlAsH-EDT₂ reagent. Images were obtained under a constant flow (2 ml/min) of a balanced salt solution containing 115 mm NaCl, 5.4 mm KCl, 1 mm MgCl₂, 2 mm CaCl₂, 10 mm glucose, and 20 mm HEPES-NaOH, pH 7.4. Fluorescent signals were acquired at 2 Hz with an IX71 inverted fluorescence microscope (see below). For [Ca²⁺]_i calibration, fluorescence imaging of F-L577 and Indo-5F was performed using the IX81 inverted microscope. [Ca²⁺]_i was calculated using the formula of Grynkiewicz et al. (27). After agonist stimulation (1 μm ATP for 2 min), the minimum and maximum fluorescence ratio of Indo-5F was obtained by treatment of the cells with 2 μm ionomycin (Br-A23187) and 5 mm EGTA followed by 2 μm ionomycin and 10 mm CaCl₂.

Imaging Equipment

Imaging in intact and permeabilized cells was performed at 37 °C using an IX71 or IX81 inverted microscope (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 fluorescent images of F-L577, Indo-5F (Dojindo), and Mag-Indo-1 (Molecular Probes), an emission splitter (W-view; Hamamatsu Photonics) was used with a light source exchanger (DG-4; Sutter Instrument Co.) on the IX71 inverted microscope. Sequential excitations of F-L577 and Indo-5F (or Mag-Indo-1) were performed using a 450-nm dichroic mirror and two excitation filters (425–445 nm for F-L577, 360 nm for Indo-5F and Mag-Indo-1). Emissions were split with a 460–510-nm filter (for F-L577, Indo-5F, and Mag-Indo-1), a long path 520-nm barrier filter (for F-L577) and two 505-nm dichroic mirrors (for all fluorophores) equipped in W-view. Images were acquired at 0.25 or 2 Hz, with an exposure time of 100 or 150 ms. For [Ca²⁺]_i calibration, fluorescence imaging of F-L577 and Indo-5F was performed using the IX81 inverted microscope equipped with two excitation filters (425–445 nm for F-L577 and 333–348 nm for Indo-5F), two dichroic mirrors (450 nm for F-L577 and 400 nm for Indo-5F), two emission filters (460–510 nm for F-L577 and Indo-5F, 515–565 nm for F-L577). The filters were controlled by Lambda 10–2 (Sutter Instrument Co.) and IX2-RFACA (Olympus). Image acquisition was performed with the MetaFluor software (Molecular Devices). Offline analyses were performed with the MetaFluor Analyst software (Molecular Devices) and Igor Pro software (WaveMetrics).

RESULTS AND DISCUSSION

Construction and Characterization of Fluorescently Labeled SERCA2a

Fig. 1A shows a schematic representation of the F-L577 protein constructed in this study to investigate the conformational changes linked with SERCA2a activity in living cells. Because the N-terminal fusion of ECFP to SERCA1a has no notable effects on its ATPase activity (35), ECFP was fused to the N terminus of the A-domain of SERCA2a (Fig. 1A). The site of the TC tag insertion was determined based on the following criteria: 1) the relative distance between the donor and acceptor should change dramatically during the reaction cycle of SERCA, and 2) the site should be located within a surface loop connecting secondary structures, such as an α-helix and a β-sheet. Fig. 1B shows a schematic representation of the conformational changes during the reaction cycle of SERCA1a (28–31, 36). Because the relative orientation of the N-domain is markedly changed during the cycle, the penultimate loop between Leu-577 and Glu-578 in the N-domain was chosen as the TC tag insertion site (Fig. 1, A and B).

