Redistribution of SERCA calcium pump conformers duringintracellular calcium signalingReceived for publication, February 15, 2018, and in revised form, May 1, 2018 Published, Papers in Press, May 15, 2018, DOI 10.1074/jbc.RA118.002472

Olga N. Raguimova‡, Nikolai Smolin‡, Elisa Bovo‡, Siddharth Bhayani‡, Joseph M. Autry§, Aleksey V. Zima‡,and Seth L. Robia‡1

From the ‡Department of Cell and Molecular Physiology, Loyola University Chicago, Maywood, Illinois 60153 and the §Departmentof Biochemistry, Molecular Biology, and Biophysics, University of Minnesota, Minneapolis, Minnesota 55455

Edited by Roger J. Colbran

The conformational changes of a calcium transport ATPasewere investigated with molecular dynamics (MD) simulations aswell as fluorescence resonance energy transfer (FRET) measure-ments to determine the significance of a discrete structural ele-ment for regulation of the conformational dynamics of thetransport cycle. Previous MD simulations indicated that a loopin the cytosolic domain of the SERCA calcium transporter facil-itates an open-to-closed structural transition. To investigate thesignificance of this structural element, we performed additionalMD simulations and new biophysical measurements of SERCAstructure and function. Rationally designed in silico mutationsof three acidic residues of the loop decreased SERCA domain–domain contacts and increased domain– domain separation dis-tances. Principal component analysis of MD simulations sug-gested decreased sampling of compact conformations uponN-loop mutagenesis. Deficits in headpiece structural dynamicswere also detected by measuring intramolecular FRET of a Cer–YFP–SERCA construct (2-color SERCA). Compared with WT,the mutated 2-color SERCA shows a partial FRET response tocalcium, whereas retaining full responsiveness to the inhibitorthapsigargin. Functional measurements showed that themutated transporter still hydrolyzes ATP and transports cal-cium, but that maximal enzyme activity is reduced while main-taining similar calcium affinity. In live cells, calcium elevationsresulted in concomitant FRET changes as the population of WT2-color SERCA molecules redistributed among intermediates ofthe transport cycle. Our results provide novel insights on howthe population of SERCA pumps responds to dynamic changesin intracellular calcium.

The sarcoendoplasmic reticulum Ca2�-ATPase (SERCA)2 isthe ion transporter responsible for sequestering calcium in the

sarcoplasmic reticulum (SR) and endoplasmic reticulum (ER).Mutations in the skeletal muscle isoform SERCA1a causeBrody myopathy, with impaired muscle relaxation (1). Muta-tions of the nonmuscle SERCA2b isoform are the basis forDarier disease, a disorder characterized by epidermal lesions(2). Alterations in cardiac SERCA2a expression, activity, andregulation have been linked to cardiovascular diseases such asheart failure, hypertrophy, and senescence (3–5). SERCA istherefore a high-value therapeutic target for many diseases andcell types (6, 7), but enhancing calcium handling by SERCAgene delivery has proven challenging (8). Thus, there is a criticalunmet need in the development of small-molecule therapiesbased on modulation of endogenous SERCA function.

SERCA is composed of four major domains. The cytosolicheadpiece consists of an actuator (A) domain, a nucleotide-binding (N) domain, and an autophosphorylation (P) domain.The spatial arrangement of these domains changes duringphosphoryl transferase steps in the catalytic cycle, therebyaltering the orientation and affinity of the Ca2�-transport sitesin the transmembrane (TM) domain (9 –11). SERCA transportstwo Ca2� ions per ATP molecule hydrolyzed, with the forma-tion of a phosphoenzyme intermediate (phospho-Asp), andthus is classified as a P-type ATPase (12). The catalytic cycle ofP-type pumps was first identified for the Na�/K�-ATPase andis referred to as the Post-Albers transport mechanism, wherethe Ca2�-transport sites show alternating access (cytosolic andluminal) and alternating affinity (high and low, respectively).

To investigate the rearrangement of SERCA cytosolicdomains and identify key structural determinants we previ-ously performed a computational study of SERCA headpiecemotions (13). The analysis indicated that a specific structuralfeature of the N-domain, the N�5–�6 loop, facilitates the tran-sition of the SERCA headpiece from an open arrangement ofdomains to a more compact architecture. This short �-loop iscomposed of residues 426 – 436 (426DYNEAKGVYEK436) andcontains three negatively-charged amino acids (Asp-426, Glu-429, and Glu-435) that form salt bridges and hydrogen bondswith basic and polar residues on the surface of the A-domain.

This work was supported by the National Institutes of Health GrantsHL092321 (to S. L. R) and HL130231 (to A. V. Z.) and the Loyola Cardiovas-cular Research Institute. The authors declare that they have no conflicts ofinterest with the contents of this article. The content is solely the respon-sibility of the authors and does not necessarily represent the official viewsof the National Institutes of Health.

1 To whom correspondence should be addressed: Dept. of Cell and MolecularPhysiology, Loyola University Chicago, Maywood, IL 60153. Tel.: 708-216-2522; E-mail: srobia@luc.edu.

2 The abbreviations used are: SERCA, sarco-endoplasmic reticulum calciumATPase; 2-color SERCA, SERCA with YFP fused to the nucleotide-bindingdomain and Cer fused to the actuator domain; AAA, triple D426A/E429A/E435A mutant of SERCA; RR, ruthenium red; Cer, cerulean fluorescent pro-

tein; E1, SERCA enzyme conformation with high-affinity Ca2� transportsites oriented toward the cytoplasm; E2, SERCA enzyme conformation withlow-affinity Ca2� transport sites oriented toward the lumen; ER, endoplas-mic reticulum; Iono, ionomycin; MD, molecular dynamics; PCA, principalcomponent analysis; RyR, ryanodine receptor; TG, thapsigargin; X-Rhod,X-Rhod-1/AM; YFP, enhanced yellow fluorescent protein; TM, transmem-brane; PDB, Protein Data Bank; AMP-PCP, 5�-adenylyl-�,�-imidodiphosphate.

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These interactions help initiate domain– domain contact andeventually induce SERCA headpiece closure. When these threenegative charges were mutated in silico to Ala, contactsbetween the A- and N-domains were decreased, and headpiecedynamics were altered in short MD trajectories. This previousfinding suggested that the three negatively-charged residues ofthe N�5–�6 loop are important for SERCA headpiece closure,and we predicted that these residues are key determinants oftransport function.

In the present study, we tested this hypothesis with longerMD simulations and additional trajectory analyses, plus we per-formed physical experiments including functional assays ofN-loop mutants. We also measured FRET as an index of overallheadpiece conformation. These experiments exploited a“2-color SERCA” construct consisting of fluorescent proteintags fused to the N- and A-domains of cardiac SERCA2a (14,15). We previously used this 2-color construct as an intramo-lecular biosensor in high-throughput screening assays to iden-tify small-molecule effectors that affect pump structure andactivity (16). Here the 2-color construct was used to report theredistribution of the population of SERCA pumps among dif-ferent conformational states and to directly test the proposedrole of the N�5–�6 loop in the structural transitions of theCa2� transport cycle.

