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Originally published In Press as doi:10.1074/jbc.M308014200 on January 13, 2004

J. Biol. Chem., Vol. 279, Issue 15, 14639-14648, April 9, 2004
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Ryanodine Receptor Oligomeric Interaction

IDENTIFICATION OF A PUTATIVE BINDING REGION*

Lynda M. Blayney{ddagger}, Spyros Zissimopoulos, Emma Ralph, Eleanor Abbot, Laura Matthews, and F. Anthony Lai

From the Wales Heart Research Institute, Department of Cardiology, University of Wales College of Medicine, Heath Park, Cardiff CF14 4XN, Wales, United Kingdom

Received for publication, July 23, 2003 , and in revised form, January 13, 2004.


   ABSTRACT
TOP
 ABSTRACT
INTRODUCTION
 EXPERIMENTAL PROCEDURES
 RESULTS
 DISCUSSION
 REFERENCES
 
Specific interactions between adjacent ryanodine receptor (RyR) molecules to form ordered two-dimensional arrays in the membrane have been demonstrated using electron microscopy both in situ, in tissues and cells, and in vitro, with the purified protein. RyR interoligomeric association has also been inferred from observations of simultaneous channel gating during multi-RyR channel recordings in lipid bilayers. In this study, we report experiments designed to identify the region(s) of the RyR molecule, participating in this reciprocal interaction. Using epitope-specific antibodies, we identified a RyR tryptic fragment that specifically bound the intact immobilized RyR. Three overlapping RyR fragments encompassing this epitope, expressed using an in vitro mammalian expression system, were immunoprecipitated by RyR. To refine the binding regions, smaller RyR fragments were expressed as glutathione S-transferase (GST) fusion proteins, and their binding to RyR was monitored using a "sandwich" enzyme-linked immunosorbent assay. Three GST-RyR fusion proteins demonstrated specific binding, dependent upon ionic strength. Binding was greatest at 50–150 mM NaCl for two GST-RyR constructs, and a third GST-RyR construct demonstrated maximum binding between 150 and 450 mM NaCl. The binding at high NaCl concentration suggested involvement of a hydrophobic interaction. In silico analysis of secondary structure showed evidence of coil regions in two of these RyR fragment sequences, which might explain these data. In GST pull-down assays, these same three fragments captured RyR2, and two of them retained RyR1. These results identify a region at the center of the linear RyR (residues 2540–3207 of human RyR2) which is able to bind to the RyR oligomer. This region may constitute a specific subdomain participating in RyR-RyR interaction.


   INTRODUCTION
TOP
 ABSTRACT
 INTRODUCTION
EXPERIMENTAL PROCEDURES
 RESULTS
 DISCUSSION
 REFERENCES
 
Acting as sentinels to the sarcoplasmic reticulum (SR)1 Ca2+ store in muscle cells are ryanodine receptors (RyR), the ion channels that mediate release of SR Ca2+ into the cytosol (1–3). There are three distinct mammalian isoforms of RyR identified and characterized, with RyR1 and RyR2 being the predominant forms found in skeletal and cardiac muscle, respectively (4–9). To constitute a functional Ca2+ release channel, four RyR subunits, each of ~560 kDa, coalesce to form a stable homotetramer that surrounds the single ion channel pore (10). The unique size of this homotetrameric RyR complex, about 30 nm across (11–13), has enabled a detailed study of its morphology in the hydrated form using cryoelectron microscopy combined with image reconstruction analysis (14). A remarkable similarity is observed in topological models of three-dimensional reconstructions of RyR1, RyR2, and RyR3, each showing a structure with tetragonal symmetry (15–18).

Analysis of the intracellular organization of RyR particles using freeze-fractured, negatively stained muscle SR junctions, revealed that RyRs are packed into discrete, two-dimensional arrays termed couplons (19–22). In recent studies using RyR1 purified from rabbit muscle SR, the assembly of RyRs into a regular, chessboard-like two-dimensional array was found to be an intrinsic property of RyR oligomers and not dependent upon insertion in the SR membrane or ancillary proteins (23). The projection maps derived from these reconstituted RyR arrays resembled those described previously for junctional SR arrays observed in situ (23). Thus, RyR molecules associate together to form an ordered array, enabling individual oligomers to interact allosterically, creating an integrated microdomain structure within the SR membrane. The RyR-RyR interaction site is found within the cytoplasmic domain (23), which is highly conserved in mammalian and non-mammalian species (4–9) in regard to topology and size (14, 24), suggestive of an essential functional role (25). RyR array formation is a shared feature of the distinct isoforms because both RyR1 and RyR3, when transfected and expressed independently in a dyspedic myotube cell line, also formed similar two-dimensional arrays in situ (26, 27).

The concept of the couplon, comprising arrays of interacting RyRs (and ancillary proteins such as calmodulin, sorcin, and FK506-binding protein (FKBP12)) (20, 21), provides a model to explore the underlying structure and function relationship. The lateral dimensions of individual couplons and their distribution within the myocytes vary with muscle type (22) and may influence the kinetics of local and global Ca2+ signals. Ca2+ sparks originating from the SR were first observed in cardiac myocytes using fluo-3 confocal microscopy (28). However, it remains unclear whether a spark emanates from a finite structural entity, i.e. it equates to activation of an entire couplon, or whether it involves a fraction of the RyRs. The extent of recruitment could depend upon both the mechanisms initiating the spark and the regulatory influences modulating RyR activity (29, 30), including allosteric interactions and the disposition of other proteins (31). In cardiac muscle, the "local control" model of Ca2+ signaling ("cluster bomb" hypothesis) postulates that a small number of RyR channels interact spatially with each L-type voltage-operated Ca2+ channel (dihydropyridine receptor), and then calcium-induced calcium release operates to trigger the opening of other RyRs (20, 32–35). Combined patch clamp and confocal measurement of Ca2+ in the subsarcolemmal space estimates that each SR Ca2+ release event ("spark") requires the concerted opening of four to six RyR molecules, following Ca2+ influx via one dihydropyridine receptor in isolated cardiac myocytes (36). The mechanism of simultaneous activation of the RyR molecules within the cluster to produce a Ca2+ spark remains uncertain. Stochastic gating theory suggests that each RyR molecule activates and closes independently (20, 33). An alternative hypothesis proposes coupled gating, involving the functional interaction of adjacent RyR molecules, mediated by FKBP12 or 12.6, as demonstrated for RyR1 and RyR2 channels recorded in planar bilayer experiments, respectively (37, 38). Similar results with coupled gating of inositol trisphosphate receptors (39) suggest that this may be a common mechanism for related Ca2+ release channels. Thus, RyR-RyR oligomeric interaction may underlie a central mechanism for regulation of EC coupling in muscle cells.

