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• W2046822541 abstract "Using cryo-electron microscopy and single particle image processing techniques, we present the first three-dimensional reconstructions of isoform 3 of the ryanodine receptor/calcium release channel (RyR3). Reconstructions were carried out on images obtained from a purified, detergent-solubilized receptor for two different buffer conditions, which were expected to favor open and closed functional states of the channel. As for the heart (RyR2) and skeletal muscle (RyR1) receptor isoforms, RyR3 is a homotetrameric complex comprising two main components, a multidomain cytoplasmic assembly and a smaller (∼20% of the total mass) transmembrane region. Although the isoforms show structural similarities, consistent with the ∼70% overall sequence identity of the isoforms, detailed comparisons of RyR3 with RyR1 showed one region of highly significant difference between them. This difference indicated additional mass present in RyR1, and it likely corresponds to a region of the RyR1 sequence (residues 1303–1406, known as diversity region 2) that is absent from RyR3. The reconstructions of RyR3 determined under “open” and “closed” conditions were similar to each other in overall architecture. A difference map computed between the two reconstructions reveals subtle changes in conformation at several widely dispersed locations in the receptor, the most prominent of which is a ∼4 ° rotation of the transmembrane region with respect to the cytoplasmic assembly. Using cryo-electron microscopy and single particle image processing techniques, we present the first three-dimensional reconstructions of isoform 3 of the ryanodine receptor/calcium release channel (RyR3). Reconstructions were carried out on images obtained from a purified, detergent-solubilized receptor for two different buffer conditions, which were expected to favor open and closed functional states of the channel. As for the heart (RyR2) and skeletal muscle (RyR1) receptor isoforms, RyR3 is a homotetrameric complex comprising two main components, a multidomain cytoplasmic assembly and a smaller (∼20% of the total mass) transmembrane region. Although the isoforms show structural similarities, consistent with the ∼70% overall sequence identity of the isoforms, detailed comparisons of RyR3 with RyR1 showed one region of highly significant difference between them. This difference indicated additional mass present in RyR1, and it likely corresponds to a region of the RyR1 sequence (residues 1303–1406, known as diversity region 2) that is absent from RyR3. The reconstructions of RyR3 determined under “open” and “closed” conditions were similar to each other in overall architecture. A difference map computed between the two reconstructions reveals subtle changes in conformation at several widely dispersed locations in the receptor, the most prominent of which is a ∼4 ° rotation of the transmembrane region with respect to the cytoplasmic assembly. ryanodine receptor isoform FK506-binding protein 3-[(3-cholamidopropyl)dimethylammonio]-1-propanesulfonic acid 1,4-piperazinediethanesulfonic acid sarcoplasmic reticulum Intracellular calcium is essential for the regulation and function of many fundamental biological processes, and the release of calcium from intracellular storage compartments plays an essential role in modulating cytoplasmic Ca2+ levels (1.Berridge M.J. J. Physiol. (Lond.). 1997; 499: 291-306Crossref Scopus (916) Google Scholar). Ryanodine receptors (RyRs)1 and inositol 1,4,5-trisphosphate receptors are the two families of intracellular calcium release channels that have been characterized to date (reviewed in Refs. 2.Fleischer S. Inui M. Annu. Rev. Biophys. Biophys. Chem. 1989; 18: 333-364Crossref PubMed Scopus (442) Google Scholar, 3.Meissner G. Annu. Rev. Physiol. 