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.
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.