Three-dimensional Localization of Divergent Region 3 of the Ryanodine Receptor to the Clamp-shaped Structures Adjacent to the FKBP Binding Sites*

Jing ZhangDagger , Zheng Liu§, Haruko MasumiyaDagger , Ruiwu WangDagger , Dawei JiangDagger ||, Fei Li§, Terence Wagenknecht§**, and S. R. Wayne ChenDagger DaggerDagger

From the Dagger  Cardiovascular Research Group, Departments of Physiology and Biophysics and of Biochemistry and Molecular Biology, University of Calgary, Calgary, Alberta T2N 4N1, Canada, the § Wadsworth Center, New York State Department of Health, Albany, New York 12201, and the ** Department of Biomedical Sciences, School of Public Health, State University of New York, Albany, New York 12201

Received for publication, December 26, 2002, and in revised form, February 5, 2003

    ABSTRACT
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ABSTRACT
INTRODUCTION
EXPERIMENTAL PROCEDURES
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DISCUSSION
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Of the three divergent regions of ryanodine receptors (RyRs), divergent region 3 (DR3) is the best studied and is believed to be involved in excitation-contraction coupling as well as in channel regulation by Ca2+ and Mg2+. To gain insight into the structural basis of DR3 function, we have determined the location of DR3 in the three-dimensional structure of RyR2. We inserted green fluorescent protein (GFP) into the middle of the DR3 region after Thr-1874 in the sequence. HEK293 cells expressing this GFP-RyR2 fusion protein, RyR2T1874-GFP, were readily detected by their green fluorescence, indicating proper folding of the inserted GFP. RyR2T1874-GFP was further characterized functionally by assays of Ca2+ release and [3H]ryanodine binding. These analyses revealed that RyR2T1874-GFP functions as a caffeine- and ryanodine-sensitive Ca2+ release channel and displays Ca2+ dependence and [3H]ryanodine binding properties similar to those of the wild type RyR2. RyR2T1874-GFP was purified from cell lysates in a single step by affinity chromatography using GST-FKBP12.6 as the affinity ligand. The three-dimensional structure of the purified RyR2T1874-GFP was then reconstructed using cryoelectron microscopy and single particle image analysis. Comparison of the three-dimensional reconstructions of wild type RyR2 and RyR2T1874-GFP revealed the location of the inserted GFP, and hence the DR3 region, in one of the characteristic domains of RyR, domain 9, in the clamp-shaped structure adjacent to the FKBP12 and FKBP12.6 binding sites. COOH-terminal truncation analysis demonstrated that a region between 1815 and 1855 near DR3 is essential for GST-FKBP12.6 binding. These results provide a structural basis for the role of the DR3 region in excitation-contraction coupling and in channel regulation.

    INTRODUCTION
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ABSTRACT
INTRODUCTION
EXPERIMENTAL PROCEDURES
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DISCUSSION
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The ryanodine receptor (RyR),1 located in the sarco(endo)plasmic reticulum of a variety of cells, functions as an intracellular Ca2+ release channel and plays an essential role in various fundamental cellular processes including muscle contraction and relaxation, fertilization, and apoptosis (1). Three RyR isoforms, RyR1, RyR2, and RyR3, have been identified in mammalian tissues and have been shown to display distinct patterns of expression. RyR1 is predominantly expressed in skeletal muscle, whereas RyR2 is mainly expressed in cardiac muscle and the brain. RyR3 expression is widespread but at relatively low levels (2-5). The physiological function and mechanism of activation are also specific to each RyR isoform. RyR1 is required for excitation-contraction (EC) coupling in skeletal muscle, in which RyR1 is activated upon depolarization by the voltage sensor, the L-type Ca2+ channel (dihydropyridine receptor, DHPR), through a direct physical interaction. RyR2, on the other hand, mediates EC coupling in cardiac muscle, and it is activated by Ca2+ influx through the L-type Ca2+ channel via a mechanism known as Ca2+-induced Ca2+ release. Importantly, neither RyR2 nor RyR3 is capable of substituting for RyR1 in mediating skeletal type EC coupling (2, 6-9). The physiological role of RyR3 is unclear, but RyR3 has been shown to be involved in amplification of the Ca2+ signal generated by RyR1 in skeletal muscle (3, 10) and in regulation of the Ca2+ release activity of RyR1 or RyR2 in smooth muscle (11). Some of these functional differences among RyR isoforms are likely to be attributable to differences in their primary sequences. One of our goals is to determine which differences in the amino acid sequences of the isoforms are responsible for these functional variations.

The amino acid sequences of the three RyR isoforms share a high degree of sequence identity (66-70%) with their major variations occurring in three regions, known as divergent regions 1, 2, and 3 (DR1, DR2, and DR3) (12). The DR1 region comprises residues 4254-4631 of RyR1 and residues 4210-4562 of RyR2. The DR2 region includes residues 1342-1403 of RyR1 and residues 1353-1397 of RyR2. Residues 1872-1923 of RyR1 and residues 1852-1890 of RyR2 are denoted as the DR3 region. These divergent regions have been the focus of a number of structural and functional studies. For instance, sequence variations in the DR1 region between RyR1 and RyR2 have been shown to account for the isoforms' different sensitivities to Ca2+ inactivation (13-15), whereas the DR2 region, which is absent in RyR3, was found to be critical for skeletal muscle EC coupling, since deletion of DR2 in RyR1 abolished EC coupling (16).

The DR3 region appears to have multiple roles in RyR channel function. In RyR1, DR3 consists of a stretch of ~30 negatively charged amino acid residues that are thought to constitute a low affinity Ca2+ binding site (17). Consistent with this hypothesis, deletion of the DR3 region altered the Ca2+- and Mg2+-dependent regulation and conduction properties of the RyR1 channel (18-20). A region of RyR1 between residues 1837 and 2168, encompassing the DR3 region, has also been shown to interact with the II-III loop of the DHPR and to participate in EC coupling (21). Interestingly, the interaction between a sequence in the II-III loop of DHPR and RyR1 was enhanced by the 12-kDa FK506-binding protein (FKBP12), suggesting that FKBP12 may modulate EC coupling in skeletal muscle (22). Recently, we have shown that an NH2-terminal fragment containing the first 1937 residues of RyR2 is sufficient to enable GST-FKBP12.6 binding, whereas a shorter NH2-terminal fragment including only the first 1636 residues is unable to bind GST-FKBP12.6, indicating that the region between residues 1636 and 1937 of RyR2, encompassing the DR3 region, is required for FKBP12.6 interaction (23). Whether the corresponding region in RyR1 is essential for FKBP12-RyR1 interaction has yet to be determined. Taken together, these observations suggest that residues within or near the DR3 region may be involved in EC coupling and in channel regulation by Ca2+, Mg2+, or FKBPs. The molecular mechanisms by which the DR3 region participates in such seemingly diverse functions are, however, unknown.