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, a schematic representation of the three-dimensional structures of SERCA in its four different conformational states during the reaction cycle. The four distinct functional domains, namely the nucleotide-binding (N), phosphorylation (P), actuator (A), and transmembrane (TM) domains, are highlighted. The sequential conformational changes of SERCA are accompanied by active transport of 2 mol of Ca²⁺ per 1 mol of bound ATP, and 1 mol of ATP is hydrolyzed during one reaction cycle. Ca²⁺ binding stabilizes the conformation for binding of ATP (E1-Ca²⁺ state). The conformational change induced by phosphorylation decreases the Ca²⁺ affinity (E1-ATP state), resulting in a Ca²⁺ shift from the cytosolic side of the membrane to the luminal side. During this process, 1 mol of ATP is hydrolyzed. The Ca²⁺ affinity then decreases, and Ca²⁺ is mobilized into the ER lumen (E2P state). This Ca²⁺ mobilization process promotes the dephosphorylation of the Asp-351 phosphorylated residue, thereby making the Ca²⁺-binding site accessible to cytosolic Ca²⁺ again (E2 state).

Fig. 2A shows a Western blot analysis of F-L577 expressed in COS7 cells. The signal for F-L577 (lane 2) was detected with the expected size. The subcellular localization of F-L577 in the ER was confirmed by confocal microscopy (Fig. 2B). The ER was labeled by cotransfection with mRFP-KDEL (an ER-targeted monomeric red fluorescent protein) (34). The fluorescent signals of ECFP and FlAsH colocalized well with the mRFP-KDEL signals, indicating that the ECFP fusion and TC tag insertion did not disrupt the ER localization of F-L577.

FIGURE 2.

The characterization of F-L577. A, Western blot analysis of endogenous SERCA2 and F-L577 with an anti-SERCA2 antibody. Cell lysates (2 μl each) 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 shown on the left. B, the subcellular distribution of F-L577 in COS7 cells. Confocal images of COS7 cells expressing F-L577 and mRFP-KDEL are shown. C, acceptor photobleaching of F-L577-expressing COS7 cells. Representative emission intensities of ECFP (continuous line, donor) and FlAsH (broken line, acceptor) are shown. D, half-decay times (t½) of ATP-induced Ca²⁺ uptake of microsomal vesicles prepared from non-infected Sf9 cells (NC) and Sf9 cells expressing recombinant WT-SERCA2a or F-L577 were calculated. The error bars represent the S.D. The numbers of measurements are shown in parentheses. **, p < 0.01; n.s., not significant by Tukey-Kramer test. a.u., arbitrary units.

To confirm that FRET occurred in living COS7 cells, we performed acceptor (FlAsH) photobleaching of F-L577. After the photobleaching of FlAsH, the ECFP (donor) fluorescence intensity was increased to 130.1 ± 10.1% (mean ± S.D., n = 6) (Fig. 2C). This finding indicates that FRET occurred between the N-terminal ECFP and the FlAsH label in the N-domain in resting COS7 cells.

We measured the Ca²⁺ pump activity of F-L577 in vitro (Fig. 2D). Recombinant WT-SERCA2a and F-L577 were expressed in Sf9 insect cells. ATP-induced Ca²⁺ accumulation in the microsomal vesicles containing WT-SERCA2a or F-L577 was monitored by spectrofluorometry. A representative trace of Ca²⁺ uptake is shown in supplemental Fig. S1. The half-decay time (t½) of the ATP-induced Ca²⁺ accumulation in the microsomal vesicles prepared from cells expressing WT-SERCA2a was significantly smaller than the t½ of Ca²⁺ accumulation in the microsomal vesicles prepared from non-transfected cells (Fig. 2D). These findings indicate that the activity of the recombinant WT-SERCA2a was successfully detected in this experimental system. Overexpression of F-L577 also significantly decreased the t½ (Fig. 2D). Based on these findings, we concluded that the ECFP fusion and TC tag insertion did not abolish the SERCA2 pump activity.