The aim of this study was to identify potential sites of interaction between RyR oligomers. The strategy used was to trypsinize RyR and detect liberated peptides that specifically bound to immobilized tetramers of RyR, using site-specific antibodies for different regions of the RyR. A putative binding region was identified using this approach. The corresponding RyR protein fragments were expressed in vitro and as GST fusion proteins and then tested for binding to RyR by immunoprecipitation, ELISA, and GST pull-down assays. The results describe a potential RyR binding region near the center of the linear RyR sequence, within which were a number of potential binding domains.


   EXPERIMENTAL PROCEDURES
TOP
 ABSTRACT
 INTRODUCTION
 EXPERIMENTAL PROCEDURES
RESULTS
 DISCUSSION
 REFERENCES
 
Materials—Restriction endonucleases were purchased from Amersham Biosciences, and all biochemicals, unless indicated otherwise, were purchased from Sigma. Primary antibodies (Abs) to RyR were rabbit antisera raised against peptides derived from RyR sequences; Ab2142 was to RyR1 residues 830–845 (peptide sequence RREGPRGPHLVGPSRC), Ab34 to the RyR2 residues 2872–2893 (peptide sequence CTLTAKEKAKDREKAQDLKFL, conserved in RyR1), Ab2160 to the RyR2 C terminus residues 4951–4965 (peptide sequence FFPAGDCFRKQYEDQL, conserved in RyR1), and Ab1093 to the hRyR2 residues 4459–4478 (peptide sequence EDKGKQKLRQLHTHRYGEPEC). The GST polyclonal antibody was raised against purified, recombinant GST protein.

Preparation of hRyR2 Expression Vector Constructs—The coordinates of the expression vector constructs, corresponding to the protein sequence of human RyR2 (hRyR2), are given in Figs. 2A and 3A (6). Forward and reverse oligonucleotide primers were designed to contain appropriate restriction sites in-frame with the vectors used and were synthesized by Sigma Genosys. RyR constructs 1, 2, and 3 were inserted into a pGBKT7 vector (Clontech) for in vitro expression. For RyR construct 1 the primers were 5'-AAGGATCCACGCTTCTCTCATTGAC-3' (forward) and 5'-AGGACTCGAGTTCCAGGTCCTTAAA-3' (reverse); for RyR construct 2, 5'-CCGGATCCATAATATATGGCCAAAG-3' (forward) and 5'-CTGGCTCGAGTTTCTCCAAAG-3' (reverse); and for RyR construct 3, 5'-AGGGATCCTTAACCCACAACCT-3' (forward) and 5'-CCTGCTCGAGGTTTTCCATGGT-3' (reverse). DNA constructs encoding GST fusion proteins were made using pGEX vectors (Amersham Biosciences). GST-RyR 4, 4A, 4B, and 5 were constructed in pGEX-5X-2, whereas GST-RyR 6 used pGEX-4T-3. For GST-RyR 4 the forward primer was that used for GST-RyR construct 1, and the reverse was 5'-CAGCGTCGACAGAAACCTGGCT-3'. For GST-RyR construct 4A the forward primer was that used for RyR construct 1, and the reverse primer was 5'-GAGGCTCGAGTGTTTTTCCATCATATCGACATA-3'. For GST-RyR construct 4B the forward primer was 5'-CGCGGGATCCAGTCATCAATGGATTCTGAA-3', and the reverse primer was that used for GST-RyR construct 4. For GST-RyR 5 the primers were 5'-CCGGATCCTTGACATGAGCAATGTTA-3' (forward) and 5'-ACATGTCGACTGAGTTGTTGGAGGAAAC-3' (reverse). For GST-RyR construct 6 the forward primer was 5'-CTATGGATCCGTGATGAAGACTGGCCTGGAG-3', and the reverse primer was that used for RyR construct 2. All constructs were generated by PCR using Pfu DNA polymerase (Promega) and full-length hRyR2 DNA as the template (6) and were confirmed by DNA sequencing (ABI PRISM Big Dye kit; Applied Biosystems).



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  		FIG. 2.
Immunoprecipitation by RyR1 of recombinant constructs expressed in vitro. A, coordinates of overlapping RyR2 constructs 1, 2, and 3 (6) and the position of the Ab34 epitope. B, autoradiographs of [35S]methionine-labeled constructs 1, 2, and 3 (lanes A–D) and after incubation with RyR1, immunoprecipitation using Ab2142 (specific for RyR1 N terminus) and 15% SDS-PAGE (lanes E–H). Lane A is a control construct (RyR2 2346–2507); lanes B, C, and D are constructs 1, 2, and 3, respectively, and lane N is FKBP12, a positive control protein known to bind RyR1. Lanes E, F, G, and H are the immunoprecipitated proteins, corresponding to the control construct (2346–2507) and constructs 1, 2, and 3, respectively. Lane I is a positive control showing the immunoprecipitation of FKBP12. Lanes J, K, L, and M show the experimental control (with Ab2142 omitted). The expected molecular masses were: control construct (2346–2507), 19 kDa; construct 1, 44 kDa; construct 2, 43 kDa; construct 3, 33 kDa; and FKBP12, 12 kDa.

 



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  		FIG. 3.
Sandwich ELISAs of GST-RyR constructs. A, hRyR2 coordinates of GST-RyR constructs 4, 4A, 4B, 5, and 6 expressed and purified for ELISA. B, concentration-dependent binding of GST-RyR constructs to ELISA plates coated with an enriched RyR1 preparation (closed circles) or BSA (open circles). The binding of the GST moiety alone to either RyR1 (closed triangles) or BSA (open triangles) on the same ELISA plates is shown for each construct. The asterisks denote significant binding to RyR1 compared with BSA, p > 0.05, n = 3, ±S.E. C, Western blot analysis of the purified GST-RyR constructs and GST-FKBP12 developed with an anti-GST antibody. The expected molecular mass of each GST fusion protein (in kDa) is given beneath each blot and corresponds to the uppermost band observed.

