Ryanodine Receptors: Allosteric Ion Channel Giants Filip Van Petegem PII: DOI: Reference:

S0022-2836(14)00419-7 doi: 10.1016/j.jmb.2014.08.004 YJMBI 64533

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Journal of Molecular Biology

Received date: Revised date: Accepted date:

9 June 2014 2 August 2014 5 August 2014

Please cite this article as: Van Petegem, F., Ryanodine Receptors: Allosteric Ion Channel Giants, Journal of Molecular Biology (2014), doi: 10.1016/j.jmb.2014.08.004

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ACCEPTED MANUSCRIPT Ryanodine Receptors: Allosteric Ion Channel Giants Filip Van Petegem

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The University of British Columbia, Department of Biochemistry and Molecular Biology

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Correspondence: [email protected]

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2350 Health Sciences Mall,

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V6T 1Z3, Vancouver, BC, Canada

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Phone: +1 604 827 4267

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ABSTRACT

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The endoplasmic (ER) and sarcoplasmic reticulum (SR) form major intracellular Ca2+ stores. Ryanodine Receptors (RyRs) are large tetrameric ion channels in the SR and ER membranes that can release the Ca2+ upon triggering. With molecular weights exceeding 2.2 MDa, they represent the pinnacle of ion channel complexity. RyRs have adopted long-range allosteric mechanisms, with pore opening resulting in conformational changes over 200Å away. Together with the tens of protein and small molecule modulators, RyRs have adopted rich and complex regulatory mechanisms. Structurally related to inositol-1,4,5-trisphosphate receptors (IP3Rs), RyRs have been studied extensively using cryo-electron microscopy. Along with more recent X-ray crystallographic analyses of individual domains, these have resulted in pseudo-atomic models. Over 500 mutations in RyRs have been linked to severe genetic disorders, which underscore their role in the contraction of cardiac and skeletal muscle. Most of these have been linked to gain-of-function phenotypes, resulting in premature or prolonged leak of Ca2+ in the cytosol. This review outlines our current knowledge on the structure of RyRs at high and low resolution, their relationship to IP3Rs, an overview of the most commonly studied regulatory mechanisms, and models that relate disease-causing mutations to altered channel function.

Keywords: Excitation-contraction coupling, allostery, structural biology, calcium release, genetic disease

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ACCEPTED MANUSCRIPT INTRODUCTION

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Over four decades ago, electron dense protrusions were identified between the sarcoplasmic reticulum (SR) and transverse tubules of the plasma membrane in frog twitch fibers1. Although their identities were unknown at the time, later purification studies confirmed them to be Ryanodine Receptors (RyRs), the major Ca2+ release channels from the SR and ER2; 3. RyRs owe their name to Ryanodine, a small molecule from the south American plant Ryania speciosa with insecticidal properties4. Ryanodine preferentially interacts with open RyRs, a property that has been used widely to probe the functional state of the channel.

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Mammalian species contain three RyR isoforms (RyR1-3). Since their initial cloning5; 6; 7; 8; 9, RyRs are still the largest ion channels identified to this day, with molecular weights exceeding 2.2MDa. They form homotetrameric assemblies, with individual subunits consisting of ~5000 amino acids. RyRs are found in a wide variety of cell types, including neurons, lymphocytes, epithelial cells, and exocrine cells10, but have a claim to fame for their role in excitation-contraction (E-C) coupling. Together with L-type voltagegated calcium channels, they convert an electrical signal (depolarization of the plasma membrane) into a chemical one, by releasing the potent intracellular messenger Ca2+. RyR1 is the predominant isoform found in skeletal muscle, whereas RyR2 is mostly found in cardiac myocytes. However, the widely used names ‘skeletal muscle isoform’ and ‘cardiac isoform’ are misleading because they are expressed in many different cell types. RyRs are also found in lower organisms, where they are as large and as complex as their mammalian counterparts. Non-mammalian vertebrates express two RyR isoforms (typically called RyRα and RyRβ), whereas insects, including D. melanogaster, and other invertebrates like nematodes, sea urchin, and lobster only contain a single isoform10. Clear evidence for bona fide RyRs in non-metazoan species is lacking, but protozoa like Paramecium tetraurelium contain up to 34 genes predicted to form Ca2+ release channels. Although they lack several domains normally found in all RyRs, some of these are sensitive to pharmacological agents known to activate mammalian RyRs11. RyRs act as both signal integrators and signal amplifiers. Their large structure contains docking sites for tens of small molecules and auxiliary proteins that can provide positive or negative inputs. These input signals are integrated by the RyR, resulting in either opening or closing. Open RyRs have a large single channel conductance of ~100pS for Ca2+, but subconductance states have also been observed2; 12; 13; 14; 15. One of the signals that stimulates RyR opening is Ca2+. As such, RyRs are also signal amplifiers: by detecting small increases in cytosolic Ca2+, they can release more Ca2+ into the cytosol, through a process known as Ca2+ induced Ca2+ release (CICR) 16; 17. However, the relationship between Ca2+ and RyR opening is very complex, as larger cytosolic Ca2+ levels can trigger closing18, and RyRs respond to luminal Ca2+ as well19; 20. In skeletal muscle, it has been shown that depolarization of the plasma membrane can trigger Ca2+ release even in the absence of extracellular Ca2+. Many studies suggest that RyR1 and CaV1.1 form direct physical contacts, with CaV1.1 acting as the voltage sensor for RyR1 through direct mechanical coupling 21; 22; 23; 24; 25. RyR1 and RyR2 have been implicated in a number of serious genetic disorders of cardiac or skeletal muscle tissue. RyR2 mutations have been linked to catecholaminergic polymorphic ventricular tachycardia (CPVT)26; 27, whereas mutations in RyR1 has been linked primarily to malignant hyperthermia 2

ACCEPTED MANUSCRIPT (MH) 28; 29 30 and central core disease (CCD) 31; 32. When disease-causing mutations are not found within the RyR sequences, they are frequently observed in one of the auxiliary proteins. These disorders highlight the key role RyRs play in muscle contraction.