FRET Signals of F-L577 in Four Major Conformational States of SERCA

To examine the ability of F-L577 to reflect the various conformational changes linked to SERCA2 pump activity, we measured its FRET signals in the four major conformational states (Fig. 3A). To isolate these states, we performed state fixation of F-L577 expressed in COS7 cells. Briefly, permeabilized COS7 cells expressing F-L577 were initially perfused with an internal solution containing 5 mm EGTA to provide an arbitrary F-L577 reference state. The cells were then perfused with an internal solution containing 30 μm thapsigargin and 5 mm EGTA (E2 state) (29); 100 μm CaCl₂ (E1-Ca²⁺ state) (28); 1 mm ADP, 0.33 mm AlCl₃, 5 mm NaF, 1 mm MgCl₂, and 100 μm 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⁻ are phosphate analogues that can be used to fix SERCA1a in the E1-ATP and E2P states, respectively (30, 36). Supplemental Fig. S2 shows the relationship between the FRET signals of F-L577 and the concentration of thapsigargin, as a SERCA-specific inhibitor, in permeabilized COS7 cells. The EC₅₀ value was ∼20 μm, which was higher than that measured in vitro (37). Therefore, a saturating concentration (30 μm) of thapsigargin was used for the state fixation experiments. Fig. 3B shows representative traces of the FRET signal changes from the reference state to the four different states. The average values of the FRET signal changes (ΔR/R_base) for the four different states are shown in Fig. 3C. When the cells were fixed in the E2 state (thapsigargin-bound state), F-L577 showed a 7.3 ± 1.6% (mean ± S.D.) decrease in the FRET signal relative to the baseline signal in the reference state (Fig. 3, B and C). The FRET signals in the E1-Ca²⁺ state (Ca²⁺-bound state) and E1-ATP state (ATP-bound state) showed 11.9 ± 3.2% and 6.2 ± 1.9% decreases relative to the baseline signal, respectively (Fig. 3, B and C). In contrast, the FRET signal showed a 2.4 ± 0.86% increase following the transition from the reference state to the E2P state (Fig. 3, B and C). The overall dynamic range of the FRET signal changes of F-L577 was ∼15%.

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-state model. The components of the solutions required for state fixation are shown in parentheses. B, representative traces of the FRET signal changes of F-L577 (ΔR/R_base) in permeabilized COS7 cells. Fixation of F-L577 in the four states was performed by perfusion of solutions containing thapsigargin (E2), Ca²⁺ (E1-Ca²⁺), Ca²⁺ with Mg²⁺-ADP and AlF₄⁻ (E1-ATP) and BeF₃⁻ in the absence of Ca²⁺ (E2P) (horizontal black bars) after perfusion of an internal solution containing 5 mm EGTA. Each image was acquired at 0.25 Hz. R, ratio of FlAsH fluorescence to ECFP fluorescence; R_base, basal level of R; ΔR/R_base, (R − R_base)/R_base. C, the average values of the FRET signal changes of F-L577 in the four major conformational states. The error bars represent the S.D. The numbers of measurements are shown in parentheses.

The distances between the N terminus and Leu-577 of SERCA1a in the four major conformational states were calculated using the molecular modeling software PyMOL. The calculated distances were 39.19 Å in the E2 state (Protein Data Bank code 1IWO) (29), 69.79 Å in the E1-Ca²⁺ state (Protein Data Bank code 1SU4) (28), 36.83 Å in the E1-ATP state (Protein Data Bank code 1T5T) (30), and 33.32 Å in the E2P state (Protein Data Bank code 3B9B) (36). Therefore, the order of the distance between the N terminus and Leu-577 was E1-Ca²⁺ > E2 > E1-ATP > E2P. This order was consistent with the order of the relative amounts of the FRET signal changes of SERCA2a (Fig. 3C), suggesting that the FRET signal changes contain information about the distance between the N terminus and Leu-577. The measured FRET signal changes were predictive of the defined structural changes during the cycling of SERCA, strongly suggesting that F-L577 is capable of detecting the conformational changes of SERCA2a.

