Three-dimensional Reconstruction of the Recombinant Type 2 Ryanodine Receptor and Localization of Its Divergent Region 1 (2002)

Isoform 2 of the ryanodine receptor (RyR2) is the major calcium release channel in cardiac muscle. In the present study, two kinds of RyR2 cDNA were constructed, one encoding the wild type mouse RyR2 (RyR2wt) and the other encoding modified RyR2, into which was inserted a cDNA encoding green fluorescent protein (GFP). GFP was inserted into the divergent region 1 (DR1) of RyR2, after the Asp-4365 (RyR2D4365-GFP). HEK293 cells expressing both RyR2wt and RyR2D4365-GFPcDNAs showed caffeine- and ryanodine-sensitive calcium release, demonstrating that both wild type and modified RyR2s form functional calcium release channels. Cells expressing the fusion protein, RyR2D4365-GFP, were readily identified by their fluorescence due to the presence of GFP, indicating that the inserted GFP folded properly. Both expressed RyR2s were purified from cell lysates in a single step by affinity chromatography using a GST-FKBP12.6 as the affinity ligand. Cryoelectron microscopy of purified RyR2s showed structurally intact receptors, and three-dimensional reconstructions were obtained by single particle image processing. The three-dimensional reconstruction of RyR2wt appeared very similar to that of the native RyR2 purified from dog heart. The location of the inserted GFP, and consequently of DR1, was mapped on the three-dimensional structure of RyR2 to one of the subunit's characteristic domains, domain 3, also known as the "handle" domain. This study describes the first internal fusion of a protein into a ryanodine receptor, and it demonstrates the potential of this technology for localizing functional and structural domains on the three-dimensional structure of RyR. Isoform 2 of the ryanodine receptor (RyR2) is the major calcium release channel in cardiac muscle. In the present study, two kinds of RyR2 cDNA were constructed, one encoding the wild type mouse RyR2 (RyR2wt) and the other encoding modified RyR2, into which was inserted a cDNA encoding green fluorescent protein (GFP). GFP was inserted into the divergent region 1 (DR1) of RyR2, after the Asp-4365 (RyR2D4365-GFP). HEK293 cells expressing both RyR2wt and RyR2D4365-GFPcDNAs showed caffeine- and ryanodine-sensitive calcium release, demonstrating that both wild type and modified RyR2s form functional calcium release channels. Cells expressing the fusion protein, RyR2D4365-GFP, were readily identified by their fluorescence due to the presence of GFP, indicating that the inserted GFP folded properly. Both expressed RyR2s were purified from cell lysates in a single step by affinity chromatography using a GST-FKBP12.6 as the affinity ligand. Cryoelectron microscopy of purified RyR2s showed structurally intact receptors, and three-dimensional reconstructions were obtained by single particle image processing. The three-dimensional reconstruction of RyR2wt appeared very similar to that of the native RyR2 purified from dog heart. The location of the inserted GFP, and consequently of DR1, was mapped on the three-dimensional structure of RyR2 to one of the subunit's characteristic domains, domain 3, also known as the "handle" domain. This study describes the first internal fusion of a protein into a ryanodine receptor, and it demonstrates the potential of this technology for localizing functional and structural domains on the three-dimensional structure of RyR. The cardiac ryanodine receptor, also known as type 2 ryanodine receptor (RyR2), 1The abbreviations used for: RyR2, type 2 RyR or cardiac RyR; RyR, ryanodine receptor; RyR1 and -3, RyR isoform 1 and 3, respectively; DR1, -2, and -3, divergent region 1, 2, and 3, respectively; EM, electron microscopy; FKBP12.6, 12.6-kDa FK506-binding protein; GFP, green fluorescent protein; GST, glutathioneS-transferase; PIPES, 1,4-piperazinediethanesulfonic acid; CHAPS, 3-[(3-cholamidopropyl)dimethylammonio]-1-propanesulfonic acid. functions as a calcium release channel in the heart, where it plays a crucial role in excitation-contraction coupling (1Bers D.M. Nature. 