Skip to main content

Thank you for visiting nature.com. You are using a browser version with limited support for CSS. To obtain the best experience, we recommend you use a more up to date browser (or turn off compatibility mode in Internet Explorer). In the meantime, to ensure continued support, we are displaying the site without styles and JavaScript.

The amino-terminal disease hotspot of ryanodine receptors forms a cytoplasmic vestibule

Abstract

Many physiological events require transient increases in cytosolic Ca2+ concentrations. Ryanodine receptors (RyRs) are ion channels that govern the release of Ca2+ from the endoplasmic and sarcoplasmic reticulum1. Mutations in RyRs can lead to severe genetic conditions that affect both cardiac and skeletal muscle, but locating the mutated residues in the full-length channel structure has been difficult2,3. Here we show the 2.5 Å resolution crystal structure of a region spanning three domains of RyR type 1 (RyR1), encompassing amino acid residues 1–559. The domains interact with each other through a predominantly hydrophilic interface. Docking in RyR1 electron microscopy maps4,5 unambiguously places the domains in the cytoplasmic portion of the channel, forming a 240-kDa cytoplasmic vestibule around the four-fold symmetry axis. We pinpoint the exact locations of more than 50 disease-associated mutations in full-length RyR1 and RyR2. The mutations can be classified into three groups: those that destabilize the interfaces between the three amino-terminal domains, disturb the folding of individual domains or affect one of six interfaces with other parts of the receptor. We propose a model whereby the opening of a RyR coincides with allosterically coupled motions within the N-terminal domains. This process can be affected by mutations that target various interfaces within and across subunits. The crystal structure provides a framework to understand the many disease-associated mutations in RyRs that have been studied using functional methods, and will be useful for developing new strategies to modulate RyR function in disease states.

Your institute does not have access to this article

Relevant articles

Open Access articles citing this article.

Access options

Buy article

Get time limited or full article access on ReadCube.

$32.00

All prices are NET prices.

Figure 1: Overall structure of the RyR1 A, B and C domains.
Figure 2: Docking of RyR1 ABC in the 9.6 Å RyR1 cryo-EM map.
Figure 3: Disease-associated mutations in RyR1 and RyR2.
Figure 4: The

Accession codes

Primary accessions

Protein Data Bank

Data deposits

Atomic coordinates and structure factors for the RyR1 ABC structure have been deposited with the Protein Data Bank (http://www.rcsb.org) under accession code 2XOA.

References

  1. Giannini, G., Conti, A., Mammarella, S., Scrobogna, M. & Sorrentino, V. The ryanodine receptor/calcium channel genes are widely and differentially expressed in murine brain and peripheral tissues. J. Cell Biol. 128, 893–904 (1995)

    CAS  Article  Google Scholar 

  2. Betzenhauser, M. J. & Marks, A. R. Ryanodine receptor channelopathies. Pflügers Arch. 460, 467–480 (2010)

    CAS  Article  Google Scholar 

  3. Robinson, R., Carpenter, D., Shaw, M. A., Halsall, J. & Hopkins, P. Mutations in RYR1 in malignant hyperthermia and central core disease. Hum. Mutat. 27, 977–989 (2006)

    CAS  Article  Google Scholar 

  4. Ludtke, S. J., Serysheva, I. I., Hamilton, S. L. & Chiu, W. The pore structure of the closed RyR1 channel. Structure 13, 1203–1211 (2005)

    CAS  Article  Google Scholar 

  5. Samsó, M., Wagenknecht, T. & Allen, P. D. Internal structure and visualization of transmembrane domains of the RyR1 calcium release channel by cryo-EM. Nature Struct. Mol. Biol. 12, 539–544 (2005)

    Article  Google Scholar 

  6. MacLennan, D. H. & Chen, S. R. Store overload-induced Ca2+ release as a triggering mechanism for CPVT and MH episodes caused by mutations in RYR and CASQ genes. J. Physiol. (Lond.) 587, 3113–3115 (2009)

    CAS  Article  Google Scholar 

  7. Amador, F. J. et al. Crystal structure of type I ryanodine receptor amino-terminal β-trefoil domain reveals a disease-associated mutation ‘hot spot’ loop. Proc. Natl Acad. Sci. USA 106, 11040–11044 (2009)

    ADS  CAS  Article  Google Scholar 

  8. Lobo, P. A. & Van Petegem, F. Crystal structures of the N-terminal domains of cardiac and skeletal muscle ryanodine receptors: insights into disease mutations. Structure 17, 1505–1514 (2009)