Results

All-atom molecular dynamics simulations of SERCA mutants

Our previous MD simulations of SERCA structural dynamicspredicted that the open-to-closed transition of the SERCAcytosolic headpiece is facilitated by electrostatic and hydrogenbond interactions between basic and polar residues in theA-domain and acidic residues in the N-domain N�5–�6 loop(13). The contacts help to pierce a structured and poorly diffus-ible water layer between the domains. These residue–residueinteractions also support a stable, ordered arrangement of theN- and A-domains during the transition from an open to aclosed headpiece conformation. In silico triple mutation of thethree negatively-charged loop residues to Ala abolished thesestabilizing interactions and decreased the likelihood of sponta-neous transition of the SERCA headpiece from an open archi-tecture to a closed, compact structure, as assessed by 40-ns MDsimulations (13). Here we extended these simulations to 100 ns,ran new simulations of single-point mutants of the SERCAN�5–�6 loop, and performed additional structural analyses onall trajectories. Fig. 1A shows the starting X-ray crystal struc-ture of SERCA used for all-atom MD simulations (PDB acces-sion code 1SU4) (17), including the A-, N-, P-, and TM-do-mains, plus the N�5–�6 loop of the N-domain and its threeacidic residues, highlighted in orange. For triple mutant AAA(D426A/E429A/E435A) or single substitutions of N�5–�6 loopresidues, we observed similar root mean square deviation tra-jectories in N-domain motions and insignificant differences inresidue-specific root mean square fluctuations, and the angularautocorrelation of the cytoplasmic domains was not detectablydifferent (data not shown); thus, the overall structure anddynamics of SERCA are not disrupted by single or triple muta-tions. Covariance matrices were only modestly different for

WT- and AAA-SERCA, although there was a decrease in neg-atively correlated motions of the N- and A-domains (Fig. 1B,dotted box outlines) for AAA. The most pronounced effect ofN�5–�6 loop mutation observed in the present MD simula-tions is a reduction in contacts between the N- and A-domains(Fig. 1, C and D). In particular, substitution of three N�5–�6loop residues (Asp-426, Glu-429, and Glu-435) to Aladecreased domain– domain contacts (Fig. 1, C and D, AAA),resulting in greater separation of the domains over the course ofthe trajectories (Fig. 1E, AAA). Fig. 1F reveals the general trendof a negative dependence of domain separation distance on thenumber of domain– domain contacts, whereby WT shows ashort distance between N- and A-domains stabilized by a largenumber of contacts, whereas AAA shows a large domain–domain separation and fewer domain– domain contacts. Sin-gle-point mutants yielded intermediate values or were compa-rable with WT (Fig. 1, C–F). The data are consistent with theproposed role of the three acidic N-loop residues in the open-to-closed structural transition (13).

Comparison between structural ensembles of WT–SERCA andAAA–SERCA

We performed principal component analysis (PCA) of theensembles of WT– and AAA–SERCA trajectories (n � 6 each)and analyzed how the ensemble trajectory of each constructsampled the first two principal components. The most domi-nant motion PC1 (48% of all motions) was SERCA headpieceopening/closing, and the second major component PC2 (15% ofall motions) was twisting of the cytosolic domains. Fig. 1G sum-marizes the positive and negative deflections of the A- andN-domains along components 1 and 2, with the origin (0, 0)representing the starting equilibrated structure. With respectto PC1, WT-SERCA sampled values ranging from a minimumof �36 Å (closed) to a maximum of �25 Å (open) (Fig. 1H,black). AAA–SERCA trajectories (Fig. 1H, red) showed a simi-lar range along PC1 (minimum of �30 Å and maximum of �31Å), but with relatively more population of open structures at theexpense of closed conformations. This result is consistent withthe observed increased minimum N- to A-domain– domaindistance for AAA compared with WT (Fig. 1, E and F). WTshowed several trajectories that populated the extremes of thePC2 axis (twisting of the N- and A-domains) (Fig. 1H), rangingfrom �27 to �23 Å, which is 56% greater than the range ofAAA–SERCA along PC2 (from �14 to �18 Å). Thus, the AAAmutant showed smaller twisting motions of the N- and A-do-mains (Fig. 1G). Overall, PCA indicates that the AAA loopmutations decrease the range of motions of SERCA cytosolicdomains (with respect to PC2), and shift the population ofstructures toward a more open architecture (with respect toPC1).

The role of the N�5–�6 loop in SERCA ATPase andCa2�-transport activities

To determine the functional significance of the N�5–�6 loopin Ca2�-activated ATP hydrolysis, we prepared ER microsomesfrom HEK-293 cells expressing 2-color WT–SERCA or AAA–SERCA (Fig. 2, A–C). Fig. 2A shows an example experimentmeasuring Ca2�-activated ATP hydrolysis by SERCA con-

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structs. The apparent Ca2� affinity (KCa) of AAA–SERCA wasnot significantly different from that of WT–SERCA, yieldingKCa values of 7.0 � 0.2 and 7.1 � 0.3, respectively (n � 4, p �0.61) (Fig. 2B). However, the maximal activity of AAA–SERCAwas decreased by 63 � 5% (n � 4, p � 0.015) (Fig. 2C). Thisresult is compatible with the previous finding by the Inesi labthat the single-point mutation of Asp-426 to Ala decreasesSERCA ATPase activity similarly by 61% (18). Overall, the datademonstrate that the SERCA cycling rate is decreased by muta-tion of acidic loop residues, yet the mutated transporter stillretains the ability to perform Ca2�-activated ATP hydrolysiswith the same Ca2� affinity as WT–SERCA.

To determine the rate of Ca2� transport by the AAA mutant,we quantified intracellular Ca2� uptake in the ER of HEK-293cells permeabilized with saponin (Fig. 2, D–F). For each cell,Cer-SERCA expression (WT or AAA) was determined from theemission intensity of Cer fusion tag. To provide pharmacolog-ical control over ER Ca2� content, cells were co-transfectedwith the cardiac ryanodine receptor (RyR), an SR Ca2� channelthat opens in response to application of caffeine (19). ER Ca2�

content of HEK-293 cells was measured with a genetically-encoded low-affinity Ca2� sensor R-CEPIA1er (20). The com-bination of a high level of Ca2�-transport activity fromexogenous Cer-SERCA together with the ER Ca2�-load depen-

Figure 1. MD simulations of SERCA structural dynamics. For C–E, WT–SERCA (black), AAA (red), D426A (blue), E429A (green), E435A (pink) are shown. Datarepresent average of 6 MD run productions. A, SERCA starting X-ray structure of PDB 1SU4 for simulations showing the actuator (A), nucleotide-binding (N),phosphorylation (P), and TM domains. The N�5–�6 loop is highlighted in orange, and three negatively charged residues Asp-426, Glu-429, and Glu-435 arelabeled in the magnified inset. B, covariance matrices C� atoms for WT–SERCA (upper left) and AAA–SERCA (lower right). Covariance analysis of WT–SERCAresidue dynamics as measured from C� revealed positively (red) and negatively (blue) correlated motions. Dotted boxes highlight regions of covariance of theN- and A-domains. For AAA–SERCA, covariance analysis indicated similar global dynamics yet reduced anti-correlated N- and A-domain motions comparedwith WT–SERCA. Data show representative of 6 MD runs. C, number of contacts between the N- and A-domains during MD trajectories. D, quantification ofresults in C. E, AAA–SERCA shows an increase in N- to A-domains separation distance compared with WT-SERCA. F, negative correlation of separation distanceon domain– domain contacts, from results in D and E. G, first and second principal components of SERCA domain motions. H, relative sampling of the top twoprincipal components by WT–SERCA (black) and AAA–SERCA (red) trajectories. Each point represents a conformation extracted from the MD trajectories at aninterval of 0.1 ns. For comparison, gray dots represent X-ray structures with open (1SU4), closed (1VFP), and intermediate (3W5B) headpiece conformations.