2002; 415: 198-205Crossref PubMed Scopus (3352) Google Scholar). Influx of Ca2+through the cardiac L-type voltage-gated Ca2+ channel on the plasma membrane triggers RyR2 to release Ca2+ from the sarcoplasmic reticulum into the cytoplasm (2Fabiato A. J. Gen. Physiol. 1985; 85: 247-289Crossref PubMed Scopus (685) Google Scholar). The resulting increase in cytoplasmic [Ca2+] activates the myofilaments to generate cardiac muscle contraction. Recently, mutations of RyR2 have been implicated in some heart diseases (3Marks A.R. J. Mol. Cell Cardiol. 2001; 33: 615-624Abstract Full Text PDF PubMed Scopus (155) Google Scholar). At least 21 RyR2 mutations are now linked to genetic forms of sudden cardiac death: catecholaminergic polymorphic ventricular tachycardia (4Priori S.G. Napolitano C. Tiso N. Memmi M. Vignati G. Bloise R. Sorrentino V. Danieli G.A. Circulation. 2001; 103: 196-200Crossref PubMed Scopus (1152) Google Scholar, 5Priori S.G. Napolitano C. Memmi M. Colombi B. Drago F. Gasparini M. DeSimone L. Coltorti F. Bloise R. Keegan R. Cruz Filho F.E.S. Vignati G. Benatar A. DeLogu A. Circulation. 2002; 106: 69-74Crossref PubMed Scopus (952) Google Scholar), familial polymorphic ventricular tachycardia (6Laitinen P.J. Brown K.M. Piippo K. Swan H. Devaney J.M. Brahmbhatt B. Donarum E.A. Marino M. Tiso N. Viitasalo M. Toivonen L. Stephan D.A. Kontula K. Circulation. 2001; 103: 485-490Crossref PubMed Scopus (623) Google Scholar), and arrhythmogenic right ventricular cardiomyopathy type 2 (7Tiso N. Stephan D.A. Nava A. Bagattin A. Devaney J.M. Stanchi F. Larderet G. Brahmbhatt B. Brown K. Bauce B. Muriago M. Basso C. Thiene G. Danieli G.A. Rampazzo A. Hum. Mol. Genet. 2001; 10: 189-194Crossref PubMed Scopus (696) Google Scholar). In addition, Marx and co-workers have proposed that hyperphosphorylation of RyR2 by protein kinase A, which causes dissociation of the regulatory FKBP12.6 subunit from RyR2, affects RyR2 functioning and contributes to the ventricular arrhythmias that often occur during heart failure (8Marx S.O. Reiken S. Hisamatsu Y. Jayaraman T. Burkhoff D. Rosemblit N. Marks A.R. Cell. 2000; 101: 365-376Abstract Full Text Full Text PDF PubMed Scopus (1677) Google Scholar). Two other mammalian isoforms of the ryanodine receptor (RyR), in addition to RyR2, have been identified and cloned (9Takeshima H. Nishimura S. Matsumoto T. Ishida H. Kangawa K. Minamino N. Matsuo H. Ueda M. Hanaoka M. Hirose T. Numa S. Nature. 1989; 339: 439-445Crossref PubMed Scopus (867) Google Scholar, 10Zorzato F. Fujii J. Otsu K. Phillips M. Green N.M. Lai F.A. Meissner G. MacLennan D.H. J. Biol. Chem. 1990; 265: 2244-2256Abstract Full Text PDF PubMed Google Scholar, 11Otsu K. Willard H.F. Khanna V.K. Zorzato F. Green N.M. MacLennan D.H. J. Biol. Chem. 1990; 265: 13472-13483Abstract Full Text PDF PubMed Google Scholar, 12Nakai J. Imagawa T. Hakamata Y. Shigekawa M. Takeshima H. Numa S. FEBS Lett. 1990; 271: 169-177Crossref PubMed Scopus (288) Google Scholar, 13Hakamata Y. Nakai J. Takeshima H. Imoto K. FEBS Lett. 1992; 312: 229-235Crossref PubMed Scopus (351) Google Scholar). RyR isoform 1 (RyR1) is the major calcium release channel in skeletal muscle, and RyR isoform 3 (RyR3) is widely expressed in many tissues. These RyR isoforms, encoded by three different