One approach to understanding the roles of individual regions or domains in RyR channel function is to map their three-dimensional locations in the channel structure. The three-dimensional structures of all three RyR isoforms have been determined to ~30-40 Å by cryoelectron microscopy (cryo-EM) combined with single particle image processing (24, 25). The three RyR isoforms display virtually identical three-dimensional structures, as expected based on their high degree of sequence identity. The three-dimensional structure of RyR is essentially composed of two major components: a cytoplasmic assembly and a transmembrane assembly. The cytoplasmic assembly contains at least 10 domains (26). Large conformational changes, particularly in the clamp-shaped structure that includes domains 5, 6, 9, and 10, and is located at the corners of the cytoplasmic assembly, have been observed when the channel was switched from the closed to the open state (25, 27). The three-dimensional locations of several ligand binding sites, including those for calmodulin, FKBP12, FKBP12.6, and Imperatoxin A, have been identified (24). We have recently mapped the three-dimensional locations of the NH2-terminal and DR1 regions. The NH2 terminus is located at the corners within the clamp structure, whereas the DR1 region maps to domain 3, also known as the "handle" domain, adjacent to the calmodulin binding site (28, 29).

In the present study, in order to gain insight into the structural basis and functions of the DR3 region, we have constructed and expressed in HEK293 cells a RyR2 fusion protein in which green fluorescence protein (GFP) was inserted into the DR3 region after residue Thr-1874 in the sequence (RyR2T1874-GFP). The RyR2T1874-GFP protein was purified and then subjected to cryo-EM and single-particle image processing. Three-dimensional reconstruction of RyR2T1874-GFP revealed the localization of GFP, and therefore of DR3, to a region in domain 9 in the clamp-shaped structure, adjacent to the known FKBP12 and FKBP12.6 binding sites. These results suggest a role of the DR3 region in conformational changes and in EC coupling, and they further demonstrate the utility of this approach in correlating linear sequence to three-dimensional structure, thereby allowing an understanding of the structural basis of RyR function.

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Materials-- Restriction endonucleases and DNA-modifying enzymes were purchased from New England BioLabs Inc. The anti-RyR and anti-GFP antibodies were obtained from Affinity BioReagents (Golden, CO). The anti-c-Myc antibody was kindly provided by the Immunology Core Facility at the Wadsworth Center of the New York State Department of Health. Soybean phosphatidylcholine was obtained from Avanti Polar Lipids, Inc. CHAPS and other reagents were purchased from Sigma.

Cell Culture and DNA Transfection-- HEK293 cells were maintained in Dulbecco's modified Eagle's medium as described previously (30). HEK293 cells grown on 100-mm tissue culture dishes for 18-20 h after subculture were transfected with 6-12 µg of wild type (WT) or mutant RyR cDNAs using Ca2+ phosphate precipitation (31).

Construction of RyR2T1874-GFP-- The cloning and construction of the 15-kb full-length cDNA encoding the mouse cardiac RyR2 has been described previously (32). The DNA encoding GFP flanked by Gly-rich spacers and an AscI site was obtained by PCR as described previously (29). The AscI site was introduced into the DR3 region of RyR2 after Thr-1874 in the sequence by overlap-extension using PCR (33). The "outer" primers used were as follows: forward, 5'-GATCCTCTGCAGTTCATGTCCCTC-3'; reverse, 5'-TCCATCACTGTCTCATGCATCCC-3'. The primers for introducing the AscI site were as follows: forward, 5'-AGTATAGAAGACGGGCGCGCCGAAGGCGAAGAAGAAGCC-3'; reverse, 5'-TTCTTCGCCTTCGGCGCGCCCGTCTTCTATACTGATCTC-3'. The Bsu36I (4900)-EcoRV (6439) PCR fragment containing the AscI site was used to replace the Bsu36I (4900)-EcoRV (13,873) fragment in the full-length RyR2. The missing EcoRV (6439)-EcoRV (13,873) fragment was then added back to yield the full-length RyR2 with the inserted AscI site. The AscI-AscI fragment containing GFP and the spacers was then subcloned into the full-length RyR2 at the introduced AscI site. The sequences of all PCR fragments and the orientation of the inserted GFP cDNA were verified by DNA sequencing analysis.

Construction of COOH-terminal Truncation Mutants of RyR2-- Construction of the 1-1937 and 1-1636 COOH-terminal truncation mutants was described earlier (23). PCR was used to introduce a stop codon after residues 1895, 1855, 1815, 1775, and 1735 to generate the COOH-terminal truncation mutants, 1-1895, 1-1855, 1-1815, 1-1775, and 1-1735, respectively. The forward primer used for all truncation mutants was 5'-GATCCTCTGCAGTTCATGTCCCTC-3'. The reverse primers, each containing a stop codon followed by an EcoRV site were 5'-ATGATATCTCACATCTGGAGCAAGCCTTCCTT-3' (1895-stop), 5'-ATGATATCTCAAGCTTCCTTAAACACACTGGG-3' (1855-stop), 5'-ATGATATCTCACGTGGTCCCACCCACAGGATC-3' (1815-stop), 5'-GATATCCTAGTCGTTGCTAATGCTCACGA-3' (1775-stop), and 5'-GATATCCTACTTTGTCTCCTCTGTCATAG-3' (1735-stop). The Bsu36I (4900)-EcoRV (introduced after the stop codon at various sites) PCR fragments were used to replace the Bsu36I (4900)-EcoRV (13,873) fragment in the full-length RyR2 cDNA to yield the various COOH-terminal truncation mutants. All truncation mutants were confirmed by DNA sequencing.