FRET Signal Changes of F-L577 Directly Reflect Ca²⁺ Pump Activity

To confirm that the FRET signal changes of F-L577 reflect the Ca²⁺ pump activity in living cells, we performed time-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 loaded with a low affinity fluorescent Ca²⁺ indicator, Mag-Indo-1 AM (K_d = ∼35 μm) using a method described previously (33, 34). For time-lapse imaging during Ca²⁺ pumping, a baseline was initially established by perfusion with an internal solution containing 1 μm Ca²⁺ without MgATP. Under these ATP-free conditions, the E2 and E1-Ca²⁺ states should dominate (Fig. 3A). To initiate Ca²⁺ accumulation in the ER, 1 mm MgATP was added to the perfusate. Following the application of MgATP, the FRET signal of F-L577 increased by 3–6%, accompanied by an increase in the ER luminal Ca²⁺ concentration (Fig. 4A). The increase in the FRET signal suggests that the fractions of the E1-ATP and E2P states, which showed higher FRET signals than the E2 and E1-Ca²⁺ states (Fig. 3C), are increased when SERCA2a is activated.

FIGURE 4.

FRET signal changes of F-L577 reflect the Ca²⁺ uptake activity. A, representative traces of the FRET signal changes of F-L577 (ΔR/R_base) and the fluorescent signal changes of Mag-Indo-1 loaded in the ER (ΔF/F_base) induced by MgATP. An increase in ΔF/F_base represents Ca²⁺ uptake from the cytosol to the 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 indicate the presence of 1 μm Ca²⁺. Each image was acquired at 0.25 Hz. R, ratio of FlAsH fluorescence to ECFP fluorescence; R_base, basal level of R; ΔR/R_base, (R − R_base)/R_base; F, fluorescence of Mag-Indo-1 at 450–495 nm; F_base, basal level of F; ΔF/F_base, (F − F_base)/F_base. B, superimposition of the first derivatives of ΔR/R_base (d(ΔR/R_base)/dt; continuous lines) and ΔF/F_base (d(ΔF/F_base)/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 ΔR/R_base and ΔF/F_base in the presence of 1 μm Ca²⁺. The data represent means ± S.D. The numbers of measurements are shown in parentheses. D, the correlation between ΔR/R_base and ΔF/F_base. 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 (ΔF/F_base) of F-L577-expressing (+) and non-expressing (−) cells under the three conditions 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.

To determine the timing of the FRET changes relative to Ca²⁺ uptake, we compared the first derivatives of the two responses (Fig. 4B). The peak responses were detected at the same sampling points, indicating that the timing of the conformational changes of F-L577 was coincident with Ca²⁺ uptake into the ER.

The Ca²⁺ pump activity of SERCA2 is dependent on the ATP concentration (38). To measure the ATP dependence of the FRET signals of F-L577, we repeated the above experiments in the presence of 0.01, 0.1, or 1 mm ATP (Fig. 4C). The results demonstrated that the FRET signals of F-L577 showed ATP concentration-dependent changes (Fig. 4C, left) and that the ATP dependence of the FRET signals paralleled the accumulation of Ca²⁺ into the ER lumen (Fig. 4C, right).

We further analyzed the correlation between the F-L577 FRET and Mag-Indo-1 responses (Fig. 4D). Interestingly, the F-L577 (ΔR/R_base) and Mag-Indo-1 (ΔF/F_base) responses were significantly correlated (r = 0.767, n = 61). We also explored the effect of the cytosolic Ca²⁺ concentration on the F-L577 response (supplemental Fig. S3). The FRET signal changes of F-L577 and the Mag-Indo-1 signal changes were significantly correlated even when the cytosolic Ca²⁺ concentration varied from 0.13–1.0 μm (r = 0.651, n = 38). These results suggest that the positive FRET signal changes of F-L577 are directly correlated with the instantaneous Ca²⁺ pump activity of SERCA2a.

