Lethal Arg9Cys phospholamban mutation hindersCa2þ-ATPase regulation and phosphorylationby protein kinase AKim N. Haa, Larry R. Mastersona,b, Zhanjia Houc, Raffaello Verardia, Naomi Walsha,Gianluigi Vegliaa,b,1, and Seth L. Robiac,1

aDepartment of Biochemistry, Molecular Biology, and Biophysics, and bDepartment of Chemistry University of Minnesota, Minneapolis, MN 55455; andcDepartment of Cell and Molecular Physiology, Loyola University Chicago, Maywood, IL 60153

Edited* by David H. MacLennan, University of Toronto, Canada, and approved December 27, 2010 (received for review September 17, 2010)

The regulatory interaction of phospholamban (PLN) with Ca2þ-ATPase controls the uptake of calcium into the sarcoplasmic reticu-lum, modulating heart muscle contractility. A missense mutationin PLN cytoplasmic domain (R9C) triggers dilated cardiomyopathyin humans, leading to premature death. Using a combination ofbiochemical and biophysical techniques both in vitro and in livecells, we show that the R9C mutation increases the stability ofthe PLN pentameric assembly via disulfide bridge formation,preventing its binding to Ca2þ-ATPase as well as phosphorylationby protein kinase A. These effects are enhanced under oxidizingconditions, suggesting that oxidative stress may exacerbate thecardiotoxic effects of the PLNR9C mutant. These results reveal aregulatory role of the PLN pentamer in calcium homeostasis, goingbeyond the previously hypothesized role of passive storage foractive monomers.

SERCA ∣ ventricular dilatation ∣ calcium regulation ∣ heart failure ∣membrane proteins

Heart failure (HF) is the leading cause of morbidity and mor-tality worldwide (1, 2). The most prominent disorder leading

to HF is dilated cardiomyopathy (DCM), a disease characterizedby left ventricular dilatation and impaired systolic function (1, 2).DCM has both acquired and genetic etiologies (1, 2). Recent gen-ome sequencing has revealed a high incidence of DCM-asso-ciated mutations in cytoskeletal, nuclear, as well as sarcomericproteins (3). A number of mutations have been indentified in cal-cium handling proteins, which play a central role in the mechanicsof heart muscle contractility (3–6).

Cardiac muscle contraction (systole) begins when an actionpotential causes membrane depolarization, activating the sarco-lemmal L-type calcium (Ca2þ) channels. Ca2þ flows through theL-type Ca2þ-channels into the cytosol. This increase in Ca2þ con-centration induces a large-scale release of Ca2þ into the cytosolfrom intracellular stores by the sarcoplasmic reticulum (SR)Ca2þ-release channels (or ryanodine receptors). Ca2þ then movestoward the contractile apparatus, where it binds the troponincomplex and initiates contraction. Muscle relaxation (diastole)occurs when Ca2þ is sequestered into the SR by the SR Ca2þ-ATPase (SERCA) (7) a membrane-embedded Ca2þ pump (8).SERCA is regulated by phospholamban (PLN), which reducesits apparent Ca2þ affinity (9, 10). PLN’s inhibition is reversedby cAMP-dependent protein kinase A (PKA), which phosphory-lates PLN at Ser16, enhancing cardiac contractility and reestab-lishing Ca2þ flux (11).

PLN is a single-pass membrane protein, which comprises threestructural domains (12–14), further subdivided into four dynamicdomains [cytoplasm: domain Ia (residues 1–16), loop (residues17–22), domain Ib (residues 23–30); transmembrane: domain II(residues 31–52)] (15) (Fig. S1). In membranes, PLN formshomopentamers arranged in a pinwheel topology that are inequilibrium with monomers (16, 17) that bind SERCA with 1∶1stoichiometry (6, 18–21). Also, it has been proposed that the PLN

monomer-pentamer equilibrium plays a central role in SERCAregulation (6).