1994; 56: 485-508Crossref PubMed Scopus (836) Google Scholar, 4.Ogawa Y. Crit. Rev. Biochem. Mol. Biol. 1994; 29: 229-274Crossref PubMed Scopus (228) Google Scholar, 5.Sorrentino V. Sorrentino V. Ryanodine Receptors. CRC Press, Boca Raton, FL1995: 85-100Google Scholar, 6.Franzini-Armstrong C. Protasi F. Physiol. Rev. 1997; 77: 699-729Crossref PubMed Scopus (591) Google Scholar, 7.Mikoshiba K. Curr. Opin. Neurobiol. 1997; 7: 339-345Crossref PubMed Scopus (160) Google Scholar, 8.Shoshan-Barmatz V. Ashley R.H. Int. Rev. Cytol. 1998; 183: 185-271Crossref PubMed Google Scholar, 9.Ogawa Y. Kurebayashi N. Murayama T. Adv. Biophys. 1999; 36: 27-64Crossref PubMed Scopus (61) Google Scholar). Both RyRs and inositol 1,4,5-trisphosphate receptors exist as homotetrameric protein complexes composed of an unusually large subunit, ≈560 kDa for RyRs and ≈313 kDa for inositol 1,4,5-trisphosphate receptors. Frequently an ≈12-kDa isoform of FK506-binding protein (FKBP, an immunophilin) binds with sufficient affinity to the receptors so as to co-purify with them, and the immunophilin may therefore be considered as a component of the receptors (10.Wagenknecht T. Radermacher M. Grassucci R. Berkowitz J. Xin H.-B. Fleischer S. J. Biol. Chem. 1997; 272: 32463-32471Abstract Full Text Full Text PDF PubMed Scopus (151) Google Scholar, 11.Qi Y. Ogunbunmi E.M. Freund E.A. Timerman A.P. Fleischer S. J. Biol. Chem. 1998; 273: 34813-34819Abstract Full Text Full Text PDF PubMed Scopus (40) Google Scholar, 12.Copello J.A. Jeyakumar L. Fleischer S. Biophys. J. 1999; 76: A469Abstract Full Text Full Text PDF PubMed Scopus (946) Google Scholar).Three different isoforms of RyRs, each encoded by a different gene, have been identified in mammals, designated as skeletal (RyR1), cardiac (RyR2), and brain (RyR3). Recent studies have shown that the receptors are much more widely expressed than is indicated by the nomenclature (13.Giannini G. Conti A. Mammarella S. Scrobogna M. Sorrentino V. J. Cell Biol. 1995; 128: 893-904Crossref PubMed Scopus (477) Google Scholar, 14.Conti A. Gorza L. Sorrentino V. Biochem. J. 1996; 316: 19-23Crossref PubMed Scopus (97) Google Scholar, 15.Sutko J.L. Airey J.A. Physiol. Rev. 1996; 76: 1027-1071Crossref PubMed Scopus (364) Google Scholar). For instance, all three isoforms are expressed in brain, but with distributions unique to each. RyR3, the subject of this study, is present at low levels in a variety of excitable and nonexcitable cells, the richest source being diaphragm muscle where it is co-expressed at about 2.5% of the level of RyR1 (16.Jeyakumar L.H. Copello J.A. O'Malley A.M. Wu G.-M. Grassucci R. Wagenknecht T. Fleischer S. J. Biol. Chem. 1998; 273: 16011-16020Abstract Full Text Full Text PDF PubMed Scopus (79) Google Scholar).RyR1 and RyR2 are sufficiently enriched in skeletal and heart muscle, respectively, to have allowed their purification in sufficient quantities for biochemical and biophysical characterization (17.Inui M. Saito A. Fleischer S. J. Biol. Chem. 1987; 262: 15637-15642Abstract Full Text PDF PubMed Google Scholar, 18.Inui M. Saito A. Fleischer S. J. Biol. Chem. 1987; 262: 1740-1747Abstract Full Text PDF PubMed Google Scholar). RyR1 and RyR2 are key components of the multicomponent complexes that are responsible for excitation-contraction coupling in striated muscle (6.Franzini-Armstrong C. Protasi F. Physiol. Rev. 1997; 77: 699-729Crossref PubMed Scopus (591) Google Scholar). Our understanding of RyR3 has lagged behind that of the other isoforms because of difficulty in isolating sufficient quantities, and only recently have protocols been developed that allow commencement of its characterization (16.Jeyakumar L.H. Copello J.A. O'Malley A.M. Wu G.