Cryoelectron Microscopy and Image Processing-- The expression and purification of RyR2T1874-GFP was carried out as described previously (29). The purified RyR2T1874-GFP was diluted 5-10-fold with EM dilution buffer (20 mM Na-PIPES, (pH 7.2), 400 mM KCl, 3 mM EGTA, 0.5% CHAPS, 2 mM dithiothreitol, and 2 mg/ml leupeptin). Grids were prepared for cryo-EM according to standard methods (34). Micrographs were recorded using low dose protocols on a Philips EM420, equipped with low dose kit and a GATAN 626 cryotransfer holder, at a magnification of 38,600 × (±2%) as verified by a tobacco mosaic virus standard. Each exposure corresponded to an electron dose of ~10 e-2. Micrographs were checked for drift, astigmatism, and presence of Thon rings by optical diffraction. Selected electron micrographs were digitized on a Zeiss/Imaging scanner (Z/I Imaging Corp., Huntsville, AL) with a step size of 14 µm. Images were processed using the SPIDER/WEB software package (35), and three-dimensional reconstructions were obtained through use of the projection matching procedure (29, 36). The final three-dimensional reconstructions of WT RyR2 and RyR2T1874-GFP were computed, respectively, from 4365 and 4374 particles. 4-Fold symmetry was enforced in both three-dimensional reconstructions. The final resolutions of both reconstructions were estimated to be 34 Å by Fourier shell correlation using a cut-off value of 0.5 (37). The difference map was calculated by subtracting the three-dimensional volume of the WT RyR2 from the RyR2T1844-GFP volume.

Ca2+ Release and [3H]Ryanodine Binding Assays-- Measurements of free cytosolic Ca2+ concentrations in the transfected HEK293 cells, using fluorescence Ca2+ indicator dye fluo-3, and of equilibrium [3H]ryanodine binding to cell lysate were done as described previously (38).

GST-FKBP12.6 Pull-down, Immunoprecipitation, and Immunoblotting Analyses-- GST-FKBP12.6 pull-down, immunoprecipitation, and immunoblotting were carried out as described previously (23, 29).

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Insertion of Green Fluorescent Protein into the DR3 Region of RyR2-- To understand how the DR3 region participates in channel function and regulation, we attempted to map the location of the DR3 region onto the three-dimensional structure of RyR2. To this end, we constructed a RyR2 fusion protein (RyR2T1874-GFP) in which GFP was inserted into the middle of the DR3 region, after residue Thr-1874 in the sequence (Fig. 1). The strategy was to use GFP, a relatively small protein (238 amino acids, compared with the ~5000 amino acids of RyR), as a structural marker (39, 40). Through comparison of the three-dimensional reconstructions of the WT RyR2 and GFP-inserted RyR2, it would then be feasible to map the location of GFP and thus the region into which GFP was inserted. To minimize potential perturbation of the channel folding and function of RyR2, we added two glycine-rich spacers on either side of GFP (40).


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Fig. 1.   Insertion of GFP into RyR2 after residue Thr-1874 in the sequence. The amino acid sequences of the three RyR isoforms differ primarily in three regions known as the divergent regions (DR1, DR2, and DR3) shown by the shaded areas. GFP flanked by two Gly-rich spacers was inserted into the DR3 region after Thr-1874, as indicated by a filled box. The linear sequence of RyR2 is denoted by an open rectangle. The hatched area depicts the transmembrane domain (TM). The phosphorylation site (S2808), the calmodulin binding site (CaM; 3614-3643), the proposed Ca2+ sensor (E3987), and the proposed pore-forming segment (4820-4829) are also shown.

Expression and Functional Characterization of RyR2T1874-GFP-- Since the fluorescence of GFP depends on the proper folding of its own structure and the structure of the inserted region (40, 41), GFP could be used not only as a structural marker but also as an indicator of proper protein folding. Fig. 2A shows phase-contrast and fluorescent microscopic images of the HEK293 cells transfected with RyR2T1874-GFP. Green fluorescent HEK293 cells were readily detected after transfection (Fig. 2A, a), whereas no fluorescence was observed in HEK293 cells transfected with the WT RyR2 (data not shown). The green fluorescence indicates that the inserted GFP and its neighboring region are likely to have folded properly. A reticular pattern of distribution of RyR2T1874-GFP was observed in most of the fluorescent cells, as expected based on its function as an intracellular Ca2+ release channel (Fig. 2B). In some cells, however, the fluorescence appeared as an amorphous mass due to heavy expression of the RyR2T1874-GFP protein.


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Fig. 2.   Expression and functional characterization of RyR2T1874-GFP. HEK293 cells grown on glass coverslips were transfected with RyR2T1874-GFP cDNA. Transfected cells were fixed with 1% formaldehyde 24 h after transfection. Fluorescence (a) and phase-contrast images (b) were recorded under the fluorescence microscope at ×40 magnification (A). B, HEK293 cells were transfected with 12 µg of RyR2T1874-GFP cDNA. Fluorescence intensity of the fluo-3-loaded transfected cells was monitored continuously before and after the addition of 2.5 mM caffeine (C) or 50 µM of ryanodine (R). The sharp decreases in fluorescence intensity immediately after the second and third additions of caffeine were due to fluorescence quenching by caffeine. Similar results were obtained from three separate experiments.

To further demonstrate that RyR2T1874-GFP retains proper folding, we assessed its functional properties. We measured Ca2+ release from transfected HEK293 cells using the fluorescent Ca2+ indicator dye fluo-3. As shown in Fig. 2B, HEK293 cells transfected with RyR2T1874-GFP cDNA displayed multiple Ca2+ release events in response to repeated stimulation by caffeine (Fig. 2B, a), an activator of RyRs. Transfected cells pretreated with ryanodine, on the other hand, responded only to the first and not the second or third caffeine stimulation (Fig. 2B, b), suggesting that RyR2T1874-GFP is sensitive to ryanodine modification (42). The immediate drop in fluorescence after the second and third caffeine stimulations was caused by fluorescence quenching by caffeine (42). Similar Ca2+ release responses to caffeine and ryanodine were observed in HEK293 cells transfected with WT RyR2 (42). No caffeine- or ryanodine-induced Ca2+ release was detected in nontransfected HEK293 cells or in cells transfected with pCDNA3 vector DNA (data not shown). Thus, RyR2T1874-GFP is able to form a caffeine- and ryanodine-sensitive Ca2+ release channel in HEK293 cells.

