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Two potential calmodulin-binding sequences inthe ryanodine receptor contribute to a mobile,intra-subunit calmodulin-binding domain

Xiaojun Huang1,2, Ying Liu1, Ruiwu Wang3, Xiaowei Zhong3, Yingjie Liu3, Andrea Koop3, S. R. Wayne Chen3,Terence Wagenknecht1,2 and Zheng Liu1,*1Wadsworth Center, New York State Department of Health, Albany, New York 12201, USA2Department of Biomedical Sciences, School of Public Health, State University of New York at Albany, Albany, New York, 12201, USA3Libin Cardiovascular Institute of Alberta, Department of Physiology and Pharmacology, and of Biochemistry and Molecular Biology, University ofCalgary, Calgary, Alberta, T2N 4N1, Canada

*Author for correspondence (liuz@wadsworth.org)

Accepted 25 June 2013Journal of Cell Science 126, 4527–4535� 2013. Published by The Company of Biologists Ltddoi: 10.1242/jcs.133454

SummaryCalmodulin (CaM), a 16 kDa ubiquitous calcium-sensing protein, is known to bind tightly to the calcium release channel/ryanodinereceptor (RyR), and modulate RyR function. CaM binding studies using RyR fragments or synthetic peptides have revealed the presenceof multiple, potential CaM-binding regions in the primary sequence of RyR. In the present study, we inserted GFP into two of these

proposed CaM-binding sequences and mapped them onto the three-dimensional structure of intact cardiac RyR2 by cryo-electronmicroscopy. Interestingly, we found that the two potential CaM-binding regions encompassing, Arg3595 and Lys4269, respectively, arein close proximity and are adjacent to the previously mapped CaM-binding sites. To monitor the conformational dynamics of these

CaM-binding regions, we generated a fluorescence resonance energy transfer (FRET) pair, a dual CFP- and YFP-labeled RyR2(RyR2R3595-CFP/K4269-YFP) with CFP inserted after Arg3595 and YFP inserted after Lys4269. We transfected HEK293 cells with theRyR2R3595-CFP/K4269-YFP cDNA, and examined their FRET signal in live cells. We detected significant FRET signals in transfected cellsthat are sensitive to the channel activator caffeine, suggesting that caffeine is able to induce conformational changes in these CaM-

binding regions. Importantly, no significant FRET signals were detected in cells co-transfected with cDNAs encoding the single CFP(RyR2R3595-CFP) and single YFP (RyR2K4269-YFP) insertions, indicating that the FRET signal stemmed from the interaction betweenR3595–CFP and K4269–YFP that are in the same RyR subunit. These observations suggest that multiple regions in the RyR2 sequence

may contribute to an intra-subunit CaM-binding pocket that undergoes conformational changes during channel gating.

Key words: Ryanodine receptor/calcium release channel, Calmodulin, Cryo-EM, FRET

IntroductionCalmodulin (CaM) is a 16 kDa ubiquitous calcium-sensing

protein. It can bind to more than 30 proteins and enzymes that

are involved in growth, apoptosis, motility, mitosis and

development (Bähler and Rhoads, 2002). CaM consists of four

EF-hand calcium-binding motifs, which give CaM the ability of

binding four Ca2+ in total. Two of these EF-hand motifs form the

N-terminal lobe of CaM, while the other two form the C-terminal

lobe. In muscle excitation–contraction coupling, CaM modulates

the major participating transporters, including the ryanodine

receptor (RyR) in sarcoplasmic reticulum (SR) membrane and

the L-type calcium channel (DHPR) in the transverse tubule

(Aracena et al., 2005).

RyR is a calcium release channel that controls calcium flow

from the lumen of the SR to the cytosol. It plays a key role in

excitation–contraction coupling, during which an electrical

stimulus (from a neuron) triggers muscle contraction. The

electrical stimulus induces depolarization of the muscle plasma

membrane, and activation of the DHPR. RyR is the next target to

be activated, resulting in an increase in the concentration of Ca2+

in the cytosol. The increased cytosolic Ca2+ concentration

triggers sliding filament movements within the myofibril and

results in the contraction of muscle. Mammalian RyRs mainly

exist in three isoforms: RyR1 and RyR2 are predominantly

distributed in skeletal and cardiac muscle, respectively; RyR3

was first cloned from brain, but is also expressed in the

diaphragm, smooth muscle and several abdominal organs

(Kushnir et al., 2005).

CaM regulates the RyRs by a direct interaction and by an

indirect phosphorylation through CaM-dependent kinase II (Ai

et al., 2005). Both apo-CaM (no Ca2+ bound) and Ca2+-CaM

(four Ca2+ bound) can bind to all three RyR isoforms with high

affinity (Kd value in the nanomolar range) and regulate RyR

channel functions. At low (resting) Ca2+ concentration

(,100 nM), CaM exists mainly as apo-CaM, and apo-CaMactivates RyR1 (Buratti et al., 1995; Damiani and Margreth,

2000; Tripathy et al., 1995) and RyR3 (Chen et al., 1997;

Yamaguchi et al., 2005), but inhibits RyR2 (Balshaw et al., 2001;

Xu and Meissner, 2004). At high Ca2+ concentration (,1 mM),most CaM exists as Ca2+-CaM, and Ca2+-CaM inhibits all three

RyR isoforms (Buratti et al., 1995; Chen et al., 1997; Xu and

Meissner, 2004; Yamaguchi et al., 2005). Interestingly, apo and

Research Article 4527

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Ca2+ forms of CaM bind at distinct but overlapping locations

in RyR1 according to three-dimensional (3D) cryo-electron

microscopy (cryo-EM) results from our laboratory (Samso and

Wagenknecht, 2002; Wagenknecht et al., 1997), implying that

CaM moves on the surface of RyR1 by ,30 Å when it switchesbetween Ca2+-free and Ca2+-bound states. Recently, using a

mutant CaM that is incapable of binding Ca2+, we confirmed the

distinct binding sites for CaM in RyR1 and demonstrated that the

binding location switch is due to binding of Ca2+ to CaM as

opposed to effects of Ca2+ itself on the conformation of RyR1

(Huang et al., 2012). Interestingly, apo-CaM binds to RyR2 at a

similar binding site to that of Ca2+-CaM on RyR1, in seeming

agreement with the inhibitory effects of these two forms of CaM

on their respective receptors (Huang et al., 2012).

To identify CaM-binding sequences in the primary structure of

RyR, CaM overlays on RyR1- and RyR2-derived peptides were

used to test the binding capacity of CaM on RyR fragments,

which were expressed from RyR cDNA fragments generated

from available endonuclease restriction sites (Balshaw et al.,

2001; Chen and MacLennan, 1994; Guerrini et al., 1995;

Menegazzi et al., 1994). A number of sequences have been

reported to be potential CaM-binding sequences (Fig. 1). In

RyR1, these include residues 921–1173 (Chen and MacLennan,

1994), 1975–1999 (Zhang et al., 2003a), 2063–2091 (Chen and

MacLennan, 1994), 2804–2930 and 2961–3084 (Chen and

MacLennan, 1994), 2937–3225 (Menegazzi et al., 1994), 3042–

3057 (Guerrini et al., 1995), 3225–3662 (Balshaw et al., 2001),

3546–3655 (Menegazzi et al., 1994), 3611–3642 (Chen and

MacLennan, 1994), 3614–3643 (Rodney et al., 2001; Xiong et al.,

2002; Zhu et al., 2004), 3617–3634 (Guerrini et al., 1995), 4302–

4430 (Balshaw et al., 2001), 4303–4328 (Chen and MacLennan,

1994), 4425–4621 (Menegazzi et al., 1994) and 4540–4557

(Guerrini et al., 1995). In RyR2, these include residues 263–614,

2724–3016, 3007–3023, 3298–3961, 3583–3603, 4480–4497 and

4548–4748 (Balshaw et al., 2001; Guerrini et al., 1995;

Yamaguchi et al., 2003). The evidence supporting these

hypotheses of CaM regulation is mostly based on studies of

synthetic RyR peptides or RyR fragments rather than on the

intact, full-length RyR. Although the use of RyR peptides helps

simplify the experiments, the results gained from RyR peptide

studies may not represent what occurs in the intact RyR, and thus

some internal, buried RyR sequences are likely to be detected as

potential CaM-binding sequences. CaM, as a soluble protein,

however, can only bind to sequences exposed on the surface of

RyR.