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RyRs are related to inositol-1,4,5-trisphosphate receptors (IP3Rs), another class of Ca2+ release channels found predominantly (but not exclusively) in the ER membrane. In this paper I will review the latest insights from a structural biologist’s viewpoint, outlining what we’ve learned about RyR function using high and low resolution structural methods, along with the similarities and dissimilarities to IP3Rs.

OVERALL STRUCTURE

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Because of their large size, membrane protein nature, and inherent ability to adopt multiple conformational states, full-length RyRs are notoriously difficult targets for high-resolution methods like X-ray crystallography. Instead, their large size has made them popular targets for cryo-electron microscopy (cryo-EM). Numerous papers have reported RyR reconstructions, and reassuringly agree on the overall structure33. The highest quality reconstructions are for rabbit RyR1, with reported resolutions in the 10-12Å range34; 35; 36; 37.

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The most prominent feature is the overall shape of a mushroom, with a large cap located in the cytosol, and the stem containing the transmembrane area and the intraluminal portion (Figure 1a,b). The cap has dimensions of ~270Å x 270Å x 100Å and corresponds to ~80% of the overall density. It is connected to the stem via electron dense columns, which appear crucial in allosteric coupling with the pore region. Notably, the cytosolic cap is not one massive block, but consists for ~50% of solvent channels that maximize the amount of exposed surface area - up to 500,000Å2 for the entire RyR. This allows for ample binding sites for auxiliary proteins and small molecules. In order to help with structural descriptions, the cap is further divided in regions called the clamps, handle, and the central rim. Several globular masses can be identified, which have received identifying numbers (Figure 1a,b)34; 38. The related IP3 receptor has also been studied extensively via cryo-EM, but has been the topic of substantial controversy, as multiple groups have reached 3D reconstructions that are largely incompatible39; 40; 41; 42; 43; 44; 45. This may be due, in part, by the different procedures in solubilization and purification leading to a large amount of sample variability. The highest resolution reconstruction, previously reported at ~10Å45, is now estimated at ~17Å by using new criteria for reporting resolution37. This method consists of refining two models independently, one for each half of the data, allowing socalled gold standard Fourier Shell Correlation (FSC) curves to be calculated46; 47. This reconstruction is likely the closest to the true IP3R structure, as recently discussed37, but also in part because it is the only one that satisfies the similarities in pseudo-atomic models for IP3Rs and RyRs (see section on the Nterminal region). Similar to RyRs, IP3Rs also resemble the shape of a mushroom, albeit with a much smaller cap (Figure 1c-f). The main difference seems to lie in the clamp and handle regions, which are absent in the IP3Rs. ARRAYS 3

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RyRs do not act independent of one another. Indirectly, Ca2+ released by one channel will of course affect neighboring receptors. However, it has been found that cardiac and skeletal muscle RyRs can form regular arrays in their native environment, forming a 2D-crystal often referred to as a checkerboard48; 49. Allosteric movements within one RyR can thus be transmitted mechanically to the neighboring RyRs. Indeed, it has been observed that RyRs in planar lipid bilayers can undergo coupled gating, whereby two or more channels seem to open instantaneously50; 51.

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Studies on purified RyR1 have shown that the formation of arrays is an intrinsic property of the channel52. Further 2D crystallization studies have shown that subregion 6, an area in the clamp, is responsible for the crystal contacts53 (Figure 2). Although the 2D crystals offer potential for higher resolution images using diffraction studies, the current resolution limit from these experiments is still at ~20Å.

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OVERALL DOMAIN ARRANGEMENT

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The 2D arrays seem to form spontaneously, but not all RyRs experience the same arrangement in their native context54. The 2D crystal contacts can change in the presence of higher Mg2+ levels, leading to a denser packing55 , and it has recently been shown that both Mg2+ levels and phosphorylation can lead to altered RyR2 arrangements within ventricular myocytes54.

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In a one-dimensional view, RyRs consist of a modular arrangement of several domains (Figure 3). Given the ~5000 residue length per subunit, it is surprising that RyRs only use a select number of unique folds, several of which are duplicated. These include: 1) two β-trefoil domains, 2) an Armadillo repeat domain, 3) three SPRY domains (SplA Kinase Ryanodine Receptor domain), 4) two tandem repeat domains, 5) an EF-hand domain, 6) a pore-forming domain. Together, these domains cover ~40% of the sequence. In addition, ~11% of the sequence is covered by three so-called divergent regions (DR1-3), areas with largely dissimilar sequences among RyR1-3, and which are predicted to be intrinsically disordered. The remainder of the sequence cannot be unambiguously assigned to a known fold, but is predicted to be α-helical rich. Several of these stretches bear distant resemblance to armadillo repeat proteins, which contain a ~42 amino acid motif composed of three α helices56. In tandem, these repeats can form superhelices57 and may thus act as long rigid tethers like the columns that connect the cap and stem of the RyR mushroom. The sequence upstream of the pore-forming region likely contains additional transmembrane helices. The overall consensus is that there are 6 or 8 transmembrane helices per subunit58, and cryo-EM studies have detected 5 or 6 of these34; 36. Overall, RyRs and IP3Rs share ~17% sequence identity. In IP3Rs, the sequence seems to be devoid of SPRY and tandem repeat domains. It is therefore likely that these domains are located in the extended clamp and handle regions that are unique to the RyR structure.

OPENING AND CLOSING 4

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The RyR pore-forming region bears sequence homology with pores of tetrameric potassium and sodium channels. Movement of the inner helices has been reported as a mechanism to control opening and closing for many channel types, but this model was called into question for RyRs36. A cryo-EM reconstruction of RyR1, presumed to be in the closed state, shows distinct electron densities corresponding to bent inner helices, implying that their bending is not what controls channel opening. In contrast with this, two other cryo-EM reports at similar resolution suggested that the inner helices are straight in the closed state34; 35.