 
In Vitro Expression of RyR Fragments and Immunoprecipitation by RyR—A TNT T7 Quick-coupled transcription/translation system (Promega) was used to incorporate [35S]methionine and [35S]cysteine (Promix, Amersham Biosciences) into RyR constructs 1, 2, and 3. A 10-µl aliquot of TNT Quick Master Mix was incubated with 1 µg of DNA vector template and 1 µl of Promix for 90 min at 30 °C to synthesize proteins in vitro.5 µl of the reaction mix was then added to native RyR1 protein and incubated for 16 h at 4 °C before immunoprecipitation. The final immunoprecipitation reaction mix contained 250 µg of enriched RyR1 protein, 100 mM NaCl, 20 mM PIPES buffer, pH 7.1, 0.5% CHAPS, and 1% bovine serum albumin (BSA), 20 µl of Protein G PLUS-agarose beads (Santa Cruz Biotechnology), and 10 µl of Ab2142. The Protein G PLUS beads were then washed five times with the immunoprecipitation buffer and twice more with phosphate-buffered saline (PBS: 137 mM NaCl, 8 mM Na2HPO4, 1.5 mM KH2PO4, and 2.7 mM KCl, pH 7.4) and then boiled in SDS sample buffer (0.2 M Tris-HCl, pH 6.8, 0.3 M dithiothreitol (DTT), 15 mM EDTA, 6% SDS, 30% glycerol, and bromphenol blue) for 1 min. Samples were loaded and separated electrophoretically on 12% SDS-polyacrylamide gels. The gel was fixed for 30 min (40% methanol, 10% acetic acid in H2O) and then incubated with Amplify enhancer solution (Amersham Biosciences) for 30 min. The gel was dried and exposed to x-ray film at –80 °C. Ab2142 was omitted from the controls to estimate nonspecific binding to the Protein G PLUS-agarose beads and plasticware.

Preparation of GST-RyR Fragments—The protocol was based on the manufacturer's instructions for pGEX vectors (Amersham Biosciences). GST-RyR fusion protein expression in bacterial cultures (1l, 25 °C, Escherichia coli BL21 Rosetta; Novagen) was induced by 0.1 mM isopropyl {beta}-D-1-thiogalactopyranoside when the absorbance (A600) reached 0.5. Cells were harvested after 3 h and the pellets frozen at –80 °C until required for purification. The batch purification protocol with glutathione-Sepharose 4B beads and elution with reduced glutathione was used (Amersham Bioscience). Reduced glutathione was removed by overnight dialysis of the eluted recombinant protein against PBS, pH 7.4. Purified GST fusion proteins were stored at –80 °C until use.

GST-RyR Fusion Protein Pull-down Assays, Silver Stain, and Immunoblot Analysis—Pull-down assays using GST-RyR fusion proteins were performed with native RyR1- or RyR2-enriched preparations. 20 µg of GST fusion protein was mixed gently overnight at 4 °C with 40 µl of glutathione-Sepharose 4B and 200 µg of RyR1 or RyR2 in binding buffer (20 mM PIPES, pH 7.1, 0.5% CHAPS, 1% BSA, 200 mM NaCl) in a total volume of 800 µl. The beads were washed four times with PBS, eluted with SDS sample buffer, and boiled for 1 min. The sample was separated by 5.5% SDS-PAGE and either silver stained (Bio-Rad silver stain kit) or transferred to polyvinylidene difluoride membrane for 4 h at 20 V using a semidry blotter (Bio-Rad) and the recommended transfer solution (39 mM glycine, 48 mM Tris, 0.0375% SDS, 20% methanol). The membrane was blocked overnight at 4 °C with blocking buffer (0.2% Tween 20, 5% milk proteins). The remaining blotting procedure was carried out at 22 °C, using primary antibodies Ab2142 (1:1,000) and Ab1093 (1:500) to detect RyR1 and RyR2 (1 h), respectively. Horseradish peroxidase-conjugated secondary Ab (1:10,000) was used; the blots were developed with Supersignal West Dura reagent (Perbio) and the images recorded on a ChemiDoc acquisition system (Bio-Rad).

GST-RyR Fusion Protein Overlay Assays—To dissociate the RyR1 tetramers to monomers, enabling analysis on native polyacrylamide gels, the enriched RyR1 preparations were incubated for 1 h at 4 °C with zwittergent (0.9%) (10). Zwittergent-treated RyR1 preparations were run on native 5.5% SDS gels, as described above, except that SDS was omitted from the gel and sample and running buffers, and DTT from the sample buffer. Proteins were transferred to polyvinylidene difluoride membrane as above for 4 h (>200 kDa) or for 30 min (<200 kDa). A Bio-Rad antibody screening apparatus was used to create distinct wells covering the length of the membrane, which was incubated with 500 µl of 40 µg/ml GST-RyR fusion proteins in binding buffer (as in the above GST pull-down assays, except 5% milk protein replaced 1% BSA) with rocking at 4 °C overnight. The membrane was washed three times with binding buffer and developed with either anti-RyR Ab2142 or anti-GST as described above.

Binding of RyR Tryptic Fragments to Immobilized RyR—Solubilized RyR from skeletal muscle microsomes was purified by sucrose gradient centrifugation (23). To lower the NaCl concentration to 200 mM, the RyR peak fraction was diluted with 20 mM PIPES buffer, pH 7.1, 0.5% CHAPS, 100 µM EGTA, 150 mM CaCl2, 2 mM DTT, and protease inhibitors (0.2 mM AEBSF, 0.5 mM benzamidine, 1 mM iodoacetamide) and concentrated to ~1.5 mg/ml protein using centrifugal concentrators (Amicon). Protease inhibitors were omitted from gradients when used for trypsinization experiments, and DTT was omitted from preparations used to couple RyR1 to agarose. Trypsin digestion was performed in 20 mM PIPES buffer, pH 7.1, 0.5% CHAPS, 100 µM EGTA, 150 mM CaCl2, 2 mM DTT, 200 mM NaCl, with 800 µg of RyR protein and 2 µg of trypsin (protein:trypsin ratio 400:1 w/w (40)). At specific intervals up to 30 min, trypsin digests were stopped by the addition of 100 µg of trypsin inhibitor and AEBSF to 0.2 mM.

RyR1 was coupled to an agarose support using Sulfolink coupling gel according to the manufacturer's protocol (Perbio). The coupling buffer used to equilibrate a 2-ml bed volume column was 50 mM Tris, pH 8.5, 5 mM EDTA, 1 M NaCl, 0.5% CHAPS, and 5% sucrose. 1 ml of this buffer was combined with 1 ml of RyR1 preparation (1.5 mg of protein/ml) and then mixed with coupling gel for 15 min and allowed to settle for 30 min at room temperature. The coupling gel was washed with 5 bed volumes of coupling buffer. To quench unbound coupling groups, 2 ml of 50 mM cysteine was added and mixed as before. A control column lacking RyR protein was quenched in a similar way. The coupling gel columns were washed four times with 2 bed volumes of 1 M NaCl in PBS to remove unbound protein and equilibrated with 5 bed volumes of 20 mM PIPES buffer, pH 7.1, 0.5% CHAPS, 100 µM EGTA, 150 mM CaCl2, 2 mM DTT, 200 mM NaCl, and 0.2 mM AEBSF.

A 30-min tryptic digest of 1.2 ml of RyR1 was loaded into the RyR and control columns, which were capped and incubated at room temperature for 1 h and then washed with 5 bed volumes of equilibration buffer. Bound protein was eluted with 1 ml of the same buffer but containing 1 M NaCl. Eluates were concentrated using centrifugal concentrators, desalted using Microbiospin chromatography columns (Bio-Rad) to reduce the NaCl concentration, and the eluates were boiled with sample buffer and subjected to SDS-PAGE and Western blotting with Abs 2142, 34, and 2160.