-M. Grassucci R. Wagenknecht T. Fleischer S. J. Biol. Chem. 1998; 273: 16011-16020Abstract Full Text Full Text PDF PubMed Scopus (79) Google Scholar, 19.Murayama T. Ogawa Y. J. Biol. Chem. 1997; 272: 24030-24037Abstract Full Text Full Text PDF PubMed Scopus (64) Google Scholar, 20.Chen S.R.W. Li X. Ebisawa K. Zhang L. J. Biol. Chem. 1997; 272: 24234-24246Abstract Full Text Full Text PDF PubMed Scopus (143) Google Scholar, 21.Chen S.R.W. Ebisawa K. Li X. Zhang L. J. Biol. Chem. 1998; 273: 14675-14678Abstract Full Text Full Text PDF PubMed Scopus (107) Google Scholar, 22.Murayama T. Oba T. Katayama E. Oyamada H. Oguchi K. Kobayashi M. Otsuka K. Ogawa Y. J. Biol. Chem. 1999; 274: 17297-17308Abstract Full Text Full Text PDF PubMed Scopus (78) Google Scholar). These studies have shown that RyR3 has similar, but not identical, conductance and pharmacological properties to those found for the other isoforms. For example, RyR3 is activated by Ca2+ (micromolar) and by millimolar ATP and is inhibited by millimolar Mg2+ and by Ca2+ above millimolar levels. All of the isoforms bind with high affinity the plant alkaloid, ryanodine, which locks the receptors into similar subconductance states. As isolated, RyR1 and RyR2 contain FKBP12 and FKBP12.6, respectively. RyR1 and RyR3 are capable of binding both FKBP isoforms, whereas RyR2 binds only FKBP12.6. Purified RyR2 and RyR3 differ from RyR1 by their lack of modulation of channel activity by FKBP12 and FKBP12.6 (12.Copello J.A. Jeyakumar L. Fleischer S. Biophys. J. 1999; 76: A469Abstract Full Text Full Text PDF PubMed Scopus (946) Google Scholar, 22.Murayama T. Oba T. Katayama E. Oyamada H. Oguchi K. Kobayashi M. Otsuka K. Ogawa Y. J. Biol. Chem. 1999; 274: 17297-17308Abstract Full Text Full Text PDF PubMed Scopus (78) Google Scholar, 23.Qi Y. Jeyakumar L. Xin H.-B. Fleischer S. Biophys. J. 1998; 74: A322Google Scholar). Despite the similarities of the isolated RyR isoforms, they are not functionally interchangeablein vivo, particularly with regard to their roles in e-c coupling (24.Takeshima H. Yamazawa T. Ikemoto T. Takekura H. Nishi M. Noda T. Iino M. EMBO J. 1995; 14: 2999-3006Crossref PubMed Scopus (128) Google Scholar, 25.Takeshima H. Ikemoto M. Nishi M. Nishiyama N. Shimuta M. Sugitani Y. Kuno J. Saito I. Saito H. Endo M. Iino M. Noda T. J. Biol. Chem. 1996; 271: 19649-19652Abstract Full Text Full Text PDF PubMed Scopus (150) Google Scholar, 26.Yamazawa T. Takeshima H. Sakurai T. Endo M. Iino M. EMBO J. 1996; 15: 6172-6177Crossref PubMed Scopus (50) Google Scholar, 27.Tarroni P. Rossi D. Conti A. Sorrentino V. J. Biol. Chem. 1997; 272: 19808-19813Abstract Full Text Full Text PDF PubMed Scopus (75) Google Scholar, 28.Yamazawa T. Takeshima H. Shimuta M. Iino M. J. Biol. Chem. 1997; 272: 8161-8164Abstract Full Text Full Text PDF PubMed Scopus (75) Google Scholar).Comparison of the amino acid sequences of ryanodine receptor isoforms reveals three regions that are much more variable than the overall ≈70% identity among the isoforms (29.Sorrentino V. Volpe P. Trends Pharmacol. Sci. 1993; 14: 98-103Abstract Full Text PDF PubMed Scopus (272) Google Scholar). These hypervariable regions are thought to partially account for the functional differences among the isoforms. Indeed, two of these regions, termed D2 (residues 1303–1406 of RyR1) and D3 (residues 1872–1923), seem to be involved in establishing the properties of excitation-contraction coupling that are characteristic of skeletal muscle vis-à-viscardiac muscle (30.Nakai J. Ogura T. Protasi F. Franzini-Armstrong C. Allen P.D. Beam K.G. Proc. Natl. Acad. Sci. U. S. A. 1997; 94: 1019-1022Crossref PubMed Scopus (91) Google Scholar, 31.Nakai J. Sekiguchi N. Rando T.A. Allen P.D. Beam K.G. J. Biol. Chem. 