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Which conclusion is correct? One issue here is resolution, and different criteria have been used by the two groups to report the resolution value. Using Fourier Shell Correlations or FSC cutoffs at ~0.1534; 35 versus 0.535 yields ~4Å differences in resolution for these maps. The more recently accepted gold standard for reporting resolution now has the map, favoring the non-bending hypothesis, at ~12Å37. An updated resolution has not been reported for the other maps.

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Another issue lies with the sample preparation. For example, monovalent cations and anions have been shown to affect RyR activity, so differences in the buffer compositions may impact the overall conformation. It has also been observed that binding of ryanodine to RyRs is strongly affected by detergents, and that the presence of phospholipids are required for maximum binding activity3. These observations underline the importance of lipids in maintaining a fully native RyR structure. Although the RyRs may have been in conditions that should promote a closed state in a lipid bilayer, there is no guarantee that they remain closed upon extraction with detergents and during the conditions used for purification and freezing. As such, it is probably safer to just analyze the structure and decide a posteriori what state has been captured. This is hard to know with only one reconstruction, but one study has systematically compared two states, using EGTA or PCB95 to promote the closed or open states of RyR1, respectively35. Because of the otherwise identical treatment of the samples, the observed structural changes are most likely to represent rearrangements that can occur during channel gating. Based on the latter study, these can be summarized as follows (Figure 4). Upon channel opening: 1) the pore expands, possibly due to bending of the inner helices; 2) the inner branches, located immediately above the pore and surrounded by the columns, move apart; 3) the cytosolic cap undergoes a large tilting motion, whereby the central rim moves up and outward, and the clamp corners move downward towards the ER/SR membrane. The correlated nature, whereby most motions can be described as one large tilt, suggests that these differences are not due to any noise, which would have caused random non-correlated movements. The largest motions during opening are observed at the clamp regions, with downward motions towards the ER/SR up to 8Å. Interestingly, these are the areas that make inter-RyR contacts within the 2D arrays, such that downward movements in one RyR may pull the corresponding region in neighboring RyRs, forcing their opening. The different cryo-EM structures thus confirm the RyR as a huge allosteric protein, with motions in the transmembrane region being coupled to areas >200Å away. The implication is that any protein or small molecule that can bind to the cytosolic cap can potentially interfere with channel opening, by having differential affinity for the open or closed state of the cytosolic cap. However, it is overly simplistic to 5

ACCEPTED MANUSCRIPT just think of a two-state model for RyRs. Indeed, FRET studies using different RyR activators have shown that multiple conformations are possible within the cytosolic cap59.

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THE N-TERMINAL AREA

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In the past 5 years, crystal structures have been reported for a select number of domains which now cover ~15% of the overall sequence. Several studies have focused on the N-terminal area60; 61; 62; 63; 64; 65; 66 , with the largest structures encompassing three domains (labeled A, B, C) in RyR161 and RyR263(Figure 5a,b). Domains A and B form β-trefoil folds and consist of 12 β strands each. Domain C folds up in a helical bundle that resembles armadillo repeat motifs. These three domains interact through a series of salt bridges, forming a compact arrangement that is conserved in all available crystal structures. In RyR2, a central salt bridge network connecting the three domains is replaced by a chloride binding site built up by three Arginine residues. The central chloride ion is crucial for stability and proper relative domain orientation63.

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The availability of both cryo-EM maps and crystal structures allows for the generation of pseudo-atomic models. The N-terminal area was thus found to be located at the cytosolic face near the 4-fold symmetry axis, where it interacts with neighboring N-terminal regions, forming a central vestibule (Figure 6a,b). Docking of crystal structures into cryo-EM maps is relatively straightforward using exhaustive 6-dimensional searches (3 rotational and 3 translational parameters)67. For each position and orientation, a correlation coefficient is calculated, and the top ranked hit is often assumed to be the correct one. Important however is the validation of these top hits. For example, is the correlation coefficient of the top hit significantly higher compared to the next hits in the ranking? If it isn’t, then the correlation coefficient just reflects background noise. Previous reports on docking of homology models into RyR cryo-EM maps have ignored this important feature, leading to completely different conclusions on the location of the N-terminal region38; 68. Part of the initial controversy around the N-terminal location arose from difference cryo-EM studies, whereby intact RyRs were fused to GST or GFP in several areas33. Fusion within the N-terminal area led to difference density in the clamp region69; 70, but the GST fusion likely interfered with folding, as indicated by the observed aggregation69. This implies that difference density may also be due to longrange conformational changes far away from the insertion sites. In addition, 10 residue linkers were used, which, together with the dimensions of GST or GFP, would allow difference density far away (>80Å) from the insertion sites61. The position in the central rim has recently been confirmed experimentally via difference cryo-EM studies that used shorter, 5 residue linkers for insertions in domains B and C71. The location is also compatible with recent FRET measurements for insertions in the N-terminal region and FKBP72. Intersubunit FRET for donors and acceptors inserted in the N-terminal domains also confirms that the N-terminal region is located next to the four-fold symmetry axis71. The IP3R N-terminal region has received an equal amount of crystallographic investigation, in part because it contains the binding site for IP3 73; 74; 75; 76; 77. Similar to RyRs, this area folds up into 3 domains with the same fold and overall domain arrangement (Figure 5c). IP3 binds in a cleft between domains B 6

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and C, together also known as the IP3 binding core, and comparisons between the apo- and IP3-bound states shows that IP3 induces a clamp-like closure76; 77. Docking of this crystal structure into the 17Å IP3R cryo-EM map yields a strikingly similar arrangement compared to RyRs: the N-terminal region also forms a ring around the 4-fold symmetry axis, and the same loops appear to be involved in the intersubunit contacts (Figure 6c,d). The overall similarity between both channels is further supported by a functional chimera, in which domain A from the IP3R was replaced by the corresponding RyR domain and which still displays IP3 sensitivity76. These observations underscore the idea that IP3Rs and RyRs have evolved from a common ancestor78.