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dence of the exogenous RyR opening (21) results in spontane-ous Ca2� release events, followed by refilling of the ER Ca2�

stores by SERCA (Fig. 2D: 8 release/uptake events betweent � 0 –15 s). Cells expressing AAA–SERCA showed similaraverage basal ER Ca2� content compared with WT, with fewspontaneous Ca2� release events (Fig. 2D). Spontaneous Ca2�

release was not observed in nontransfected cells (Ctrl) (Fig. 2D),as these cells express a low amount of native SERCA and lackendogenous RyR (22).

To determine the Ca2� uptake rate by SERCA pumps, caf-feine (10 mM) was applied to empty the ER Ca2� stores (Fig. 2D,Caf). Once caffeine was washed out, the RyR inhibitor ruthe-nium red (10 �M) was applied to stop Ca2� release and allowmeasurement of the rate of luminal [Ca2�]ER recovery. At theend of each experiment, the R-CEPIA1er signal was calibratedby addition of the Ca2� ionophore ionomycin (Iono) (21). ERCa2� recovery, monitored by [Ca2�]ER accumulation, was ana-lyzed to determine the maximum ER Ca2� uptake rate (i.e.SERCA transport rate) and maximum ER Ca2� load. ER Ca2�

uptake in cells expressing WT–SERCA was determined to be0.14 � 0.02 mM/s, a 47-fold increase over control cells (0.003 �0.0005 mM/s) (Fig. 2E). AAA–SERCA Ca2� uptake rate was0.05 � 0.007 mM/s, a 3-fold reduction in Ca2� transport rateversus WT. Furthermore, AAA–SERCA generated a �25%lower maximal ER Ca2� load. The differences in Ca2� uptakerate and ER maximal load were not due to differential expres-sion of WT– and AAA–SERCA, because the Cer fluorescenceemission was similar for these two groups: 73 � 5.0 arbitraryunits of WT and 86 � 11 arbitrary units in AAA–SERCA. Thelocalization of 2-color SERCA was not significantly altered bythe AAA mutation, as determined from confocal microscopy.Moreover, we observed similar levels of SERCA protein inER microsomal preparations as evaluated by comparingexogenous Cer-labeled SERCA with endogenous SERCA by

Western blotting. In HEK-293 cell microsomes, exogenousWT–SERCA expression was 68 � 7% of total SERCA, whereasAAA-SERCA expression was 78 � 7% of total SERCA. Ponceaustaining of the blot also indicated similar amounts of WT– andAAA–SERCA, 6.8 � 0.6 and 6.5% � 0.7% of total protein,respectively. The data suggest that translation and localizationof SERCA in the ER membrane was similar for WT and AAA.We conclude that AAA–SERCA exhibits lower Ca2� transportactivity (65% less) than WT–SERCA (Fig. 2, D and E), which isconsistent with the decreased ATPase rate by AAA–SERCA(63% inhibition) relative to WT (Fig. 2, A and C).

Quantification of 2-color SERCA FRET in ER microsomes fromHEK-293 cells

We have previously used intermolecular FRET to quantifySERCA regulatory interactions with phospholamban and sar-colipin (23–27), and intramolecular FRET to detect SERCAstructural transitions (14 –16). The latter experiments utilized adoubly labeled SERCA with fluorescent proteins fused to the N-and A-domains (2-color SERCA). Here we prepared micro-somal membranes from cells expressing WT 2-color SERCA toquantify the FRET response to Ca2� binding using confocalfluorescence microscopy.

Fig. 3A shows that FRET increased with Ca2� concentration,with an EC50 of 1.25 � 0.22 �M and a Hill coefficient (n) of 0.76.The apparent lack of cooperativity is compatible with previousstudies that suggest the E2–E1 structural transition of the cyto-solic headpiece is complete after binding of the first Ca2� totransport site I (28 –33). Overall, the data are consistent withour previous observation that ionophore treatment of HEK-293cells expressing 2-color SERCA caused accumulation of 2-colorSERCA in a high FRET state over the course of a few minutes(14).

Figure 2. N-domain �5–�6 loop triple mutation decreases SERCA function. A, calcium-dependent ATPase activity of cells expressing WT– or AAA–SERCA,or nontransfected cells (Ctrl). B, Ca2� sensitivity of ATPase activity (KCa), as in A, p � 0.61. C, maximal Ca2�-dependent ATPase rate (Vmax), as in A, *, p � 0.015.D, triple-transfected cells: SERCA, RyR, and R-CEPIA1er. ER Ca2� was depleted by caffeine (Caf) addition, followed by ER Ca2� store recovery in the presence ofRyR blocker ruthenium red (RR). E, maximal Ca2� uptake rate, as in D. *, p � 0.016 for AAA versus WT and p � 0.002 for Ctrl versus WT. F, maximal ER Ca2� load,as in D. *, p � 4 � 10�5 versus WT.

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SERCA conformational states (“conformers”) were stabilizedwith substrates to characterize the enzymatic intermediates ofthe Ca2� transport cycle (9). Importantly, whereas the statedesignations applied here are widely used in the field todescribe the biochemical states stabilized by particular con-ditions, it is likely that significant structural heterogeneityexists for all ligand-stabilized biochemical states (34). Forexample, our previous time-resolved fluorescence measure-ments have shown that SERCA bound to the inhibitor TGcan sample at least two major conformations (15), eventhough X-ray crystallography and EM have identified onlyone structural state of SERCA bound to TG (35, 36). Thepresent measurements capture the average FRET of the pop-ulation ensemble.

We observed generally low FRET for 2-color SERCA in HEK-293 microsomes in solution conditions under which E2 con-formers (low Ca2� affinity) are expected to predominate (Fig.3B). For example, E2 (protonated) SERCA yielded �10% FRET,as did E2–thapsigargin (E2–TG), a potent inhibitor that locksSERCA in the calcium-free E2 state (37, 38). E2–AlF4

� andE2–Vi biochemical intermediates (analogs of the E2P phos-phoenzyme intermediate) also showed low FRET (�8%) (Fig.3B). In contrast, we observed generally high FRET (�15%) for2-color SERCA in microsomes in solution conditions underwhich E1 conformers (high Ca2� affinity) are expected to pre-dominate, such E1–ATP, E1–2Ca, 2Ca–AMP-PCP, andE1–2Ca–ADP–AlF4

� (Fig. 3B). The FRET values for each stateare summarized in Table 1, and these results provide the basisfor quantitative analysis of the population distribution ofSERCA in HEK-293 cells (see next section). Fluorescent proteinseparation distances (calculated from measured FRET effi-ciency) correlated well with distances measured from X-raycrystal structures (Fig. 3C), with the exception of the E1–2Cacrystal structure (PDB code 1SU4). We conclude that SERCAE2 conformers have more open headpiece structures (lowerFRET), whereas E1 conformers have more closed headpiecestructures (higher FRET).

SERCA structural dynamics in HEK-293 cells

For initial investigation of SERCA structural changes inresponse to Ca2� elevations in live cells, we quantified intramo-lecular FRET of 2-color SERCA by widefield fluorescencemicroscopy. Compared with WT, 2-color AAA–SERCA

showed decreased basal FRET (Fig. 4A) in live HEK-293 cells,indicating a more open headpiece architecture compared withWT. We regard this result as consistent with our MD simula-tions, which showed fewer contacts between N- and A-domainsfor AAA-SERCA (Fig. 1D). To a lesser degree, the single-pointmutations of acidic N�5–�6 loop residues also reduced FRETin live HEK-293 cells (Fig. 4A). The more moderate phenotypesof D426A, E429A, and E435A are also compatible with MDsimulations, which showed domain contacts of the pointmutants are intermediate between WT and AAA, or close toWT (Fig. 1D).