genes, share a high degree of sequence homology. For example, the amino acid sequence of rabbit RyR2 is 67 and 70% identical to RyR1 and RyR3, respectively (11Otsu K. Willard H.F. Khanna V.K. Zorzato F. Green N.M. MacLennan D.H. J. Biol. Chem. 1990; 265: 13472-13483Abstract Full Text PDF PubMed Google Scholar, 13Hakamata Y. Nakai J. Takeshima H. Imoto K. FEBS Lett. 1992; 312: 229-235Crossref PubMed Scopus (351) Google Scholar). Therefore, the isoforms are expected to have essentially identical three-dimensional structures, except in a few limited regions corresponding to locations of high primary sequence diversity. Three such regions of high variability have been identified (14Sorrentino V. Volpe P. Trends Pharmacol. Sci. 1993; 14: 98-103Abstract Full Text PDF PubMed Scopus (274) Google Scholar) and named divergent regions 1, 2, and 3 (DR1, DR2, and DR3). DR1 is the largest of the divergent regions, comprising residues 4254–4631 of RyR1 and residues 4210–4562 of RyR2 (14Sorrentino V. Volpe P. Trends Pharmacol. Sci. 1993; 14: 98-103Abstract Full Text PDF PubMed Scopus (274) Google Scholar). DR1 includes at least one transmembrane domain, one intraluminal loop, and one cytoplasmic loop, according to the predicted transmembrane topologies (9Takeshima H. Nishimura S. Matsumoto T. Ishida H. Kangawa K. Minamino N. Matsuo H. Ueda M. Hanaoka M. Hirose T. Numa S. Nature. 1989; 339: 439-445Crossref PubMed Scopus (867) Google Scholar, 10Zorzato F. Fujii J. Otsu K. Phillips M. Green N.M. Lai F.A. Meissner G. MacLennan D.H. J. Biol. Chem. 1990; 265: 2244-2256Abstract Full Text PDF PubMed Google Scholar, 11Otsu K. Willard H.F. Khanna V.K. Zorzato F. Green N.M. MacLennan D.H. J. Biol. Chem. 1990; 265: 13472-13483Abstract Full Text PDF PubMed Google Scholar, 12Nakai J. Imagawa T. Hakamata Y. Shigekawa M. Takeshima H. Numa S. FEBS Lett. 1990; 271: 169-177Crossref PubMed Scopus (288) Google Scholar). In a previous study, we overexpressed RyR3 in HEK293 cells and purified the functional protein for structural studies (15Liu Z. Zhang J. Sharma M. Li P. Chen S.R.W. Wagenknecht T. Proc. Natl. Acad. Sci. U. S. A. 2001; 98: 6104-6109Crossref PubMed Scopus (57) Google Scholar). In the present study, mouse RyR2 was expressed in HEK293 cells, purified in a single step by affinity chromatography, and studied by cryoelectron microscopy (cryo-EM) and three-dimensional reconstruction. As shown by our previous study on RyR3, an advantage of using cloned receptors for quantitative EM is that genetic manipulations can be made that effectively create sequence-specific labels. Here we have explored the utility of insertional gene fusions involving green fluorescent protein (GFP) for this purpose. GFP has been well established as a reporter protein for monitoring gene expression and protein targeting (16Tsien R.Y. Annu. Rev. Biochem. 1998; 67: 509-544Crossref PubMed Scopus (4944) Google Scholar), and it appears to be a good choice as an insertional fusion protein, because its characteristic green fluorescence depends on the proper folding of the protein (17Doi N. Yanagawa H. FEBS Lett. 1999; 457: 1-4Crossref PubMed Scopus (50) Google Scholar, 18Niwa H. Inouye S. Hirano T. Matsuno T. Kojima S. Kubota M. Ohashi M. Tsuji F.I. Proc. Natl. Acad. Sci. U. S. A. 1996; 93: 13617-13622Crossref PubMed Scopus (399) Google Scholar). Indeed, this approach has been shown to be feasible for structural analysis of surface-exposed regions of icosahedral viruses by cryo-EM (19Kratz P.A. Böttcher B. Nassal M. Proc. Natl. Acad. Sci. U. S. A. 1999; 96: 1915-1920Crossref PubMed Scopus (230) Google Scholar). Proteins of the size of GFP (molecular mass 28 kDa) are easily visualized and mapped by image