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PHOSPHORYLATION DOMAIN

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RyRs are targets for multiple kinases. Each RyR1 subunit contains 15 sites and there may be as many as 35 per RyR2 subunit (140 per tetramer) (obtained from www.phosphosite.org). The number of combinations of phosphorylated states that can be made with tetrameric proteins is astronomically high (e.g. 2140 = 1.39 x 1042 for RyR2 !). In most cases, the specific sites have not yet been studied, and it is not known to what degree their phosphorylation contributes to channel gating. However, the few that have been studied have sparked what is undoubtedly the greatest controversy in the RyR field79; 80.

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Several kinases (PKA, PKG, CaMKII) and phosphatases (PP1, PP2A, PDE4D3) have been found to target RyRs. Scaffolding proteins can anchor them to the RyR structure, allowing for compartmentalized regulation81. Phosphorylation by PKA has been widely studied, because it links RyRs with stress signaling through activation of β-adrenergic receptors. PKA was found to target S2808 in RyR2 (and the corresponding S2843 in human RyR1). Under conditions of heart failure, it was proposed that PKA hyperphosphorylates this residue82, a confusing but widely used term to indicate that most RyR2 subunits are phosphorylated at this site. This event was then found to cause dissociation of FKBP12.6, a small protein that stabilizes the closed state (see section on FKBPs). The removal of FKBP12.6 would thus lead to facilitated channel opening. However, multiple groups have failed to reproduce both the hyperphosphorylation and the dissociation79; 80, with some claiming that S2030 is instead the major PKA target residue83. In addition to PKA, CaMKII has also been widely studied as a RyR2 regulator. It was found to be acting on S2808, just like PKA, but also on the nearby S281484. Crystal structures are available for the domain containing the controversial sites85; 86 (Figure 5d). It is built up by two pseudo-symmetrical halves (tandem repeats), which are likely the result of an ancient gene duplication. They are separated by a flexible loop that contains the suggested PKA and CaMKII sites. Intrinsically disordered segments are often great targets for phosphorylation, because the absence of steric hindrance makes them readily accessible to kinases. In RyR3, where no phosphorylation of residues in the phosphodomain has been identified, the disordered loop is largely replaced by an α helix. A mass spectrometry approach showed that both PKA and CaMKII could modify multiple residues in the RyR2 phosphorylation loop85. It remains to be found to what extent each residue in the loop is phosphorylated and what other kinases may target them, but because of their

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ACCEPTED MANUSCRIPT proximity, it is clear that phosphorylation of each site individually will impinge on the same domaindomain or protein interaction, and thus have the same functional effect.

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Docking of the phosphorylation domain into RyR1 cryo-EM maps suggests a location in the corner region (Figure 6)85; 86. The contrast between the top solution and the next solutions in the ranking is much lower than for the N-terminal region, so the confidence is lower. It is compatible with a difference cryoEM study whereby GFP was fused into the phosphorylation loop87. However, because 10 residue linkers were used to fuse GFP to an already disordered loop, difference density may again be observed far away from the insertion site. Its location remains to be confirmed experimentally.

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Multiple studies suggest that phosphorylation by PKA or CaMKII increases the Po of RyRs in planar lipid bilayers (e.g. see84; 88). It is currently unknown what other domain interacts with the phosphorylation domain, and as such, the mechanism whereby it leads to altered channel gating remains obscure. If speculation is allowed, its putative location in the corner region, which is near 2D crystal contacts within RyR arrays, would suggest it may interfere with inter-RyR interactions. Interestingly, a recent report has shown that phosphorylation of RyR2 in ventricular myocytes shifted them from a mixture of arrangements into a predominantly checkerboard configuration54.

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DISEASE MUTATIONS

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Many reports have linked RyR1 and RyR2 to a series of life-threatening genetic conditions89. RyR1 mutations are mostly linked to malignant hyperthermia (MH) and Central Core Disease (CCD). MH is known as a pharmacogenetic disorder, because both a mutation and an external trigger are needed to cause symptoms. MH is typically triggered by volatile halogenated anesthetics (e.g. isoflurane, halothane) or by succinylcholine, a muscle relaxant. The link between MH and RyR1 was established through a mutation in pigs that suffer from the related porcine stress syndrome (PSS)28. The corresponding human mutation (R614C) was soon after found to cause MH in humans30. Many MH mutations have been described for RyR1, but some additional mutations have been found in the skeletal muscle L-type calcium channel (CaV1.1)90. Central core disease (CCD) is another condition linked to RyR1,31; 91 characterized by progressive muscle weakness and the presence of cores (metabolically inactive tissue) within the muscle fibers. MH and CCD are often linked, and the same mutation can sometimes be found to cause both. The related multimini core disease (MmD) is another congenital myopathy, characterized by multiple non-well-defined cores in muscle fibers, and is also mainly associated with mutations in RyR192. In RyR2, mutations are frequently linked to catecholaminergic polymorphic ventricular tachycardia (CPVT)26. Similar to MH, a combination of a mutation and a trigger is needed to develop a phenotype. In this case, exercise or emotional stress are the usual triggers for the resulting cardiac arrhythmia, and can result in sudden cardiac death. In addition to RyR2, CPVT mutations have also been found in several of its auxiliary proteins, including calsequestrin93, triadin94, and calmodulin95. RyR2 mutations have also been associated with arrhythmogenic right ventricular dysplasia (ARVD), a condition in which the right 8

ACCEPTED MANUSCRIPT ventricular muscle is gradually replaced by fibrofatty deposits96. Finally, since RyR2 is also widely expressed in the central nervous system, it is likely that mutations have neuronal effects. A mouse model has shown that RyR2 mutations can indeed give rise to seizures97.

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Since their initial identification, over 500 suspected disease-causing mutations have been found in RyR1 and RyR2 combined. Many mutations cluster within distinct disease hot spots, located in the N-terminal region (~residues 1-600), a central region (~2100-2500), and the C-terminal area (~3900-end). RyR1 mutations are increasingly found in between these (Figure 3), but so far the hot spots remain clearly visible in RyR2. The general observation is that MH and CPVT confer gain-of-function phenotypes, whereby channel opening is facilitated through increased sensitivity to triggering agents (e.g.98; 99). Several studies have shown that the mutant RyRs are also sensitive to Ca2+ overload in the SR, leading to store-overload induced calcium release (SOICR)100; 101; 102. CCD mutations are harder to conceptualize, since opposing functional effects have been found. CCD mutations in the cytosolic region are mostly gain-of-function103, whereas loss-of-function CCD mutations are found within the pore region, likely interfering with Ca2+ permeation104; 105.