2-Color SERCA FRET is markedly decreased with binding toTG (14), and here we found that all of the mutants, includingAAA–SERCA, responded normally to this SERCA inhibitor(Fig. 4B). Increasing intracellular Ca2� by application of iono-mycin to cells expressing WT 2-color SERCA resulted in abiphasic response (Fig. 4C, black trace), with a rapid decrease inFRET (phase 1), followed by a slower increase in FRET (phase2), which recovers to around the initial high FRET value (pre-Iono). Interestingly, the point mutants all showed a normaltwo-phase FRET response to Ca2� (like WT) (Fig. 4C, blue,green, and magenta traces). However, mutation of all threeacidic loop residues (AAA-SERCA) abolished the slow FRETincrease in phase 2 (Fig. 4C, red trace). Proposed mechanis-tic origins of the two-phase response are detailed under“Discussion.”

Overall, the widefield fluorescence microscopy data are con-sistent with the MD simulation analyses, showing that muta-tion of one negative residue is moderately tolerated, but that

Figure 3. Ligand-induced control of SERCA structure in ER microsomes. A, WT 2-color SERCA shows increased FRET with increasing Ca2�, as detected byconfocal fluorescence microscopy. Black error bars represent S.E., whereas gray bars represent S.D. (n � 6 experiments). B, FRET of WT 2-color SERCA stabilizedin key enzymatic states. *, p � 0.008 compared with H�. C, calculated FRET distances compared with distances between the fluorescent protein fusion sitesmeasured from X-ray crystal structures. Select structures are labeled for comparison, other data are identified in Table 1.

Table 1FRET values for SERCA E1 and E2 conformersIntramolecular FRET was measured in ER microsomes from HEK-293 cells express-ing 2-color SERCA. Yellow highlights E1 states, while green highlights E2 states.Data represent at least 5 independent measurements. FRET distances are comparedto distances measured between residues 1 and 510 (fusion sites for fluorescentprotein tags) in the corresponding X-ray crystal structure.

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mutation of three acidic residues from the N�5–�6 loopimpairs N- to A-domain contacts (Fig. 1D), thereby decreasingthe likelihood of forming a more compact headpiece conforma-tion. Importantly, the observation that AAA–SERCA can stillrespond to TG in live cells (Fig. 3B) indicates that the proteinstructure is intact and TG-induced changes in headpiece struc-tural dynamics are preserved after loop mutation. Thus, wepropose that the impaired second-phase of Ca2� response andthe reduced activity of AAA–SERCA are not due to a grossstructural defect (such as misfolding), but instead due to thelack of the N-domain structural determinant that is responsiblefor the stable interaction with the A-domain during the SERCAheadpiece transition from an open-to-closed conformation.These FRET experiments serve as the foundation for assessingthe redistribution of SERCA calcium pump conformers duringdynamic intracellular Ca2� signaling.

SERCA structural dynamics in response to changes inintracellular Ca2� concentration

We have previously quantified FRET in cardiac myocytes(15, 39, 40), but motion artifacts of actively contracting cellsmake quantification of dynamic changes in fluorescencechallenging. As an alternative, we reconstituted aspects ofmuscle cell Ca2� handling with co-expression of RyR2 andSERCA2a in live HEK-293 cells, and subjected the proteinsto confocal microscopy.

In these experiments, Ca2� in the cytosol or ER lumen wasmonitored with X-Rhod (X-Rhod-1/AM) or R-CEPIA1er,respectively. SERCA conformational changes were quantifiedby excitation of Cer at 458 nm and observed as anti-correlatedchanges in the fluorescence intensities of Cer and enhancedyellow fluorescent protein (YFP) (Fig. 5A). The ratio of YFP/Cerfluorescence was taken as a measure of relative intramolecularFRET in the SERCA headpiece (Fig. 5, B, C, and E–L). Based onmicrosomal membrane experiments that showed high FRETfor E1 states (Fig. 3B), and the eventual accumulation of a highFRET state after ionomycin treatment of cells (Fig. 4C), weanticipated that spontaneous Ca2� release events would beaccompanied by increases in intramolecular FRET. Instead,Ca2� elevations in the cytosol corresponded to reductions inFRET, as indicated by decreases in the ratio of YFP/Cer fluores-cence (Fig. 5B). Likewise, depletions of ER Ca2� occurredsimultaneously with decreases in SERCA intramolecular FRET(Fig. 5C). We noted that the recovery of basal SERCA FRET was

complete before full restoration of ER Ca2� stores, as shown bySERCA returning to the low FRET state in the middle of thesawtooth profile in ER Ca2� content detected by R-CEPIA1er(Fig. 5C). In contrast, cytosolic Ca2� elevations were squaresteps that closely mirrored SERCA FRET depressions (Fig. 5B).The result suggests that the ER continues to fill even after cyto-solic Ca2� is already back to baseline and the population ofSERCA has returned to the basal high FRET conformation.This may be due to store-operated Ca2� entry mechanisms thatlargely bypass the bulk cytoplasm.

The FRET fluctuations were not due to changes in inter-molecular FRET between different SERCA molecules (39).Although we detected intermolecular FRET between Cer–SERCA and YFP–SERCA under these experimental conditions(Fig. 5D), intermolecular FRET did not change in responseto ionomycin (Fig. 5E) or spontaneous Ca2� release events(Fig. 5F).

Despite the surprising response to intracellular Ca2� release,WT–SERCA intramolecular FRET still showed a biphasicresponse (decrease, then increase) in these confocal micros-copy experiments after addition of ionomycin. Fig. 5G com-pares these contrasting results in a single trace: transient eleva-tions of Ca2� resulted in transiently decreased FRET, but asustained increase in Ca2� after ionomycin addition caused alagging increase in FRET. As an alternative, we activated theco-expressed RyR with caffeine, and observed transientlyincreased cytosolic Ca2� followed by rapid equilibration to alow concentration of cytosolic Ca2� (Fig. 5H). Remarkably, thisevent was mirrored by a FRET change (decrease, then increase)that had similar kinetics.

In light of the poor responsiveness of AAA–SERCA to iono-mycin-induced Ca2� influx (Fig. 4C, red), we were also sur-prised to find AAA–SERCA responded to spontaneous cytoso-lic Ca2� elevations (Fig. 5I) with no apparent deficit comparedwith WT (Fig. 5B), but again, Ca2� influx after ionomycin addi-tion yielded a sustained decrease in FRET (Fig. 5J) instead of thebiphasic FRET response seen for WT (Fig. 5G). The FRETresponse of AAA–SERCA to caffeine (Fig. 5K) was similar tothat of WT–SERCA (Fig. 3H); a transient decrease followed bya sustained increase in FRET.

Discussion

In the present study we aimed to quantify SERCA dynamicsduring calcium signaling in live cells to examine the role of a

Figure 4. Ca2�-dependent redistribution of SERCA conformers in live cells. A, FRET of 2-color SERCA constructs expressed in basal HEK-293 cells, asdetected by acceptor-sensitized fluorescence microscopy, *, p � 0.005. B, TG-induced E2 conformers of WT 2-color SERCA and the four mutant constructs showreduced FRET in HEK-293 cells, whereas DMSO vehicle-only control (Ctrl, dark blue) had no significant effect. C, addition of the Ca2� ionophore Iono resulted ina biphasic FRET response (1 � quick decrease, 2 � slow recovery) for WT and single-point mutants, as detected using epifluorescence microscopy. AAA–SERCA(red) showed only the first phase: a quick decrease in FRET. Data represent the average of n � 6 –25 cells for each condition.