    CAS  Article  Google Scholar 

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

    Article  Google Scholar 

  10. Garzon, J. I., Kovacs, J., Abagyan, R. & Chacon, P. ADP_EM: fast exhaustive multi-resolution docking for high-throughput coverage. Bioinformatics 23, 427–433 (2007)

    CAS  Article  Google Scholar 

  11. Chacón, P. & Wriggers, W. Multi-resolution contour-based fitting of macromolecular structures. J. Mol. Biol. 317, 375–384 (2002)

    Article  Google Scholar 

  12. Serysheva, I. I., Hamilton, S. L., Chiu, W. & Ludtke, S. J. Structure of Ca2+ release channel at 14 Å resolution. J. Mol. Biol. 345, 427–431 (2005)

    CAS  Article  Google Scholar 

  13. Liu, Z. et al. Three-dimensional reconstruction of the recombinant type 3 ryanodine receptor and localization of its amino terminus. Proc. Natl Acad. Sci. USA 98, 6104–6109 (2001)

    ADS  CAS  Article  Google Scholar 

  14. Wang, R. et al. Localization of an NH2-terminal disease-causing mutation hot spot to the ‘clamp’ region in the three-dimensional structure of the cardiac ryanodine receptor. J. Biol. Chem. 282, 17785–17793 (2007)

    CAS  Article  Google Scholar 

  15. Baker, M. L. et al. The skeletal muscle Ca2+ release channel has an oxidoreductase-like domain. Proc. Natl Acad. Sci. USA 99, 12155–12160 (2002)

    ADS  CAS  Article  Google Scholar 

  16. Wriggers, W. & Chacón, P. Modeling tricks and fitting techniques for multiresolution structures. Structure 9, 779–788 (2001)

    CAS  Article  Google Scholar 

  17. Serysheva, I. I. et al. Subnanometer-resolution electron cryomicroscopy-based domain models for the cytoplasmic region of skeletal muscle RyR channel. Proc. Natl Acad. Sci. USA 105, 9610–9615 (2008)

    ADS  CAS  Article  Google Scholar 

  18. Bhuiyan, Z. A. et al. Expanding spectrum of human RYR2-related disease: new electrocardiographic, structural, and genetic features. Circulation 116, 1569–1576 (2007)

    Article  Google Scholar 

  19. Marjamaa, A. et al. Search for cardiac calcium cycling gene mutations in familial ventricular arrhythmias resembling catecholaminergic polymorphic ventricular tachycardia. BMC Med. Genet. 10, 12 (2009)

    Article  Google Scholar 

  20. Bellinger, A. M. et al. Hypernitrosylated ryanodine receptor calcium release channels are leaky in dystrophic muscle. Nature Med. 15, 325–330 (2009)

    CAS  Article  Google Scholar 

  21. Durham, W. J. et al. RyR1 S-nitrosylation underlies environmental heat stroke and sudden death in Y522S RyR1 knockin mice. Cell 133, 53–65 (2008)

    CAS  Article  Google Scholar 

  22. Eu, J. P., Sun, J., Xu, L., Stamler, J. S. & Meissner, G. The skeletal muscle calcium release channel: coupled O2 sensor and NO signaling functions. Cell 102, 499–509 (2000)

    CAS  Article  Google Scholar 

  23. Xu, L., Eu, J. P., Meissner, G. & Stamler, J. S. Activation of the cardiac calcium release channel (ryanodine receptor) by poly-S-nitrosylation. Science 279, 234–237 (1998)

    ADS  CAS  Article  Google Scholar 

  24. Aracena-Parks, P. et al. Identification of cysteines involved in S-nitrosylation, S-glutathionylation, and oxidation to disulfides in ryanodine receptor type 1. J. Biol. Chem. 281, 40354–40368 (2006)

    CAS  Article  Google Scholar 

  25. Ikemoto, N. & Yamamoto, T. Regulation of calcium release by interdomain interaction within ryanodine receptors. Front. Biosci. 7, d671–d683 (2002)

    CAS  Article  Google Scholar 

  26. Bosanac, I. et al. Structure of the inositol 1,4,5-trisphosphate receptor binding core in complex with its ligand. Nature 420, 696–700 (2002)

    ADS  CAS  Article  Google Scholar 

  27. Bosanac, I. et al. Crystal structure of the ligand binding suppressor domain of type 1 inositol 1,4,5-trisphosphate receptor. Mol. Cell 17, 193–203 (2005)