To further test these potential CaM-binding sequences, in this

study we used site-specific GFP insertion coupled with 3D cryo-

EM, to map two of the proposed CaM-binding sequences. 3D

localization of inserted GFP is based on structural differences

between 3D reconstructions of GFP-inserted RyR2 and no-

insertion RyR2 control. The two binding sequences that we

investigated were: sequence 3581–3612 in RyR2 (corresponding

to sequence 3614–3643 in RyR1) and sequence 4261–4286 in

RyR2 (4303–4328 in RyR1). Fig. 1 illustrates schematically the

two GFP insertion sites in the primary sequence and their

correlation to the reported CaM-binding sequences. The DNA

sequence of GFP was inserted after Arg3595 for mapping

sequence 3581–3612 (henceforth named RyR2R3595-GFP), and

after Lys4269 for the sequence 4261–4286 (henceforth named

RyR2K4269-GFP), respectively. Extensive experimental evidence

supports the view that amino-acid residues within the sequence

3614–3643 in RyR1 and the corresponding sequence 3581–3610

in RyR2 are involved in binding CaM (Balshaw et al., 2001;

Chen and MacLennan, 1994; Guerrini et al., 1995; Menegazzi

et al., 1994; Rodney et al., 2001; Xiong et al., 2002; Zhang et al.,

2003a; Zhu et al., 2004). Sequence 4261–4286 in RyR1 (4303–

4328 in RyR2) was first detected as a CaM-binding sequence in

our 125I-labeled CaM binding study (Chen and MacLennan,

Fig. 1. Proposed CaM-binding sites in the

primary sequences of RyR1 and RyR2.

Regions proposed for Ca2+-CaM binding are

shown as red boxes, whereas regions

proposed for both apo-CaM and Ca2+-CaM

binding are shown as pink boxes. [Figure

adapted from Balshaw et al. (Balshaw et al.,

2001) with modification.] The two green

circles indicate positions where GFP was

inserted (see Materials and Methods) into

the sequence of RyR2 in two of the proposed

CaM-binding regions, specifically, after

residues Arg3595 and Lys4269.

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1994), and later confirmed in a 35S-labeled CaM binding study

(Balshaw et al., 2001). This sequence is also close to several otherproposed CaM-binding sequences (Balshaw et al., 2001; Guerriniet al., 1995) (see Fig. 1). Our 3D localization of GFP in RyR2R3595-GFP and RyR2K4269-GFP demonstrated that both sequence 3581–3612 and 4261–4286 could be CaM-binding sequences, since bothof them are adjacent to the CaM-binding sites determinedpreviously (Huang et al., 2012; Samsó and Wagenknecht, 2002;

Wagenknecht et al., 1997). Interestingly, the two GFP locations arealso close to one another, allowing us to generate a fluorescenceresonance energy transfer (FRET) probe to characterize the

conformational changes around the CaM-binding site when theRyR switches between the closed and open states. Furthermore,our FRET study has also established that there are interactions

between CaM-binding sequences 3581–3612 and 4261–4286 fromthe same subunit within the tetrameric RyR molecule. Thus, ourdata suggest that CaM-binding sequences 3581–3612 and 4261–

4286 form an intra-subunit binding pocket for CaM.

ResultsConstruction of a GFP structural marker in the CaM-binding sequences

In previous cryo-EM analyses of RyR1/RyR2–CaM interactions,the physical locations of CaM binding were determined (Huanget al., 2012; Samsó and Wagenknecht, 2002; Wagenknecht et al.,

1997). To gain insight into which regions of the RyR amino-acidsequence are potentially involved in CaM binding, we used site-specific GFP insertion coupled with 3D localization of inserted

GFP by cryo-EM techniques to map the CaM-binding sequencesonto the 3D architecture of RyR2. Briefly, we genetically taggedthe residue of interest with GFP, and identified the GFP location asan extra mass in the density map that was derived from cryo-EM.

To minimize potential disruptive effects of the insertion on foldingof both GFP and RyR2, two Gly-rich spacers (ten residues and nineresidues) flanked the inserted GFP (Kratz et al., 1999). Most of our

cryo-EM mappings using this method are consistent with otherstudies. For instance, 3D localization of Tyr2801 in thephosphorylation domain (Meng et al., 2007) is consistent with

the docking models from two groups in which a crystal structure ofRyR fragments containing the phosphorylated reside was fitted intoa RyR cryo-EM structure (Sharma et al., 2012; Yuchi et al., 2012);3D localization of Tyr846 (Wang et al., 2011) is consistent with a

docking model based on a pseudo-atomic structure of the N-terminal fragment (Zhu et al., 2013); and 3D localization ofArg626 in the FK506-binding protein (FKBP)-adjoining domain

by combining FRET and cryo-EM studies (Wang et al., 2011) isconsistent with binding assays that implicate this region in theRyR–FKBP interaction (Girgenrath et al., 2013). However, a

shortcoming of the long linkers is that they may allow GFP to moveaway from the target sites. In our previous mapping of Ser437 inthe N-terminal region, the inserted GFP containing long linkers

was located between domain 5 and 9 (Wang et al., 2007), roughly60 Å from the insertion site that is predicted from the dockingmodel of N-terminal ABC domains (Tung et al., 2010). The offsetmay come from the flexibility of linkers, from the flexibility of the

loop structure of RyR2 where the Ser437 located, and from thecylindrical shape of GFP with an overall length of nearly 50 Å.

In this study, GFP mappings were done with our refined insertion

protocol, in which both linkers are truncated by five or six residues,such that the entire GFP sequence, with a short Gly-rich linker (fourresidues) on each side, can be inserted into two of the proposed

CaM-binding regions in RyR2: after Arg3595 for mapping thesequence 3581–3612, and after Lys4269 for the sequence 4261–

4286. With the short linkers, we will be able to map the surface-exposed residues of RyR2 more precisely because of the reducedextendibility and flexibility from the linkers. If the GFP-labeled

sequence is involved in CaM binding to RyR1, the GFP in the 3Dreconstruction should be at or adjacent to one of the previouslyreported CaM-binding sites. The recombinant RyR2-GFP cDNAswere then expressed in HEK293 cells. The RyR2 function was

characterized by measuring Ca2+ release induced by sequentialadditions of increasing concentrations of caffeine in HEK293 cellstransfected with wild-type RyR2 (RyR2WT), RyR2R3595-GFP or

RyR2K4269-GFP, using the fluorescent Ca2+ indicator dye fluo-3 AM.

Fig. 2 shows that both RyR2R3595-GFP and RyR2K4269-GFP formfunctional Ca2+-release channel in HEK293 cells (Fig. 2B,C), with

significantly reduced response to caffeine (Fig. 2H).

Two-dimensional and three-dimensional localization ofR3595-GFP in RyR2

Purified RyR2R3595-GFP was imaged by cryo-EM (Fig. 3A). In theimages taken without tilting the specimen, RyR2R3595-GFP showed

a typical tetrameric RyR shape, which is a square with a centralcross and four protruding corners (Fig. 3A). Images of untiltedRyR2R3595-GFP particles were aligned and averaged to increase the

signal-to-noise ratio (Fig. 3B), and compared with the averagedimages of RyR2WT control (no-GFP-insertion; Fig. 3C). Foursymmetrically related strong positive regions were apparent after

subtracting the averaged images of the RyR2WT control from thatof RyR2R3595-GFP (Fig. 3D). Statistical analysis (t-tests) showedthese positive regions to be significant at more than 99.9%confidence levels. We ascribe these regions to the inserted GFP

locations on RyR2R3595-GFP. Since the two-dimensional (2D)analysis described above was only done with untilted particles, allin the fourfold symmetric orientation, the localization provided

XY coordinates for the GFP but the coordinate along the Z-axis(defined as parallel to the fourfold symmetry axis of RyR) wasindeterminate (Fig. 3E), hence the need for 3D reconstructions.