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discrete structural element that we previously hypothesizedwas an important determinant of SERCA headpiece structuraltransitions. Our new experiments confirm and extend our pre-vious results (13). In particular, new MD experiments furtherdefined the structural role of the acidic residues in the N�5–�6loop, which is to establish inter-domain contacts that deter-mine the range of domain movements and facilitate headpiececlosure. The functional significance of this structural element isindicated by in vitro ATPase assays and live cell Ca2� uptakeassays, which showed that mutations of key loop residuesreduced ATPase activity and Ca2� transport kinetics. In addi-tion, simultaneous FRET and Ca2� measurements providedinsight into the dynamic redistribution of SERCA conformersin the physiologically relevant context of the cell (Fig. 5). Tointerpret the results of Ca2� dynamics experiments, we alsoquantified the average headpiece conformation of ligand-stabilized enzymatic intermediates (Fig. 3B), which was use-ful for gauging the average conformation of the population ofSERCA molecules for each biochemically defined interme-diate state.

Fig. 6A shows a simplified Post-Albers reaction cycle, withhigh Ca2�-affinity states highlighted in yellow and low Ca2�-affinity states highlighted in green. The relative distribution of

SERCA molecules among the conformational states dependson whether ligands such as Ca2� are abundant or limiting.Importantly, when ligands are not limiting, the distribution ofstates depends on the relative kinetics of partial reactions (41),with an increased population of states preceding slow steps.Major physiological states and transitions are shown in black(Fig. 6A), plus an additional nonphysiological state (TG-inhib-ited) and an alternate pathway (Ca2� binding prior to ATP) areshown in gray. States with similar headpiece conformations aregrouped in blue boxes annotated with the FRET efficiencyobserved for those conformations in steady-state experiments(Fig. 3). In this context, we may interpret the observed changesin FRET in live cells.

Intracellular Ca2� signaling and SERCA conformationalchanges

Redistribution of SERCA conformers in response to changesin the concentration of cytosolic Ca2� during signaling is sum-marized in the schematic diagram of Fig. 6B. When cells are atrest (e.g. nonstimulated HEK-293 cells or noncontracting myo-cytes), Ca2� is low (�100 –200 nM) and ATP is saturating (3–5mM), so the major populated conformation of SERCA isE1–ATP. Pre-bound ATP shifts the Ca2� binding E2–E1 (apo)

Figure 5. SERCA structural dynamics measured by FRET in HEK-293 live cells. A, anti-correlated changes in Cer and YFP fluorescence intensity indicaterhythmic FRET fluctuations in intact cells. B, the ratio of YFP/Cer, as in A, was used as an index of FRET (gray). FRET (bottom panel, gray) was inversely correlatedto cytosolic Ca2�, as measured by X-Rhod fluorescence (black, top panel). WT–SERCA FRET decreased during cytosolic Ca2� elevations due to spontaneous ERCa2� release through RyR. C, a decrease in 2-color SERCA intramolecular FRET (bottom trace) occurs simultaneously with depletion of ER Ca2� stores (top trace).D, quantification of intermolecular FRET between Cer–SERCA and YFP–SERCA using progressive acceptor photobleaching (started at black arrow). Datapresented are the mean F/F0 of Cer and YFP fluorescence measured in 6 cells. E, SERCA–SERCA intermolecular FRET did not change in response to ionomycinaddition. F, SERCA–SERCA intermolecular FRET did not change with spontaneous Ca2� release events (top trace). The data indicate that changes in 2-colorSERCA FRET are due to changes in intramolecular FRET rather than changes in intermolecular FRET. G, after addition of ionomycin, both cytosolic Ca2� andWT–SERCA FRET increased, as detected by confocal fluorescence microscopy. H, addition of caffeine (Caf) transiently increased cytosolic Ca2� and decreasedWT–SERCA FRET. I, AAA–SERCA FRET decreased during spontaneous cytosolic Ca2� elevations. J, in contrast to WT–SERCA FRET, AAA–SERCA FRET decreasedwith addition of ionomycin. K, AAA–SERCA FRET increased in response to caffeine, similar to WT–SERCA FRET (H). L, addition of ionomycin causes an increasein ER Ca2� content, but this increase occurs more slowly than the second phase of the observed FRET response of 2-color SERCA. We conclude that the phase2 FRET increase of 2-color SERCA is not due to saturation of SERCA luminal Ca2�-binding sites (i.e. low affinity E2 orientation).

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population toward the high affinity E1 (apo)–ATP conformerready to bind Ca2� (42–44). This state exhibited high FRET invitro, and indeed we observed high FRET for WT 2-colorSERCA in HEK-293 cells in the basal state. Then, when Ca2� isreleased from intracellular stores into the cytosol, FRET is tran-siently decreased (Fig. 6B). This FRET change occurs as a con-sequence of redistribution of the population of transporters tomore open headpiece conformations. Specifically, at high Ca2�

the pumps are continuously cycling, and all states of catalyticcycle are populated, with a relative build-up of E1P–2Ca and E2conformers before the slow interconversion step of the trans-port sites, plus the E2P conformer before the slow phosphoen-zyme hydrolysis and release (Fig. 6A) (32, 45– 48). For SERCA,these are isomerization steps, the E2 to E1 transition and therate-limiting transition from E1P to E2P (41). Therefore, whenCa2� increases to micromolar concentrations the populationshifts from being predominantly E1–ATP (high FRET) to a pre-dominant mixture of E1P–2Ca (high FRET) and E2 (low FRET)(Fig. 6A) (32, 45– 48). The result of accumulation of the lowFRET E2 population decreased overall FRET. Such conditionsoccur during contractions of cardiac or skeletal muscle cells, orin nonmuscle cells during intracellular Ca2� waves (as in Figs.2D and 5B). Termination of Ca2� release and restoration ofbasal Ca2� returns the pumps to the resting condition, inwhich the majority conform to E1–ATP. Thus, the redistri-bution of SERCA conformers during Ca2� signaling is some-what counterintuitive: the E1–ATP state prevails during

periods of low Ca2�, and the E2 state population increases athigh Ca2�.