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With several crystal structures available, multiple disease mutations can be mapped and analyzed at high resolution. Although at first glance the positions for disease mutations seem to scatter everywhere on the structures, there is a common theme, as most of them are located at domain-domain interfaces. In the phosphorylation domain, the disease mutations are found on two opposing faces, with the bulk of them clustered around the phosphorylation loop85. This clustering suggests that both phosphorylation and disease mutations affect the same interface. It is currently unknown which domains interact with the phosphorylation domain, and could thus be affected by the mutations. The N-terminal region corresponds to one of the previously identified disease hot spots. The largest cluster of mutations is found at the intersubunit interface, where they would affect interactions between domains A and B of neighboring subunits (Figure 6). Docking of the crystal structure in the open and closed state cryo-EM maps suggests that this interface is labile: upon channel opening, the Nterminal regions from neighboring subunits move apart, possibly leading to disruption of the interface64 (Figure 7a). Recently, the outward movement predicted from these pseudo-atomic models has been confirmed via FRET experiments. By placing FRET donor and acceptor in domains A and B of different subunits, it was observed that the FRET distance increases with triggers for channel opening, thus supporting a widening of the intersubunit gap upon channel opening71. One attractive hypothesis is that the N-terminal region therefore acts as a gating ring (Figure 8). The intersubunit contacts stabilize the closed state through interactions between domains A and B across subunits. Weakening those interactions through disease-causing mutations then destabilizes the closed state, making it easier for triggering agents to open the channel. Several disease mutant versions of the RyR1ABC or RyR2ABC crystal structures have been reported60; 62; 63; 64; 66 . The largest structural changes are observed for mutations affecting salt bridges between domains A, B, and C (Figure 7b). The mutations affect relative domain orientations, resulting in positional shifts ~2.5Å. Interestingly, IP3 binding to the IP3R N-terminal region also induces relative domain reorientations76; 77 (Figure 7c). One possible implication is that this misalignment of the N9

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terminal domains results in incorrect placement at the intersubunit interface, which would indirectly weaken their interaction. Just like mutations found at the intersubunit interface, these others would then lead to facilitated channel opening (Figure 8). The mechanism through which IP3 affects channel opening in IP3Rs is likely very similar: by realigning the domains, it indirectly weakens an intersubunit interface involved in channel gating. Although the absolute extent of the domain reorientations may appear small compared to the dimensions of the intact channels, movements of a few Ångström are more than sufficient to disrupt salt bridges or hydrogen bonds, and to thus have an energetic impact.

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Figure 8 presents a model for how the N-terminal region in both RyRs and IP3Rs acts as a gating ring64. Further support for this model has come from recent studies with Calcium-binding protein 1 (CaBP1), a neuronal Ca2+ sensor protein. It was found to promote cross-linking of purified IP3R-ABC into tetramers, implying it strengthens the intersubunit interactions. According to the model in figure 8, this would make it harder for the channel to open, in agreement with the ability of CaBP1 to inhibit IP3-mediated Ca2+ release. Conversely, IP3 was found to attenuate the cross-linking ability, showing that stabilizing or destabilizing the intersubunit interactions is a primary mechanism to control Ca2+ release channel gating106.

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Could there be any ligand for RyRs that binds the N-terminal region, induces different domain orientations, and thus stimulates channel opening, similar to IP3? There may already be two candidates. The first is a chloride ion, which was found to bind three arginine residues on different domains in RyR263 (Figure 5b). The functional impact of chloride binding to this particular site remains to be described, but it is clear that it will have a large effect on the relative domain orientations, because its removal drastically destabilizes the N-terminal region and would lead to repulsive positive charges across the domain interfaces. Interestingly, the chloride binding site is the target for two disease mutations (R420Q, R420W), which seem to abolish binding63. The chloride binding site in the N-terminal region is not observed in RyR1. A second candidate is not a bona fide ligand, but rather a modification. RyRs have been shown to be sensitive to redox modification, a property due to several reactive cysteines that can be oxidized, nitrosylated, or glutathionylated107; 108; 109; 110. C35 in human RyR1 was found to be a target for glutathionylation111. It is accessible at the surface of domain A60; 65, but is completely buried at the domain A-B interface, implying that this interface can be dynamic. Once glutathionylated, these domains cannot go back to the orientation observed in the crystal structures, as there is no space for a bulky glutathione group in the interface between domains A and B61. The redox modification of C35 may then have the same functional effect as IP3 binding in IP3Rs, promoting channel opening. In addition to hundreds of point mutations, sometimes deletions of larger pieces have been reported. In particular, several reports have linked the deletion of exon 3 in RyR2 to a very severe form of CPVT112; 113; 114 . Functional experiments have shown that the mutation has an abnormal termination threshold 2+ for Ca release, leading to prolonged channel opening115. Exon 3 encodes 35 residues located in RyR2 domain A, corresponding to a β strand and α helix (Figure 9). Surprisingly, its deletion does not cause the expected misfolding that would result from disrupting the β-trefoil core. Instead, a flexible α-helix

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ACCEPTED MANUSCRIPT encoded by exon 4 undergoes a helix-to-β-strand transition and adopts the role of the deleted strand62; 66 . This exon 4 is unique to RyR2, and may play a general role in alternative splicing62.

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In the absence of additional high-resolution structures, the precise mechanisms for mutations in the central and C-terminal disease hot spots remain obscure. Previously, a hypothesis was put forward that the N-terminal and central hot spots could form physical interactions, which would be disrupted or ‘unzipped’ during channel opening116; 117. Disease mutations were proposed to cluster at their interface, thus facilitating channel opening. However, it is now clear that the bulk of the mutations in the Nterminal area are at interfaces with other N-terminal domains, either within or across subunits, and therefore unavailable for interactions with the central disease hot spot. Although some part of the Nterminal region may still bind the central hot spot, it is most certainly not the major disease mechanism.