We compared the Mag-Indo-1 signal changes of non-transfected cells and F-L577-expressing cells under three different conditions (Fig. 4E). When permeabilized COS7 cells were stimulated by 0.1 or 1 mm MgATP in an internal solution containing 1 μm Ca²⁺, the Mag-Indo-1 signal change in F-L577-expressing cells was significantly larger than that in non-transfected 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 conformational state, (2) the conformational change of F-L577 is coincident with Ca²⁺ uptake, (3) the ATP and Ca²⁺ concentration dependences of the F-L577 FRET signal are comparable with those of Ca²⁺ uptake, (4) the FRET signal change of F-L577 is correlated with Ca²⁺ uptake in permeabilized cells, and (5) F-L577 itself has Ca²⁺ pump activity in living cells. In summary, the FRET signal changes of F-L577 directly reflect the Ca²⁺ pump activity linked with its conformational changes in the SERCA reaction cycle.

Visualization of Ca²⁺ Pump Activities of SERCA During Ca²⁺ Oscillations in COS7 Cells

To investigate the dynamics of SERCA activity in living cells during ATP-evoked Ca²⁺ oscillations, we performed dual imaging of intracellular Ca²⁺ and the FRET signal changes of F-L577 in COS7 cells. Fig. 5A shows representative FRET signal changes of F-L577 (upper panel) and cytoplasmic Ca²⁺ concentration changes (lower panel) observed in COS7 cells stimulated with 1 μm ATP. The FRET signals of F-L577 showed oscillatory dynamics during Ca²⁺ oscillations. Although the oscillatory SERCA activity has been predictable, these findings comprise the first experimental evidence for the detection of oscillatory SERCA activity in living cells. The representative traces of the FRET signal changes of F-L577 (continuous line) and Ca²⁺ changes (broken line) are also shown on an enlarged time scale (Fig. 5B). There was no detectable delay between the F-L577 signals and the Indo-5F signals during either the rising or falling phases of the Ca²⁺ spikes. These findings indicate that the Ca²⁺ pump activity of SERCA2a is synchronized with cytosolic Ca²⁺ concentration changes without any detectable delay.

FIGURE 5.

FRET signal changes of F-L577 during Ca²⁺ oscillations. A, FRET signal changes of F-L577 (ΔR/R_base) and fluorescence changes of Indo-5F (ΔF/F_base) evoked by 1 μm ATP (horizontal bar) in intact F-L577-expressing COS7 cells. B, the superimposition of representative traces of the F-L577 signal (continuous line) and Ca²⁺ concentration (broken line) on an enlarged time scale. Each image was acquired at 2 Hz. C, Ca²⁺ sensitivity of the FRET signal changes of F-L577 (ΔR/R_base) in intact COS7 cells. The values for ΔR/R_base during Ca²⁺ oscillations were plotted against the calibrated Ca²⁺ concentration. Filled circles, samples in the rising phase of Ca²⁺ spikes; open circles, samples in the falling phase of Ca²⁺ spikes; daggers, samples at the peak of Ca²⁺ spikes. The estimated values for the K_d and Hill coefficient by non-linear regression with the Hill equation are 0.41 ± 0.0095 μm and 5.7 ± 0.73, respectively (means ± S.D.). The thin line shows the Hill equation with the same K_d value but a Hill coefficient of 2. The data were collected from six independent measurements with a sampling rate of 0.25 Hz.

Fig. 5C shows the relationship between the FRET signal changes of F-L577 and [Ca²⁺]_i during the period of Ca²⁺ oscillations evoked with 1 μm ATP. We found a good fit between the data and the Hill equation with an apparent affinity for Ca²⁺ of 0.41 ± 0.0095 μm and a Hill coefficient of 5.7 ± 0.73 (Fig. 5C). The apparent Ca²⁺ sensitivity of F-L577 was consistent with previously reported values (0.34 ∼ 0.45 μm) for native SERCA2a measured in vitro (15, 39, 40). In contrast, the high degree of cooperativity differed 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 the SERCA pump or fluorescent labeling, we investigated the FRET signal change of F-L577 under the steady-state condition in [Ca²⁺]_i (Fig. 6). Fig. 6 shows the relationship between [Ca²⁺]_i in the solutions applied to permeabilized cells and the FRET signal change of F-L577. We found a good fit between the data and the Hill equation with an apparent affinity for Ca²⁺ of 0.46 ± 0.26 μm and a Hill coefficient of 0.94 ± 0.41 (Fig. 6). Both the apparent Ca²⁺ sensitivity of F-L577 and the Hill coefficient were consistent with that measured in vitro (15, 39, 40). These results confirm that the highly cooperative dependence of the FRET signal changes of F-L577 on cytosolic Ca²⁺ does not arise from artifacts caused by the overexpression and/or fluorescent labeling. These findings indicate that the Ca²⁺ pump activity of SERCA2a shows a high degree of cooperativity in the physiological condition of living cells.