processing, even at moderate (∼3-nm) resolution. In this study, a modified RyR2, containing GFP inserted after Asp-4365 within DR1, and termed RyR2D4365-GFP, was characterized by cryo-EM and three-dimensional reconstruction. This approach should be generally useful for mapping the RyR2 linear sequence onto the three-dimensional structure of the receptor, thereby providing insights into the structural basis of RyR2 functions. The cloning and construction of the 15-kb cDNA encoding the mouse cardiac muscle RyR2 have been described previously (20Zhao M. Li P. Li X. Zhang L. Winkfein R.J. Chen S.R.W. J. Biol. Chem. 1999; 274: 25971-25974Abstract Full Text Full Text PDF PubMed Scopus (169) Google Scholar). The DNA encoding GFP flanked by Gly-rich linkers and an AscI site (Fig. 1) was obtained by PCR, using the following primers: 5′-GGG CGC GCC GGT GGA GGT GGA AGT GGA GGT GGA GGT ACT ATG GTG AGC AAG GGC GAG GAG CTG-3′ and 5′-GGC GCG CCC ACC ACC TCC TCC AGA TCC TCC ACC ACC CTT GTA CAG CTC GTC CAT GCC GAG-3′. TheAscI site was introduced into the DR1 of RyR2 after Asp-4365 by overlap-extension PCR, using the following primers: 5′-GAC CTG ACA GAC GGG CGC GCC CTG ACA GAG GAG AGT GAT-3′ and 5′-CTC CTC TGT CAG GGC GCG CCC GTC TGT CAG GTC TTC AGA-3′. Then the AscI fragment containing GFP and linkers was subcloned into full-length RyR2wt after Asp-4365. The sequences of all PCR fragments were verified by DNA sequencing. HEK293 cells were transfected with RyR2 cDNAs (RyR2wt or RyR2D4365-GFP) using calcium phosphate as described previously (21Chen S.R.W. Li X. Ebisawa K. Zhang L. J. Biol. Chem. 1997; 272: 24234-24246Abstract Full Text Full Text PDF PubMed Scopus (144) Google Scholar, 22Chen S.R.W. Ebisawa K. Li X. Zhang L. J. Biol. Chem. 1998; 273: 14675-14678Abstract Full Text Full Text PDF PubMed Scopus (108) Google Scholar). The transfected HEK293 cells were detergent-lysed in buffer (25 mm Na-PIPES, pH 7.2, 140 mm NaCl, 5 mm EGTA, 2.5 mm dithiothreitol, 1% CHAPS, 0.5% egg lecithin, and a protease inhibitor mix (2 mmbenzamidine, 4 μg/ml leupeptin, 2 μg/ml pepstatin A, 4 μg/ml aprotinin, 0.75 mm phenylmethylsulfonyl fluoride)) and incubated on ice for 1 h. Insoluble material was removed by sedimentation, and sucrose (400 mm, final concentration) and NaCl (400 mm, final concentration) were added to the supernatant. Glutathione-Sepharose beads (100 μl) bound with 400 μg of GST-FKBP12.6 were added to the cell lysate supernatant. The mixture was incubated at 4 °C with rotation for 2 days. The beads were collected by centrifugation and washed four times with the lysis buffer containing 0.5% CHAPS and 0.25% egg lecithin. The RyR2s were eluted from the beads by incubation with 100 mm glutathione, in buffer (25 mm Na-PIPES, pH 7.2, 400 mm NaCl, 400 mm sucrose, 1 mm EGTA, 0.2 mmCaCl2, 2.5 mm dithiothreitol, 0.4% CHAPS, 0.16% egg lecithin, 200 mm Tris/Hepes, pH 7.4, and the protease inhibitor mix) for 15 min. The elution was aliquoted, frozen in liquid nitrogen, and stored at −90 °C. Free cytosolic Ca2+ concentration in the transfected HEK293 cells was measured with the fluorescence Ca2+ indicator dye fluo-3 as described previously (21Chen S.R.W. Li X. Ebisawa K. Zhang L. J. Biol. Chem. 1997; 272: 24234-24246Abstract Full Text Full Text PDF PubMed Scopus (144) Google Scholar). The purified RyR2 proteins were denatured in Laemmli sample buffer and subjected to SDS-polyacrylamide (6%) minigel electrophoresis. The resolved proteins were transferred to nitrocellulose membranes at 30 V for 15–16 h at 4 °C in the presence of 0.01% SDS. The nitrocellulose membranes were blocked for 1 h with phosphate-buffered saline plus 0.5% Tween 20 and 5% skim milk powder, incubated 1–2 h with