Several naturally occurring mutations in the PLN gene havebeen linked to hereditary DCM (5), including a substitution ofArg9 for Cys (PLNR9C) located in the cytoplasmic domain Ia ofPLN (Fig. S1), which has been identified in several cases offamilial DCM (22). R9C cardiotoxic effects are correlated withinefficient Ca2þ handling (23) and show a dose-dependent inhi-bition of SERCA (24). Schmitt et al. hypothesized that PLNR9C

leads to DCM by binding irreversibly to the catalytic subunit ofPKA (PKA-C) and preventing PLNR9C and/or PLNwt phosphory-lation at Ser16 (22). To date, however, there are no firm conclu-sions on the molecular mechanisms that link PLNR9C to DCM.

Here, we used an array of biochemical and biophysical tech-niques both in vitro and in live cells to establish the moleculardeterminants of the cardiotoxic effects of PLNR9C. Specifically,we focused on the effects of this aberrant mutation on (i) therecognition and phosphorylation by PKA-C, (ii) the PLN mono-mer-pentamer equilibrium, and (iii) SERCA regulation. Wefound that the R9C mutation stabilizes the pentameric assembly,hindering PLN deoligomerization, phosphorylation by PKA-C,and SERCA regulation. Importantly, we discovered that theseeffects are exacerbated under oxidative environments, which arerelated to both physiological and pathophysiology conditions ofcardiac myocytes resulting from myocardial ischemia (25, 26).

ResultsOur immediate objectives were to determine the effects of R9Cmutation on (a) the PKA-C recognition and phosphorylation, (b)the PLN monomer-pentamer equilibrium, and (c) SERCA regu-lation. Toward these goals, we utilized three different PLN con-structs with and without the R9C mutation: (i) synthetic peptidesspanning cytoplasmic residues of PLN (PLNwt

1−20 or PLNR9C1−20), (ii)

full-length recombinant pentamers (PLNwt and PLNR9C), and(iii) recombinant monomeric PLN (AFA-PLN), where the threetransmembrane cysteines (Cys36, Cys41, Cys46) were mutatedinto Ala, Phe, Ala, respectively. This triple mutation abolishesPLN oligomerization without altering PLN’s inhibitory function(27). We carried out these experiments in the presence of dithio-threitol (DTT ranging from 1 to 20 mM) or hydrogen peroxide(H2O2 ranging from 1–100 μM), chemicals commonly used to mi-mic physiological redox conditions and oxidative stress (28, 29).

Author contributions: G.V. and S.L.R. designed research; K.N.H., L.R.M., Z.H., R.V., andN.W. performed research; K.N.H., L.R.M., Z.H., R.V., N.W., G.V., and S.L.R. analyzed data;and K.N.H., G.V., and S.L.R. wrote the paper.

The authors declare no conflict of interest.

*This Direct Submission article had a prearranged editor.1To whom correspondence may be addressed. Email: vegli001@umn.edu and srobia@lumc.edu.

This article contains supporting information online at www.pnas.org/lookup/suppl/doi:10.1073/pnas.1013987108/-/DCSupplemental.

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Effects of the R9C Mutations on the Phosphorylation Kinetics byPKA-C. Under reducing conditions, phosphorylation kinetics ofsynthetic PLN peptides were monitored using a coupled enzymeassay (30), standardized with a synthetic peptide corresponding tothe minimal recognition sequence for the kinase (Kemptide)(31). Under our experimental conditions, recombinant PKA-Cshows a catalytic efficiency typical for Kemptide (kcat∕KM∼0.78) (30, 32). Interestingly, we found that PKA-C is able tophosphorylate both PLNwt

1−20 and PLNR9C1−20 peptides with similar

catalytic efficiencies (Fig. 1A and Table S1). Moreover, we carriedout competitive kinetic assays in the presence of products phos-phorylated at Ser16 (pPLNwt

1−20 or pPLNR9C1−20). Our measurements

did not show any substantial product inhibition (Fig. 1A). Also,we performed the experiments under oxidative conditions, vary-ing the concentration of H2O2 from 1 to 100 μM. Because thecoupled enzyme assay is incompatible with oxidizing agents,we monitored peptide phosphorylation using electrophoreticmobility shift assay (EMSA) and identified the products withelectrospray ionization mass spectrometry (ESI-MS). We foundthat the PLN peptides are phosphorylated under both oxidizingand reducing conditions (Fig. 1B), but under oxidizing conditions,PLNR9C