1998; 273: 13403-13406Abstract Full Text Full Text PDF PubMed Scopus (156) Google Scholar), although other regions are also important (32.Leong P. MacLennan D.H. J. Biol. Chem. 1998; 273: 7791-7794Abstract Full Text Full Text PDF PubMed Scopus (99) Google Scholar, 33.Leong P. MacLennan D.H. Biochem. Cell Biol. 1999; 76: 681-694Crossref Scopus (43) Google Scholar).Three-dimensional structural information is essential to understanding how ryanodine receptors function but is difficult to obtain by X-crystallography owing to their large size and complexity, and because integral membrane proteins are inherently difficult to crystallize. Cryo-electron microscopy of purified, noncrystalline RyRs combined with three-dimensional image reconstruction offers a practical alternative to characterizing the three-dimensional structures of RyRs, albeit at less than atomic levels of resolution (34.Radermacher M. Rao V. Grassucci R. Frank J. Timerman A.P. Fleischer S. Wagenknecht T. J. Cell Biol. 1994; 127: 411-423Crossref PubMed Scopus (239) Google Scholar, 35.Serysheva I.I. Orlova E.V. Chiu W. Sherman M.B. Hamilton S.L. van Heel M. J. Struct. Biol. 1995; 2: 18-24Crossref Scopus (168) Google Scholar, 36.Sharma M.R. Penczek P. Grassucci R. Xin H.-B. Fleischer S. Wagenknecht T. J. Biol. Chem. 1998; 273: 18429-18434Abstract Full Text Full Text PDF PubMed Scopus (77) Google Scholar). Here we describe the first such reconstructions of RyR3, obtained under conditions that favor two different structural configurations of the receptor. Comparisons with previous reconstructions of the other two isoforms show that all three have a highly conserved three-dimensional organization in which about 80% of the protein mass forms a cytoplasmic assembly containing at least 10 distinguishable domains. One significant difference in morphology between RyR3 and RyR1 has been detected, and it likely corresponds to one of the regions of the sequence, the D2 region, that is hypervariable among the isoforms and is missing.DISCUSSIONThree-dimensional reconstructions of the RyR3 isoform in two different configurational states have been determined. The availability of detergent-solubilized RyR3 from bovine diaphragm terminal cisternae in sufficient amounts made these studies possible (16.Jeyakumar L.H. Copello J.A. O'Malley A.M. Wu G.-M. Grassucci R. Wagenknecht T. Fleischer S. J. Biol. Chem. 1998; 273: 16011-16020Abstract Full Text Full Text PDF PubMed Scopus (79) Google Scholar). Three-dimensional reconstructions in the 30–40 Å resolution range are now available for all three RyR isoforms (34.Radermacher M. Rao V. Grassucci R. Frank J. Timerman A.P. Fleischer S. Wagenknecht T. J. Cell Biol. 1994; 127: 411-423Crossref PubMed Scopus (239) Google Scholar, 35.Serysheva I.I. Orlova E.V. Chiu W. Sherman M.B. Hamilton S.L. van Heel M. J. Struct. Biol. 1995; 2: 18-24Crossref Scopus (168) Google Scholar, 36.Sharma M.R. Penczek P. Grassucci R. Xin H.-B. Fleischer S. Wagenknecht T. J. Biol. Chem. 1998; 273: 18429-18434Abstract Full Text Full Text PDF PubMed Scopus (77) Google Scholar).Open versus Closed RyR3 ReconstructionsReconstructions were determined for RyR3 under buffer conditions that should favor open and closed states of the receptor based upon conductance measurements on purified receptors following their incorporation into lipid bilayers (16.Jeyakumar L.H. Copello J.A. O'Malley A.M. Wu G.