anti-RyR antibody 34C or anti-GFP antibody PA1–980 (Affinity BioRegents, Golden, CO), and then washed three times for 15 min each with the same buffer. The bound antibodies were visualized by using alkaline phosphatase-conjugated anti-mouse (for anti-RyR) or anti-rabbit (for anti-GFP) IgG and the enhanced chemiluminescence Western blotting analysis system (21Chen S.R.W. Li X. Ebisawa K. Zhang L. J. Biol. Chem. 1997; 272: 24234-24246Abstract Full Text Full Text PDF PubMed Scopus (144) Google Scholar). Equilibrium [3H]ryanodine binding to cell lysate was carried out as described previously (23Du G.G. Imredy J.P. MacLennan D.H. J. Biol. Chem. 1998; 273: 33259-33266Abstract Full Text Full Text PDF PubMed Scopus (47) Google Scholar) with some modifications. [3H]ryanodine binding to the whole cell lysate was carried out in a total volume of 300 μl of binding solution containing 30 μl of cell lysate, 500 mm KCl, 25 mm Tris, 50 mm Hepes, pH 7.4, 0.8 mm CaCl2, the protease inhibitor mix, and various concentrations of [3H]ryanodine at 37 °C for 2 h. For the Ca2+ response curve, the binding solution contained 5 nm [3H]ryanodine and various Ca2+ concentrations. The binding mixture was diluted with 5 ml of ice-cold washing buffer containing 25 mm Tris-HCl (pH 8.0) and 250 mm KCl and was filtered immediately through Whatman GF/B filters presoaked with 1% polyethyleneimine. The filters were washed three times, and the radioactivity associated with the filters was measured by liquid scintillation counting. Nonspecific binding was determined by measurement of [3H]ryanodine binding in the presence of 30 μm unlabeled ryanodine. All binding assays were done in duplicate. The purified RyR2wt or RyR2D4365-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). The cryoelectron microscopy procedures have been previously described in detail (15Liu Z. Zhang J. Sharma M. Li P. Chen S.R.W. Wagenknecht T. Proc. Natl. Acad. Sci. U. S. A. 2001; 98: 6104-6109Crossref PubMed Scopus (57) Google Scholar). Selected electron micrographs were digitized using a Eurocore Hi-Scan microdensitometer (Saint-Denis, France). Image processing was performed using the SPIDER/WEB software package (24Frank J. Radermacher M. Penczek P. Zhu J. Li Y. Ladjadj M. Leith A. J. Struct. Biol. 1996; 116: 190-199Crossref PubMed Scopus (1808) Google Scholar). 4-fold symmetry was enforced for all the two-dimensional averages and three-dimensional reconstructions. Three-dimensional reconstructions were obtained by the projection matching procedure (15Liu Z. Zhang J. Sharma M. Li P. Chen S.R.W. Wagenknecht T. Proc. Natl. Acad. Sci. U. S. A. 2001; 98: 6104-6109Crossref PubMed Scopus (57) Google Scholar, 25Penczek P.A. Grassucci R.A. Frank J. Ultramicroscopy. 1994; 53: 251-270Crossref PubMed Scopus (358) Google Scholar). For the recombinant RyR2wt and RyR2D4365-GFP, totals of 10,630 and 6,163 particle images were selected from 65 and 40 micrographs, respectively. The final three-dimensional reconstructions of RyR2wt and RyR2D4365-GFP were computed from 4,365 and 3,961 particles, and the resolution in each was estimated by computing two independent reconstructions (from even- and odd-numbered particles) and using the Fourier shell correlation criteria with a cut-off of 0.5 (26Malhotra A. Penczek P. Agrawal R.K. Gabashvilli I.S. Grassucci R.A. Jünemann R. Burkhardt N. Nierhaus K.H. Frank J. J. Mol. Biol. 1998; 280: 103-116Crossref PubMed Scopus (163) Google Scholar). We designed a chimeric cDNA of RyR2 with the entire 238-amino acid GFP sequence inserted after Asp-4365 in DR1 (Fig. 1). Asp-4365 and nearby amino acids show strong hydrophilicity and are predicted to be surface-exposed on RyR2; hence, the GFP insertion