Preparation of Enriched RyR1—Native RyR1 was prepared from solubilized skeletal muscle microsomes (23) using fast protein liquid chromatography anion exchange chromatography with a 20-ml Resource Q column (Bio-Rad) in place of sucrose gradient centrifugation. RyR1 was eluted with a 125–1,000 mM NaCl gradient in 20 mM PIPES buffer, pH 7.1, 0.5% CHAPS, 100 µM EGTA, 150 mM CaCl2, 2 mM DTT, and protease inhibitors 0.2 mM AEBSF, 0.5 mM benzamidine, and 1 mM iodoacetamide. The RyR protein peak eluting at ~350 mM NaCl provided the RyR1 preparation used for all experiments other than those with trypsin digests. The protein concentration in this fraction was typically 1 mg/ml. In all of the experimental protocols described, the final NaCl concentration was calculated to allow for the 350 mM NaCl contribution of the buffer containing the enriched RyR preparation.

Preparation of Enriched RyR2—Native RyR2 protein was prepared from pig heart microsomes, solubilized, and enriched using sucrose density gradient centrifugation with the same protocol as that for the RyR1 preparation used for the trypsin digest experiments (described above).

RyR Interaction ELISAs—Either 10 µg of RyR1-enriched preparation or BSA was added to the wells of a 96-well ELISA plate (ICN) for 2hat22 °C in PBS and bicarbonate buffer (41). The wells were washed three times with PBS. 100 µl of GST-RyR fusion protein (1,000 ng/well), made up in assay buffer, 20 mM PIPES, pH 7.1, 0.5% CHAPS, 1% BSA, and 150 mM NaCl (unless otherwise indicated) was added and the plates incubated on a rocker at 4 °C, overnight. This buffer contained 1% BSA to block nonspecific binding and was used to maintain the specific binding conditions for the duration of the assay (20 h), including the addition of Abs. The wells were washed three times, and anti-GST Ab (1:10,000 dilution) was added for 1 h at 22 °C. The wells were washed three times, and secondary Ab (1:10,000 dilution) was incubated at 22 °C for 1 h. After four final washes with PBS, SigmaFASTTM OPD peroxidase substrate was added, and the reaction was stopped after 30 min with 3 N HCl and A495 determined using a plate reader.

Protein Estimation—A BCA protein assay kit (Perbio) was used for all protein estimations. RyR1 and RyR2 preparations were diluted to ensure that buffer components were reduced to concentrations tolerated by the kit.

In Silico Analysis of the RyR Protein Sequences—Alignment of RyR sequence from rabbit RyR1 and human RyR2 and RyR3 was performed using ClustalW and Dialign 2 (42). both accessed through www.ch.embnet.org. Secondary structure analysis used Jpred www.compbio.dundee.ac.uk to determine the probability of {alpha}-helix (43) and the Coils server at www.ch.embnet.org. The latter used the Lupas algorithm based on hydrophobic amino acids in the a and d positions of an {alpha}-helix heptad repeat, with a charged amino acid in the e and g positions (44). The MTIDK matrix and both weighted and unweighted options were used.


   RESULTS
TOP
 ABSTRACT
 INTRODUCTION
 EXPERIMENTAL PROCEDURES
 RESULTS
DISCUSSION
 REFERENCES
 
Binding of RyR Tryptic Peptide Fragments to RyR—To search for potential RyR-RyR binding regions, rabbit skeletal muscle RyR1 protein was digested with trypsin, and the resultant peptide fragments were tested for binding to intact RyR1 tetramers immobilized on agarose beads. The binding buffer contained 200 mM NaCl, and bound peptide(s) were eluted by buffer containing 1 M NaCl. An agarose column, without RyR1 bound, was used as the control. Domain-specific RyR antibodies, Ab2142 (N terminus), Ab34 (central region), and Ab2160 (C terminus) (Fig. 1A) were used to detect RyR1 breakdown into smaller fragments at various times after trypsin addition, shown as a series of Western blots in Fig. 1B. The RyR N terminus was rapidly proteolysed, with all Ab2142-immunoreactive bands diminished in intensity after 15 min of digest (Fig. 1B, left panel). The tryptic pattern exhibited by the central region antibody (Ab34) showed significant RyR breakdown into smaller bands by 5 min, many of which were still apparent at 15 min, but most bands were fading by 30 min (Fig. 1B, center panel). Detection with the C terminus antibody, Ab2160, resulted in the appearance of a triplet of low molecular mass bands (~41 kDa marker) within 5 min and the appearance of a lower molecular mass band (<32 kDa marker), which increased in intensity with time (Fig. 1B, right panel). Fig. 1C shows a Coomassie-stained gel of the enriched RyR1 prepared by sucrose density gradient centrifugation. Fig. 1D, lanes A–D, presents a Western blot of RyR1 using the central region antibody, Ab34, before and 30 min after trypsin digestion (lanes A and B, respectively). Trypsinized RyR corresponding to lane B was incubated with immobilized RyR1 or control agarose beads, and lanes C and D are the 1 M NaCl eluates from the RyR1 or the control column, respectively. The eluate from the RyR1 column (Fig. 1D, lane C, arrow) contains a single band at around 20–25 kDa, which was not observed in the eluate from the agarose control (lane D). Silver staining of the eluted proteins is shown in Fig. 1D, lanes E and F, corresponding to immobilized RyR1 and control column, respectively. The similar profile in both lanes reveals the presence of several nonspecifically bound proteins, thus emphasizing the requirement of specific antibodies (e.g. Ab34) to identify presence of RyR-derived fragments. In parallel experiments, neither of the other two site-directed antibodies, Ab2142 and Ab2160, was found to detect any bands in the eluate (data not shown). This paper focuses on the binding region recognized by Ab34, although there may be others outside the limited spectrum of site-specific Abs used in this study.



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  		FIG. 1.
Epitope-specific antibody mapping of trypsinized RyR. A, sequence coordinates of the site-directed antibodies 2142, 34, and 2160 raised to the N terminus, central domain, and C terminus of RyR1, respectively. B, time course of RyR1 tryptic digestion monitored using Abs 2142, 34, and 2160. At the times shown, samples were taken and subjected to SDS-PAGE, transferred to polyvinylidene difluoride membrane, and analyzed by Western blot. C, Coomassie-stained 5.5% gel of the enriched RyR1 preparation prepared by sucrose density gradient centrifugation. D, lanes A–D show a Western blot using Ab34 and a 15% gel; lanes E and F show a silver-stained gel corresponding to lanes C and D.In lane A is a 15-µg undigested enriched RyR1 preparation. Lane B shows a trypsin digest after 30 min. This digest was either incubated with RyR immobilized onto an agarose support or with a control agarose column. Lane C shows an Ab34-immunoreactive band of ~20–25 kDa (arrow) specifically eluted from the RyR column, which was absent from lane D (the control column eluate). Lanes E and F are the silver-stained gels corresponding to lanes C and D, and these show a pattern of bands very similar to each other.