Likewise, bottlenecks in catalytic cycle account for the rapiddecrease in FRET immediately after ionomycin addition toHEK-293 cells (Fig. 6B, phase 1) as the population of transport-ers redistributes to a mixture of E1P/E2. This is the point wherethe WT and mutant SERCA diverge: Fig. 6B shows thatWT–SERCA FRET rebounds (phase 2), whereas AAA–SERCAremains in predominantly low FRET conformations (Fig. 6B,dotted line). The phase 2 increase in FRET for WT-SERCA wasremarkable because cytosolic Ca2� remained elevated at milli-molar concentrations for the remainder of the experiment. Sev-eral possible mechanisms could account for this result. We con-sidered the possibility of alkalinization of the cell throughCa2�/H� exchange by ionomycin (49). If this is the origin of theapparent second phase of the FRET change it must be due spe-cifically to a change in SERCA conformation as opposed to adirect effect of pH on the fluorescent protein tags, because con-trol experiments with Cer–SERCA and YFP–SERCA showedno change in intermolecular FRET with ionomycin addition(Fig. 5E). We do not attribute the phase 2 FRET change to sat-uration of SERCA luminal Ca2� binding and accumulation ofSERCA in the E2–2Ca state, because accumulation of Ca2� inthe ER after ionomycin treatment was much slower than thephase 2 FRET change (Fig. 5L). Another possible mechanismfor the phase 2 FRET change is that full activation of SERCA inthe ionomycin-treated cells may deplete ATP and increaseADP over the course of several minutes, with consequent accu-mulation of the population of 2-color SERCA in ATP-free, highFRET conformations. In vitro measurements revealed E1–2Caand E1–2Ca–ADP–AlF4

� to be a high (15%) FRET conforma-tions (Fig. 3B). Finally, SERCA cycling is inhibited at very high(mM) Ca2� concentrations (50, 51), a condition that stabilizesthe physiologically rare E1–2Ca state (52). Thus, we proposethat the high FRET observed upon ionomycin-induced Ca2�

release (Figs. 5G and 6A) is due to stabilization of E1–2Ca(branched gray pathway in Fig. 6A). Interestingly, a recentmolecular dynamics study of SERCA at supraphysiologic Ca2�

concentration (10 mM) indicated possible Ca2� binding to theN�5–�6 loop (53).

The effect of N�5–�6 loop mutations

The failure of AAA–SERCA to undergo a phase 2 redistribu-tion to high FRET states (Fig. 6B, dotted line) illustrates theconsequences of mutation of acidic residues in the N�5–�6loop for SERCA dynamics. We observed that the triple muta-tion AAA impairs SERCA cytosolic headpiece closure insilico and we attribute the lack of a phase 2 FRET change tothe decreased kinetics of structural transition from lowFRET states (open headpiece) to high FRET states (closedheadpiece). Thus, loss of N- to A-domain contacts results ina new rate-limiting step in the SERCA catalytic cycle, the E2to E1–ATP transition (Fig. 6A). Although the mutant pumpis functional (Fig. 2), there is much greater accumulation ofthe population of cycling transporters in E2 when there is asustained elevation of Ca2�, so FRET remains low, with nosecond phase increase.

Figure 6. Redistribution of SERCA conformers during Ca2� signaling. A, asimplified Post-Albers cycle. Blue boxes enclose states with similar intramolec-ular FRET efficiency. B, schematic diagram of changes in cytosolic Ca2� (red)and changes in FRET (gray) as the population of SERCA redistributes amongstructural states, with the predominant state shown in blue. The WT FRETresponse to ionomycin shows two phases (phases 1 and 2), whereas AAAFRET shows only a decrease in FRET (dotted line).

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Importantly, impaired headpiece dynamics did not preventapparently normal AAA–SERCA FRET changes during spon-taneous Ca2� release events and subsequent Ca2� re-uptakeinto the ER lumen (Fig. 5I). In particular, we did not detect anyapparent delay in the upstroke of the FRET signal that occursconcomitantly with the decrease in cytosolic Ca2�. This sur-prising phenomenon may now be understood as another indi-cation that SERCA function is decreased, but not abolished, byloop mutations. Termination of Ca2� release allows reseques-tration of Ca2� by the combined efforts of AAA–SERCA andendogenous SERCA. After withdrawal of Ca2�, flux throughthe transport cycle ceases, permitting return of AAA–SERCAback to the high FRET basal conformation, E1–ATP. Thekinetic delay due to disruption of structural determinants in theloop is not resolvable as a slow FRET transition on this times-cale; it is only detectable as a bottleneck step in actively cyclingAAA-SERCA.

Comparison with other structural studies

The present results are in harmony with X-ray crystallogra-phy of SERCA stabilized by ligands in different biochemicalstates, with relatively open crystal structures (PDB 1IWO (54),3W5C (55), 5A3Q (56), and 2O9J (57)) corresponding to lowFRET states observed here and compact crystal structures(PDB 1VFP (58), 1T5S (11), and 2Z9R (60)) corresponding tohigh FRET states (Table 1). A notable exception is the crystalstructure of E1–2Ca (PDB 1SU4) (17), which showed awidely open headpiece conformation. We measured highFRET values for this state in vitro and under conditions thatshould stabilize this state in cells, consistent with a closedheadpiece. Fig. 3C shows the close correlation of the calcu-lated FRET distance and the distance between fluorescentprotein fusion sites, as measured from X-ray structures. Theregression of these data (excluding outlier 1SU4) yielded a yintercept of 36 Å. We attribute this offset to the additionaldistance conferred by the difference between the fusion siteand the chromaphore at the center of the fluorescent protein�-barrel.

A recent single-molecule FRET study of a related ATPaserevealed a similar trend of high FRET between A- and P-do-mains for E1 conformers and low FRET for A- and P-domainsfor E2 conformers (61). Although it is difficult to compareFRET measurements taken from different labeling sites, overallthe studies are in harmony. Both suggest E1 conformationshave a compact, ordered headpiece, whereas E2 states are char-acterized by an open, disordered architecture.

Summary

The goals of this study were 2-fold. The first was to quan-tify how the overall conformation of the SERCA cytosolicheadpiece changes as the transporter steps through thestructural transitions of the catalytic cycle. Second, wesought to test directly the hypothesis that residues in a loopof the SERCA N-domain are key determinants of transportfunction (13). The present results are compatible with thisproposed mechanism, as mutations of the loop residuesresulted in altered headpiece dynamics, and functional mea-surements revealed a consequent decrease in ATP hydrolysis

rate and Ca2� transport. The results support the proposedrole of the loop in facilitating SERCA headpiece closure dur-ing functional enzymatic cycling. As a discrete structuralelement, the N-loop may be a worthwhile target for develop-ment of small molecules to enhance (16) or inhibit (62–64)SERCA function in vivo.

Experimental procedures

All-atom molecular dynamics simulations

All-atom MD simulations were performed as described (13).Briefly, the GROMACS software package (65, 66) withCHARMM 27 forcefield (67) and TIP3P water model (68) wereused to carry out MD simulations. The reference Ca2�-boundcrystal structure of SERCA (17) was used to run WT simula-tions and to introduce N�5–�6 loop mutations. For all con-structs, energy minimization was performed using the steepestdescent method for 1000 steps. Then the structures wereembedded into a POPC lipid bilayer and solvated in a rectan-gular water box with dimension sizes 130 � 130 � 160 Å. Na�

and Cl� ions were added to the solution to a concentration of150 mM. The Berendesen method (69) with relaxation time of0.1 ps was used to increase the temperature of the system to 300K and reach the pressure of 1 bar. After 1-ns equilibration, theproduction run was performed in the NPT assemble using theNose-Hoover thermostat (70, 71) and the Parrinello-Rahmanbarostat (72) with a relaxation time of 1 ps. Six independentproduction runs for each WT or mutant construct were startedwith a different set of assigned velocities at 300 K. The integra-tion time was 2 fs, and atom coordinates were saved every 1 ps.Production runs were carried out for 100 ns (n � 6 for WT– andAAA–SERCA).

Structural analysis and visualization

The VMD program (73) was used for visualization and ren-dering snapshots. The GROMACS program (65, 66) was usedfor the computational analysis of MD production runs. TheN- to A-domain– domain distance was defined as the mini-mum distance between any atom of the A-domain (residuesnumber 1– 40 and 128 –241) and any atom of the N-domain(residues 360 – 603). The number of contacts was calculatedwith a 4-Å cutoff between atoms of the N- and A-domains.One contact of a N-domain atom with multiple atoms ofA-domain was counted as one contact (i.e. instead of multi-ple contacts). The first 10 ns of MD simulations were con-sidered as equilibration time and not included in structuralanalyses.