    CAS  Article  Google Scholar 

  28. Chan, J. et al. Ligand-induced conformational changes via flexible linkers in the amino-terminal region of the inositol 1,4,5-trisphosphate receptor. J. Mol. Biol. 373, 1269–1280 (2007)

    CAS  Article  Google Scholar 

  29. Kabsch, W. XDS . Acta Crystallogr. D 66, 125–132 (2010)

    CAS  Article  Google Scholar 

  30. McCoy, A. J. et al. Phaser crystallographic software. J. Appl. Cryst. 40, 658–674 (2007)

    CAS  Article  Google Scholar 

  31. Van Petegem, F., Clark, K. A., Chatelain, F. C. & Minor, D. L., Jr Structure of a complex between a voltage-gated calcium channel β-subunit and an α-subunit domain. Nature 429, 671–675 (2004)

    ADS  CAS  Article  Google Scholar 

  32. Langer, G., Cohen, S. X., Lamzin, V. S. & Perrakis, A. Automated macromolecular model building for X-ray crystallography using ARP/wARP version 7. Nature Protocols 3, 1171–1179 (2008)

    CAS  Article  Google Scholar 

  33. Emsley, P. & Cowtan, K. Coot: model-building tools for molecular graphics. Acta Crystallogr. D 60, 2126–2132 (2004)

    Article  Google Scholar 

  34. Murshudov, G. N., Vagin, A. A. & Dodson, E. J. Refinement of macromolecular structures by the maximum-likelihood method. Acta Crystallogr. D 53, 240–255 (1997)

    CAS  Article  Google Scholar 

  35. Laskowski, R. A., Macarthur, M. W., Moss, D. S. & Thornton, J. M. PROCHECK: a program to check the stereochemical quality of protein structures. J. Appl. Cryst. 26, 283–291 (1993)

    CAS  Article  Google Scholar 

  36. Volkmann, N. Confidence intervals for fitting of atomic models into low-resolution densities. Acta Crystallogr. D 65, 679–689 (2009)

    CAS  Article  Google Scholar 

  37. Volkmann, N. & Hanein, D. Docking of atomic models into reconstructions from electron microscopy. Methods Enzymol. 374, 204–225 (2003)

    CAS  Article  Google Scholar 

Download references

Acknowledgements

We thank the staff at beamline 08ID-1 of the Canadian Light source, the Stanford Synchrotron Radiation Lightsource, K. Beam for the rabbit RyR1 clone, K. Lau for assistance with preparing the figures and the movie file, and C. Ahern, E. Moore and R. Pancaroglu for comments on the manuscript. F.V.P. is funded by the Canadian Institutes of Health Research (CIHR) and the Heart and Stroke Foundation of Canada, and is a CIHR new investigator and a Michael Smith Foundation for Health Research Scholar.

Author information

Authors and Affiliations

Authors

Contributions

C.-C.T. expressed, purified and crystallized the protein, and collected diffraction data. P.A.L. cloned several initial constructs and assisted with the melting curve analysis. L.K. prepared the disease-associated mutation, purified the corresponding protein and measured the melting curves. F.V.P. designed and supervised the experiments, collected diffraction data, solved the structure, performed the docking experiments, and wrote the manuscript.

Corresponding author

Correspondence to Filip Van Petegem.

Ethics declarations

Competing interests

The authors declare no competing financial interests.

Supplementary information

Supplementary Information

The file contains a Supplementary Discussion, Supplementary Tables 1-3 and Supplementary Figures 1-7 with legends. (PDF 3564 kb)

Supplementary Movie 1

This movie file shows a rotation of the docked RyR1ABC crystal structure inside the 9.6Å cryoEM map of RyR1 in the closed state. (MOV 9601 kb)

PowerPoint slides

Rights and permissions

Reprints and Permissions

About this article

Cite this article

Tung, CC., Lobo, P., Kimlicka, L. et al. The amino-terminal disease hotspot of ryanodine receptors forms a cytoplasmic vestibule. Nature 468, 585–588 (2010). https://doi.org/10.1038/nature09471

Download citation

  • Received:

  • Accepted:

  • Published:

  • Issue Date:

  • DOI: https://doi.org/10.1038/nature09471

Further reading

Comments

By submitting a comment you agree to abide by our Terms and Community Guidelines. If you find something abusive or that does not comply with our terms or guidelines please flag it as inappropriate.

Search

Quick links

Nature Briefing

Sign up for the Nature Briefing newsletter — what matters in science, free to your inbox daily.

Get the most important science stories of the day, free in your inbox. Sign up for Nature Briefing