3D reconstruction of RyR2R3595-GFP identified a square prismshape, the standard RyR2 appearance (Liu et al., 2002; Sharmaet al., 1998) composed of 14 cytoplasmic domains connecting to

a transmembrane region (Fig. 4A,B). Compared with the no-GFP-insertion RyR2WT control, RyR2R3595-GFP contained fourmajor excess density regions, each located between domains 3

and 8a (highlighted with red circles in Fig. 4A,B). Subtraction ofthe 3D volume of RyR2WT from RyR2R3595-GFP provided a clearpicture of where the excess density regions were located

(Fig. 4C,D). Because these regions were within the areasdetected by 2D analysis and there were no other substantialexcess densities nearby, we attribute them to be the locations ofinserted GFP in RyR2R3595-GFP. This location is close to the

previously mapped locations of both apo-CaM- and Ca2+-CaM-binding sites on RyR1, and of apo-CaM on RyR2 (Huang et al.,2012; Samsó and Wagenknecht, 2002; Wagenknecht et al.,

1997), and is therefore consistent with evidence for the RyR2sequence 3581–3612 being involved in binding CaM.

Two-dimensional and three-dimensional localization ofK4269-GFP in RyR2

We next investigated the GFP location in the RyR2K4269-GFP.Purified RyR2K4269-GFP was imaged and analyzed using identicalmethods to those used for RyR2R3595-GFP. Cryo-EM showed the

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normal, standard RyR shape (Fig. 3F). 2D analysis (Fig. 3G–I)

revealed four significant, symmetrically related positive peaks in

the difference map, which represented the GFP location.

Comparing the 3D reconstruction of RyR2K4269-GFP with

RyR2WT control, we found that the two reconstructions are

similar except for the extra masses representing the inserted GFP,which were clearly present on the side of domain 3 facing domain7 (Fig. 4E–H). This location is also close to both the previously

mapped Ca2+-CaM-binding site in RyR1 and the apo-CaM-bindingsite in RyR2 (Huang et al., 2012; Wagenknecht et al., 1997).

FRET characterization of dynamic conformational changesaround the CaM-binding site

Based on the structural information from cryo-EM, wedemonstrated that both sequences, 3581–3612 and 4261–4286 inRyR2 (3614–3643 and 4303–4328 in RyR1), are adjacent to the

CaM-binding sites. Fortuitously, the two GFP reporters are locatedin close proximity, the distance between them being ,40 Å, aspatial arrangement that is favorable for characterizing

conformational dynamics by FRET. We first replaced the GFPinsertion in the RyR2R3595-GFP and RyR2K4269-GFP with a cyanfluorescent protein (CFP) and a yellow fluorescent protein (YFP),

respectively, to generate two single-insertion FRET probes,RyR2R3595-CFP and RyR2K4269-YFP. Subsequently, we constructeda cDNA with dual fluorescent protein insertions in the RyR2primary sequence, RyR2R3595-CFP/K4269-YFP (Fig. 5A,B). We next

carried out FRET analyses in HEK293 cells that were transfectedwith the cDNAs to probe for conformational changes around theCaM-binding site. Note that HEK293 cells expressing RyR2R3593-CFP,

RyR2K4269-YFP, RyR2R3595-CFP/K4269-YFP, or co-expressingRyR2R3593-CFP and RyR2K4269-YFP form functional Ca

2+ releasechannels (Fig. 2D–G). However, similar to GFP insertions, the CFP

and YFP insertions also significantly suppressed the response of theRyR2 channel to caffeine (Fig. 2H).

Fig. 5C shows the results of acceptor photobleaching experimentsto measure FRET efficiency in the HEK293 cells that expressed

RyR2R3595-CFP/K4269-YFP. Cyan (donor) and yellow (acceptor)fluorescence images were recorded before and afterphotobleaching of the acceptor and used to calculate the FRET

efficiency. As shown in Fig. 5D, the average FRET efficiency in thecells that expressed RyR2R3595-CFP/K4269-YFP in the absence of anytreatment was 22.161.1% (mean 6 s.e.m., n533 cells). The additionof 5 mM caffeine, a pharmacological activator of RyR channelactivity, significantly decreased the FRET efficiency to 8.760.5%(n530, P,0.001). The strong FRET that we observed supports thecryo-EM results that place residues R3595 and K4269 in close

proximity, and the decrease in FRET efficiency upon caffeinetreatment indicates that the two putative CaM-binding sequencesseparate from one another when the RyR channel switches from the

closed to the open conformation (see model in Fig. 6).

When the two cDNAs (RyR2R3595-CFP and RyR2K4269-YFP) areco-expressed in HEK293 cells, various hybrid tetrameric RyR2scould form (see models in Fig. 5B). FRET pairs will be formed

only when the following two criteria are both satisfied: (1) thedonor (CFP) and acceptor (YFP) are within a certain distance ofeach other (normally below 100 Å); (2) the two domains

containing CFP and YFP must belong to two neighboringRyR2 subunits. The models in the lower panel in Fig. 5Bhighlight FRET pairs that occur only when the two domains form

an inter-subunit interaction. No FRET will be detected in the caseof an intra-subunit interaction (top panel in Fig. 5B), because thedistance between CFP and YFP in the two neighboring subunits

is over 180 Å, which is far beyond the distance limitationbetween donor and acceptor for energy transfer. Little or nosignificant FRET (2–3%) or caffeine-induced reduction in FRET

Fig. 2. Functional characterization of RyR2s labeled with GFP, CFP or

YFP. HEK293 cells were transfected with RyR2WT (A), RyR2R3595-GFP (B),

RyR2K4269-GFP (C), RyR2R3595-CFP (D), RyR2K4269-YFP (E), or

RyR2R3595-CFP/K4269-YFP (F), or co-transfected with both RyR2R3595-CFP and

RyR2K4269-YFP cDNAs (G). Fluorescence intensity of fluo-3-AM-loaded transfected

cells was monitored before and after the additions of various concentrations of

caffeine. The numbers (under the traces) indicate caffeine concentrations. Traces

shown are from representative experiments. (H) Ca2+ release in relation to

cumulative caffeine concentration in transfected HEK293 cells. Data shown are

means 6 s.e.m. (n53). Except for those labeled ‘NS’ (not significant), all other data

points of GFP-, CFP- or YFP-labeled RyR2 are significantly different from that of

RyR2WT for a given caffeine concentration (P,0.05).

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was observed in the cells co-expressing cDNAs of RyR2R3595-CFPand RyR2K4269-YFP (Fig. 5D). These residual, non-specific FRET

signals most probably arise from the diffusion of nearby

RyR2R3595-CFP into the photobleached region, because FRET

was measured in live cells instead of fixed cells to maintain near

physiological conditions. Using a fluorescence recovery after

photobleaching (FRAP) experiment, we showed that the diffusion

of RyR2 in the live cells would not substantially interfere with

the FRET efficiency determined by the photobleaching approach,

but a 3–5% RyR2 diffusion indeed exists in live cells (Liu et al.,

2010). The observation of little or no FRET in the cells co-

transfected with RyR2R3595-CFP and RyR2K4269-YFP indicates that

these two closely located CaM-binding sequences are from the

same subunit within the homo-tetrameric RyR.

DiscussionSequences not directly involved in CaM binding

As indicated in Fig. 1, numerous regions in the sequences of

RyR1 and RyR2 have been implicated in binding CaM. The

physical locations of Ca2+-CaM and apo-CaM binding on the

surface of RyR1 and of apo-CaM on RyR2 have previously been

established by 3D cryo-EM (Huang et al., 2012; Samsó and

Wagenknecht, 2002; Wagenknecht et al., 1997). For this study

we focused on the two CaM-binding sequences that contain

residues Arg3595 and Lys4269 (RyR2 sequence numbers)

because the proposed sites that are located N-terminally to

residues 3581–3610 in RyR2 (3614–3643 in RyR1) are

considered unlikely on the basis of subsequent experimental

findings, particularly several cryo-EM analyses of GFP-insertions

and docking models of crystal structures of RyR fragments. The

proposed RyR2 CaM-binding sequence 263–615 (Balshaw et al.,

2001) is incompatible with the EM data of Wang et al. and the X-

ray crystallography/molecular docking results from Tung et al.