1−20 forms dimers, which can still be fully phosphorylatedby PKA-C. Phosphorylation reactions were repeated with full-length PLNR9C and PLNwt. Interestingly, we did not detect anyphosphorylation for pentameric PLNR9C under either reducing

or oxidizing conditions (Fig. 1B). The absence of phosphorylationof pentameric PLNR9C was confirmed by EMSA and MALDI-TOFmass spectrometry (Fig. S2). In contrast, we found completephosphorylation for the PLNwt and monomeric AFA-PLNR9C.The observed gel shift (Fig. 1B) is typical of AFA-PLN phos-phorylation at Ser16 (33). Based on these results, we concludethat phosphorylation by PKA-C is impaired only for the R9Cpentamer.

SERCA Activity Assays. To characterize the efficacy of PLNR9C tobind and reduce SERCA’s apparent Ca2þ affinity (pKCa), we per-formed coupled enzyme activity assays in reconstituted lipids. Inagreement with Schmitt et al. (24), we found that monomericAFA-PLNR9C is a loss-of-function (LOF) mutant, with a partialinhibitory effect on SERCA; i.e., slight reduction in pKCa (Fig. 1C,Left). Phosphorylation at Ser16 relieves the inhibitory effect forboth AFA-PLNwt and AFA-PLNR9C (Fig. 1C, Center). Remark-ably, the PLNR9C pentameric species is a total LOF (Fig. 1C,Right). Taken together, these results suggest that if PLNR9C wereto deoligomerize, it would be able to reversibly inhibit SERCA.

Stability of PLN Pentamer and Cys Accessibility. Based on the resultsabove, we deduced that PLNR9C could in principle regulate SER-CA, although with a reduced degree of inhibition. However,EMSA and mass spectrometry data show that the pentamericspecies is not phosphorylated at Ser 16. Therefore, the cardio-toxic species is possibly the pentameric assembly, which preventsphosphorylation and hampers the monomer-pentamer equili-brium necessary for the regulation of SERCA. To further test thishypothesis, we measured the stability of the pentamers (PLNwt

and PLNR9C) using thermal unfolding and gel electrophoresis.At 25 °C and in the presence of 10 mM DTT, the pentamer tomonomer ratio detected by SDS-PAGE for PLNwt is approxi-mately 4∶1 (78% pentamer). Fitting of the densitometry datafrom the SDS gels gave a melting temperature (Tm) of45� 1 °C for the wild-type pentamer. Under the same conditions,the pentamer/monomer ratio for PLNR9C increases noticeably(92% pentamer) and its Tm is 51� 1 °C (Fig. 2). The thermo-stability of the mutant is even more pronounced under oxidativeconditions (100 μM H2O2). We obtained Tm values of 52� 2 °Cand 67� 6 °C for PLNwt and PLNR9C, respectively. The SDS gelsof the oxidized pentamers (Fig. 2) show some important features:(i) PLNR9C pentamers have slightly less mobility than the PLNwt,which suggests a change in the protein structural topology (i.e.,hydrodynamic radius), and (ii) the presence of heat-resistantdimers. The latter was also observed in the oxidation studies ofPLNwt carried out by Froehlich et al. (34) (Fig. 2). To test whetherthe formation of the dimers is due to the cytoplasmic (Cys 9) ortransmembrane (Cys36, Cys41, Cys46) cysteines, we carried outthe same experiments with AFA-PLNR9C. Under reducing con-ditions, AFA-PLNR9C runs as a monomer on an SDS-PAGE gel;whereas under oxidizing conditions it forms dimers (Fig. 3A).PLNR9C

1−20 behaves in a similar manner, suggesting a marked ten-dency of Cys9 to form intermolecular disulfide bridges. To furthersupport the formation of disulfide bridges, we probed the pre-sence of free thiols for the PLN variants with Ellman’s reagent(5,5′-dithiobis-(2-nitrobenzoic) acid; DTNB). For PLNR9C

