Flex your ryanodine receptor research muscles right here.

Due to their role in many disorders, ion channels are the focus of many drug discovery initiatives. Since the determination of the first ion channel structure, KcsA, by the MacKinnon Lab in 1998(1), we have witnessed many great advancements in the field of ion channel structural biology. Recent examples of some of these important ion channel structures include the TRPV1 channel(2), the Cav1.1 channel(3), the Nav1.7 channel(4), and the TREK-2 channel(5), to name a few. 

One important family of ion channels are ryanodine receptors. These large (> 2000 kDa), homotetrameric, intracellular ligand-gated calcium channels mediate the rapid release of calcium from the sarcoplasmic reticulum into the cytosol leading to muscle contraction in cardiac and skeletal tissue(6). Mutations in these channels lead to a number of disorders, including cardiac arrhythmias and muscular dystrophy. Three different isoforms of ryanodine receptors exist: RyR1, which is expressed mainly in skeletal muscle; RyR2, which is expressed in the heart and smooth muscle; and RyR3, which is expressed in the brain and other tissues. The activity of all three ryanodine receptor proteins is regulated by a number of proteins and small molecules including Ca2+, ATP, caffeine, Cav1.1, calstabin, calmodulin, and ryanodine(7).

Work on obtaining the structure of the ryanodine receptor has been ongoing for many years, with the deamination of the first low resolution map by negative stain EM in 1989(8). The year 2005 saw the publication of multiple cryo-EM structures of RyR1, in both open and closed conformations, at resolutions ranging from 14 Å to 9.6 Å(9, 10, 11) allowing for a greater understanding of the mechanism of the ryanodine receptor channels. 

Thanks to the major advancements in the field of single-particle electron cryo-microscopy, the past two years have seen an explosion of high-resolution ryanodine receptor structures. At the beginning of 2015, three different groups published cryo-EM structures of RyR1 simultaneously: 

  • The Hendrickson, Frank, and Marks labs at Columbia University determined the closed-state structure of RyR1 in complex with FKBP12.6 (calstabin2) to 4.8 Å (PDB: 3J8E)(12)
  • The Yan lab at Tsinghua University determined the closed-state structure of RyR1 in complex with its modulator FKBP12 (calstabin1) to 3.8 Å resolution (PDB: 3J8H)(13)
  • The Raunser lab at the Max Planck Institute of Molecular Physiology determined both the closed-state (PDB: 4UWA) and open-state (PDB: 4UWE) structures of RyR1 to 6.1 Å and 8.5 Å resolution, respectively(14)

Together, these structures provided a detailed look at the architecture of the RyR1 channel, including the six-helix transmembrane pore, the calcium binding domain, and allowed assignment of domains in the large cytosolic N-terminal domain. Additionally, the information gleaned from these structures also permitted the refinement of hypotheses of the mechanism allosteric regulation of the channel. A detailed table of how these proteins were prepared for EM studies is presented below.

Building upon these groundbreaking structures, a number of additional structures of RyR1 in various states were recently published helping to further elucidate the mechanism(15, 16). In September, 2016, the Hendrickson, Marks, and Frank labs determined over 25 structures of both open and closed state RyR1 in the presence of multiple activators, including Ca2+, ATP, caffeine, and ryanodine, providing the structural basis for stochastic gating and activation by this channel(17)

Lastly, in September, 2016, the first structures of both the closed (PDB: 5GO9) and open (PDB: 5GOA) states of the RyR2 channel were determined by the Yan Lab at Tsinghua University in China(18). These structures were determined to 4.4 Å and 4.2 Å, respectively. Comparison of the open and closed state structures of RyR2 suggest that the opening and closing of the channel is controlled via conformational changes within the central domain. 

Anatrace Detergents
In these notable publications, CHAPS, DOPC, Digitonin (GDN), Tween-20, Amphipol A8-35, and fluorinated octyl maltoside, were used in solubilization, purification, and/or EM studies. At Anatrace, we’re seriously committed to developing and supplying the industry’s finest high-purity products – and equally committed to the high standards that make it possible. Our standards have made Anatrace an internationally-recognized leader in manufacturing reagents for membrane protein studies and custom chemical synthesis. And those same standards mean you’ll have the confidence to aim higher, too.

 Table of ryanodine receptor structures:
PDB Name Ref.  Protein Preparation
 3J8E RyR1 – calstabin2, closed  (12) Membranes from rabbit skeletal muscle were solubilized and purified using CHAPS.  The final buffer for EM contained CHAPS and DOPC
 3J8H RyR1 – calstabin, closed  (13)

Membranes containing RyR1 were solubilized using CHAPS and soybean phospholipids, and the protein purified using Tween-20

 4UWA
 4UWE
RyR1, closed
RyR1, open
 (14)

Sarcoplasmic reticulum vesicles were solubilized using CHAPS and soy lecithin, and the protein purified using CHAPS.  For EM, RyR1 was reconstituted into nanodiscs.  0.2 % fluorinated OM was used to improve vitrification. 

 5GKY
 5GL1
RyR1 - calstabin, closed
RyR1 - calstabin, open
 (15)

Membranes containing RyR1 were solubilized using CHAPS and soybean phospholipids, and the protein purified using Tween-20.  The open-state structure was purified using CHAPS.

 5J8V RyR1, open  (16)

Solubilized in CHAPS and soybean lecithin.  Purified in CHAPS and exchanged into Amphipol A8-35 for Cryo- EM

 5GO9
 5GOA
RyR2, closed
RyR2, open
 (18)

Sarcoplasmic reticulum membranes were solubilized in CHAPS and soybean lecithin.  RyR2 was purified in Digitonin.

 5TB0 RyR1 – calstabin2, multiple states  (17)

Membranes from rabbit skeletal muscle were solubilized and purified using CHAPS.  The final buffer for EM contained CHAPS and DOPC. 

 

References:
  1. Doyle, D. A., et al. (1998) Science 280(5360), 69-77.
  2. Liao, M., et al. (2013) Nature 504(7478), 107-112.
  3. Wu, J., et al. (2015) Science 350(6267), aad2395.
  4. Ahuja, S., et al. (2015) Science 350(6267), aac5464.
  5. Dong, Y. Y., et al. (2015) Science 347(6227), 1256-1259.
  6. Baker, M. R., Fan, G., and Serysheva, I. I. (2015) Eur J Transl Myol 25(1), 35-48.
  7. Clarke, O. B. and Hendrickson, W. A. (2016) Curr Opin Struct Biol 39, 144-152.
  8. Wagenknecht, T., et al. (1989) Nature 338(6211), 167-170.
  9. Serysheva, I. I., et al. (2005) J Mol Biol. 345(3), 427-431.
  10. Samsó, M., Wagenknecht, T., and Allen, P. D. (2005) Nat Struct Mol Biol. 12(6), 539-544.
  11. Ludtke, S. J., et al. (2005) Structure 13(8), 1203-1211.
  12. Zalk, R., et al. (2015) Nature 517(7532), 44-49.
  13. Yan, Z, et al. (2015) Nature 517(7532), 50-55.
  14. Efremov, R. G., et al. (2015) Nature 517(7532), 39-43.
  15. Bai, X. C., et al. (2016) Cell Research 26(9), 995-1006.
  16. Wei, R., et al. (2016) Cell Research 26(9), 977-994.
  17. des Georges, A., et al. (2016) Cell 167(1), 145-157.
  18. Peng, W., et al. (2016) Science Sep 22/science.aah5324.