-M. Grassucci R. Wagenknecht T. Fleischer S. J. Biol. Chem. 1998; 273: 16011-16020Abstract Full Text Full Text PDF PubMed Scopus (79) Google Scholar, 19.Murayama T. Ogawa Y. J. Biol. Chem. 1997; 272: 24030-24037Abstract Full Text Full Text PDF PubMed Scopus (64) Google Scholar, 20.Chen S.R.W. Li X. Ebisawa K. Zhang L. J. Biol. Chem. 1997; 272: 24234-24246Abstract Full Text Full Text PDF PubMed Scopus (143) Google Scholar, 22.Murayama T. Oba T. Katayama E. Oyamada H. Oguchi K. Kobayashi M. Otsuka K. Ogawa Y. J. Biol. Chem. 1999; 274: 17297-17308Abstract Full Text Full Text PDF PubMed Scopus (78) Google Scholar). However, we cannot be certain that the solubilized RyR3 used for cryo-microscopy behaves exactly as do the receptors in a bilayer environment. Evidence that strongly suggests that solubilized receptors do indeed mimic reconstituted receptors when exposed to modulatory ligands comes from ryanodine binding experiments, which have been done using solubilized RyR3. Ryanodine is known to preferentially bind to the open form of the receptor over the closed form (41.Chu A. Diaz-Muñoz M. Hawkes M.J. Brush K. Hamilton S.L. Mol. Pharmacol. 1990; 37: 735-741PubMed Google Scholar), and for solubilized RyR3, ryanodine binding is modulated by known receptor activators and inhibitors in much the same manner as channel gating in bilayer experiments (19.Murayama T. Ogawa Y. J. Biol. Chem. 1997; 272: 24030-24037Abstract Full Text Full Text PDF PubMed Scopus (64) Google Scholar,22.Murayama T. Oba T. Katayama E. Oyamada H. Oguchi K. Kobayashi M. Otsuka K. Ogawa Y. J. Biol. Chem. 1999; 274: 17297-17308Abstract Full Text Full Text PDF PubMed Scopus (78) Google Scholar).It is reassuring that many of the differences that we found between the reconstructions of open and closed RyR3 are similar to changes described recently by another laboratory for closed and open RyR1 (40.Orlova E.V. Serysheva I.I. van Heel M. Hamilton S.L. Chiu W. Nat. Struct. Biol. 1996; 3: 547-552Crossref PubMed Scopus (146) Google Scholar,42.Serysheva I.I. Schatz M. van Heel M. Chiu W. Hamilton S.L. Biophys. J. 1999; 77: 1936-1944Abstract Full Text Full Text PDF PubMed Scopus (88) Google Scholar). The differences between open and closed RyR3 that are similar to those reported for RyR1 include the following.Transmembrane AssemblyFor RyR3 we found a 4° rotation of the transmembrane assembly in the open relative to the closed state when viewed from the SR junctional face (Fig. 3 (middle panels) and Fig. 4 A (left panel)). For RyR1, a rotation of the same magnitude and handedness was seen for the ryanodine-induced state (40.Orlova E.V. Serysheva I.I. van Heel M. Hamilton S.L. Chiu W. Nat. Struct. Biol. 1996; 3: 547-552Crossref PubMed Scopus (146) Google Scholar) but not when natural activators (nucleotide and Ca2+) were used. We suggest that this rotation, as well as the apparent expansion in the overall size of the transmembrane region that we find for the open form of RyR3 (Fig. 3), should not be interpreted literally, rather, they result from underlying changes in the relative orientations of the transmembrane domains that are contributed by each of the four receptor subunits. At the limited resolution of the current three-dimensional reconstructions, these structural rearrangements themselves are not resolved but may still manifest themselves as visible changes, such as apparent rotations or changes in shape of the transmembrane region. Support for this interpretation is presented in Fig. 4 B (and for RyR1 in the study by Serysheva et al. (42.Serysheva I.I. Schatz M. van Heel M. Chiu W. Hamilton S.L. Biophys. J. 1999; 77: 1936-1944Abstract Full Text Full Text PDF PubMed Scopus (88) Google Scholar)) in which a high density threshold has been used to display the reconstructions of open and closed receptors. There it appears that the four transmembrane domains change their orientation with respect to the symmetry axis of the receptor so that regions at the distal end of the transmembrane assembly move away from the symmetry axis, perhaps thereby creating a pathway for ions to travel when the receptor is in its native environment. A