1−20, wedetected the formation of disulfide bridges even in the presenceof moderate amounts of reducing agent (DTT < 1 mM). Understronger reducing conditions (DTT > 10 mM), however, thePLNR9C

1−20 runs as a monomer (Fig. 3A) and forms a 1∶1 adduct(DTNB: PLNR9C

1−20) after ∼80 min (Fig. 3B). Under oxidizing con-ditions (ranging from 1 to 100 μMH2O2), PLNR9C

1−20 forms dimers,with the Cys residues becoming completely inaccessible toDTNB. We repeated these measurements with PLNR9C andPLNwt and found that under reducing conditions PLNR9C is muchmore reactive with DTNB than PLNwt. We monitored the DTNB

Fig. 1. PKA-C phosphorylation reaction and SERCA inhibition assays forwild-type and R9C constructs of PLN. (A) Steady state phosphorylationkinetics of PLNwt

1−20 and PLNR9C1−20 and competition assays. (Left) Plot of the

initial rates as a function of substrate concentration. (Right) Plot of the ap-parent KM as a function of phosphorylated products (pPLNwt

1−20 and pPLNR9C1−20).

(B) SDS-PAGE gels for the phosphorylation reactions of PLNwt1−20, PLN

R9C1−20, AFA-

PLN, AFA-PLNR9C, PLNwt, and PLNR9C carried out under oxidizing (100 μMH2O2) and reducing (10 mM DTT) conditions. The number of phosphatesper peptide was detected by ESI mass spectrometry. (C) Histograms showingthe change in apparent Ca2þ affinity (ΔpKCa) of SERCA in the presence ofphosphorylated and unphosphorylated PLN monomers (AFA-PLN andAFA-PLNR9C) and pentameric PLNwt and PLNR9C constructs.

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reaction up to ∼90 min. To reach completion, however, the re-action requires more than 18 h (35). Because the membrane-embedded Cys residues of PLNwt react with DTNB very slowly(>300 min), we assigned the fast rise of the binding curve forPLNR9C to the reactivity of Cys 9 (Fig. 3B), which is more exposedto the soluble DTNB. Under oxidizing conditions, both PLNR9C

and PLNwt behave identically, with very sluggish reaction kineticswith DTNB. The latter is in quantitative agreement with previouscysteine accessibility measurements carried out by Karim et al.(35). Overall, these data suggest that oxidation of Cys 9 resultsin formation of stable dimers and confers greater thermostabilityto PLNR9C oligomers.

Probing PLN Oligomerization and SERCA Binding Using QuantitativeFluorescence Resonance Energy Transfer (FRET). We probed theoligomerization of the PLNwt and PLNR9C pentamers in liveAAV-293 cells using quantitative FRET measurements betweenfluorescent protein tags fused to PLN’s cytoplasmic domains (36,37). Specifically, to detect intrapentameric FRET (i.e., FRETbetween protomers in each pentamer), we engineered PLNwt orPLNR9C with cerulean fluorescent protein (Cer) and yellow fluor-escent protein (YFP), respectively, and coexpressed them inAAV-293 cells (36, 37). For SERCA binding assays, we tagged

SERCA with cyan fluorescent protein (CFP) and measuredFRET with YFP-PLN constructs. To quantify the dependenceof FRETon PLN expression levels, we carried out a cell-by-cellsurvey of quantitative FRETand fluorescence intensity (an indexof protein concentration). For both PLN constructs, we foundthat intrapentameric FRET increased with protein concentrationto a maximum value (FRETmax) (Fig. 4A). For PLNR9C, FRETmaxis slightly higher than PLNwt (p < 0.05). This corresponds to amodest decrease in the average distances between the Cer-YFPprobes within the PLNR9C pentamer relative to PLNwt, whichmay reflect the formation of disulfide bridges in the pentamericmutant (Table S2). The curve representing the concentration de-pendence for PLNR9C is left-shifted with respect to that of PLNwt