 
Immunoprecipitation of in Vitro Expressed RyR Fragments by RyR—Three overlapping constructs, which encompass the Ab34 binding region (Fig. 2A), were prepared in a T7 vector to enable in vitro expression. In addition, a RyR control construct from outside this region (hRyR2 2346–2507), encoding the putative FKBP12 binding site (45, 46) and therefore precluding direct binding to RyR1, and a construct of FKBP12, a confirmed RyR1-binding protein (47), were used as negative and positive controls, respectively. The proteins were synthesized in a mammalian in vitro expression system and then analyzed for interaction with intact RyR by immunoprecipitation with the N terminus-specific RyR antibody (Ab2142). Lanes A–D and lane N of the autoradiograms in Fig. 2B show robust in vitro expression of [35S]methionine-labeled protein for the RyR control and RyR fragments 1, 2, and 3 and FKBP12, respectively. Lanes E–H correspond to lanes A–D after incubation with RyR and immunoprecipitation by RyR antibody. A band corresponding to the RyR control was not observed in lane E, indicating that the RyR control fragment did not interact with intact RyR. However, in lanes F–H, bands are observed which correspond to immunoprecipitation of RyR fragments 1, 2, and 3, respectively, suggesting that intact RyR bound to these polypeptides directly. Lane I confirms immunoprecipitation of the RyR-associated, positive control protein, FKBP12, thus validating the effectiveness of the RyR interaction assay. Lanes J–M are identical experiments to lanes E–I but were performed in the absence of Ab2142, illustrating the negligible background binding of the expressed RyR fragments to the Protein G beads. The results demonstrate that all three RyR fragments can specifically interact with RyR1, analogous to FKBP12, a known regulatory protein that binds to RyR1. Because all three RyR fragments are able to bind intact RyR, this suggests that there is either one site present within the overlapping region or that all three regions contain distinct RyR binding domains (Fig. 2A).

RyR Interaction with GST-RyR Fusion Proteins—To localize the specific site of interaction further, smaller RyR fragments (4, 4A, 4B, 5, and 6) were produced as GST fusion proteins (Fig. 3A). To assess fusion protein binding criteria, a sandwich ELISA was developed where RyR1, RyR2, or BSA was coated onto the wells of an ELISA plate, and various GST-RyR fusion proteins were then added, in the presence of 1% BSA, and incubated overnight. To assess any nonspecific interaction by the GST moiety, the binding of recombinant GST alone was performed in parallel. Fig. 3B indicates a dose response for binding of each of the GST-RyR fusion proteins to both RyR1 (filled symbols) and BSA (open symbols). A significant difference between fusion protein binding to RyR1 and to BSA is indicated with an asterisk (* p > 0.05, n = 3, ±S.E.), and comparison with the parallel GST control experiment is shown (triangles). GST-RyR 4 exhibited specific binding to RyR1, which was significantly greater than binding to BSA and greater than that displayed by GST. Fusion protein 4A showed no specific binding to RyR1 greater than that to BSA, nor that of GST. GST-RyR 4B and 5 were both found to bind to RyR considerably greater than did GST, but similar levels of binding to BSA indicated that this association was not specific for RyR1. With GST-RyR 6, although background binding to BSA compared with GST was present, there was significant binding to RyR1 (* p > 0.05, n = 3, ±S.E.). Fig. 3C shows Western blots of each of the purified GST-RyR constructs detected with anti-GST antibody. All GST-RyR constructs showed a predominant band at the expected fusion protein molecular mass. All GST-RyR fusion proteins also displayed varying quantities of a band comigrating with GST protein (26 kDa), indicating that some proteolysis had occurred. GST-RyR 4B routinely showed a pattern of multiple small bands, suggesting a greater degree of lability.

Ionic Strength Dependence of RyR Binding to GST-RyR Fusion Proteins—Previous experiments have identified the RyR-RyR homotetramer interaction to be sensitive to the salt concentration, requiring near physiological ionic strength buffers for arrays to form (23). GST-RyR fusion proteins were therefore used to examine the effect of NaCl concentration on binding to RyR1 using the sandwich ELISA. The results in Fig. 4A demonstrate the binding of fusion proteins 4, 5, 6, and GST to RyR1 or BSA with different NaCl concentrations in the binding buffer. The specific binding of each fusion protein (binding to RyR minus binding to BSA) is given in Fig. 4B using open symbols assigned as for 4, 5, 6 and GST, respectively. The asterisks demonstrate a significant difference in binding compared with GST alone (*p > 0.05, n = 5, ±S.E.). GST-RyR fusion proteins 4 and 5 showed the greatest binding at 50–100 mM NaCl. In addition, for GST-RyR 5 there was a "shoulder" of binding apparent at around 450 mM NaCl. GST-RyR 6, in contrast, showed maximum binding between 150 and 450 mM NaCl, possibly indicating different mechanisms of interaction with RyR1.



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  		FIG. 4.
Effect of NaCl concentration on the binding of GST-RyR constructs to RyR1 and RyR2. A, sandwich ELISA of the binding of GST-RyR constructs 4 (upright triangles), 5 (downward triangles),6(circles), and GST (squares) to RyR1 (solid symbols) or BSA (open symbols) with different NaCl concentrations in the binding buffer. The specific binding of each construct (binding to RyR–binding to BSA) is given in B using open symbols assigned as for GST-RyR constructs 4, 5, 6, and GST, respectively. C, histogram depicting the binding of GST and GST fusion proteins 4, 5, and 6 to enriched preparations of RyR2 at 150 mM NaCl (black shading) and 450 mM NaCl (light shading). The asterisks demonstrate a significant difference in binding compared with GST alone (*p > 0.05, Student's paired t test) n = 5, ±S.E. or n = 3, ±S.E. for the RyR1 and RyR2 experiments, respectively.

 
Those GST-RyR fusion proteins binding to RyR1 (4, 5, and 6) were tested further for binding to RyR2 at NaCl concentrations 150 and 450 mM NaCl. Fig. 4C shows that GST-RyR fusion proteins 4 and 5 significantly bound RyR2 at 150 mM NaCl (*p > 0.05, n = 3, ±S.E.) but not at 450 mM NaCl. GST-RyR 6 showed significant binding compared with GST at both 150 and 450 mM NaCl (*p > 0.05, n = 3, ±S.E.). Thus, the profile of RyR2 fusion protein binding to native RyR2 was similar to that observed for binding to native RyR1.