Principal component analysis

To identify the major motions of the SERCA headpiece dur-ing MD trajectories, we aligned SERCA structures using the10-helix TM domain as a reference and used PCA (74 –76). Tocompare structural ensembles with respect to the same eigen-vectors, we combined three SERCA reference X-ray crystalstructures (PDB 1SU4 (17), 3W5B (55) and 1VFP (58)), six MDtrajectories of WT–SERCA, and six MD trajectories of AAA–SERCA into a single trajectory. To obtain sets of eigenvectorsand eigenvalues corresponding to principal components, we

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built covariance matrixes of the atomic fluctuations in GRO-MACS (65, 66). The diagonalization of matrixes yielded theeigenvectors (which are principal components) and their asso-ciated eigenvalues.

Molecular biology and cell culture

The engineering and functional characterization of 2-colorSERCA was previously described (14 –16). We used a canineSERCA2a construct labeled with Cer on the N terminus and anYFP intrasequence tag inserted before residue 509 in the N-domain for FRET experiments (transient transfection). ForATPase assay experiments, we used an analogous 2-colorSERCA construct, with a red fluorescent protein (tagRFP) onthe N terminus and an enhanced GFP inserted before residue509 in the N-domain. The Cer–YFP pair has a Forster distance(R0) of 49.8 Å, and the GFP–tagRFP pair has an R0 of 58.3 Å (77).We introduced Ala mutations in N�5–�6 loop residues withQuikChange Lightning Site-directed Mutagenesis Kit (AgilentTechnologies, Stratagene, La Jolla, CA) according to the man-ufacturer’s protocol. Single mutations and a triple mutationwere made: D426A, E429A, E435A, and D426A/E429/E435A(AAA). Adenoviral vectors of tagRFP–GFP–SERCA (WT andAAA) were produced by the Loyola Cardiovascular ResearchInstitute virus production facility.

HEK-293 cells were cultured in Dulbecco’s modifiedEagle’s medium cell culture medium supplemented with 10%fetal bovine serum (ThermoScientific, Waltham, MA) andtransiently transfected using MBS mammalian transfectionkit (Agilent Technologies, Stratagene), as described previ-ously (14). The transfected cells were trypsinized (Thermo-Scientific) and replated onto poly-D-lysine-coated glass bot-tom chambers and allowed to adhere for 1–2 h prior toimaging.

Wide-field acceptor sensitization fluorescence microscopy tomeasure SERCA intramolecular FRET

Wide-field fluorescent microscopy was done as describedpreviously (26). Briefly, cells were imaged with an invertedmicroscope (Nikon Eclipse TE2000-U) equipped with a metalhalide lamp and a back-thinned CCD camera (iXon 887: AndorTechnology, Belfast, Northern Ireland). For each sample,acquisition of field was performed with a �60 1.49 N.A. objec-tive with 100 or 150 ms exposure for each channel: Cer, YFP,and FRET. Fluorescence intensity was automatically quantifiedwith a multiwavelength cell scoring application in MetaMorphsoftware (Molecular Devices, Sunnyvale, CA). FRET efficiencywas calculated according to E � G/(G � 3.2 � FCer), where G �FFRET � a � FYFP � d � FCer (26, 40), where FFRET, FYFP, andFCer are the matching fluorescence intensities from FRET, YFP,and Cer images, respectively, and G represents FRET intensitycorrected for the bleedthrough of the channels. The parametersa and d are bleedthrough constants calculated as a � FFRET/FCer for a control sample transfected with only YFP–SERCAand d � FFRET/FCer for a control sample transfected with onlyCer–SERCA. These values were determined to be a � 0.074and b � 0.70. Apparent probe separation distance (R) was

calculated from FRET efficiency (E) according to the relation-ship (78),

R � R0��61 � E

E� (Eq. 1)

with a Forster distance R0 of 49.8 Å for the mCer and EYFPFRET pair (77). The error of the distance measurement wasestimated from the standard deviation of repeated FRETmeasurements.

Ratiometric confocal fluorescence microscopy to measureSERCA intramolecular FRET, HEK-293 cytosolic Ca2�, andHEK-293 ER luminal Ca2�

HEK-293 cells were transiently co-transfected with expres-sion plasmids containing cDNA of GFP-human ryanodinereceptor-2 fusion protein (RyR), R-CEPIA1er, and 2-colorcanine SERCA2a wildtype (WT) or triple loop mutant AAA.Transfected cells were cultured for 24 h and seeded into poly-D-lysine-coated glass-bottom chamber slides in Dulbecco’smodified Eagle’s medium plus 10% fetal bovine serum. 24 hafter seeding, cell culture medium was changed with PBS withCa2�/Mg2�, and experiments were conducted with a Leica SP5laser scanning confocal microscope equipped with a �63 waterobjective. R-CEPIA1er was excited with the 543-nm line of aHe-Ne laser, and emitted fluorescence was measured at wave-length 580 nm. 2-color SERCA fluorophores Cer and YFPwere excited with the 430 and 514 nm lines of an argon laser,respectively, and emitted fluorescence was measured at wave-lengths 485 � 15 and 537 � 15 nm, respectively. Images wereacquired in line-scan mode for up to 8 –12 min with addition of10 mM caffeine or 100 �M ionomycin at time (t) indicated in thefigures. Ionomycin powder (Sigma) was dissolved in DMSO tomake a 13.3 mM stock solution, which was used to prepare 2�Iono solution (200 �M) in PBS. The final concentration ofDMSO applied to cells was 0.75%. Fluorescence image analysiswas performed with ImageJ software (79).

To load cells with the low-affinity Ca2� indicator X-Rhod-1/AM (X-Rhod) (80), cells were incubated with 10 �M X-Rhod(ThermoScientific) for 15 min in PBS (�Ca/�Mg), and thenwashed twice with PBS (�Ca/�Mg) to remove X-Rhod fromthe cell culture medium.

HEK-293 cell microsomal membrane preparation

ER microsomal membranes expressing SERCA were isolatedfrom HEK-293 cells infected with adenovirus encoding 2-colorWT–SERCA or AAA–SERCA (for ATPase assay) or trans-fected with 2-color SERCA constructs (FRET measurement) asdescribed (81). Cells were grown to confluence on 150 mm2

dishes for 2 days, washed twice with PBS, harvested by scraping,and pelleted at 1000 � g for 10 min at 4 °C. To prepare cellhomogenates, the cell pellets were 1) resuspended in coldhomogenizing solution (0.5 mM MgCl2, 10 mM Tris-HCl, pH7.5, plus EDTA-free complete protease inhibitor mixture(Santa Cruz Biotechnology, Inc., Dallas, TX); 2) disrupted by 10strokes in a Potter-Elvehjem homogenizer; 3) supplementedwith an equal volume of sucrose solution (100 mM MOPS, pH7.0, 500 mM sucrose, plus EDTA-free complete protease inhib-

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itor mixture); and 4) passed through a 27-gauge needle 10times. To prepare microsomal membranes, 1) cell homoge-nates were centrifuged at 1,000 � g for 10 min at 4 °C; 2) thelow-speed supernatants were centrifuged at 126,000 � g for 30min at 4 °C; 3) the high-speed membrane pellets were resus-pended in a 1:1 mixture of homogenizing and sucrose solutions;and 4) the membrane suspensions were passed through a27-gauge needle 10 times. The protein concentration of micro-somal membranes were determined with a Pierce BCA assay kit(ThermoScientific). ATPase assay was performed the same dayas membrane preparation; otherwise, microsomal membraneswere aliquoted, snap-frozen in liquid nitrogen, and stored at�80 °C.