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Despite this, the so-called zipper hypothesis is still widely cited. The main support came from small RyRderived peptides that were shown to interfere with channel opening. However, the folding state of these peptides in relation to their native structure within RyRs is mostly unknown, and at least in one case it is clear that it must be different. A peptide in the N-terminal region of RyR2 (residues 163-195) has been shown to increase leak of Ca2+ through RyR2, and was interpreted to bind to the central hot spot118. Unknown at the time of the study, the peptide is now found to be an integral part of the βtrefoil core of domain A60; 63, and taken out of context it is extremely unlikely to adopt a native conformation. Its location near the inter-RyR subunit interface also makes it impossible for the corresponding native peptide region to interact with the central disease hot spot. For other reported peptides there is no corresponding high-resolution structure, but one should consider the possibility that the observed increase in RyR activity may not be due to a mimic of native interactions between Nterminal and central disease hot spots.

LIGANDS AND AUXILIARY PROTEINS

A Ca2+ permeable channel could be made with only a few transmembrane α helices. However, since the rapid release of Ca2+ delivers a very potent message, RyRs have been under evolutionary pressure to maintain a large structure that allows positive or negative regulation via tens of signaling molecules. All three RyR isoforms can be triggered to open by Ca2+. Plotting the activity of RyR1 versus cytosolic Ca2+ concentration shows a bell-shaped curve, whereby low Ca2+ concentrations (~1µM) activate, and high concentrations (~1mM) inhibit the channel18. Mg2+ has been found to inhibit channel activity. The exact identities of these regulatory ion binding sites remain to be confirmed, but several reports have indicated the predicted EF hands in the C-terminal area (Figure 3) as possible Ca2+ sensors, with an apparent affinity for Ca2+ of ~60µM Kd119; 120. A glutamate residue just upstream of the EF hands (E3987) has been proposed as an activating Ca2+ binding site121 . In addition, luminal Ca2+ levels in the SR or ER are also known to affect RyR activity. Under conditions of increased Ca2+ load, luminal Ca2+ can trigger opening in a process known as store-overload induced Ca2+ release (SOICR)19; 100. A recent report strongly indicates that a glutamate residue (E4872 in RyR2) at the bundle crossing of the pore-forming region is the luminal Ca2+ sensor102. Mutation of this residue prevents activation by high luminal Ca2+ and 11

ACCEPTED MANUSCRIPT is protective against CPVT, supporting the idea that spontaneous Ca2+ release plays a major role in the pathophysiology of this disorder.

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In the next two sections, I discuss two classes of regulatory proteins, EF hand containing proteins and FKBPs, for which structural information on RyR binding is available. In addition to those, several other proteins and small molecules have been found to regulate RyRs (Figure 10). This includes proteins on the luminal side. Calsequestrin (CASQ) is a major Ca2+ buffering protein in the SR lumen. It can bind Ca2+ with high capacity (40-50 ions), but with low affinity (Kd ~1mM), which mediates its oligomerization122. In complex with junctin and triadin, it has been proposed to directly affect RyR activity123. Histidine rich Ca2+ (HRC)-binding protein is another luminal protein that can bind triadin124 . Homer 1c is an adaptor protein that can activate RyR1 and inhibit RyR2.125; 126; 127; 128 Small molecule modulators include ATP and cADPR (activators), toxins like natrin (inhibitor) and imperatoxin (activator). A more complete list of modulators is shown in figure 10.

EF HAND CONTAINING PROTEINS

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In addition to direct Ca2+ sensing, several EF-hand containing proteins can further fine-tune the response of RyRs to changing Ca2+ levels. The most widely studied is Calmodulin (CaM), a ~17kDa protein containing four EF hands in two domains (often referred to as N-lobe and C-lobe). It binds directly to RyRs, but the functional effect is dependent on Ca2+ and on the exact RyR isoform. At high Ca2+ levels, CaM can inhibit both RyR1 and RyR2. At low Ca2+ levels, it activates RyR1 but inhibits RyR2129; 130; 131; 132. The importance of this modulation is underscored by the fact that mutations in CaM can also give rise to CPVT, likely the result of altered regulation of RyR295. The binding site for CaM has been studied extensively via cryo-EM133; 134. These reports show that one CaM binds per RyR subunit, although it is possible that weaker binding sites exist, not captured by the cryo-EM studies. Both ApoCaM and Ca2+/CaM bind to the edge of the cytosolic cap, but their location is ~30Å apart in RyR1 (Figure 11c). This implies that CaM is always present as a resident Ca2+ sensor, likely grabbing a different segment when Ca2+ levels rise. Their location in the cryo-EM may hint at a mechanism of inhibition. The clamp region undergoes substantial movements downwards (towards the SR membrane) during channel opening, and Ca2+/CaM sits directly in a position where it could hamper this movement. Apo-CaM is bound higher up towards the cytosolic face of RyR1, and is therefore less likely to obstruct the movement. In RyR2, apoCaM seems to bind to roughly the same location as Ca2+/CaM in RyR1, in agreement with the notion that apoCaM inhibits RyR2, just like Ca2+/CaM inhibits RyR1134. In primary sequence, several binding sites for CaM have been proposed based on pull-downs and overlay assays135; 136; 137; 138; 139; 140; 141; 142; 143. However, some of these sites appear to be unavailable for binding, as they are hidden within the phosphorylation domain85. Others are incompatible with cryo-EM studies134, and only three segments remain as putative CaM sites144. These three CaM binding domains (CaMBDs) are encoded by RyR1 residues 1975-1999 (CaMBD1), 3614-3640 (CaMBD2), and 4295-4325 (CaMBD3). CaMBD1 and 2 are located within the α-helical rich regions and are highly conserved among 12