GST-RyR Fusion Protein Pull-down Assays of RyR—The studies described above demonstrate that interactions between intact RyR and smaller RyR fragments, expressed either by an in vitro expression system or as GST fusion proteins, can be monitored by immunoprecipitation or a sandwich ELISA, respectively. With each of these techniques, the RyR fragment was the assay component detected, i.e. via [35S]methionine incorporation or via GST tag detection. To examine whether the component within RyR preparations specifically interacting with the fragments was RyR itself, GST-RyR fusion protein pull-down assays were performed and the pellets analyzed for presence of the native RyR. Fig. 5A shows Coomassie-stained gels of the enriched RyR1 (lane A) and RyR2 (lane B) preparations fractionated by fast protein liquid chromatography and sucrose density gradient centrifugation, respectively. Fig. 5B shows Western blots for RyR1 and RyR2 isoforms after incubation with GST-RyR fusion proteins, followed by precipitation with glutathione-Sepharose 4B beads. The two panels of Fig. 5B illustrate the RyR1 and RyR2 protein, migrating at ~560 kDa, detected within the pellet after GST-RyR pull-down. Fig. 5B (upper panel) shows that RyR1 binding was observed to both GST-RyR 4 and 6 and the control protein GST-FKBP12, but that no interaction was detected with GST-RyR 4A and 5, nor with the GST moiety alone. Similarly, RyR2 also exhibited binding to GST-RyR 4 and 6 (Fig. 5B, lower panel) and to GST-RyR 5. As for RyR1, no interaction was found between RyR2 and GST-RyR 4A nor GST alone. These results confirm the detection of a valid association between native, solubilized RyR and specific RyR fragments expressed in both bacteria and in mammalian cell systems.



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  		FIG. 5.
GST-RyR overlay and pull-down assays. A, Coomassiestained gel of enriched RyR1 (lane A) and RyR2 (lane B) prepared by fast protein liquid chromatography or sucrose density centrifugation, respectively. B, GST pull-down assays using GST, GST-FKBP12, and GST-RyR constructs 4, 4A, 4B, 5, and 6 (see Fig. 3A) incubated with either RyR1 (upper panel) or RyR2 (lower panel) preparations. The Prep lane corresponds to 15 µg of enriched RyR. In the Control lane, GST protein was omitted. C, blot of zwittergent-treated RyR1 overlaid with GST proteins as indicated above. The sections above and below the line represent transfer times of 4 h and 30 min, respectively. The lanes labeled Coomassie (pre-transfer) and PT (post-transfer) illustrate electrophoresed protein bands before and after membrane transfer. The lanes labeled RyR Ab and C were incubated with anti RyR2412 and buffer, respectively. D, silver-stained gels of the proteins present in RyR1 pull-down assays, as described in B, separated by 5.5 and 15% SDS-PAGE. The numbers on the right or left of A, C, and D indicate the molecular mass markers.

 
GST-RyR Fusion Protein Overlay Assays of RyR—To determine whether contaminating proteins in the enriched RyR1 preparations contributed to the binding of GST fusion proteins, overlay assays with GST fusion protein and silver staining of the fusion protein pull-down assays were performed.

Native protein gels, overlaid with GST-RyR fusion proteins, were used to identify the relative gel mobility of the interacting protein(s). The RyR tetramer is too large to enter a 5.5% acrylamide gel, so it was dissociated into monomers (560 kDa) by incubation with zwittergent (10). Comparison of the lane labeled PT (post-transfer) with the Coomassie lane (pre-transfer) shows that proteins above 200 kDa were only partially transferred to the polyvinylidene difluoride membrane, whereas those of lower molecular mass were transferred efficiently (Fig. 5C). The positive control lane (labeled RyR Ab) using anti-RyR Ab2142 revealed the mobility of the RyR monomer above the 200 kDa marker (Fig. 5C). The GST fusion protein overlay assay results (labeled GST Ab) show that the strongest binding was demonstrated by GST-FKBP12 and GST-RyR constructs 4 and 6 specifically in the region of the gel corresponding to the mobility of RyR. No other protein of lower molecular mass exhibited any specific binding to the GST fusion proteins.

The proteins precipitated by GST pull-down assays were subjected to silver stain analysis. Fig. 5D shows the proteins detected after pull-down assays with GST, and GST-RyR constructs 4 and 6 analyzed by 5.5% or 15% polyacrylamide gels, to illustrate higher or lower molecular mass proteins, respectively. Both gels show a band around 66 kDa, which corresponds to the BSA used to block nonspecific binding and which is present in all lanes except that of the enriched RyR preparation, where it was omitted for clarity. Another prominent band was observed in the 5.5% gel at about 100 kDa and also appeared in all lanes. These bands were therefore nonspecifically bound to the beads. The only other band apparent in the 5.5% gel is close to the top of the gel with a mobility corresponding to the RyR and can be discerned only in lanes 4 and 6, but not in the GST lane. The 15% gel confirms the presence of GST fusion protein bands corresponding to the appropriate molecular masses shown in the Western blots in Fig. 3C.

The results from the overlay assays and silver stain analysis of GST pull-down assays suggest that although the enriched RyR preparations contain several other proteins, RyR is the principal interactant with selected GST fusion proteins.


   DISCUSSION
TOP
 ABSTRACT
 INTRODUCTION
 EXPERIMENTAL PROCEDURES
 RESULTS
 DISCUSSION
REFERENCES
 
Evidence for RyR-RyR oligomeric interaction in the SR membrane is apparent from studies in situ (22), has also been demonstrated by two-dimensional arrays formed in vitro with purified RyR1 (23), and is consistent with functional studies of RyR channels incorporated into lipid bilayers (37, 38). The precise physiological role that interoligomeric association plays in excitation-contraction coupling remains to be elucidated, but its existence presents an exciting new parameter to consider in addressing the mechanism of RyR function and regulation (29). Although the interaction between RyRs has been reliably observed, the nature of this homo-oligomeric association remains undefined. The aim of this study was to search for specific regions of the RyR protein which may be involved directly in the interoligomeric interaction.

RyR functions as a homotetramer, and the four subunits remain stably bound in a complex upon solubilization of the RyR from SR membranes using high ionic strength buffers and CHAPS detergent (48). Analysis of the RyR homotetramer topology reveals the RyR molecule to be highly folded (14, 15). Thus, there may be a variety of interacting regions within the RyR molecule which maintain both the intrasubunit tertiary structure and intersubunit associations binding the tetrameric complex together. To minimize identification of intramolecular and intersubunit binding regions and to enable a specific focus on the surface-exposed areas where RyR-RyR interoligomer binding domains would be situated, our experimental rationale was to employ the native RyR tetramer as the interactant in disparate binding interaction assays.