(Fig. 4A). The calculated dissociation constant (Kd1) was approxi-mately 53% lower than that of PLNwt, suggesting the formation ofmore stable pentamers for the R9C mutant (Table S2). Interest-ingly, the left-shift of the intrapentamer FRETcurve for PLNR9C

corresponds to a right-shift of SERCA∕PLNR9C binding curve(Fig. 4B), with the calculated apparent dissociation constant(Kd2) approximately 130% greater than that of PLNwt. We con-clude that the PLNR9C has much lower affinity for SERCA thanPLNwt and SERCA is not able to deoligomerize PLNR9C as effi-ciently as PLNwt (18, 19). This explains the LOF character ofPLNR9C measured by ATPase activity (22). Notably, we did notobserve a significant difference in FRETmax for SERCA com-plexes with either PLNwt or PLNR9C, which suggests that bothspecies probably bind SERCA in a similar manner.

Because patients with DCM carry both the wild-type and themutant alleles, with the latter showing a dominant inheritancepattern (22, 24), we also tested the stability of mixed

Fig. 2. Thermostability of PLNwt and PLNR9C pentamers using electro-mobility shift assay. (A) SDS-PAGE gels for PLNwt and PLNR9C incubated atdifferent temperatures under reducing (Upper) and oxidizing (Lower) condi-tions. (B) Plots of the percent pentamers obtained from densitometryanalysis versus temperature for both PLNwt and PLNR9C under reducing(Upper) and oxidizing (Lower) conditions.

Fig. 3. Electromobility shift and DTNB cysteine accessibility assays forwild-type and R9C constructs of PLN. (A) SDS-PAGE gels in Tris buffer, 1 mMDTT, and 100 μM H2O2 of PLNR9C

1−20 (Left), and AFA-PLNR9C (Right). (B) Plots ofcysteine reactivity over time. Cysteine reactivity is measured by moles TNBproduced per peptide or full-length protomer from the reaction with DTNBunder reducing and oxidizing conditions. (Upper) Black squares, PLNwt

1−20 inTris buffer; red squares, PLNR9C

1−20 in Tris buffer; open red squares, PLNR9C1−20

in 1 mM DTT; and red crosses, PLNR9C1−20 in 100 μMH2O2. (Lower) Black squares,

PLNwt in Tris buffer; red squares, PLNR9C in Tris buffer; open black squares,PLNwt in 100 μM H2O2; and open red circles, PLNR9C in 100 μM H2O2.

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PLNwt∕PLNR9C pentamers. We coexpressed Cer-PLNR9C andYFP-PLNwt and measured FRET between them. Fig. 4C showsthat FRETmax is reduced for the mixed pentamers compared toPLNwt homopentamers. This is consistent with a clustering ofCer-PLNR9C cytoplasmic domains away from YFP-PLNwt oligo-mers. Notably, mixed pentamers showed a small but reproduciblereduction of Kd1 compared to that PLNwt, suggesting that theinteractions between the R9C mutants within the mixed penta-mers prevent deoligomerization. To determine FRET specificity,we carried out competition assays with increasing amounts of acompetitor PLN that cannot serve as a FRETacceptor. We foundthat by increasing the competitor concentration we obtained areduction in FRETmax to a minimum value of 4% (Fig. 4D). Thisvalue represents the amount of nonspecific FRET that is sub-tracted from total FRET for interprobe distance calculations(Table S2).

In vitro assays reported in the previous sections and workfrom other groups (26, 34) demonstrate that both PLNR9C andPLNwt are sensitive to oxidation. Therefore, we treated theAAV-293 cells with 100 μM H2O2. Upon addition of H2O2,Cer-PLNR9C fluorescence decreased by approximately 4% overthe course of 5 min (Fig. 4E), with approximately 5% increasein the emission of YFP-PLNR9C. The FRET ratio YFP/Cer in-creased by 10% for PLNR9C (Fig. 4F and Movie S1), whereasno increase was detected for PLNwt and AFA-PLN (Fig. 4F).To quantify the relative contributions to the observed FRETratioof protein oligomerization and changes in distance between thefluorescent probes, we measured FRETmax at regular intervalsand estimated Kd1 after H2O2 treatment. Fig. 4G shows that bothFRETmax and Kd1 changed after addition of H2O2, with a ∼40%reduction in Kd1 and a ∼10% increase in FRETmax. This suggestsan increase in PLN oligomerization with a slightly more compactconformation of the pentameric assembly. Note that we didnot detect any large-scale aggregation of PLNR9C either before orafter treatment with H2O2. Such aggregation would appear asfluorescent puncta, which would be visible by wide-field fluores-cence microscopy (Fig. 4H) or total internal reflection fluores-cence (Fig. S3).