The functional properties of RyR2T1874-GFP were further characterized by [3H]ryanodine binding studies, which have been widely used as a functional assay for RyR channel activity, since ryanodine has access to its binding site only when the channel is open (43). Fig. 3A shows [3H]ryanodine binding to a cell lysate prepared from HEK293 cells transfected with RyR2T1874-GFP in the presence of a wide range of Ca2+ concentrations. Analysis of the Ca2+ dependence of [3H]ryanodine binding by the Hill equation yielded an EC50 of 0.20 ± 0.02 µM (mean ± S.E., n = 3). This EC50 value is similar to that reported for the WT RyR2 (29). We also performed binding in the presence of various concentrations of [3H]ryanodine (Fig. 3B). Scatchard analysis of these binding data revealed that RyR2T1874-GFP exhibited high affinity [3H]ryanodine binding with a Kd of 2.75 ± 0.43 nM (n = 3), which is also similar to the behavior of [3H]ryanodine binding to WT RyR2 reported previously (38). No ryanodine binding activity was detected in lysates from HEK293 cells transfected with either pCDNA3 vector DNA or no DNA. Taken together, these results suggest that the insertion of GFP after Thr-1874 does not significantly alter the structure or function of RyR2.


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Fig. 3.   Ca2+ dependence and [3H]ryanodine binding properties of RyR2T1874-GFP. A, [3H]ryanodine binding to cell lysate prepared from HEK293 cells transfected with RyR2T1874-GFP was carried out at various concentrations of Ca2+ in the presence of 5 nM [3H]ryanodine. B, [3H]ryanodine binding to RyR2T1874-GFP transfected cell lysate was performed at various concentrations of [3H]ryanodine in the presence of 100 µM Ca2+. Data shown are from representative experiments, each repeated three times. The inset in B shows a Scatchard plot. The binding affinity (Kd) is 2.75 ± 0.43 (mean ± S.E., n = 3). The binding density (Bmax) is 0.25 ± 0.06 pmol/mg protein (mean ± S.E., n = 3).

Purification of Recombinant RyR2T1874-GFP Protein by GST-FKBP12.6 Affinity Chromatography-- RyR2T1874-GFP retains the ability to interact with GST-FKBP12.6. Taking advantage of this property, we were able to purify the RyR2T1874-GFP protein from transfected HEK293 cell lysate by affinity chromatography in a single step using GST-FKBP12.6 as the affinity ligand. The purified protein was analyzed by SDS-PAGE and immunoblotting. A single high molecular weight band, which migrated at a slightly slower rate than the WT RyR2 (as expected due to the addition of GFP), was detected in the purified sample of RyR2T1874-GFP (Fig. 4A). This band was recognized by both the anti-RyR and anti-GFP antibodies and hence corresponds to RyR2T1874-GFP. On the other hand, the purified WT RyR2 protein was recognized only by the anti-RyR antibody and not by the anti-GFP antibody (Fig. 4, B and C). RyR2T1874-GFP appears to be expressed in an intact form; no degradation products were detected.


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Fig. 4.   Purification of RyR2T1874-GFP by GST-FKBP12.6 affinity chromatography. The RyR2T1874-GFP protein was purified from cell lysate by affinity chromatography using GST-FKBP12.6 as the affinity ligand. The purified RyR2T1874-GFP and WT RyR2 proteins were solubilized and separated in SDS-PAGE. The gel was then stained with Coomassie Brilliant Blue (CBB) (A). A similar SDS-PAGE gel was transferred to nitrocellulose membrane. The membrane was probed either with the anti-RyR antibody (B) or the anti-GFP antibody (C) for Western blotting. Note that the RyR2T1874-GFP protein migrated at a slightly slower rate in SDS-PAGE than did the WT RyR2, due to the addition of GFP. Also note that the anti-GFP antibody reacted with RyR2T1874-GFP but did not recognize the WT RyR2, indicating that the antibody is specific.

Cryo-EM and Three-dimensional Reconstruction of RyR2T1874-GFP-- The purified RyR2T1874-GFP proteins were preserved in a thin layer of vitreous ice and imaged by cryo-EM. Fig. 5 shows a typical electron micrograph of frozen-hydrated RyR2T1874-GFP in which individual RyR2T1874-GFP particles are readily visualized. The particles displayed an apparently intact structure with multiple orientations similar to those observed in the WT RyR2 (29).


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Fig. 5.   Cryoelectron microscopy of RyR2T1874-GFP. A portion of a cryo-EM micrograph of the purified RyR2T1874-GFP proteins embedded in a thin layer of vitreous ice is shown. The tetrameric structure of RyR2T1874-GFP is well preserved. Several individual RyR2T1874-GFP particles are marked with white circles. Scale bar, 500 Å.

Single particle image processing was then applied to obtain a more detailed structure of RyR2T1874-GFP. Fig. 6A shows a surface representation of the three-dimensional reconstruction of RyR2T1874-GFP in three orientations (top, bottom, and side views). The overall form of the reconstructed RyR2T1874-GFP structure resembles a mushroom in shape and consists of two major components: a large squarelike cytoplasmic assembly (290 × 290 × 130 Å) encompassing 10 distinct domains (labeled by numerals in Fig. 6) and a smaller transmembrane assembly (120 × 120 × 70 Å, labeled TA).


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Fig. 6.   Three-dimensional surface representation of RyR2T1874-GFP and difference map. A, the three-dimensional reconstruction of RyR2T1874-GFP is shown in green. B, difference map (RyR2T1874GFP - WT RyR2) shown in green is superimposed on the three-dimensional reconstruction of the WT RyR2 (in blue). The asterisk indicates the equivalent location of the FKBP12 binding site. The three-dimensional reconstructions are shown in three views. Left, top views from the cytoplasmic surface, which in situ would face the transverse-tubule; middle, views toward the bottom of the receptor (i.e. as it would appear if viewed from the lumen of the sarcoplasmic reticulum; right, side views. The numerals on the cytoplasm assembly indicate the distinguishable domains.

Overall, the three-dimensional reconstruction of RyR2T1874-GFP is very similar to that of the recombinant WT RyR2 obtained previously (29), but close examination reveals some small differences. The most noticeable difference was found on domain 9, one of the domains located within the "clamp" structures that form each of the corners of the RyR2 square-shaped cytoplasmic assembly (25, 27). Specifically, the volume of domain 9 of RyR2T1874-GFP appears to be larger than the volume in the corresponding domain in WT RyR2. These differences could result either directly from the GFP insertion or from conformational changes caused by GFP insertion. To more precisely determine the differences, we generated a three-dimensional difference map by subtracting the three-dimensional volume of WT RyR2 from that of RyR2T1844-GFP. The difference regions were displayed in green and superimposed on the three-dimensional reconstruction of WT RyR2 (shown in blue) using nearly the same threshold as was used in the display of RyR2T1874-GFP and WT RyR2 (Fig. 6B). This difference map clearly indicates four significant differences located in each domain 9 in the cytoplasmic assembly. This significant difference, repeated four times in the difference map, is not only due to the 4-fold symmetry enforcement, since RyR2 is a homotetramer composed of four identical monomers. Because GFP was inserted into each RyR2 monomer, the difference as a result of GFP insertion would be expected to repeat four times in the three-dimensional difference map.