(Wang et al., 2007; Tung et al., 2010). The predicted CaM-

binding sequence 921–1173 in RyR1 (Chen and MacLennan,

1994) is also incompatible with experimental and modeling

studies on a fragment that overlaps this sequence (Zhu et al.,

2013). Proposed CaM-binding sequences in the vicinity of 2724–

3016 in RyR2 (Balshaw et al., 2001) and 2804–2930 in RyR1

(Chen and MacLennan, 1994) contain a phosphorylation site

Fig. 3. Two-dimensional analysis of RyR2R3595-GFP and RyR2K4269-GFP. (A,F) Cryo-EM CCD image of RyR2R3595-GFP and RyR2K4269-GFP, respectively,

showing protein particles embedded in a thin layer of vitreous ice. The tetrameric structure of RyR2 is well preserved, and several individual particles are marked

with circles. Scale bars: 500 Å. (B,G), 2D average of RyR2R3595-GFP and RyR2K4269-GFP, respectively, determined from 321 and 300 selected individual

particles with defocus value ranges between 22.8 and 23.2 mm and between 22.8 and 23.6 mm, respectively. (C,H), 2D average of RyR2WT control from 380and 333 particles in the same defocus range as were used for B and G, respectively. (D,I) The difference map obtained by subtracting the image in C from the

image in B and the image in H from G, respectively. Bright white spots represent positive differences (one spot is highlighted by an asterisk), which correspond to

the four extra masses contributed by GFP insertion. The images shown in B–D and G–H represent the projection of RyR as seen from the lumen side of the

sarcoplasmic reticulum, which is shown for 3D models in E and J. The asterisk highlights the location of GFP within the plane of projection. The width of frames

in B–J is 544 Å.

Fig. 4. Three-dimensional reconstruction of

RyR2R3595-GFP and RyR2K4269-GFP. (A,B,E,F) Surface

representations of the 3D reconstruction of RyR2R3595-GFP(A,B) and RyR2K4269-GFP (E,F), shown in a side view

(A,E) and in bottom view (B,F). The side view shows the

surface that would be perpendicular to the plane of the

sarcoplasmic reticulum membrane, and the bottom view

shows the surface parallel to the sarcoplasmic reticulum

membrane, which in situ would face the sarcoplasmic

reticulum lumen. Red and orange circles highlight the

locations of the extra masses representing GFP. (C,D,G,H)

Surface representations of the 3D reconstruction of RyR2

control shown in blue, and the difference map [subtraction

of 3D volume of RyR2 control from that of RyR2R3595-GFP(C and D) or RyR2K4269-GFP (G and H)] shown in green and

superimposed on the 3D reconstruction of RyR2 control.

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that, according to cryo-EM (Meng et al., 2007) and X-ray

crystallography and molecular docking results (Yuchi et al.,

2012) is not near the known CaM-binding locations.

One of the proposed CaM-binding sequences located N-

terminally to the sequence 3614–3643 in RyR1 deserves further

consideration. Zhang et al. found that a peptide corresponding to

RyR1 residues 1975–1999 binds apo-CaM and that this same

region was protected from tryptic digestion in the presence of

apo-CaM (Zhang et al., 2003a). Several attempts have been made

by our laboratory to insert GFP into the corresponding region of

RyR2, but these were unsuccessful, apparently because they

interfered with binding the accessory protein FKBP12.6 to RyR2,

which we make use of to purify the GFP-modified RyR2.

However, cryo-EM of RyR2 containing GFP insertions that lie on

either side of 1975–1999 (1942–1966 in RyR2), specifically at

Thr1874 and Thr2023, showing that these insertions are not in the

vicinity of known CaM-binding sites (Jones et al., 2008; Zhang

et al., 2003b), but further study of this region is needed to

ascertain whether or not the sequence 1975–1999 is involved in

direct interactions with CaM.

Sequence 4261–4286

Several proposed CaM-binding sequences are on the C-terminal

side of the well-established CaM-binding sequence, 3614–3643

in RyR1 (3581–3610 in RyR2). These sequences fall within the

range 4480–4748 in RyR2 and within 4302–4621 in RyR1

(Fig. 1). Previously, we used GFP insertion and cryo-EM to map

residue Asp4365 in RyR2 (corresponding to Asp4413 in RyR1)

to a region in domain 3 that was within a few nanometers of the

apo-CaM-binding location (Liu et al., 2002). Here we have

inserted GFP at Lys4269 in RyR2, which corresponds to RyR1

residue 4311, which lies within the proposed CaM-binding

Fig. 5. Dynamic interaction between two CaM-binding sequences characterized by FRET analysis. (A) Structural model of the dual insertion

RyR2R3595-CFP/K4269-YFP. The cyan and yellow spheres represent CFP and YFP, respectively. Each RyR2 molecule contains four FRET pairs (highlighted by green

ellipses). Red curves represent a possible boundary between the four subunits. FRET signals will be detected regardless of whether two structural domains bearing

R3595-CFP and K4269-YFP are contained within one RyR2 subunit (i.e. an intra-subunit interaction, top panel) or belong to two different subunits (i.e. an inter-

subunit interaction, bottom panel). (B) Models of six possible hybrid RyR2 tetramers when two cDNAs encoding RyR2R3595-CFP and RyR2K4269-YFP are co-

expressed. The top row shows six RyR2 tetramer structures for the situation in which two structural domains bearing R3595-CFP and K4269-YFP are contained

within one RyR subunit. In this case, no FRET signal will be detected, since the distance between CFP and YFP in the two neighboring subunits is over 180 Å.

The bottom row shows six RyR2 tetramer structures for the case in which the two structural domains bearing R3595-CFP and K4269-YFP belong to two different

subunits. In this case, four out of six structures have at least one FRET pair (highlighted by green ellipses). Numbers between two rows are the mathematical

probability ratios for the six possible structures, assuming that RyR2 tetramers formed by random assembly of the two differently labeled subunits. (C) Confocal

images of a HEK293 cell expressing RyR2R3595-CFP/K4269-YFP cDNA, showing cyan and yellow fluorescence before and after photobleaching. The green ellipse

demarcates the area selected for photobleaching. Scale bar: 10 mm. (D) FRET efficiency determined by photobleaching of acceptor. Data are means 6 s.e.m., withthe number of the cells indicated on the bars. ***P,0.001; n.s., not significant.

Journal of Cell Science 126 (19)4532

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sequences proposed by Chen and MacLennan (4303–4328) andby Balshaw et al. (4302–4430) in RyR1 (Balshaw et al., 2001;

Chen and MacLennan, 1994).

The localization of 4261–4286 was quite unambiguous. Both2D analysis and 3D reconstruction of RyR2K4269-GFP clearlyresolved the inserted GFP, which localized to the bottom of

domain 3 and facing domain 7, a location that is within a fewnanometers of the known apo-CaM- and Ca2+-CaM-bindinglocations. However, biochemical evidence supporting the

involvement of 4261–4286 of RyR2 (4303–4328 of RyR1) inbinding of CaM is insufficient. Only two [125I]CaM and[35S]CaM overlay experiments (Balshaw et al., 2001; Chen and

MacLennan, 1994), which detected the binding of RyR1immobilized fragments to radio-labeled CaMs, showed that4303–4328 and 4302–4430 of RyR1 were able to bind CaM. Thebinding of a RyR fragment to CaM does not necessarily indicate

that CaM binds to that specific sequence within the intact RyR,because RyR folding might restrict exposure of the sequence and/or its conformation. Here we have shown for an intact RyR,

rather than a RyR fragment, that GFP inserted within this regionis probably at least partly surface-exposed as opposed to beinginternal (buried). If the site is internal, the insertion would

probably disrupt the structure of RyR2, at least locally, andprevent GFP localization. Additionally, the fact that the GFP inRyR2K4269-GFP is located close to the actual CaM-binding site

(Huang et al., 2012; Wagenknecht et al., 1997), supports theinvolvement of 4261–4286 of RyR2 in CaM binding. However,strong evidence against the involvement of this sequence is thelack of an effect on CaM binding to RyR1 when a region

containing residues 4274–4535 is deleted by site-directedmutagenesis (Yamaguchi et al., 2001). Also, mutations withinthe sequence 3581–3612 are known to eliminate CaM binding to

RyR (discussed below), suggesting that the sequence 4274–4535is not capable of independent CaM binding. It is possible that thesequence 4274–4535 is capable of binding CaM with a much

lower affinity than the 3581–3612 region, but at present there isno evidence for this, or for any physiologically relevant reasonfor such a secondary CaM-binding site. Nevertheless, we cannotexclude this region of RyR from playing some role in CaM

binding.