In our initial study, we generated RyR fragments from enriched RyR1 preparations by typsinization and mixed the resultant fragments with RyR tetramers bound to a solid support. These binding studies were performed using 200 mM NaCl because regular two-dimensional arrays of RyR were formed only at concentrations below 250 mM (23). Using epitope-specific RyR antibodies, only a ~25-kDa tryptic fragment detected by Ab34 was identified in the immobilized RyR1 column eluate, suggesting that this specific region was able to bind directly to the intact RyR oligomer (Fig. 1). The pattern of proteolytic cleavage monitored by RyR antibodies suggested that the RyR N terminus was extremely vulnerable to trypsin because Ab2142-immunoreactive bands were removed most rapidly within 30 min. With Ab34, the immunoreactive bands were similar at 5 and 15 min and, although diminished, these were still visible after 30 min. The C terminus appeared to degrade slowly, with the Ab2160-reactive triplet consistently observed from 5 to 30 min, with gradual accumulation of a <32 kDa band. These data agree with previous work where proteolysis of SR proteins detected with site-directed RyR Abs (49) indicated a protected RyR C terminus, consistent with colocation of a hydrophobic region comprising the predicted RyR transmembrane domain that is SR membrane-embedded. This C terminus protection apparent with our solubilized RyR1 preparations indicates that this region may be buried within the protein or that the detergent annulus confers a similar protective effect. A previous RyR proteolysis study identified a 33-kDa fragment, involving trypsin cleavage at Arg-2840 and Arg-3119 (50), similar to the ~25-kDa fragment detected by Ab34 in our studies. This central region of RyR also coincides with a cleavage-sensitive zone demonstrated with trypsin (49) and also with Lys-C and Glu-C proteases (51) and has thus been interpreted as being surface-exposed. Thus, our data are consistent with that of previous reports in identifying a region predicted to have a surface topology in the RyR complex and is therefore a good candidate region for one site participating in the RyR-RyR oligomeric interaction. However, it is important to note that there may be other regions of RyR involved in oligomeric interactions that are outside of the limited range of the epitope-specific antibodies used in this study.

To refine further the location of the binding region, three overlapping constructs RyR 1, 2, and 3 were prepared spanning the Ab34 binding region (Fig. 2). Expression and direct immunoprecipitation with native RyR1 of all three RyR fragments suggest either that the overlapping region (Ab34 epitope) is the unique binding domain or that there are several binding domains within this region. In vitro expression provided the advantage of expressing mammalian RyR in a mammalian cell-free system, but the quantitative yield of protein was experimentally limiting. Sandwich ELISAs were therefore devised to provide a more flexible method for RyR fragment binding. Prokaryotic expression of GST fusion proteins covering the potential interacting region (Fig. 3) indicated native RyR1 binding to two fragments. Specificity of GST-RyR fusion protein binding to RyR1-enriched preparations was defined as significantly greater binding than that observed to BSA as well as compared with binding of the GST moiety alone. Thus, GST-RyR constructs 4 and 6 were found to exhibit significant binding to RyR1 (Figs. 3 and 4). Direct comparison between the binding observed for different GST-RyR fusion proteins was not feasible because of the persistent presence of variable amounts of 26-kDa protein, presumed to be GST produced by degradation of the fusion protein.

The ELISA also enabled a profile of the ionic strength dependence of GST-RyR fusion protein binding. GST-RyR fusion proteins 4 and 5 bound optimally in the range of 50–100 mM NaCl, whereas for GST-RyR 6 maximal binding was shifted to 150–450 mM NaCl. Binding of these same constructs to RyR2 at 150 and 450 mM NaCl showed the same pattern of binding for the GST-RyR constructs. Previous in vitro RyR-RyR interactions observed with purified RyR were not retained above 250 mM NaCl, indicating that high ionic strength buffers may be disrupting an electrostatic interaction between RyR molecules (23), entirely consistent with requirement of 1 M NaCl for efficient RyR solubilization from native membranes (48). The results for GST-RyR fusion proteins 4 and 5 support this idea, but the binding profile for GST-RyR 6 suggests that other types of interaction may also be important. In silico analysis of the RyR sequences in the candidate binding region revealed three regions with probability of a coil secondary structure (Fig. 6). In the classical coil, hydrophobic amino acids reside in the a and d positions of a 7-amino acid stretch of an {alpha}-helix (labeled a–g) called a heptad, which is often repeated and which provides the coil backbone (44). The specificity of interaction is provided by key amino acids at other positions in the heptad repeat, particularly the e and g positions, and these are often charged (52, 53). The COILS server identified a strong coil probability of a 28-amino acid stretch of four heptad repeats in GST-RyR 6 (Table I). The coil probability within repeat region 3 (GST-RyR 5) was more variable and dependent upon the weighting for a and d amino acids and charged amino acids at e and g (Table I), despite the close sequence homology between the isoforms. The maximum binding of GST-RyR 6 at high salt concentration and the shoulder at 450 mM NaCl for GST-RyR 5 suggest a hydrophobic component to the binding mechanism. Coiled-coil interactions are often quite complex and are not always defined by a 7-amino acid heptad repeat; amino acid stutters and staggers in between heptads have been reported, and 11 amino acid sequences have also been observed (52, 53). Charged amino acids play a role in establishing and maintaining the coiled coil interaction and in its specificity (53). In our experiments, the presence of the GST moiety in the RyR fusion proteins enhance solubility and ease of purification but may also influence the response to NaCl. An additional consideration is that our data do not only reflect the binding properties of the fusion protein, but also of the native RyR complex used as the binding partner. Evidence for an {alpha}-helix secondary structure was displayed by all of these regions when examined on the Sspro (54) and Impred servers (43), and the latter also identified coil 3 in GST-RyR 6 (Table I and Fig. 6). A limitation of our experiments is that they do not address whether the potential binding domains in this region interact with themselves or with a distinct complementary binding region of the RyR, as illustrated in Fig. 6C. Adjacent RyR molecules within an array should interact with their protein sequences running in opposite directions, and thus any involvement of coiled coils would have to be antiparallel (55). The in silico data provide potential insights into the mechanism involved in RyR-RyR interaction but require further investigation.



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  		FIG. 6.
Features of the putative RyR-RyR binding region. A, hRyR2 coordinates of GST constructs used in the sandwich ELISAs. B illustrates some of the notable sequence characteristics of hRyR2 in this region, including the repeat regions (dotted lines) (6), the Ab34 binding site (filled box), and three coil regions (striped boxes labeled 1–3) identified by the ISREC-Server using the COILS program based on the Lupas algorithm. The region above coil 3 shows the relevant amino acid sequence, corresponding to a region within GST-RyR construct 6. This 4-heptad repeat, labeled a–g, is highly conserved in all three RyR isoforms. The amino acid sequence presented below coils 1 and 2 shows the corresponding residues that are present in GST-RyR construct 5. This sequence is also well conserved in all three RyR isoforms, although Table I indicates that the coil prediction in this region is variable. The lowermost sequence compares coil 1 of repeat region 4 with the homologous sequence of repeat region 3, which gave no coil prediction. Sequence comparison using Dialign suggests a high probability for structural similarity for this region in all three RyR isoforms (data not shown). The second coil region of repeat region 4 compared with repeat region 3, however, showed less similarity. C, two adjacent RyR molecules represented by squares. The orientation arrows indicate that at any point of contact the protein strands will interact back to back. This is illustrated further in the parallel line diagram, which demonstrates that interacting binding domains (shaded boxes) must have complementary partners.