FIGURE 6.

FRET signal changes of F-L577 under the steady-state condition. Ca²⁺ sensitivity of the FRET signal changes of F-L577 (ΔR/R_base) in permeabilized F-L577-expressing COS7 cells is shown. FRET signal changes of F-L577 were induced by 1 mm MgATP in the presence of 0.13, 0.4, 1.0, 10, and 30 μm Ca²⁺ as shown in Fig. 4. The values for ΔR/R_base under the equilibrium condition were plotted against the Ca²⁺ concentration of each internal solution. The estimated values for the K_d and Hill coefficient by non-linear regression with the Hill equation are 0.46 ± 0.26 μm and 0.94 ± 0.41, respectively (means ± S.D.). The numbers of measurements are shown in parentheses.

We assumed that the FRET signal of F-L577 was proportional to the instantaneous SERCA pump activity based on the linear relationship between the FRET signal of F-L577 and the Mag-Indo-1 signal reflecting the ER luminal Ca²⁺ concentration ([Ca²⁺]_ER) (Fig. 4, B and D). To investigate the possibility that the high degree of cooperativity is caused artificially by a nonlinear relationship between the Mag-Indo-1 signal and [Ca²⁺]_ER (Fig. 7A), we evaluated the effect of the nonlinearity on the Hill coefficient as described below. Because [Ca²⁺]_ER is difficult to calibrate, we could not estimate the range of Ca²⁺ concentrations observed in the ER Ca²⁺ imaging experiments precisely. Therefore, we assumed several possible ranges in which the Mag-Indo-1 signal maximally changed by 30% relative to the basal level (Fig. 4D and supplemental Fig. S3). Fig. 7B shows the relationship between the Mag-Indo-1 signal change and [Ca²⁺]_ER change when the basal Mag-Indo-1 signal was varied from 0.01 to 0.7 of the maximum. On the basis of the linear relationship between the Mag-Indo-1 signal and the FRET signal of F-L577 (Fig. 4D), we obtained the relationship between [Ca²⁺]_ER change and the FRET signal change of F-L577 (Fig. 7C). By using this relationship, we estimated the cytosolic Ca²⁺ dependence of the SERCA pump activity, which was directly correlated with [Ca²⁺]_ER change (Fig. 7D). We found that the Hill coefficient of the cytosolic Ca²⁺ dependence of the SERCA pump activity was almost constant regardless of the value of the basal Mag-Indo-1 signal (Fig. 7E). These results clearly demonstrate that the high cooperativity was not caused by a nonlinear relationship between the Mag-Indo-1 signal and [Ca²⁺]_ER.

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 normalized Ca²⁺ 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 K_d value of 35 μm. B, the relationship between the [Ca²⁺]_ER change (Δ[Ca²⁺]_ER) and the normalized Mag-Indo-1 signal relative to the baseline level. Six different baseline levels (F_base = 0.01, 0.1, 0.3, 0.5, 0.6, and 0.7 of the maximum) were used. C, the relationship between Δ[Ca²⁺]_ER and the normalized FRET signal change of 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 ΔF/F_base to ΔR/R_base. 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 Δ[Ca²⁺]_ER when F_base = 0.5. The measured values of ΔR/R_base of F-L577 shown in Fig. 5C were converted into Δ[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 the cytosolic Ca²⁺ dependence of the SERCA2a pump activity assessed by Δ[Ca²⁺]_ER was plotted against the assumed F_base value. The dashed line represents the 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 the presence of different [Ca²⁺]_i (y = 2.5996, x + 0.0881) (supplemental Fig. S3) was used to convert ΔF/F_base to ΔR/R_base. The error bars represent the S.D.