Sequence 3581–3612

The interpretation of the results for GFP inserted within thesequence 3581–3612 was not straightforward because of itslocation in a cleft bordered by several RyR domains. GFP was

found to locate between domains 3 and 8a (Fig. 4C,D), which isclose to all three of the CaM-binding positions that have beendetermined (apo-CaM- and Ca2+-CaM-binding sites on RyR1 andapo-CaM-binding site on RyR2) (Huang et al., 2012; Samsó and

Wagenknecht, 2002; Wagenknecht et al., 1997). This location isconsistent with 3581–3612 of RyR2 (and by inference thehomologous sequence 3615–3644 in RyR1) as an authentic CaM-

binding sequence, but there was still uncertainty as to whetherR3595-GFP was linked to domain 3 or domain 8a. Consideringthe reported locations of apo-CaM and Ca2+-CaM on RyR1 as

well as apo-CaM on RyR2 (Huang et al., 2012; Samsó andWagenknecht, 2002; Wagenknecht et al., 1997), we favor thedomain 3 location for the sequence 3581–3612 because it is more

compatible with both the Ca2+-CaM and apo-CaM physicalbinding locations. In addition, our FRET result indicatessequences 3581–3612 and 4261–4286 from the same subunit

are located in close proximity in the tetrameric RyR2 molecule.

This finding increases the possibility that both sequences occuron the same domain (domain 3). Future studies to determine thelocation of 3581–3612 more precisely could be crucial for

understanding the regulatory mechanism of CaM.

Unlike sequence 4302–4328 of RyR1 (4261–4286 of RyR2),the sequence 3614–3643 of RyR1 (3581–3612 of RyR2) as aCaM-binding sequence is well supported by several types of

studies, including amino-acid sequence prediction (Takeshimaet al., 1989), CaM overlays (Balshaw et al., 2001; Chen andMacLennan, 1994; Guerrini et al., 1995; Menegazzi et al., 1994),

point mutation and deletion mutation (Yamaguchi et al., 2001;Yamaguchi et al., 2003) as well as tryptic digestion protection(Moore et al., 1999). Sequence 3614–3643 of RyR1 (3581–3612of RyR2) was also shown to be involved in binding both apo-

CaM and Ca2+-CaM (Moore et al., 1999). We identified thissequence at the region of overlap between the apo-CaM-bindingsite on RyR1 and the apo-CaM-binding site on RyR2 (similar to

the Ca2+-CaM-binding site on RyR1) as determined by cryo-EM(Huang et al., 2012; Samsó and Wagenknecht, 2002;Wagenknecht et al., 1997), and this is fully consistent with all

available evidence that supports this sequence as being essentialfor CaM binding to RyRs.

Conformational changes in the CaM-binding region of RyRand translocation of CaM

Previous functional studies showed that apo-CaM activates RyR1but inhibits RyR2, whereas Ca2+-CaM inhibits both isoforms

(Balshaw et al., 2001; Buratti et al., 1995; Damiani and Margreth,2000; Rodney et al., 2000; Tripathy et al., 1995; Yamaguchi et al.,2003). Our previous cryo-EM studies showed distinctly differentbinding locations of apo-CaM and Ca2+-CaM on RyR1 (Samsó

and Wagenknecht, 2002; Wagenknecht et al., 1997).Interestingly, apo-CaM binds to RyR2 at a similar location tothat of Ca2+-CaM on RyR1, in seeming agreement with the

inhibitory effects of these two forms of CaM on their respectivereceptors (Fig. 6) (Huang et al., 2012). Using a mutant CaM thatis incapable of binding calcium, we have determined that the

mutant CaM binds to RyR1 at the apo-CaM site, regardless of thecalcium concentration. The existence of the two overlapping butdistinct binding sites for apo-CaM and Ca2+-CaM on RyR1 implythat the binding location switch is due to Ca2+ binding to CaM, as

opposed to direct effects of Ca2+ on RyR1, and that the switch tothe alternative apo-CaM site on RyR1 (but not on RyR2) isinvolved in the activation effect of apo-CaM on RyR1 (Huang

et al., 2012).

In this study, we demonstrated by cryo-EM that twohypothesized CaM-binding sequences in RyR2, 3581–3612 and4261–4286 (corresponding to 3614–3643 and 4302–4328 in

RyR1) are both in close proximity to the previously foundlocations of CaM. In addition, we found that when the RyR2channel is activated, the structural domains bearing these two

sequences move apart, indicating that conformational changesassociated with channel activation occur in the vicinity ofreceptor-bound CaM. We suggest that these conformational

changes also affect CaM binding, and vice versa, which would beconsistent with the known influences CaM on channel gating.Clearly, more detailed studies of conformational changes in this

region of RyR are needed. RyR activation is induced either bydirect interaction with DHPR (for RyR1), by a small amount ofCa2+ influx from DHPR (for RyR2) or by Ca2+ released from SR

Mapping CaM-binding sequences in RyR 4533

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stores through nearby RyRs (RyR1 and RyR2). Global

conformational changes underlie RyR activation, including

changes in the clamp region (Tian et al., 2013), in the

transmembrane region that result in ion channel activation

(Samsó et al., 2009), and, as we show here for the first time, in

the CaM-binding region of RyR1. Upon RyR activation the two

fluorophore-labeled sequences move apart, a structural change

that is likely to be influenced by CaM. We hypothesize that for

RyR2, the presence of either apo-CaM or Ca2+-CaM inhibits the

conformational change, whereas for RyR1 apo-CaM facilitates

and Ca2+-CaM inhibits it, thereby accounting for the regulatory

effects of CaM on the activity of RyR2 and RyR1. Additional

biophysical characterization, by FRET and other techniques, of

this conformational change on both RyR2 and RyR1 and in the

presence of CaM and calcium should allow us to refine or reject

this hypothesis. It is also possible that the conformational change

we have discovered plays a role in the termination of Ca2+

release, particularly for RyR2 for which the mechanism by which

Ca2+ release is terminated during excitation–contraction coupling

in the heart is not understood (Cannell and Kong, 2012; Stern and

Cheng, 2004).

Materials and MethodsMaterials and chemicals

The detergent CHAPS, the redox reagent DTT, and the protease inhibitor leupeptinwere purchased from CalBiochem (La Jolla, CA, USA). All the other chemicals

were purchased from Sigma-Aldrich (St. Louis, MO, USA). GFP-inserted mouseRyR2s were constructed, expressed in HEK293 cells and purified as described

previously (Liu et al., 2002).

Construction of GFP-tagged RyR2s

The cloning and construction of the 15-kb full-length cDNA encoding the mouseRyR2 has been described previously (NCBI reference sequence NP_076357.2,GI:124430578) (Zhao et al., 1999). The cDNA encoding GFP with Gly-rich linkers

(four residues) on each side and an AscI site was obtained by PCR. The AscI sitewas introduced into a cDNA fragment of RyR2 after a specific residue (Arg3595

or Lys4269) by overlap extension using PCR. The fragment with the AscI site wassubcloned into the full-length RyR2 cDNA. The AscI–AscI fragment containingGFP and the linkers were then subcloned into the full-length RyR2 at theintroduced AscI site. The sequences of all PCR fragments and the orientation of theinserted GFP cDNA were verified by DNA sequencing analysis.