 


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  		TABLE I
Predicted coils in RyR isoforms in the Ab34 binding region

The coils correspond to the hRyR sequences shown diagramatically in Fig. 6B. The coordinates are given as the first and last three amino acids in the corresponding hRyR2 sequence. The maximum value for probability of coil participation assigned to corresponding sequences for RyR1, 2, and 3 is listed. Wt and UnWt, respectively, refer to whether the COILS program was weighted for hydrophobic residues in the a and d positions and charged amino acids in positions e and g of a heptad repeat or not. See "Experimental Procedures."

 
Dividing GST-RyR 4 into 4A and 4B did not assist in further refining the binding domain. RyR 4A showed relatively little binding over and above that of the GST moiety alone, but GST-RyR 4B showed high levels of nonspecific binding. Preparations of GST-RyR 4B consistently produced multiple small bands on Western blots, indicating cleavage into subfragments. A problem inherent in producing smaller fusion proteins is the dependence of secondary or tertiary structure of binding domains on the flanking sequence. Thus, it is possible that the apparently weak binding of GST-RyR 5, which is shorter than GST-RyR 4 and 6, might reflect the loss of conformation essential to binding. Further constructs may be necessary to determine whether it is possible to optimize the coordinates (and conformation) to gain precise localization of the sequences dictating binding within this central region of the RyR.

It has been implicit throughout these experiments that binding of RyR2 constructs to RyR1 tetramers was legitimate as an experimental approach. This was considered reasonable at the outset because the characteristics of both physical interaction in situ and functionally with coupled gating in vitro were observed for both isoforms (37, 38). Moreover, the sequence of all three RyR isoforms in this central region is highly conserved. To address this issue experimentally, ELISAs and the GST pull-down assays were performed with both RyR1 and RyR2 preparations. GST-RyR 4 and 6 captured both RyR1 and RyR2, and, in addition, RyR2 was precipitated by GST-RyR 5. In general, interactions with GST-RyR 5 were weaker and showed inconsistencies, and in the GST pull-down assays there were differences in the proportionate quantities of each RyR isoform captured by each construct. This might suggest that the interaction of GST-RyR 5 for RyR2 was better than with RyR1, as control experiments were consistent for both RyR1 and RyR2 isoforms. Taken as a whole, the data suggest that using native RyR1 for interaction assays with RyR2 fragments provides a stringent test for binding regions that are conserved between RyR isoforms and therefore unlikely to produce a false positive.

The enriched RyR preparations contained a number of other proteins. To eliminate the possibility of their participation in nonspecific binding to the GST fusion proteins, the size of the interacting protein was determined. The enriched RyR preparation was separated on a native acrylamide gel to minimize disruption of secondary and tertiary protein structure. It was however necessary to treat the preparation first with zwittergent to separate the RyR into monomers because the tetramer (~2,300 kDa) was too large to enter the gel. Overlay assays with GST fusion proteins (Fig. 5C) showed that the GST-FKBP12 fusion protein only associated with the RyR band, thus confirming the validity of the overlay protocol. Similarly, the GST-RyR fusion proteins 4 and 6 also bound strongly to the RyR band. Running native gels often leaves protein complexes intact, so it was possible that another protein acted as a intermediary between the RyR and fusion protein. However, treating the enriched RyR preparation with zwittergent, which breaks the association between RyR subunits, probably destroyed any ancillary protein binding also. Furthermore, any ancillary proteins would be present as lower molecular mass contaminants, and no binding of fusion proteins was seen in this region. Finally, the silver-stained gels (Fig. 5D) showed only one major contaminant at about 100 kDa, which was probably the SR Ca2+-ATPase (10). This, however, was present in the recombinant GST control lane and was possibly binding the glutathione-Sepharose 4B beads used for the pull-down assay. Thus, although none of the different binding techniques used was singularly definitive, the combined data from the variety of approaches employed consistently suggest that RyR and GST-RyR fusion proteins 4 and 6 (and possibly 5) interact in a specific fashion.

In conclusion, we have provided several lines of evidence for the Ab34 binding region of the RyR (hRyR2 2540–3207) as a likely participant in the RyR-RyR oligomeric interaction. Recombinant constructs corresponding to this region, expressed in both a mammalian expression system and as bacterial fusion proteins, demonstrate specific binding to enriched preparations of RyR1 and RyR2 homotetramers, respectively. In pull-down assays, both RyR2 and RyR1 are captured by the GST-RyR fusion proteins. The binding was dependent upon the NaCl concentration in the binding buffer for some GST-RyR fusion proteins. Those constructs demonstrating binding at a relatively high NaCl concentration were shown by in silico analysis to contain a coil secondary structure, which would favor the hydrophobic interaction of a coiled coil backbone. Moreover, in independent studies, this central RyR region has been shown to be surface-exposed (49, 51). Further studies will be necessary to establish whether this region binds to itself or whether other parts of the RyR molecule participate as a complementary binding region.


   FOOTNOTES
 
* This work was supported by British Heart Foundation Grants PG99087 and PG 0303915274. The costs of publication of this article were defrayed in part by the payment of page charges. This article must therefore be hereby marked "advertisement" in accordance with 18 U.S.C. Section 1734 solely to indicate this fact. Back

{ddagger} To whom correspondence should be addressed. Tel.: 44-29-2074-4256; Fax: 44-29-2074-3500; E-mail: blayney{at}cf.ac.uk.

1 The abbreviations used are: SR, sarcoplasmic reticulum; Ab, antibody; AEBSF, 4-(2-aminoethyl)benzenesulfonyl fluoride; BSA, bovine serum albumin; CHAPS, 3-[(3-cholamidopropyl)dimethylammonio]-1-propanesulfonic acid; DTT, dithiothreitol; ELISA, enzyme-linked immunosorbent assay; FKBP12, FK506-binding protein; GST, glutathione S-transferase; hRyR, human ryanodine receptor; PBS, phosphate-buffered saline; PIPES, 1,4-piperazinediethanesulfonic acid; RyR, ryanodine receptor. Back



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 ABSTRACT
 INTRODUCTION
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 RESULTS
 DISCUSSION
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