similar, but not identical, interpretation has been described for RyR1 (42.Serysheva I.I. Schatz M. van Heel M. Chiu W. Hamilton S.L. Biophys. J. 1999; 77: 1936-1944Abstract Full Text Full Text PDF PubMed Scopus (88) Google Scholar).Cytoplasmic AssemblyFor RyR1 a pronounced weakening or separation of the apparent connection between domains 9 and 10 in the clamp assemblies that form the corners of the cytoplasmic assembly was observed (40.Orlova E.V. Serysheva I.I. van Heel M. Hamilton S.L. Chiu W. Nat. Struct. Biol. 1996; 3: 547-552Crossref PubMed Scopus (146) Google Scholar, 42.Serysheva I.I. Schatz M. van Heel M. Chiu W. Hamilton S.L. Biophys. J. 1999; 77: 1936-1944Abstract Full Text Full Text PDF PubMed Scopus (88) Google Scholar). For RyR3 we do not observe a distinct separation of these domains, although there is a slight weakening, of uncertain significance, of the connection between them (cf. left panels of Figs. 3, a and b, and 5). Perhaps this difference between the isoforms is related to the unique role that RyR1 plays in excitation-contraction coupling in mature skeletal muscle. In situ, RyR1 is thought to interact with the voltage sensing dihydropyridine receptors via the clamp regions of its cytoplasmic assembly (discussed in the following section), but RyR3 apparently cannot undergo this interaction.Another change in the clamp regions of the cytoplasmic assembly is in the disposition of domain 6, which makes the cytoplasmic assembly appear somewhat thicker in the direction parallel to the 4-fold symmetry axis (Fig. 3, right panels) and reduces the apparent separation between domains 5 and 6 when the receptor is viewed onto the cytoplasmic face (cf. Fig. 3, left panels). Again, quite similar changes were reported for RyR1.The remarkable similarities of RyR1 and RyR3 with regard to structural changes associated with the open and closed conditions are strong evidence that these changes are not artifactual and that they indeed correspond to authentic perturbations of the structure associated with receptor gating. An intriguing aspect of the structural differences between the open and closed states of both RyR1 and RyR3 is their global nature. The changes associated with the clamp assemblies are over 100 Å from the likely location of the channel gating mechanism in the transmembrane assembly. Previous work from our laboratory has shown that proteins that are known to modulate channel gating of RyR1 (calmodulin, FK506-binding protein, and imperatoxin A) also bind far from the transmembrane region (10.Wagenknecht T. Radermacher M. Grassucci R. Berkowitz J. Xin H.-B. Fleischer S. J. Biol. Chem. 1997; 272: 32463-32471Abstract Full Text Full Text PDF PubMed Scopus (151) Google Scholar, 43.Samso M. Trujillo R. Gurrola G.B. Valdivia H.H. Wagenknecht T. J. Cell Biol. 1999; 146: 493-499Crossref PubMed Scopus (63) Google Scholar). Thus, very long range structural rearrangements, presumably underlying allosteric communication between functional sites, seem to be a general feature of ryanodine receptors.Comparison of RyR3 and RyR1 Three-dimensional ArchitectureOverall RyR3 and RyR1 are nearly identical in structure at the resolutions attained in the three-dimensional reconstructions reported thus far. Analysis of the sequences has shown that one of the three hypervariable regions, named D2 (residues 1303–1406 in RyR1) by Sorrentino and Volpe (29.Sorrentino V. Volpe P. Trends Pharmacol. Sci. 1993; 14: 98-103Abstract Full Text PDF PubMed Scopus (272) Google Scholar), is completely absent from the RyR3 sequence. A deletion of this magnitude should be easily detectable in a comparison of the RyR1 and RyR3 reconstructions, assuming that in RyR1 the D2 region forms a rigid, globular fold and that other perturbations of