Caffeine-induced Ca2+ release measurements

Free cytosolic Ca2+ concentration in transfected HEK293 cells was measured usingthe fluorescent Ca2+ indicator dye fluo-3 AM as described previously (Li andChen, 2001).

Cryo-grid preparation

The expression and purification of RyR2R3595-GFP and RyR2K4269-GFP was carriedout as described previously (Liu et al., 2002). Cryo-grids prepared for 2D and 3Dlocalization of GFP-inserted RyR2s were prepared as follows. Purified GFP-inserted RyR2 (0.8 ml at a concentration about 0.2 mg/ml in 20 mM NaMOPS,pH 7.4, 1 M NaCl, 1% CHAPS, 2 mM DTT, 2 mg/ml leupeptin) was diluted with4 ml buffer (20 mM NaMOPS, pH 7.4, 400 mM KCl, 3.0 mM EGTA, 0.5%CHAPS, 2 mM DTT, 2 mg/ml leupeptin). A 4-ml volume was applied to 300 meshgrids coated with a thin continuous carbon film suspended over a thick holeycarbon film for 30 seconds. The grids were blotted with Whatman no. 1 filterpaper for about 3 seconds before plunging into liquid ethane.

Cryo-electron microscopy and image processing

Cryo-EM data collection and image processing were performed in a similarmanner to that described previously (Huang et al., 2012). The final 3Dreconstructions of RyR2R3595-GFP (resolution 30 Å) and RyR2K4269-GFP(resolution 31 Å) were computed from 8002 and 6934 particles, respectively.

FRET measurement

For FRET analysis, the GFP insertion in the RyR2R3595-GFP and RyR2K4269-GFPwere replaced by CFP and YFP, respectively. In addition to the single insertions,a dual insertion RyR2R3595-GFP/K4269-YFP was constructed as describedpreviously (Liu et al., 2010). FRET measurements in the HEK293 cellsexpressing RyR2R3595-CFP/K4269-YFP, or co-expressing RyR2R3595-CFP andRyR2K4269-YFP were performed as described previously (Liu et al., 2010).

AcknowledgementsWe gratefully acknowledge the 3D-EM and Advanced LightMicroscopy and Image Analysis Core Facilities at the WadsworthCenter.

Author contributionsZ.L., T.W. and S.R.W.C. designed the research; X.H., Ying L., R.W.,X.Z., Yingjie L., A.K. and Z.L. performed experiments; X.H.,Yingjie L., S.R.W.C. and Z.L. analyzed data; X.H., S.R.W.C., T.W.and Z.L. wrote the manuscript.

FundingThis work was supported by the National Institutes of Health [grantnumbers R01HL095541 to Z.L and R01AR040615 to T.W.]; and bythe Canadian Institutes of Health Research and Heart and StrokeFoundation of Canada to S.R.W.C. Deposited in PMC for releaseafter 12 months.

ReferencesAi, X., Curran, J. W., Shannon, T. R., Bers, D. M. and Pogwizd, S. M. (2005).

Ca2+/calmodulin-dependent protein kinase modulates cardiac ryanodine receptor

phosphorylation and sarcoplasmic reticulum Ca2+ leak in heart failure. Circ. Res. 97,

1314-1322.

Aracena, P., Hidalgo, C. and Hamilton, S. L. (2005). RyR1 modulation by calmodulin.

In Ryanodine Receptors: Structure, Function and Dysfunction in Clinical Disease (ed.

X. H. T. Wehrens and A. R. Marks), pp. 163-168. New York, NY: Springer.

Bähler, M. and Rhoads, A. (2002). Calmodulin signaling via the IQ motif. FEBS Lett.

513, 107-113.

Balshaw, D. M., Xu, L., Yamaguchi, N., Pasek, D. A. and Meissner, G. (2001).

Calmodulin binding and inhibition of cardiac muscle calcium release channel

(ryanodine receptor). J. Biol. Chem. 276, 20144-20153.

Buratti, R., Prestipino, G., Menegazzi, P., Treves, S. and Zorzato, F. (1995). Calcium

dependent activation of skeletal muscle Ca2+ release channel (ryanodine receptor) by

calmodulin. Biochem. Biophys. Res. Commun. 213, 1082-1090.

Cannell, M. B. and Kong, C. H. T. (2012). Local control in cardiac E-C coupling.

J. Mol. Cell. Cardiol. 52, 298-303.

Fig. 6. Hypothetical model of RyR conformation changes in the CaM-

binding region and translocation of CaM when the RyR channel switches

from closed to open. The transparent purple mass represents the apo-CaM-

binding site, and the transparent red mass is the Ca2+-CaM-binding site in

RyR1. The orange mass indicates the apo-CaM-binding site in RyR2. Two

structural domains bearing CaM-binding sequences 3615–3644 and 4303–

4328 in RyR1 are highlighted in green. Green arrows demonstrate that the two

domains move apart when the RyR channel is activated, and the purple-red

arrow indicates the shift of CaM from the apo-binding site to the Ca2+ binding

site that occurs in RyR1 but not in RyR2. Figure adapted from Samsó and

Wagenknecht and Huang et al., with modifications (Samsó and Wagenknecht,

2002; Huang et al., 2012).

Journal of Cell Science 126 (19)4534

http://dx.doi.org/10.1161/01.RES.0000194329.41863.89http://dx.doi.org/10.1161/01.RES.0000194329.41863.89http://dx.doi.org/10.1161/01.RES.0000194329.41863.89http://dx.doi.org/10.1161/01.RES.0000194329.41863.89http://dx.doi.org/10.1016/S0014-5793(01)03239-2http://dx.doi.org/10.1016/S0014-5793(01)03239-2http://dx.doi.org/10.1074/jbc.M010771200http://dx.doi.org/10.1074/jbc.M010771200http://dx.doi.org/10.1074/jbc.M010771200http://dx.doi.org/10.1006/bbrc.1995.2238http://dx.doi.org/10.1006/bbrc.1995.2238http://dx.doi.org/10.1006/bbrc.1995.2238http://dx.doi.org/10.1016/j.yjmcc.2011.04.014http://dx.doi.org/10.1016/j.yjmcc.2011.04.014

Journ

alof

Cell

Scie

nce

Chen, S. R. and MacLennan, D. H. (1994). Identification of calmodulin-, Ca(2+)-, andruthenium red-binding domains in the Ca2+ release channel (ryanodine receptor) ofrabbit skeletal muscle sarcoplasmic reticulum. J. Biol. Chem. 269, 22698-22704.

Chen, S. R. W., Li, X., Ebisawa, K. and Zhang, L. (1997). Functional characterizationof the recombinant type 3 Ca2+ release channel (ryanodine receptor) expressed inHEK293 cells. J. Biol. Chem. 272, 24234-24246.

Damiani, E. and Margreth, A. (2000). Pharmacological clues to calmodulin-mediatedactivation of skeletal ryanodine receptor using [3H]-ryanodine binding. J. Muscle Res.Cell Motil. 21, 1-8.

Girgenrath, T., Mahalingam, M., Svensson, B., Nitu, F. R., Cornea, R. L. and

Fessenden, J. D. (2013). N-terminal and central segments of the type 1 ryanodinereceptor mediate its interaction with FK506-binding proteins. J. Biol. Chem. 288,16073-16084.

Guerrini, R., Menegazzi, P., Anacardio, R., Marastoni, M., Tomatis, R., Zorzato,

F. and Treves, S. (1995). Calmodulin binding sites of the skeletal, cardiac, and brainryanodine receptor Ca2+ channels: modulation by the catalytic subunit of cAMP-dependent protein kinase? Biochemistry 34, 5120-5129.

Huang, X., Fruen, B., Farrington, D. T., Wagenknecht, T. and Liu, Z. (2012).Calmodulin-binding locations on the skeletal and cardiac ryanodine receptors. J. Biol.Chem. 287, 30328-30335.

Jones, P. P., Meng, X., Xiao, B., Cai, S., Bolstad, J., Wagenknecht, T., Liu, Z. and

Chen, S. R. W. (2008). Localization of PKA phosphorylation site, Ser(2030), in thethree-dimensional structure of cardiac ryanodine receptor. Biochem. J. 410, 261-270.