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the RyR isoforms. CaMBD3 is located in a divergent region (DR1), with little sequence conservation among the isoforms. All three have been shown to bind CaM with 1:1 stoichiometry using isothermal titration calorimetry144. Notably, the presence of CaMBD2 greatly increases the affinity of the C-lobe for Ca2+, such that it is mostly Ca2+ bound even at resting Ca2+ levels141

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A crystal structure is available for Ca2+/CaM bound to the RyR1 CaMBD2, showing both CaM lobes wrapped around an amphipatic helix in an antiparallel arrangement (Figure 11a)145. The main hydrophobic anchors, Trp3620 and Phe3636, have an unusual 1-17 spacing and the CaM lobes therefore do not interact with one another. NMR data show that the Ca2+/N-lobe is only loosely bound to the peptide, in agreement with ITC data showing that the N-lobe affinity is very weak in the presence of a pre-bound C-lobe144. It is therefore likely that the Ca2+/N-lobe binds elsewhere in the channel, possibly to CaMBD1 or CaMBD3. No high-resolution structure is available for apoCaM bound to an RyR peptide, but both CaMBD2 and CaMBD3 are able to associate with apoCaM, with the highest affinity (~5µM Kd) observed for CaMBD3. Interestingly, CaMBD2 has also been found as a target for the isolated EF hands119, but it is unclear whether these two regions are sufficiently close within the intact RyR.

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Two CaMBDs could be bridged by the two lobes of CaM, but with three CaMBDs per RyR subunit, there would be at least one too many in order to be compatible with the proposed CaM:RyR stoichiometry. Mutagenesis studies have clearly shown that CaMBD2 is required for modulation by CaM, suggesting it is a physiologically relevant site146. Cryo-EM studies with GFP insertions near CaMBD2 also show that it is located close to the observed density for CaM and apoCaM147. Deletion of a ~260aa fragment encompassing CaMBD3 has shown that RyR1 can still bind and be modulated by CaM and apoCaM148, indicating that this segment is likely not involved in functional regulation. However, the role of this segment in RyR2 remains to be investigated. S100A1 is an EF-hand containing protein from the S100 protein family. It forms homodimers, with each subunit containing a high- and a low-affinity EF hand. It promotes opening of both RyR1 and RyR2149; 150; 151; 152 . An NMR structure of the Ca2+ bound S100A1 in complex with RyR1 CaMBD2 shows that it engages the same aromatic anchor as the Ca2+/C-lobe , Trp3620, implying there is competition between S100A1 and Ca2+/CaM for the same binding site (Figure 11b)153. Its primary mechanism may therefore be the removal of inhibition by Ca2+/CaM. Another RyR modulator is sorcin, a 22kDa protein with five EF hands . Its exact binding site is unknown, but it can associate with RyR2 at high Ca2+ concentrations and mediate inhibition154; 155.

FKBP12 and FKBP12.6 FKBP12 and FKBP12.6 are small proteins (12 and 12.6 kDa, respectively) that are targeted by the immunosuppressive agents rapamycin and FK506, and owe their name to binding the latter (FK506 binding protein). They have a compact fold156 and co-purify readily with RyRs in a 4:1 stoichiometry (one FKBP per RyR subunit)157; 158. Surface plasmon resonance experiments have shown that FKBPs have a very high affinity for RyRs, with a ~1nM Kd for open channels, but orders of magnitude higher for closed

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ACCEPTED MANUSCRIPT channels159. Although other studies have revealed lower affinities in the ~100nM range88; 160, there is general agreement that the affinity is high.

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It was shown that FKBP12 can decrease the appearance of subconductance states in RyR1, and generally makes it harder for the channel to open15. This is consistent with the finding that FKBP12 binds stronger to closed than to open channels159, but others have found opposing effects161. Another feature of FKBP12 and FKBP12.6 is that they have been suggested to promote coupled gating, the phenomenon whereby two or more RyRs in a planar lipid bilayer can open simultaneously50; 162

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Cryo-EM studies of FKBP12 or FKBP12.6 bound to RyR1 and RyR2 showed them unambiguously on the periphery of the cytosolic cap35; 163; 164. Figure 11c shows a superposition of closed RyR1 cryo-EM maps with and without FKBP12, showing a large difference density that readily fits FKBP1234; 35. Interestingly, the FKBP12 crystal structure can also be docked into the full-length RyR1-FKBP12 map without any bias, reaching the same location (Figure 11c,d). Comparison of the closed RyR1 with and without FKBP12 bound shows an upward movement of the clamp region when FKBP12 is present. Since the clamps move downward upon opening, counter to the movement induced by FKBP12, I postulate that this may be the mechanism through which FKBP12 stabilizes the closed state. Rapamycin, a small molecule that binds FKBP12, occupies part of the surface that engages RyR1, explaining why this molecule disrupts FKBP-RyR interactions (Figure 11d). Although the overall location for FKBP12/12.6 is known, the exact parts of the RyR primary sequence that are involved in the high affinity binding remain unknown. The cryo-EM maps suggest that the binding site is built up by multiple globular domains that contribute, and these may not be contiguous in the sequence. As such, it is unlikely that a single high-affinity binding site will be found by using individual segments or domains from the RyR sequence as bait. It has been proposed that CPVT-causing mutations in RyR2 decrease the affinity for FKBP12.6, particularly under conditions of increased RyR2 phosphorylation88. As noted in the section about phosphorylation, unbinding of FKBP12.6 is a very controversial topic, with different groups reaching different conclusions. However, since CPVT mutations inherently facilitate opening, and since FKBPs bind stronger to closed channels, it is only logical that they cause decreased FKBP12.6 affinity, provided the affinity is tested under specific conditions. In conditions promoting a distribution of both open and closed RyR2, any mutation that inherently favors the open state will lead to fewer high-affinity closed channels, resulting in overall apparent decreased FKBP12.6 affinity. However, under more extreme conditions whereby all channels are fully closed or fully open, the CPVT mutation will have no effect on the number of open channels, and the apparent affinity for FKBP12.6 should remain the same for wild type and mutant. Perhaps this simple notion, which also applies to phosphorylation, is the reason why different groups either can or cannot reproduce the unbinding of FKBP12.6 in vitro. Importantly however, in this scenario the apparently reduced affinity of FKBP12.6 is just an epi-phenomenon, and not the root cause of the facilitated channel opening. The only exception would be RyR mutations that do not inherently affect RyR opening, but only impinge on FKBP12.6 binding. Given the large number of mutations all across the RyR sequences, this most likely only applies to a small subset of the mutations.