DiscussionBased on coimmunoprecipitation experiments, Schmitt et al. (22)proposed that PLNR9C binds PKA-C irreversibly, creating dead-end complexes that deplete the local reservoir of kinase. Thelatter would reduce phosphorylation levels of PLN, with conco-mitant dysregulation of SERCA, leading to DCM. This interpre-tation accounts for the observed weak adrenergic responsivenessand dominant effect of PLNR9C in heterozygous patients (22).In the present study, we directly tested this hypothesis using bothin vitro and in cell experiments. We found that PKA-C was ableto phosphorylate both a truncated peptide and monomericAFA-PLNR9C, which is still able to reversibly inhibit SERCA,although with lower efficacy than PLNwt or AFA-PLN. Mostimportantly, kinetic assays under reducing conditions show thatPKA-C is able to quantitatively phosphorylate PLNR9C

1−20 with thesame catalytic efficiency of PLNwt

1−20. Under oxidizing conditions,PLNR9C

1−20 is able to be phosphorylated in a similar manner toPLNwt

1−20. Thus, we did not find evidence that this single mutationat the P-7 site of the recognition sequence of PKA-C interfereswith the phosphorylation reaction. However, we found that phos-phorylation of pentameric PLNR9C is significantly impaired,which is consistent with previous reports (24). Therefore, thestabilization of the pentamer by this Arg to Cys substitution pre-vents PLN phosphorylation. This finding emphasizes the role ofmonomer-pentamer equilibrium in the SERCA regulatory me-chanism by PLN. The latter is supported by in vivo studies carriedout by Kranias and coworkers in mice models, which demonstratethe importance of PLNwt over the monomeric mutant PLNC41F

for the optimal relaxation of cardiomyocytes (38).Disulfide bridges in the cytoplasmic domains of PLNR9C sta-

bilize the pentamer, making it practically inaccessible to PKA-Cand unable to deoligomerize and regulate SERCA. An importantfinding is the presence of dimers in oxidized PLNR9C. The latterhas been previously observed by Froehlich et al. upon PLNoxidation by nitroxyl radicals, which promote the formation ofdisulfide bonds in the transmembrane region, generating nonin-hibitory oligomers that prevent SERCA regulation (34). Underoxidative conditions, PLNR9C oligomerization is enhanced. Thisis important given that ischemic oxidative stress conditions are

Fig. 4. In cell FRET measurements of the pentamer stability and SERCA regulation for wild-type and R9C constructs of PLN. (A) Intrapentameric FRET versusprotein expression level for Cer-PLNwt∕YFP-PLNwt and Cer-PLNR9C∕YFP-PLNR9C (B) Percent of FRET efficiency from CFP-SERCA to YFP-PLNwt and YFP-PLNR9C.(C) Percent of intrapentameric FRET efficiency for Cer-PLNwt∕YFP-PLNwt homoligomers and Cer-PLNR9C∕YFP-PLNwt heteroligomers. (D) Plot of FRETmax versuscompetitor to quantify nonspecific FRET. (E). Plot of Cer F∕F0 versus time for YFP-PLNR9C and Cer-PLNR9C. Arrows indicate the time of addition of 100 μMH2O2.(F) YFP/Cer FRET ratio versus time. Arrows indicate the time of addition of 100 μMH2O2. (G) Plots of Kd1 (arbitrary units) and FRETmax upon addition of 100 μMH2O2 (arrow). (H) Fluorescence microscopy images of cells expressing Cer-PLNR9C and YFP-PLNR9C. Scale bar ¼ 20 μm. Ratio color scale ¼ 0.4–2.6.