Measuring Ca2�-ATPase activity in ER microsomes fromHEK-293 cells

2-Color SERCA ATPase activity was measured in ER micro-somes from HEK-293 cells by spectrophotometric determina-tion of the rate of NADH consumption as a function of Ca2�

concentration using the enzyme-coupled activity assay in96-well plate (82, 83). The time dependence of the absorbancedecrease was measured at 340 nm at 25 °C in a PHERAstar FSXmicroplate reader (BMG Labtech, Cary, NC). Each well con-tained 3– 4 �g of microsomal membranes in 200 �l of assaysolution containing 50 mM MOPS, pH 7.0, 100 mM KCl, 5.0 mM

MgCl2, 1.0 mM EGTA, 2.5 mM ATP, 0.2 mM NADH, 1 U ofpyruvate kinase, 1 U of lactate dehydrogenase, 0.5 mM phos-phoenol pyruvate, and 0.7 �g of Ca2� ionophore (A23187).Chemicals were obtained from Sigma. Free Ca2� concentra-tions were calculated using a Ca/Mg/ATP/EGTA calculatorfrom Theo Schoenmakers’ Chelator (59). Data were fittedusing the Hill function,

V � Vmax/1 10�n(pKCa�pCa) (Eq. 2)

where V is the ATPase rate at a specific Ca2� concentration(pCa), n is the Hill coefficient, pKCa is the apparent Ca2� disso-ciation constant, and Vmax was obtained from the fit of the Hillequation at saturating Ca2� concentrations.

Measuring ER Ca2� uptake in permeabilized HEK-293 cells

Changes in [Ca2�]ER were measured with laser scanning con-focal microscopy (Radiance 2000 MP, Bio-Rad) equipped with a�40 oil-immersion objective lens (NA � 1.3). R-CEPIA1er wasexcited with the 543 nm line of a He-Ne laser and fluorescencewas measured at wavelengths �600 nm. Fluorescence measure-ments were acquired in line-scan mode (20 ms per scan; pixelsize 0.12 �m). HEK-293 cells co-transfected with GFP-RyR,R-CEPIA1er, and Cer-SERCA were washed in Ca2�-free solu-tion containing 150 mM K-aspartate, 0.25 mM MgCl2, 0.1 mM

EGTA, 10 mM HEPES, pH 7.2. The plasma membrane of HEK-293 cells was permeabilized with 0.005% saponin to controlcytosolic environment replacement with a saponin-free solu-tion containing 100 mM K-aspartate, 15 mM KCl, 5 mM

KH2PO4, 5 mM MgATP, 0.35 mM EGTA, 0.12 CaCl2, 0.75 mM

MgCl2, 10 mM phosphocreatine, 2% (w/v) dextran (Mr 40,000),and 10 mM HEPES, pH 7.2 (KOH). Free [Ca2�] and [Mg2�] ofthis solution were calculated to be 250 nM and 1 mM, respec-

tively. Activation of RyR with caffeine (10 mM) was used tocompletely deplete [Ca2�]ER. Once caffeine was removed, theRyR inhibitor ruthenium red (RR � 10 �M) was applied to mea-sure the rate of ER Ca2� uptake. Changes in [Ca2�]ER werecalculated by the formula: [Ca2�]ER � Kd � [(F � Fmin)/(Fmax �F)], where F is the R-CEPIA1er fluorescence; Fmax and Fmin arethe fluorescence level at 10 mM Ca2�/Iono before and afterdepletion of ER Ca2� with caffeine (10 mM), respectively. TheR-CEPIA1er Ca2� dissociation constant (Kd) is 390 �M basedon in situ calibrations (21). At the end of each experiment, theR-CEPIA1er signal (Fmax) was calibrated with addition of Iono(100 �M). ER Ca2� uptake (i.e. SERCA transport rate) was cal-culated as the time-dependent change of [Ca2�]ER after RyRinhibition on a cell-to-cell basis (mM Ca2�/s), and the maximalER Ca2� load was determined for each individual cell (mM

Ca2�). The reported Ca2� uptake rate and maximal ER Ca2�

load were calculated as mean � S.D.

Intramolecular FRET measurements of 2-color SERCAexpressed in ER microsomes from HEK-293 cells

To stabilize SERCA in ligand-stabilized biochemical inter-mediates, various solutions were prepared by addition ofcorresponding substrates to the calcium-free base solution,which includes 100 mM KCl, 5 mM MgCl2, 2 mM EGTA, and10 mM imidazole, pH 7.0. The following ligands were used toprepare specific solutions corresponding to SERCA bio-chemical state (in parentheses): 100 �M thapsigargin (E2–TG), 3 mM ATP (E1–ATP), 2.1 mM CaCl2 (E1–2Ca) with free[Ca2�]i � 100 �M (59); 2.1 mM CaCl2 and 3 mM nonhydro-lyzable ATP analog AMP-PCP (E1–2Ca-AMPPCP); 2.1 mM

CaCl2, 3 mM ADP, 3 mM KF, and 50 �M AlCl3 (E1–2Ca–ADP–AlF4

�); 0.1 mM orthovanadate (E2–Vi); and 50 �M

AlCl3 and 3 mM KF (E2–AlF4�). Chemicals were obtained

from Sigma.To measure SERCA intramolecular FRET in ligand-stabi-

lized biochemical intermediates, 1 �l of membrane prepara-tions (7–10 �g of total protein) was mixed with 9 �l of ligandsolution on a coverslip and immediately imaged using confocalfluorescent microscopy, as described above.

Statistical analyses

Data are presented as the mean � S.D. of n 3 experiments.All statistical tests were performed using OriginPro 9.1 (Origin-Lab Corporation, Northampton, MA). Student’s t test was usedto compare differences between two groups, and one-way anal-ysis of variance was used to compare the difference betweenthree or more groups. One-way analysis of variance was fol-lowed by Tukey’s post hoc test. A probability (p) value of �0.05was considered significant. Specific values are provided in fig-ure panels or figure legends.

Author contributions—O. N. R., N. S., and S. L. R. conceptualization;O. N. R. data curation; O. N. R., J. M. A., A. V. Z., and S. L. R. formalanalysis; O. N. R., E. B., S. B., and A. V. Z. investigation; O. N. R.,N. S., and S. L. R. methodology; O. N. R. and S. L. R. writing-originaldraft; O. N. R., J. M. A., and S. L. R. writing-review and editing; N. S.and A. V. Z. resources; N. S., A. V. Z., and S. L. R. supervision; S. L. R.funding acquisition; S. L. R. project administration.

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Acknowledgments—We are grateful for helpful suggestions from How-ard S. Young. This work was also supported by equipment and facil-ities provided by National Institute of Health “Loyola Research Com-puting Core” Grant 1G20RR030939. This work used the ExtremeScience and Engineering Discovery Environment (XSEDE), which issupported by National Science Foundation Grant ACI-1548562, aswell as Stampede and Stampede2 at the Texas Advanced ComputingCenter (TACC) through XSEDE allocation TG-MCB130108.

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Aleksey V. Zima and Seth L. RobiaOlga N. Raguimova, Nikolai Smolin, Elisa Bovo, Siddharth Bhayani, Joseph M. Autry,

signalingRedistribution of SERCA calcium pump conformers during intracellular calcium

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