was anticipated to have a minimal effect on the structure and function of RyR2. To further minimize potential disruptive effects of the insertion on folding of both GFP and RyR2, two Gly-rich spacers were added to flank either side of the GFP (19Kratz P.A. Böttcher B. Nassal M. Proc. Natl. Acad. Sci. U. S. A. 1999; 96: 1915-1920Crossref PubMed Scopus (230) Google Scholar). The cDNAs corresponding to the fusion protein RyR2D4365-GFP and to RyR2wt were expressed in HEK293 cells. Fig. 2 shows phase-contrast and fluorescence microscopy images of the HEK293 cells expressing the RyR2D4365-GFP. The green fluorescent color indicates that the inserted GFP folded properly. RyR2wt expressed in HEK293 cells has been shown to function as a Ca2+ release channel (20Zhao M. Li P. Li X. Zhang L. Winkfein R.J. Chen S.R.W. J. Biol. Chem. 1999; 274: 25971-25974Abstract Full Text Full Text PDF PubMed Scopus (169) Google Scholar, 27Chen S.R.W. Li P. Zhao M. Li X. Zhang L. Biophys. J. 2002; 82: 2436-2447Abstract Full Text Full Text PDF PubMed Scopus (59) Google Scholar). To determine whether RyR2D4365-GFP is functional as well, we measured the Ca2+ release response to ryanodine and caffeine in transfected HEK293 cells using the fluorescence Ca2+indicator dye fluo-3. As shown in Fig.3 A, RyR2D4365-GFP-transfected HEK293 cells that had not been pretreated with ryanodine responded to repeated caffeine stimulations as expected, since caffeine is a known activator of RyR channel activity. In contrast, treatment of HEK293 cells with ryanodine alone did not cause Ca2+ release, but cells pretreated with ryanodine did respond to the first caffeine stimulation, although they did not respond to second and third caffeine stimulations (Fig.3 B). The of this is as S.R.W. Li P. Zhao M. Li X. Zhang L. Biophys. J. 2002; 82: 2436-2447Abstract Full Text Full Text PDF PubMed Scopus (59) Google Scholar). RyR2s in HEK293 cells should be in a or a at the cytoplasmic Ca2+ they not be by because ryanodine with the in the (23Du G.G. Imredy J.P. MacLennan D.H. J. Biol. Chem. 1998; 273: 33259-33266Abstract Full Text Full Text PDF PubMed Scopus (47) Google Scholar, M. A. S. J. Biol. Chem. Full Text PDF PubMed Google Scholar). The first caffeine RyR2 to and then modified by which in a and the Ca2+ thereby Ca2+ release with further caffeine The in fluorescence after the second and third caffeine stimulations was by fluorescence by caffeine S.R.W. Ebisawa K. Li X. Zhang L. J. Biol. Chem. 1998; 273: 14675-14678Abstract Full Text Full Text PDF PubMed Scopus (108) Google Scholar, 27Chen S.R.W. Li P. Zhao M. Li X. Zhang L. Biophys. J. 2002; 82: 2436-2447Abstract Full Text Full Text PDF PubMed Scopus (59) Google Scholar). These are similar to with cells (Fig. 3, The fluorescence increase that was obtained for RyR2wt can be for by the of expression obtained for to RyR2wt to an effect of the on the Ca2+ activity. Ca2+ release response to ryanodine and caffeine was in HEK293 cells or in cells transfected with DNA not These that RyR2D4365-GFP functions as a and this Ca2+ release is caffeine- and we determined whether RyR2D4365-GFP high ryanodine binding affinity and whether binding of ryanodine is by out [3H]ryanodine binding assays on cell [3H]ryanodine binding has been widely used as a functional for RyR channel since ryanodine to the channel in its (23Du G.G. Imredy J.P. MacLennan D.H. J. Biol. Chem. 1998; 273: 33259-33266Abstract Full Text Full Text PDF PubMed Scopus (47) Google Scholar, M. A. S. J. Biol. Chem. Full Text PDF PubMed Google Scholar), and the binding of ryanodine to RyR is also on Ca2+ concentration Biophys. 