We believe that these differences are directly attributable to the excess mass contributed by the GFP insertion in RyR2T1874-GFP, because they are the only significant differences that appear when the three-dimensional difference map is displayed at a density threshold nearly the same as used to image the RyR2T1874-GFP and WT RyR2 structures. Furthermore, the calculated volume of each of the four difference features in Fig. 6B corresponds to a molecular mass of 28 kDa, assuming a protein density of 1.37 g/cm3 (26), and this volume agrees well with the molecular mass of GFP. Other minor differences are small and insignificant, both in volume and density, and are unlikely to correspond to the inserted GFP. Hence, we conclude that the DR3 region, as indicated by the inserted GFP, is located in domain 9, one of the characteristic domains that form the cytoplasmically located clamp structures of RyR2.

The FKBP12 binding site was previously mapped to a region at or near the junction of domains 3 and 9 in the three-dimensional structure of RyR1 (indicated by an asterisk in Fig. 6B) (44, 45). Recently, the FKBP12.6 binding site has also been mapped to a similar region in the three-dimensional structure of RyR2.2 Interestingly, the three-dimensional location of the DR3 region, as indicated by the localization of the inserted GFP, is in close proximity to the FKBP12 and FKBP12.6 binding sites.2 The distances between the DR3 location and the binding sites for FKBP12 and FKBP12.6 are ~3 and 2.5 nm, respectively. The close localization of the DR3 region and FKBP binding sites in the three-dimensional structure of RyR suggests that residues near the DR3 region may be involved in FKBP12/12.6 interaction.

A Region Upstream of DR3 Is Essential for FKBP12.6 Binding-- We have previously shown that the first 1937 NH2-terminal amino acid residues of RyR2, a span that includes the DR3 region, are sufficient for FKBP12.6 binding (23). To examine the role of the DR3 region in FKBP12.6 interaction, we constructed a series of c-Myc-tagged COOH-terminal truncation mutants by inserting a stop codon between residues 1636 and 1937 (Fig. 7A). These COOH-terminal truncation mutants were then expressed in HEK293 cells and their expression was ascertained by immunoprecipitation using the anti-c-Myc antibody, whereas their ability to interact with FKBP12.6 was assessed by a GST-FKBP12.6 pull-down assay. As shown in Fig. 7B, NH2-terminal fragments containing the first 1815 or fewer residues are unable to bind GST-FKBP12.6, whereas NH2-terminal fragments including the first 1855 or more residues are sufficient for FKBP12.6 binding. These data indicate that the DR3 region (1852-1890) is unlikely to be critical for FKBP12.6 binding, whereas a region between residues 1815 and 1855 upstream of DR3 is essential for FKBP12.6 interaction. The GST-FKBP12.6 binding-deficient NH2-terminal fragment, 1-1815, is able to restore the function of an NH2-terminal truncation mutant, D1-1636 (Fig. 8A). HEK293 cells transfected with the 1-1815 NH2-terminal fragment and the D1-1636 NH2-terminal truncation mutant alone exhibited no caffeine-sensitive Ca2+ release activity, whereas caffeine-induced Ca2+ release was readily observed in cells co-transfected with both the 1-1815 and D1-1636 fragments (Fig. 8B). These observations indicate that the 1-1815 NH2-terminal fragment is functional when co-expressed with an overlapping COOH-terminal fragment and that the lack of GST-FKBP12.6 binding to this fragment is unlikely to result from abnormal expression or gross structural alterations.


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Fig. 7.   GST-FKBP12.6 binding to COOH-terminal truncation mutants of RyR2. A, COOH-terminal truncation mutants are depicted by rectangles (constructs 1-7). The remaining NH2-terminal residues of each COOH-terminal truncation mutant are shown on the left of the corresponding rectangle. All truncation mutants were tagged with the c-Myc antibody epitope near the NH2 terminus, as indicated by small open boxes. B, HEK293 cells were transfected with truncation mutants as indicated. The c-Myc-tagged mutant proteins were precipitated from cell lysates by the anti-c-Myc antibody and by GST-FKBP12.6 glutathione-Sepharose. The precipitates were solubilized and separated in SDS-PAGE and stained with Coomassie Brilliant Blue. The expression of and GST-FKBP12.6 binding to mutants 1-1937 and 1-1636 have been shown previously (23).


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Fig. 8.   Co-expression of overlapping NH2- and COOH-terminal fragments leads to the synthesis of functional RyR2 channels in HEK293 cells. The NH2-terminal fragment containing residues 1-1815 and the COOH-terminal fragment missing the first 1636 NH2-terminal residues are shown by filled boxes in A. B, HEK293 cells were transfected with NH2-terminal fragment 1-1815 and the COOH-terminal fragment D1636 either individually (12 µg each) (Ba and Bb) or in combination (8 µg each) (Bc). Fluorescence intensity of the fluo-3-loaded transfected cells was monitored continuously before and after the addition of 2.5 mM caffeine, indicated by C. A transient increase in fluorescence was detected only in cells co-transfected with mutants 1-1815 and D1636, indicating caffeine-induced Ca2+ release from intracellular stores. The drops in fluorescence signals immediately after the addition of caffeine in Ba and Bb were due to fluorescence quenching by caffeine. Similar results were obtained from three separate experiments.