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prevailing features of pathological states such as heart failure(39, 40). Additionally, oxidative stresses are also frequent underacute β-adrenergic stimulation and even in nonpathologicalconditions, where transient oxidative stress could cause cumula-tive damage (41). Therefore, it is possible that deteriorating re-dox conditions in PLNR9C-induced heart failure would reinforceanomalous PLN oligomerization and exacerbate the mutation’seffects on calcium cycling. Furthermore, we found that for mixedPLNwt∕PLNR9C pentamers enhanced oligomerization is a domi-nant effect. This is consistent with the observed R9C phenotypesfor both heterozygous mice (24) and human patients (22).

Based on the above considerations, we propose a model forPLN-SERCA disruption by the R9C mutation (Fig. 5). The prin-cipal effect of R9C is the formation of interprotomer disulfidebonds in the cytoplasmic domains, which is transient under redu-cing conditions and increases upon oxidation, stabilizing the PLNpentamer and rendering the recognition site for the kinase inac-cessible. This also prevents PLN dissociation into monomers andformation of the regulatory complex (PLN:SERCA). Theseeffects (enhanced SERCA activity and diminished phosphory-lation) are reminiscent of ablation of PLN observed by Kraniasand coworkers (42). The formation of the disulfide bridges in thepentamer hinders PLN phosphorylation by PKA, and possibly in-duces conformational or topological changes in PLN.We proposethat these combined effects are involved in the development ofDCM. An important corollary of this study is the emerging role ofthe PLN pentamer and the monomer-pentamer equilibrium (43).This lethal mutation revealed that oligomerization and deoligo-merization of the PLN pentamer within the membrane is directlyinvolved in the SERCA regulatory process.

Experimental ProceduresSample Preparation and Kinetic Assays. Recombinant PKA-C wasexpressed, purified, and assayed as previously reported (32, 44).All peptides (PLNwt

1−20 and PLNR9C1−20) were synthesized on a

Liberty 12-channel Automated Microwave Synthesizer fromCEM (Matthews, NC) (see SI Text). Phosphorylation reactionswere performed at 25 °C and monitored by following coupledenzyme (12 units lactate dehydrogenase and 4 units pyruvatekinase) mediated consumption of NADH at 340 nm using aSpectromax microplate reader (Molecular Devices) (30) as pre-viously reported (32, 44). Reaction solutions contained 50 mM

3-(N-morpholino) propanesulfonic acid (MOPS) (pH 7.0),64 nM PKA-C, 5 mM ATP, and 10 mM MgCl2, with substrateconcentrations ranging between 20–300 μM (32, 44). Inhibitorstudies were performed with the addition of phosphorylatedPLNwt

1−20 or PLNR9C1−20 (0.5 or 1.0 mM) to the reaction solution.

For phosphorylation reactions in the presence of H2O2, 300 μMof substrate was incubated for 10 min in the reaction solution be-fore initiating phosphorylation with 64 nM PKA-C. The reactionswere stopped after 10 min by the addition of 0.5% TFA, and ana-lyzed by EMSA (25% SDS-PAGE gels) stained by CoomassieBlue and ESI-MS after desalting with a C8 Zip-Tip (Millipore).

Thermostability of the Pentamer. PLN pentamer thermostabilitywas monitored by SDS-PAGE gels. For the reducing conditions,PLNwt and PLNR9C samples contained 100 mM Tris buffer, pH6.8, 3% sodium dodecyl sulfate, 8% (v∕v) glycerol, using a rangeof 5 to 10 mMDTT; whereas for oxidizing conditions the sampleswere incubated with 100 μM H2O2 for 20 min at each tempera-ture. Each sample (3 μg total mass) was loaded into 5%Next Gels(Amresco) or a 14% Tris-tricine SDS-PAGE gels. The Coomas-sie-stained gels were quantitated using ImageJ software (45).

SERCA Activity Assays. PLN variants were coreconstituted withpurified SERCA (46, 47) in lipid bilayer membranes (1,2-dioleoyl-sn-glycero-3-phosphocholine: 1,2-dioleoyl-sn-glycero-3-phosphoethanolamine, DOPC:DOPE, 4∶1) at molar ratios of10∶1 PLN:SERCA and 700∶1 lipids:SERCA. The Ca2þ depen-dence of the ATPase activity was measured using a coupled en-zyme assay at 37 °C (48) and monitored as for the PKA-C assays.Initial rates of SERCA was measured as a function of calciumconcentration (pCa), and data were fit to the Hill equation (48).