Kratz, P. A., Böttcher, B. and Nassal, M. (1999). Native display of complete foreignprotein domains on the surface of hepatitis B virus capsids. Proc. Natl. Acad. Sci.USA 96, 1915-1920.

Kushnir, A., Mollah, A. K. M. M. and Wehrens, X. H. T. (2005). Evolution of theryanodine receptor gene family. In Ryanodine Receptors: Structure, Function andDysfunction in Clinical Disease (ed. X. H. T. Wehrens and A. R. Marks), pp. 1-8.New York, NY: Springer.

Li, P. and Chen, S. R. W. (2001). Molecular basis of Ca(2)+ activation of the mousecardiac Ca(2)+ release channel (ryanodine receptor). J. Gen. Physiol. 118, 33-44.

Liu, Z., Zhang, J., Li, P., Chen, S. R. W. and Wagenknecht, T. (2002). Three-dimensional reconstruction of the recombinant type 2 ryanodine receptor andlocalization of its divergent region 1. J. Biol. Chem. 277, 46712-46719.

Liu, Z., Wang, R., Tian, X., Zhong, X., Gangopadhyay, J., Cole, R., Ikemoto, N.,

Chen, S. R. W. and Wagenknecht, T. (2010). Dynamic, inter-subunit interactionsbetween the N-terminal and central mutation regions of cardiac ryanodine receptor.J. Cell Sci. 123, 1775-1784.

Menegazzi, P., Larini, F., Treves, S., Guerrini, R., Quadroni, M. and Zorzato,F. (1994). Identification and characterization of three calmodulin binding sites of theskeletal muscle ryanodine receptor. Biochemistry 33, 9078-9084.

Meng, X., Xiao, B., Cai, S., Huang, X., Li, F., Bolstad, J., Trujillo, R., Airey, J.,

Chen, S. R. W., Wagenknecht, T. et al. (2007). Three-dimensional localization ofserine 2808, a phosphorylation site in cardiac ryanodine receptor. J. Biol. Chem. 282,25929-25939.

Moore, C. P., Rodney, G., Zhang, J.-Z., Santacruz-Toloza, L., Strasburg, G. andHamilton, S. L. (1999). Apocalmodulin and Ca2+ calmodulin bind to the sameregion on the skeletal muscle Ca2+ release channel. Biochemistry 38, 8532-8537.

Rodney, G. G., Williams, B. Y., Strasburg, G. M., Beckingham, K. and Hamilton,

S. L. (2000). Regulation of RYR1 activity by Ca(2+) and calmodulin. Biochemistry39, 7807-7812.

Rodney, G. G., Moore, C. P., Williams, B. Y., Zhang, J.-Z., Krol, J., Pedersen, S. E.

and Hamilton, S. L. (2001). Calcium binding to calmodulin leads to an N-terminalshift in its binding site on the ryanodine Receptor. J. Biol. Chem. 276, 2069-2074.

Samsó, M. and Wagenknecht, T. (2002). Apocalmodulin and Ca2+-calmodulin bind toneighboring locations on the ryanodine receptor. J. Biol. Chem. 277, 1349-1353.

Samsó, M., Feng, W., Pessah, I. N. and Allen, P. D. (2009). Coordinated movement ofcytoplasmic and transmembrane domains of RyR1 upon gating. PLoS Biol. 7, e85.

Sharma, M. R., Penczek, P., Grassucci, R., Xin, H.-B., Fleischer, S. and

Wagenknecht, T. (1998). Cryoelectron microscopy and image analysis of thecardiac ryanodine receptor. J. Biol. Chem. 273, 18429-18434.

Sharma, P., Ishiyama, N., Nair, U., Li, W., Dong, A., Miyake, T., Wilson, A., Ryan,

T., MacLennan, D. H., Kislinger, T. et al. (2012). Structural determination of thephosphorylation domain of the ryanodine receptor. FEBS J. 279, 3952-3964.

Stern, M. D. and Cheng, H. (2004). Putting out the fire: what terminates calcium-

induced calcium release in cardiac muscle? Cell Calcium 35, 591-601.

Takeshima, H., Nishimura, S., Matsumoto, T., Ishida, H., Kangawa, K., Minamino,

N., Matsuo, H., Ueda, M., Hanaoka, M., Hirose, T. et al. (1989). Primary structure

and expression from complementary DNA of skeletal muscle ryanodine receptor.

Nature 339, 439-445.

Tian, X., Liu, Y., Liu, Y., Wang, R., Wagenknecht, T., Liu, Z. and Chen, S. R. W.

(2013). Ligand-dependent conformational changes in the clamp region of the cardiac

ryanodine receptor. J. Biol. Chem. 288, 4066-4075.

Tripathy, A., Xu, L., Mann, G. and Meissner, G. (1995). Calmodulin activation and

inhibition of skeletal muscle Ca2+ release channel (ryanodine receptor). Biophys. J.

69, 106-119.

Tung, C. C., Lobo, P. A., Kimlicka, L. and Van Petegem, F. (2010). The amino-

terminal disease hotspot of ryanodine receptors forms a cytoplasmic vestibule. Nature

468, 585-588.

Wagenknecht, T., Radermacher, M., Grassucci, R., Berkowitz, J., Xin, H.-B. and

Fleischer, S. (1997). Locations of calmodulin and FK506-binding protein on the

three-dimensional architecture of the skeletal muscle ryanodine receptor. J. Biol.

Chem. 272, 32463-32471.

Wang, R., Chen, W., Cai, S., Zhang, J., Bolstad, J., Wagenknecht, T., Liu, Z. and

Chen, S. R. W. (2007). Localization of an NH(2)-terminal disease-causing mutation

hot spot to the ‘‘clamp’’ region in the three-dimensional structure of the cardiac

ryanodine receptor. J. Biol. Chem. 282, 17785-17793.

Wang, R., Zhong, X., Meng, X., Koop, A., Tian, X., Jones, P. P., Fruen, B. R.,

Wagenknecht, T., Liu, Z. and Chen, S. R. W. (2011). Localization of the

dantrolene-binding sequence near the FK506-binding protein-binding site in the

three-dimensional structure of the ryanodine receptor. J. Biol. Chem. 286, 12202-

12212.

Xiong, L.-W., Newman, R. A., Rodney, G. G., Thomas, O., Zhang, J.-Z., Persechini,

A., Shea, M. A. and Hamilton, S. L. (2002). Lobe-dependent regulation of ryanodine

receptor type 1 by calmodulin. J. Biol. Chem. 277, 40862-40870.

Xu, L. and Meissner, G. (2004). Mechanism of calmodulin inhibition of cardiac

sarcoplasmic reticulum Ca2+ release channel (ryanodine receptor). Biophys. J. 86,

797-804.

Yamaguchi, N., Xin, C. and Meissner, G. (2001). Identification of apocalmodulin and

Ca2+-calmodulin regulatory domain in skeletal muscle Ca2+ release channel,

ryanodine receptor. J. Biol. Chem. 276, 22579-22585.

Yamaguchi, N., Xu, L., Pasek, D. A., Evans, K. E. and Meissner, G. (2003).

Molecular basis of calmodulin binding to cardiac muscle Ca(2+) release channel

(ryanodine receptor). J. Biol. Chem. 278, 23480-23486.

Yamaguchi, N., Xu, L., Pasek, D. A., Evans, K. E., Chen, S. R. W. and Meissner,

G. (2005). Calmodulin regulation and identification of calmodulin binding region of

type-3 ryanodine receptor calcium release channel. Biochemistry 44, 15074-15081.

Yuchi, Z., Lau, K. and Van Petegem, F. (2012). Disease mutations in the ryanodine

receptor central region: crystal structures of a phosphorylation hot spot domain.

Structure 20, 1201-1211.

Zhang, H., Zhang, J.-Z., Danila, C. I. and Hamilton, S. L. (2003a). A noncontiguous,

intersubunit binding site for calmodulin on the skeletal muscle Ca2+ release channel.