    DISCUSSION
TOP
ABSTRACT
INTRODUCTION
EXPERIMENTAL PROCEDURES
RESULTS
DISCUSSION
REFERENCES

By applying cryo-EM and three-dimensional reconstruction to recombinant RyR2 containing a GFP insertion at position 1874, near the center of the divergent region, DR3, we have mapped DR3 to a subregion within domain 9 in the cytoplasmic assembly of the receptor. Also, new evidence has been presented to further implicate a region of the RyR2 sequence that is just upstream of the DR3 region as being required for the binding of the accessory protein, FKBP12.6. Consistent with this interpretation, previous mapping studies by cryo-EM of FKBP12-RyR1 and FKBP12.6-RyR2 complexes have placed the FKBP12/FKBP12.6 binding sites adjacent to domain 9 in both receptor isoforms. Although our mapping of the DR3 region was obtained on RyR2, we are confident that essentially the same result would have been obtained for RyR1 and RyR3, because the high degree of sequence identity (~70%) among the isoforms means that their tertiary structures should also be very similar. Indeed, the three-dimensional reconstructions that have been reported for the three RyR isoforms show strict conservation of the domain architecture of the cytoplasmic region and few significant differences at the 2-4-nm resolution level (24). Thus, we conclude that the DR3 region maps to domain 9 in all three RyR isoforms. Much of the following discussion focuses on the skeletal muscle receptor, RyR1, which has been more intensively characterized than RyR2, with respect to the function of DR3.

Implications for Excitation-Contraction Coupling in Skeletal Muscle-- Comparison of the three-dimensional reconstructions of the closed and open states of RyR1 reveals that several domains, including 5, 6, 9, and 10, in the clamp-shaped structures at the corners of the cytoplasmic assembly, undergo large conformational changes and/or movements (25, 27). Based on these findings and on the observation that the spacing between the receptor's clamp structures is similar to that of a group of four DHPRs (also known as the tetrad) that abut on RyR1 in situ at triad junctions, it has been proposed that the clamp structures undergo direct physical interaction with DHPRs (25, 27, 46). The amino acid sequences that make up domains 5, 6, 9, and 10 are, however, unknown. Several regions in the linear sequence of RyR1, including the DR2 and DR3 regions (16, 21), and regions between residues 1076 and 1112 (47) and between residues 2659 and 3720 (48) have been implicated as being important for EC coupling or for interactions between RyR1 and DHPR. Until now, it was not known with a high level of confidence which, if any, of these sequences is located in the clamp-shaped structure. This work reveals, for the first time, that the DR3 region forms part of domain 9, one of the domains comprising the clamp-shaped structure, and raises the possibility that DR3 is involved in the conformational changes in RyRs that are associated with channel gating. This interpretation is supported by comparative studies of RyR1 and RyR3 in closed and open states, which reveal structural differences between the two states in the vicinity of domain 9 (25, 27, 49). Thus, our observations further support the notion that domain 9 is important for EC coupling.

DR3 Is Located Near the FKBP12/FKBP12.6 Binding Sites on RyRs-- Our results are also suggestive of a structural link between EC coupling and the binding of FKBP12, which is tightly associated with RyR1 (50). FKBP12 has been suggested to be an important regulator of EC coupling, in part because FK506, an immunosuppressant drug that dissociates FKBP12 from RyR1, has been shown to impair EC coupling in skeletal muscle (51). Moreover, the interaction between RyR1 and the II-III loop of DHPR is apparently potentiated by FKBP12 (22). The molecular mechanism by which FKBP12 enhances DHPR-RyR1 interaction and modulates EC coupling is, however, unclear. We found that a region including residues 1815-1855, upstream of the DR3 region, is essential for GST-FKBP12.6 binding and that the three-dimensional location of the DR3 region is very close to the FKBP12 and FKBP12.6 binding sites. It is thus possible that FKBP12, by binding to a site near the DR3 region may either stabilize a conformation required for DR3-DHPR interaction, as suggested previously (22), or may stabilize the DR3-DHPR interaction directly.

DR3 and Regulation of RyRs by Ca2+ and Mg2+-- The DR3 region encompasses a highly negatively charged sequence that is thought to form the low affinity Ca2+ binding site. Deletion of the DR3 region affected both Ca2+-dependent activation and inactivation and Mg2+ inhibition of the RyR1 channel (17-20). Mg2+ is an essential regulator of EC coupling in skeletal muscle. It has been proposed that the initial activation of RyR1 by the voltage sensor during EC coupling involves the removal of the inhibitory effect of cytoplasmic Mg2+ (52). However, the molecular mechanisms underlying Mg2+ inhibition of RyR channel activity and EC coupling are unknown. Localization of the DR3 region, and consequently the putative Mg2+ inhibitory site, to domain 9 within the clamp structure suggests certain hypotheses as to the role of Mg2+ in conformational changes and in EC coupling. For instance, it is possible that Mg2+ binds to domain 9, stabilizing RyR1 in an inactive conformation, and that the II-III loop of DHPR may bind to the same domain, relieving Mg2+ inhibition and resulting in channel openings. Binding of Mg2+ to domain 9 may also affect the binding of FKBP, which binds adjacent to domain 9 (45). This may explain the observation that Mg2+ influences the interaction between FKBP and RyR (53, 54). It will be of great interest to determine whether mutations in the DR3 region, particularly among the negatively charged residues, alter Mg2+ inhibition and skeletal muscle EC coupling.

DR3 and Channel Conduction-- The DR3 region appears to be involved not only in channel gating but also in channel conduction, since deletion of the DR3 region has been shown to alter the conduction properties of RyR1 (20). The DR3 region is located along the edge of the cytoplasmic assembly, far away from the channel pore-forming region located in the transmembrane assembly, so how this region influences channel conduction is unclear. It is unlikely that the DR3 region is directly involved in the formation of the ion conduction pathway. It is, however, possible that conformational changes in the DR3 region may have long range effects on both gating and conduction.

In summary, domain 9, which is involved in the structural changes that occur upon channel activation and is also in close contact with bound FKBPs, appears to encompass, within its DR3-containing region, a binding site for the II-III loop of DHPR and a low affinity cation binding site for Ca2+ and Mg2+. Accordingly, we propose that domain 9 constitutes an essential part of the macromolecular apparatus that carries out EC coupling. It is probable that multiple regions or domains are involved in EC coupling. The most likely additional candidates for EC coupling are the DR2 region and the regions between residues 1076 and 1112 and between residues 2659 and 3720 or domains 5, 6, and 10, located within the clamp-shaped structure (16, 25, 27, 46-48). The DR2 region is completely missing in RyR3, and comparison of the three-dimensional reconstructions of RyR1 and RyR3 revealed a significant difference in domain 6, suggesting that domain 6 may include the DR2 region (49). The three-dimensional locations of residues 1076-1112 and residues 2659-3720 and the sequence identity of domains 5 and 10 are unknown. A systematic and comprehensive study by cryo-EM and single particle image processing of modified RyR proteins containing GFP insertions into various regions in RyR will allow us to further define the structural basis of EC coupling and other functions of the RyR channel.