The cooperative dependence of SERCA on cytosolic Ca²⁺ is an important parameter for the generation of complex Ca²⁺ signals such as Ca²⁺ oscillations because small changes in this parameter had a large impact on the behavior of the Ca²⁺ dynamics. Similar to the action potential, which is generated by the combination of opposite membrane potential changes promoted by fast voltage-gated sodium channels and slowly activated 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 and Ca²⁺ pumps, respectively. Therefore, our findings provide us with evidence to reconsider not only the SERCA pump activity but also the Ca²⁺ release activity in living cells to gain a better understanding of the mechanism underlying the generation of Ca²⁺ dynamics. In addition, Ca²⁺ is one of the most important cytosolic signals in living cells, but a prolonged elevation of the cytosolic Ca²⁺ concentration results in irreversible damage, as observed during cardiac or cerebral ischemia (42). The high cooperativity of SERCA should work to rapidly take up intracellular Ca²⁺ to reduce its cytotoxicity.

Currently, we do not know the exact reason for the discrepancy between the Hill coefficients measured in intact cells (n_H = 5.7 ± 0.73) (Fig. 5C) and in vitro assays (n_H ≈ 2) (16). To explore the mechanism for the highly cooperative dependence on cytosolic Ca²⁺, we carried out a simulation analysis based on the six-state SERCA model described by Yano et al. (43) (supplemental Fig. S4A). The model includes the regulation of SERCA pump activity by both [Ca²⁺]_i and [Ca²⁺]_ER. The ATPase activity of the SERCA pump was calculated for a cycle of cytosolic Ca²⁺ oscillation with a peak value of 1 μm and the baseline concentration of 150 nm with a sinusoidal decrease in [Ca²⁺]_ER (44). [Ca²⁺]_ER was set as 100 μm at the resting state, with the minimal [Ca²⁺]_ER set as 50 μm during the cycle. We found that nonlinear modulation of the rate of the transition from the E2 state to E1 state by cytosolic Ca²⁺ can produce the highly cooperative dependence of SERCA activity on cytosolic Ca²⁺ (supplemental Fig. S4B). We measured the intracellular distribution of F-L577 in COS7 cells before and after ATP stimulation (supplemental Fig. S5A) and found that the distribution of F-L577 was not largely changed after ATP stimulation, even though the FRET signal was changed significantly (supplemental Fig. S5C). These results suggest that the fluorescent signal changes of F-L577 are caused mainly by intramolecular FRET, rather than intermolecular FRET. Therefore, the high cooperativity obtained in this study is not derived from oligomeric interaction of SERCA pumps. These results suggest that factors such as Ca²⁺-dependent SERCA binding proteins, which can be excluded by the permeabilization treatment, are involved in the modulation of the transition rate from the E2 state to the E1 state for the generation of the high cooperativity in living cells. In fact, there is a report that an interacting modulator, such as phospholamban, alters the cooperativity of Ca²⁺ binding to SERCA (45). The mechanism of the highly cooperative dependence of SERCA2a pump activity on cytosolic Ca²⁺ is an interesting 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 FRET signal changes reflect the instantaneous Ca²⁺ pump activity. We found that the Ca²⁺ pump activity of SERCA2a is synchronized with cytosolic Ca²⁺ concentration changes without delay during Ca²⁺ oscillations observed in COS7 cells stimulated with ATP. 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 μm and a Hill coefficient of 5.7 ± 0.73. The marked Ca²⁺ dependence allows SERCA2a to act as a switch to refill the Ca²⁺ stores efficiently. The F-L577 protein constructed in this study will be useful for future studies on Ca²⁺ signaling in normal and abnormal cellular processes that involve SERCA pump activity.