Cysteine Accessibility Measurements. Free thiols were assayed viatitrations with 5,5′-dithiobis(2-nitrobenzoic acid) (DTNB) (49).Samples (typically 100 μM) were dissolved in 60 mM Tris buffer(pH 8.0) and 1 mMEDTA and added to a reaction vessel contain-ing 100 mM Tris and 0.3 mM DTNB. The reactions were mon-itored at a wavelength of 412 nm (49). For oxidized and reducedconditions, we incubated PLN samples for 20 min in 100 μMH2O2 and 1 mM DTT, respectively. The excess of reducing agentwas eliminated with NaAsO2 (50).

Dynamic FRET. Transfected cells were washed with phosphatebuffered saline (PBS) and imaged by epifluorescence imagingat 1 min time intervals with excitation at 427∕10 nm and emissionat 472∕30 nm (for Cer) or 542∕27 nm (for YFP). After 5 min ofacquisition, the buffer was replaced with 100 μMH2O2 in PBS andacquisition continued for 15 min. Mean F∕F0 of all cells (�SE)at each time point was calculated for each filter configuration.The FRET ratio was calculated as the quotient of ðCerF∕F0Þ∕ðYFPF∕F0Þ, with a combined error of

ffiffiffiffiffiffiffiffiffiffiffiffiffiffiffiffiffiffiffiffiffiffiffiffiffiffiffiffiffiffiffiffiffiffiffiffiffiffiffiðseCFPÞ2 þ ðseYFPÞ2

p.

Images of YFP fluorescence were divided by images of Cer fluor-escence (both 427∕10 nm excitation) using ImageJ software (45).

Quantitative FRET. FRETmax and dissociation constants for thecomplexes were determined as described previously (36, 37). Theobserved FRET was calculated for each cell from the extent ofdonor fluorescence enhancement after acceptor photobleaching,according to E ¼ 1 − ðFprebleach∕FpostbleachÞ. For repetitive, non-destructive measurements, FRETwas quantified with a “3-cube”method (E-FRET) (36, 51). FRET efficiency of each cell wascompared to that cell’s starting YFP fluorescence (an index ofprotein concentration). FRET concentration dependence wasfit by a hyperbolic curve (36). Regulatory complex probe separa-tion distance was calculated usingR ¼ ðR0Þ½ð1∕FRETmaxÞ − 1Þ�1∕6(52). The distance between fluorescent probes in PLN pentamerswas calculated according to a ring-shaped oligomer model aspreviously described (36), with a Förster radius (R0) of 49.2 Å

Fig. 5. Proposed model of the effects of R9C mutation on the SERCA reg-ulatory cycle. The cardiotoxic effects are due to the stability of the PLNR9C

pentameric assembly, which prevents deoligomerization, phosphorylationby PKA-C, and regulation of SERCA by the monomeric species. Oxidativestress pushes the equilibrium toward the pentamer, making the PLNR9C

pentamer more stable and causing the formation of dimeric species forboth PLNwt and PLNR9C (not shown in the model) that are probably unableto regulate SERCA.

Ha et al. PNAS ∣ February 15, 2011 ∣ vol. 108 ∣ no. 7 ∣ 2739

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for Cer-YFPenergy transfer (53, 54). Non-specific FRET betweenunbound donors and acceptors was determined by measuring thereduction in FRET from YFP − PLNwt to mCherry − PLNwt incells coexpressing increasing amounts of competing CFP − PLNwt

(Fig. S4).

ACKNOWLEDGMENTS. Many thanks to Zhihong Hu, Eileen Kelly, AnthonyClementz for technical assistance, and Howard Young and NathanielTraaseth for helpful discussions. This work was supported by NationalInstitutes of Health Grants HL80081 and GM072701 (G.V.), HL09536 (K.H.),T32DE007288 (L.R.M.), and HL092321 and EB006061 (S.L.R.).

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