the surrounding structure are minimal. Indeed, when we compared the structures of RyR1 and RyR3, which were determined under similar solution conditions and with identical methodology, a single region of excess mass present in RyR1 dominated all other differences (Fig. 6). Furthermore, the overall size of the region (≈35 × 20 Å) was reasonable for a segment containing ≈100 amino acid residues. The region is located in domain 6 along each of the exterior edges of the receptor.Further support for our proposal that the major difference between the reconstructions of RyR1 and RyR3 represents the D2 region comes from the immuno-electron microscopy study by Murayama et al.(22.Murayama T. Oba T. Katayama E. Oyamada H. Oguchi K. Kobayashi M. Otsuka K. Ogawa Y. J. Biol. Chem. 1999; 274: 17297-17308Abstract Full Text Full Text PDF PubMed Scopus (78) Google Scholar), who visualized immunocomplexes of RyR1 and a D2-specific antibody in negative stain. They observed the antibody bound to the clamp regions of the receptor, in agreement with our interpretation of the major difference between the RyR1 and RyR3 reconstructions. Furthermore, we have analyzed the images described by Murayama et al. (22.Murayama T. Oba T. Katayama E. Oyamada H. Oguchi K. Kobayashi M. Otsuka K. Ogawa Y. J. Biol. Chem. 1999; 274: 17297-17308Abstract Full Text Full Text PDF PubMed Scopus (78) Google Scholar) in our own laboratory by digitizing them and then applying limited image averaging to them (data not shown), and we conclude that the D2-specific antibodies are bound along the edges of the receptor in the vicinity of domain 6, as we would predict based upon the findings described here. However, a three-dimensional reconstruction of immunocomplexes containing a D2-specific antibody is required to prove that the binding site corresponds exactly to the region that we contend contains the D2 region, and we are currently working toward this goal.The D2 region is currently of interest because of its perceived importance in excitation-contraction coupling in skeletal and cardiac muscle (27.Tarroni P. Rossi D. Conti A. Sorrentino V. J. Biol. Chem. 1997; 272: 19808-19813Abstract Full Text Full Text PDF PubMed Scopus (75) Google Scholar). A region nearby in the sequence involving residues 1076–1122 appears also to be important and to play a role in the interaction of RyR1 with the voltage sensors/dihydropyridine receptors (32.Leong P. MacLennan D.H. J. Biol. Chem. 1998; 273: 7791-7794Abstract Full Text Full Text PDF PubMed Scopus (99) Google Scholar, 33.Leong P. MacLennan D.H. Biochem. Cell Biol. 1999; 76: 681-694Crossref Scopus (43) Google Scholar, 44.Leong P. MacLennan D.H. J. Biol. Chem. 1998; 273: 29958-29964Abstract Full Text Full Text PDF PubMed Scopus (81) Google Scholar). Based largely upon freeze-fracture studies of skeletal muscle sarcolemma/transverse tubules and SR, Franzini-Armstrong and co-workers (46.Franzini-Armstrong C. Jorgensen A.O. Annu. Rev. Physiol. 1994; 56: 509-534Crossref PubMed Scopus (342) Google Scholar) have proposed a structural model for the triad junction, regions where the transverse tubules and SR form junctions to carry out excitation-contraction coupling. In this model the dihydropyridine receptors are arranged in groups of four (tetrads), which appear to be in register with the tetrameric RyR1 s in the opposing junctional SR (6.Franzini-Armstrong C. Protasi F. Physiol. Rev. 1997; 77: 699-729Crossref PubMed Scopus (591) Google Scholar, 45.Block B.A. Imagawa T. Campbell K.P. Franzini-Armstrong C. J. Cell Biol. 1988; 107: 2587-2600Crossref PubMed Scopus (586) Google Scholar, 46.Franzini-Armstrong C. Jorgensen A.O. Annu. Rev. Physiol. 