J. Biol. Chem. 278, 8348-8355.

Zhang, J., Liu, Z., Masumiya, H., Wang, R., Jiang, D., Li, F., Wagenknecht, T. and

Chen, S. R. W. (2003b). Three-dimensional localization of divergent region 3 of the

ryanodine receptor to the clamp-shaped structures adjacent to the FKBP binding sites.

J. Biol. Chem. 278, 14211-14218.

Zhao, M., Li, P., Li, X., Zhang, L., Winkfein, R. J. and Chen, S. R. W. (1999).

Molecular identification of the ryanodine receptor pore-forming segment. J. Biol.

Chem. 274, 25971-25974.

Zhu, X., Ghanta, J., Walker, J. W., Allen, P. D. and Valdivia, H. H. (2004). The

calmodulin binding region of the skeletal ryanodine receptor acts as a self-modulatory

domain. Cell Calcium 35, 165-177.

Zhu, L., Zhong, X., Chen, S. R. W., Banavali, N. and Liu, Z. (2013). Modeling a

ryanodine receptor N-terminal domain connecting the central vestibule and the corner

clamp region. J. Biol. Chem. 288, 903-914.

Mapping CaM-binding sequences in RyR 4535

http://dx.doi.org/10.1074/jbc.272.39.24234http://dx.doi.org/10.1074/jbc.272.39.24234http://dx.doi.org/10.1074/jbc.272.39.24234http://dx.doi.org/10.1023/A:1005635330773http://dx.doi.org/10.1023/A:1005635330773http://dx.doi.org/10.1023/A:1005635330773http://dx.doi.org/10.1074/jbc.M113.463299http://dx.doi.org/10.1074/jbc.M113.463299http://dx.doi.org/10.1074/jbc.M113.463299http://dx.doi.org/10.1074/jbc.M113.463299http://dx.doi.org/10.1021/bi00015a024http://dx.doi.org/10.1021/bi00015a024http://dx.doi.org/10.1021/bi00015a024http://dx.doi.org/10.1021/bi00015a024http://dx.doi.org/10.1074/jbc.M112.383109http://dx.doi.org/10.1074/jbc.M112.383109http://dx.doi.org/10.1074/jbc.M112.383109http://dx.doi.org/10.1042/BJ20071257http://dx.doi.org/10.1042/BJ20071257http://dx.doi.org/10.1042/BJ20071257http://dx.doi.org/10.1073/pnas.96.5.1915http://dx.doi.org/10.1073/pnas.96.5.1915http://dx.doi.org/10.1073/pnas.96.5.1915http://dx.doi.org/10.1085/jgp.118.1.33http://dx.doi.org/10.1085/jgp.118.1.33http://dx.doi.org/10.1074/jbc.M208124200http://dx.doi.org/10.1074/jbc.M208124200http://dx.doi.org/10.1074/jbc.M208124200http://dx.doi.org/10.1242/jcs.064071http://dx.doi.org/10.1242/jcs.064071http://dx.doi.org/10.1242/jcs.064071http://dx.doi.org/10.1242/jcs.064071http://dx.doi.org/10.1021/bi00197a008http://dx.doi.org/10.1021/bi00197a008http://dx.doi.org/10.1021/bi00197a008http://dx.doi.org/10.1074/jbc.M704474200http://dx.doi.org/10.1074/jbc.M704474200http://dx.doi.org/10.1074/jbc.M704474200http://dx.doi.org/10.1074/jbc.M704474200http://dx.doi.org/10.1021/bi9907431http://dx.doi.org/10.1021/bi9907431http://dx.doi.org/10.1021/bi9907431http://dx.doi.org/10.1021/bi0005660http://dx.doi.org/10.1021/bi0005660http://dx.doi.org/10.1021/bi0005660http://dx.doi.org/10.1074/jbc.M109196200http://dx.doi.org/10.1074/jbc.M109196200http://dx.doi.org/10.1371/journal.pbio.1000085http://dx.doi.org/10.1371/journal.pbio.1000085http://dx.doi.org/10.1074/jbc.273.29.18429http://dx.doi.org/10.1074/jbc.273.29.18429http://dx.doi.org/10.1074/jbc.273.29.18429http://dx.doi.org/10.1111/j.1742-4658.2012.08755.xhttp://dx.doi.org/10.1111/j.1742-4658.2012.08755.xhttp://dx.doi.org/10.1111/j.1742-4658.2012.08755.xhttp://dx.doi.org/10.1016/j.ceca.2004.01.013http://dx.doi.org/10.1016/j.ceca.2004.01.013http://dx.doi.org/10.1038/339439a0http://dx.doi.org/10.1038/339439a0http://dx.doi.org/10.1038/339439a0http://dx.doi.org/10.1038/339439a0http://dx.doi.org/10.1074/jbc.M112.427864http://dx.doi.org/10.1074/jbc.M112.427864http://dx.doi.org/10.1074/jbc.M112.427864http://dx.doi.org/10.1016/S0006-3495(95)79880-0http://dx.doi.org/10.1016/S0006-3495(95)79880-0http://dx.doi.org/10.1016/S0006-3495(95)79880-0http://dx.doi.org/10.1038/nature09471http://dx.doi.org/10.1038/nature09471http://dx.doi.org/10.1038/nature09471http://dx.doi.org/10.1074/jbc.272.51.32463http://dx.doi.org/10.1074/jbc.272.51.32463http://dx.doi.org/10.1074/jbc.272.51.32463http://dx.doi.org/10.1074/jbc.272.51.32463http://dx.doi.org/10.1074/jbc.M700660200http://dx.doi.org/10.1074/jbc.M700660200http://dx.doi.org/10.1074/jbc.M700660200http://dx.doi.org/10.1074/jbc.M700660200http://dx.doi.org/10.1074/jbc.M110.194316http://dx.doi.org/10.1074/jbc.M110.194316http://dx.doi.org/10.1074/jbc.M110.194316http://dx.doi.org/10.1074/jbc.M110.194316http://dx.doi.org/10.1074/jbc.M110.194316http://dx.doi.org/10.1074/jbc.M206763200http://dx.doi.org/10.1074/jbc.M206763200http://dx.doi.org/10.1074/jbc.M206763200http://dx.doi.org/10.1016/S0006-3495(04)74155-7http://dx.doi.org/10.1016/S0006-3495(04)74155-7http://dx.doi.org/10.1016/S0006-3495(04)74155-7http://dx.doi.org/10.1074/jbc.M102729200http://dx.doi.org/10.1074/jbc.M102729200http://dx.doi.org/10.1074/jbc.M102729200http://dx.doi.org/10.1074/jbc.M301125200http://dx.doi.org/10.1074/jbc.M301125200http://dx.doi.org/10.1074/jbc.M301125200http://dx.doi.org/10.1021/bi051251thttp://dx.doi.org/10.1021/bi051251thttp://dx.doi.org/10.1021/bi051251thttp://dx.doi.org/10.1016/j.str.2012.04.015http://dx.doi.org/10.1016/j.str.2012.04.015http://dx.doi.org/10.1016/j.str.2012.04.015http://dx.doi.org/10.1074/jbc.M209565200http://dx.doi.org/10.1074/jbc.M209565200http://dx.doi.org/10.1074/jbc.M209565200http://dx.doi.org/10.1074/jbc.M213164200http://dx.doi.org/10.1074/jbc.M213164200http://dx.doi.org/10.1074/jbc.M213164200http://dx.doi.org/10.1074/jbc.M213164200http://dx.doi.org/10.1074/jbc.274.37.25971http://dx.doi.org/10.1074/jbc.274.37.25971http://dx.doi.org/10.1074/jbc.274.37.25971http://dx.doi.org/10.1016/j.ceca.2003.09.002http://dx.doi.org/10.1016/j.ceca.2003.09.002http://dx.doi.org/10.1016/j.ceca.2003.09.002http://dx.doi.org/10.1074/jbc.M112.429670http://dx.doi.org/10.1074/jbc.M112.429670http://dx.doi.org/10.1074/jbc.M112.429670

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/CreateJDFFile false /SyntheticBoldness 1.000000 /Description >>> setdistillerparams> setpagedevice