    ACKNOWLEDGEMENTS

We thank the Wadsworth Center's molecular genetics, immunology, and electron microscopy core facilities and the Resource for Visualization of Biological Complexity, the Immunology Core Facility at the Wadsworth Center of the New York State Department of Health for providing the anti-c-Myc antibody, Dr. Wayne R. Giles for continued support, Cindy Brown for excellent technical assistance, and Jeff Bolstad for a critical reading of the manuscript.

    FOOTNOTES

* This work was supported by research grants from the Canadian Institutes of Health Research and the Heart and Stroke Foundation of Alberta, N.W.T. and Nunavut (to S. R. W. C.) and by the Muscular Dystrophy Association and National Institutes of Health Grant AR40615 (to T. W.). The Resource for Visualization of Biological Complexity was supported by National Institutes of Health Biotechnological Resource Grant RR01219.The costs of publication of this article were defrayed in part by the payment of page charges. The article must therefore be hereby marked "advertisement" in accordance with 18 U.S.C. Section 1734 solely to indicate this fact.

Supported by the Uehara Memorial Foundation (Japan) and Postdoctoral Fellowships from the Heart and Stroke Foundation of Canada and the Alberta Heritage Foundation for Medical Research (AHFMR).

|| Recipient of the Alex W. Church Graduate Student Award.

Dagger Dagger AHFMR Senior Scholar. To whom correspondence should be addressed. Tel.: 403-220-4235; Fax: 403-283-4841; E-mail: swchen@ ucalgary.ca.

Published, JBC Papers in Press, February 7, 2003, DOI 10.1074/jbc.M213164200

2 M. Sharma, L. Jeyakumar, S. Fleischer, and T. Wagenknecht, unpublished results.

    ABBREVIATIONS

The abbreviations used are: RyR, ryanodine receptor; CHAPS, 3-[(3-cholamidopropyl)-dimethylammonio]-1-propane sulfonate; EC, excitation-contraction; cryo-EM, cryoelectron microscopy; GFP, green fluorescent protein; PIPES, 1,4-piperazinediethanesulfonic acid.