1994; 56: 509-534Crossref PubMed Scopus (342) Google Scholar, 47.Flucher B.E. Franzini-Armstrong C. Proc. Natl. Acad. Sci. U. S. A. 1996; 93: 8101-8106Crossref PubMed Scopus (187) Google Scholar). Accordingly, it appears that each dihydropyridine receptor within a tetrad would interact with one of the clamp assemblies in the adjoining RyR1 (39.Malhotra 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, 48.Wagenknecht T. Radermacher M. FEBS Lett. 1995; 369: 43-46Crossref PubMed Scopus (40) Google Scholar, 49.Samso M. Wagenknecht T. J. Struct. Biol. 1998; 121: 172-180Crossref PubMed Scopus (38) Google Scholar). Thus, the location that we have assigned to the D2 region in the three-dimensional structure of RyR appears to be consistent with current models of the triad junction's architecture.In conclusion, we have established that RyR3 is amenable to study by three-dimensional cryo-microscopy, and that RyR3, although similar in its overall three-dimensional architecture to the other RyR isoforms, does reveal at least one significant difference that is attributed provisionally to the D2 region of the amino acid sequence of the receptor. We also observed several structural differences at diverse locations between two conformational states of RyR3 that likely correspond to, or are related to, open and closed states of the receptor. Future studies at higher resolution will certainly resolve additional structural differences between the isoforms and clarify the structural basis of channel gating. Intracellular calcium is essential for the regulation and function of many fundamental biological processes, and the release of calcium from intracellular storage compartments plays an essential role in modulating cytoplasmic Ca2+ levels (1.Berridge M.J. J. Physiol. (Lond.). 1997; 499: 291-306Crossref Scopus (916) Google Scholar). Ryanodine receptors (RyRs)1 and inositol 1,4,5-trisphosphate receptors are the two families of intracellular calcium release channels that have been characterized to date (reviewed in Refs. 2.Fleischer S. Inui M. Annu. Rev. Biophys. Biophys. Chem. 1989; 18: 333-364Crossref PubMed Scopus (442) Google Scholar, 3.Meissner G. Annu. Rev. Physiol. 1994; 56: 485-508Crossref PubMed Scopus (836) Google Scholar, 4.Ogawa Y. Crit. Rev. Biochem. Mol. Biol. 1994; 29: 229-274Crossref PubMed Scopus (228) Google Scholar, 5.Sorrentino V. Sorrentino V. Ryanodine Receptors. CRC Press, Boca Raton, FL1995: 85-100Google Scholar, 6.Franzini-Armstrong C. Protasi F. Physiol. Rev. 1997; 77: 699-729Crossref PubMed Scopus (591) Google Scholar, 7.Mikoshiba K. Curr. Opin. Neurobiol. 1997; 7: 339-345Crossref PubMed Scopus (160) Google Scholar, 8.Shoshan-Barmatz V. Ashley R.H. Int. Rev. Cytol. 1998; 183: 185-271Crossref PubMed Google Scholar, 9.Ogawa Y. Kurebayashi N. Murayama T. Adv. Biophys. 1999; 36: 27-64Crossref PubMed Scopus (61) Google Scholar). Both RyRs and inositol 1,4,5-trisphosphate receptors exist as homotetrameric protein complexes composed of an unusually large subunit, ≈560 kDa for RyRs and ≈313 kDa for inositol 1,4,5-trisphosphate receptors. Frequently an ≈12-kDa isoform of FK506-binding protein (FKBP, an immunophilin) binds with sufficient affinity to the receptors so as to co-purify with them, and the immunophilin may therefore be considered as a component of the receptors (10.Wagenknecht T. Radermacher M. Grassucci R. Berkowitz J. Xin H.-B. Fleischer S. J. Biol. Chem. 1997; 272: 32463-32471Abstract Full Text Full Text PDF PubMed Scopus (151) Google Scholar, 11.Qi Y. Ogunbunmi E." @default.
• W2046822541 created "2016-06-24" @default.
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• W2046822541 title "Three-dimensional Structure of Ryanodine Receptor Isoform Three in Two Conformational States as Visualized by Cryo-electron Microscopy" @default.
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