1985; PubMed Scopus Google Scholar, M. P. V. Biophys. PubMed Scopus Google Scholar). As shown in A, RyR2D4365-GFP shows high ryanodine binding the and are nm and protein which are similar to the for previously P. Chen S.R.W. J. Gen. Physiol. 2001; PubMed Scopus Google Scholar). as shown in Fig. 4 the with to Ca2+ of RyR2wt and RyR2D4365-GFP are μm and μm respectively. ryanodine binding was in cell lysates that were transfected with either DNA or expressed RyR2D4365-GFP not high binding affinity for but this binding is also on both of which are of wild type RyR2. We that the insertion of GFP at Asp-4365 has not the calcium release function of RyR2D4365-GFP. The expressed RyR2wt and RyR2D4365-GFP were purified in a single step from HEK293 cell lysates by affinity chromatography using GST-FKBP12.6 as the affinity (15Liu Z. Zhang J. Sharma M. Li P. Chen S.R.W. Wagenknecht T. Proc. Natl. Acad. Sci. U. S. A. 2001; 98: 6104-6109Crossref PubMed Scopus (57) Google Scholar). Fig. 5 shows of the purified RyR2wt and RyR2D4365-GFP. high was present in for each of the purified and their of was by Western blotting using a antibody (Fig. 5 B). The protein corresponding to RyR2D4365-GFP is also by anti-GFP antibody (Fig. 5 that GFP was present in expressed RyR2D4365-GFP. For three-dimensional purified RyR2wt and RyR2D4365-GFP were in a of and images were by shows electron micrographs of RyR2wt and RyR2D4365-GFP. Both micrographs show with intact structure as by with previous on from M. V. Grassucci R. Frank J. S. Wagenknecht T. J. Cell Biol. 1994; PubMed Scopus Google Scholar, Penczek P. Grassucci R. S. Wagenknecht T. J. Biol. Chem. 1998; 273: Full Text Full Text PDF PubMed Scopus Google Scholar). Fig. shows two-dimensional averages of RyR2wt and RyR2D4365-GFP, computed using images of selected that were with their 4-fold symmetry to the images were by J. Three-dimensional of Google Scholar). the images of RyR2wt and identical to averages determined previously for native RyR2 Penczek P. Grassucci R. S. Wagenknecht T. J. Biol. Chem. 1998; 273: Full Text Full Text PDF PubMed Scopus Google Scholar) except for the presence of regions of at high and each of the of receptors in Fig. and B). This is to which is to the FKBP12.6 that to RyR2 during the affinity This is made by to the essentially identical that were previously in a study of T. Grassucci R. J. S. Biophys. J. 1996; Full Text PDF PubMed Scopus Google Scholar). The of because of in the the and of the two-dimensional of RyR2wt from that of RyR2D4365-GFP a that should the of the GFP insertion and also the two projection The shown in Fig. to the and protein mass present in the RyR2D4365-GFP but from These regions of which is by in Fig. and are within the domains by the 3 three-dimensional shown in and also known as the "handle" domains M. V. Grassucci R. Frank J. S. Wagenknecht T. J. Cell Biol. 1994; PubMed Scopus Google Scholar, T. Grassucci R. J. S. Biophys. J. 1996; Full Text PDF PubMed Scopus Google Scholar). analysis of the the two images indicates that regions show at a T. Frank J. M. K. J. J. Mol. Biol. PubMed Scopus Google Scholar). These to the mass by the GFP insertion in RyR2D4365-GFP. This is and mapped in the three-dimensional reconstructions as described The three-dimensional shown in green by the three-dimensional volume of the RyR2wt from the RyR2D4365-GFP is on the three-dimensional reconstruction of RyR2wt The regions in green to the GFP insertion in the Image In Fig. and three-dimensional reconstructions of RyR2wt and RyR2D4365-GFP are as in three The of two major a cytoplasmic of at least domains by M. V. Grassucci R. Frank J. S. Wagenknecht T. J. Cell Biol. 1994; PubMed Scopus Google Scholar) and a transmembrane The three-dimensional reconstruction of recombinant RyR2wt is