    REFERENCES
TOP
ABSTRACT
INTRODUCTION
EXPERIMENTAL PROCEDURES
RESULTS
DISCUSSION
REFERENCES

1. Berridge, M. J., Lipp, P., and Bootman, M. D. (2000) Nat. Rev. Mol. Cell. Biol. 1, 11-21[CrossRef][Medline] [Order article via Infotrieve]
2. Franzini-Armstrong, C., and Protasi, F. (1997) Physiol. Rev. 77, 699-729[Abstract/Free Full Text]
3. Sutko, J. L., and Airey, J. A. (1996) Physiol. Rev. 76, 1027-1071[Abstract/Free Full Text]
4. Ogawa, Y., Kurebayashi, N., and Murayama, T. (1999) Adv. Biophys. 36, 27-64[CrossRef][Medline] [Order article via Infotrieve]
5. Giannini, G., Conti, A., Mammarella, S., Scrobogna, M., and Sorrentino, V. (1995) J. Cell Biol. 128, 893-904[Abstract]
6. Rios, E., and Pizarro, G. (1991) Physiol. Rev. 71, 849-908[Free Full Text]
7. Fill, M., and Copello, J. A. (2002) Physiol. Rev. 82, 893-922[Abstract/Free Full Text]
8. Lamb, G. D. (2000) Clin. Exp. Pharmacol. Physiol. 27, 216-224[CrossRef][Medline] [Order article via Infotrieve]
9. Bers, D. M. (2002) Nature 415, 198-205[CrossRef][Medline] [Order article via Infotrieve]
10. Yang, D., Pan, Z., Takeshima, H., Wu, C., Nagaraj, R. Y., Ma, J., and Cheng, H. (2001) J. Biol. Chem. 276, 40210-40214[Abstract/Free Full Text]
11. Lohn, M., Jessner, W., Furstenau, M., Wellner, M., Sorrentino, V., Haller, H., Luft, F. C., and Gollasch, M. (2001) Circ. Res. 89, 1051-1057[Abstract/Free Full Text]
12. Sorrentino, V., and Volpe, P. (1993) Trends Pharmacol. Sci. 14, 98-103[CrossRef][Medline] [Order article via Infotrieve]
13. Du, G. G., and MacLennan, D. H. (1999) J. Biol. Chem. 274, 26120-26126[Abstract/Free Full Text]
14. Du, G. G., Khanna, V. K., and MacLennan, D. H. (2000) J. Biol. Chem. 275, 11778-11783[Abstract/Free Full Text]
15. Nakai, J., Gao, L., Xu, L., Xin, C., Pasek, D. A., and Meissner, G. (1999) FEBS Lett. 459, 154-158[CrossRef][Medline] [Order article via Infotrieve]
16. Yamazawa, T., Takeshima, H., Shimuta, M., and Iino, M. (1997) J. Biol. Chem. 272, 8161-8164[Abstract/Free Full Text]
17. Zorzato, F., Fujii, J., Otsu, K., Phillips, M., Green, N. M., Lai, F. A., Meissner, G., and MacLennan, D. H. (1990) J. Biol. Chem. 265, 2244-2256[Abstract/Free Full Text]
18. Bhat, M. B., Zhao, J., Hayek, S., Freeman, E. C., Takeshima, H., and Ma, J. (1997) Biophys. J. 73, 1320-1328[Abstract]
19. Hayek, S. M., Zhao, J., Bhat, M., Xu, X., Nagaraj, R., Pan, Z., Takeshima, H., and Ma, J. (1999) FEBS Lett. 461, 157-164[CrossRef][Medline] [Order article via Infotrieve]
20. Hayek, S. M., Zhu, X., Bhat, M. B., Zhao, J., Takeshima, H., Valdivia, H. H., and Ma, J. (2000) Biochem. J. 351, 57-65[CrossRef][Medline] [Order article via Infotrieve]
21. Proenza, C., O'Brien, J., Nakai, J., Mukherjee, S., Allen, P. D., and Beam, K. G. (2002) J. Biol. Chem. 277, 6530-6535[Abstract/Free Full Text]
22. O'Reilly, F. M., Robert, M., Jona, I., Szegedi, C., Albrieux, M., Geib, S., De Waard, M., Villaz, M., and Ronjat, M. (2002) Biophys. J. 82, 145-155[Abstract/Free Full Text]
23. Masumiya, H., Wang, R., Zhang, J., Xiao, B., and Chen, S. R. W. (2003) J. Biol. Chem. 278, 3786-3792[Abstract/Free Full Text]
24. Wagenknecht, T., and Samso, M. (2002) Front. Biosci. 7, d1464-74
25. Serysheva, I. I., Schatz, M., van Heel, M., Chiu, W., and Hamilton, S. L. (1999) Biophys. J. 77, 1936-1944[Abstract/Free Full Text]
26. Radermacher, M., Rao, V., Grassucci, R., Frank, J., Timerman, A. P., Fleischer, S., and Wagenknecht, T. (1994) J. Cell Biol. 127, 411-423[Abstract]
27. Orlova, E. V., Serysheva, II, van Heel, M., Hamilton, S. L., and Chiu, W. (1996) Nat. Struct. Biol. 3, 547-552[Medline] [Order article via Infotrieve]
28. Liu, Z., Zhang, J., Sharma, M. R., Li, P., Chen, S. R., and Wagenknecht, T. (2001) Proc. Natl. Acad. Sci. U. S. A. 98, 6104-6109[Abstract/Free Full Text]
29. Liu, Z., Zhang, J., Li, P., Chen, S. R., and Wagenknecht, T. (2002) J. Biol. Chem. 277, 46712-46719[Abstract/Free Full Text]
30. Chen, S. R. W., Li, X., Ebisawa, K., and Zhang, L. (1997) J. Biol. Chem. 272, 24234-24246[Abstract/Free Full Text]
31. Sambrook, J., Fritsch, E. F., and Maniatis, T. (1989) Cold Spring Harbor Laboratory , Cold Spring Harbor, NY
32. Zhao, M., Li, P., Li, X., Zhang, L., Winkfein, R. J., and Chen, S. R. (1999) J. Biol. Chem. 274, 25971-25974[Abstract/Free Full Text]
33. Ho, S. N., Hunt, H. D., Horton, R. M., Pullen, J. K., and Pease, L. R. (1989) Gene (Amst.) 77, 51-59[CrossRef][Medline] [Order article via Infotrieve]
34. Wagenknecht, T., Frank, J., Boublik, M., Nurse, K., and Ofengand, J. (1988) J. Mol. Biol. 203, 753-760[Medline] [Order article via Infotrieve]
35. Frank, J. (1996) Three-Dimensional Electron Microscopy of Macromolecular Assemblies , pp. 54-125, Academic Press, Inc., San Diego, CA
36. Penczek, P. A., Grassucci, R. A., and Frank, J. (1994) Ultramicroscopy 53, 251-270[CrossRef][Medline] [Order article via Infotrieve]
37. Malhotra, A., Penczek, P., Agrawal, R. K., Gabashvili, I. S., Grassucci, R. A., Junemann, R., Burkhardt, N., Nierhaus, K. H., and Frank, J. (1998) J. Mol. Biol. 280, 103-116[CrossRef][Medline] [Order article via Infotrieve]
38. Li, P., and Chen, S. R. (2001) J. Gen. Physiol. 118, 33-44[Abstract/Free Full Text]
39. Doi, N., and Yanagawa, H. (2002) Methods Mol. Biol. 183, 49-55[Medline] [Order article via Infotrieve]
40. Niwa, H., Inouye, S., Hirano, T., Matsuno, T., Kojima, S., Kubota, M., Ohashi, M., and Tsuji, F. I. (1996) Proc. Natl. Acad. Sci. U. S. A. 93, 13617-13622[Abstract/Free Full Text]
41. Waldo, G. S., Standish, B. M., Berendzen, J., and Terwilliger, T. C. (1999) Nat. Biotechnol. 17, 691-695[CrossRef][Medline] [Order article via Infotrieve]
42. Chen, S. R., Li, P., Zhao, M., Li, X., and Zhang, L. (2002) Biophys. J. 82, 2436-2447[Abstract/Free Full Text]
43. Tanna, B., Welch, W., Ruest, L., Sutko, J. L., and Williams, A. J. (1998) J. Gen. Physiol. 112, 55-69[Abstract/Free Full Text]
44. Wagenknecht, T., Grassucci, R., Berkowitz, J., Wiederrecht, G. J., Xin, H. B., and Fleischer, S. (1996) Biophys. J. 70, 1709-1715[Abstract]
45. Wagenknecht, T., Radermacher, M., Grassucci, R., Berkowitz, J., Xin, H. B., and Fleischer, S. (1997) J. Biol. Chem. 272, 32463-32471[Abstract/Free Full Text]
46. Serysheva, I. I., Ludtke, S. J., Baker, M. R., Chiu, W., and Hamilton, S. L. (2002) Proc. Natl. Acad. Sci. U. S. A. 99, 10370-10375[Abstract/Free Full Text]
47. Leong, P., and MacLennan, D. H. (1998) J. Biol. Chem. 273, 7791-7794[Abstract/Free Full Text]
48. Nakai, J., Sekiguchi, N., Rando, T. A., Allen, P. D., and Beam, K. G. (1998) J. Biol. Chem. 273, 13403-13406[Abstract/Free Full Text]
49. Sharma, M. R., Jeyakumar, L. H., Fleischer, S., and Wagenknecht, T. (2000) J. Biol. Chem. 275, 9485-9491[Abstract/Free Full Text]
50. Jayaraman, T., Brillantes, A. M., Timerman, A. P., Fleischer, S., Erdjument-Bromage, H., Tempst, P., and Marks, A. R. (1992) J. Biol. Chem. 267, 9474-9477[Abstract/Free Full Text]
51. Lamb, G. D., and Stephenson, D. G. (1996) J. Physiol. 494, 569-576[Abstract]
52. Lamb, G. D. (2002) Front. Biosci. 7, 834-842
53. Bultynck, G., De Smet, P., Rossi, D., Callewaert, G., Missiaen, L., Sorrentino, V., De Smedt, H., and Parys, J. B. (2001) Biochem. J. 354, 413-422[CrossRef][Medline] [Order article via Infotrieve]
54. Bultynck, G., Rossi, D., Callewaert, G., Missiaen, L., Sorrentino, V., Parys, J. B., and De Smedt, H. (2001) J. Biol. Chem. 276, 47715-47724[Abstract/Free Full Text]


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