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ACCEPTED MANUSCRIPT MECHANICAL COUPLING: CaV1.1-RyR1

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A major trigger for RyR opening is cytosolic Ca2+, which can be derived from entry through L-type calcium channels. In skeletal muscle, however, it has been shown that depolarization of the plasma membrane can trigger Ca2+ release even in the absence of extracellular Ca2+. Many studies suggest that RyR1 and CaV1.1 form direct physical contacts, with CaV1.1 acting as the voltage sensor for RyR1 through direct mechanical coupling21; 22; 23; 24; 25. In addition, retrograde signalling has been reported, whereby changes in RyR1 can affect the function of CaV1.1165; 166. In freeze-fracture studies of skeletal muscle, particles thought to correspond to CaV1.1 channels were found to be grouped into tetrads, opposite foot structures corresponding to RyR1. The general interpretation is that an RyR1 channel can interact with four CaV1.1 channels, but only every other RyR1 is associated with such a tetrad23; 167.

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The ability of two complex membrane proteins, belonging to different membrane systems, to interact and affect one another’s function is an astounding evolutionary feat. Solving this puzzle is one the largest challenges in the field of E-C coupling, and a major trial for structural biologists. Minimally, understanding the points of contact between these two membrane proteins would be a significant step forward. On the CaV1.1 side, mostly two components have been involved: a large cytosolic loop connecting domains II and III of the pore-forming α1s subunit (II-III loop) 168; 169; 170, and the C-terminal tail of the auxiliary β1a subunit171; 172, although other sites have also been suggested. The binding site on RyR1 has remained more elusive. Based on studies with chimeras, it is clear that multiple regions within RyR1 are required for coupling. One region of key importance appears to be divergent region 2, located between the SPRY2 and SPRY3 domains (Figure 3), as it is strictly required for both tetrad formation and E-C coupling173; 174. Based on pull-downs, the II-III loop was found to bind a peptide located within the SPRY2 domain175. Several studies have confirmed an interaction with the SPRY2 domain166; 167; 168; 169, but the affinity is weak and a requirement of this interaction for E-C coupling has not been demonstrated. Another interaction has been shown between the RyR1 EF hand region and the CaV1.1 IQ domain119, and the RyR1 CaMBD2 has been shown to bind the C-terminal region of CaV1.1, which also contains predicted EF hands176. However, given the promiscuity of CaMBD2 to bind EF hand proteins, and the inherent ability of the EF hand region to bind to known CaM binding regions, it remains to be determined whether these interactions can also occur within the full-length proteins. Undoubtedly, the RyR-CaV interaction and its implications for allosteric coupling will remain an intriguing topic for many years to come.

PERSPECTIVES Despite decades of active investigation, RyRs are still to reveal some of their most intimate secrets. Even very fundamental questions, regarding the mechanisms for primary triggers, remain unanswered. The exact binding sites for CaV1.1 and Ca2+ at the cytosolic side are still not unambiguously assigned, so we are still far away from a mechanistic description of the triggering process. The same is true for the tens of other auxiliary proteins and small molecules that somehow affect the allosteric coupling between the pore and the cytosolic cap. There has been a huge effort in understanding the effects of 15

ACCEPTED MANUSCRIPT phosphorylation on RyR activity, but with most debates still circling around which sites matter, we are still far away from knowing which proteins or domains are interacting directly with those sites.

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More high-resolution images are obviously in need, and these are likely to come from multiple methods. New technical advances in cryo-EM have recently been able to procure electron density maps for the TrpV1 channel that allowed tracing of the backbone177. There is great potential for new RyR cryo-EM studies at resolutions well beyond the 10Å barrier. In the short term, more high-resolution crystal structures of individual domains or domain clusters are likely to follow, but a full-length RyR crystal structure has thus far remained elusive. Anecdotally, RyR1 is able to crystallize, which is perhaps not surprising given its inherent ability to form 2D crystals in vivo. The diffraction limit seems to remain the major culprit, and because the best RyR1 preps are derived from native material, one cannot apply the routinely used protein engineering techniques to expedite high-quality crystal generation.

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The ability of RyRs to form regular arrays has been clearly demonstrated, but how exactly the size and the distribution of the arrays within the cell affect Ca2+ sparks and waves remains to be determined. How is the activity of individual RyRs altered when present within an array and to what extent is this affected by their exact position within the lattice? These are difficult questions to answer.

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All in all, there will still be much to come on the structure-function relationships in RyRs. Even when high-resolution structures of full-length RyRs arise, much will remain to be learned about the allosteric movements in this membrane protein giant.

ACKNOWLEDGEMENT

I’d like to thank Drs. Z. Liu and T. Wagenknecht (Wadsworth centre) for sharing of the cryo-EM maps of CaM-bound RyR1, and R. Pancaroglu for carefully proof reading the manuscript. This work is supported by a grant from the CIHR to FVP (#259009).

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Cryo-EM structures of RyR and IP3R. Shown are isocontour maps for cryo-EM reconstructions of RyR (EMDB accession number 1275)36 and the IP3R (EMDB 5278)45. A,B) Top (from the cytosol facing the ER/SR membrane) and side views of the RyR. The numbers indicate subregions, a nomenclature that has been used extensively in literature. Dotted lines indicate the central rim, clamp, and handles. C,D)Top and side views of the IP3R. E,F) Top and side views of a superposition of the RyR (red mesh) and the IP3R (solid gray).

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RyR arrays. Model of the RyR checkerboard pattern, showing inter-RyR interactions involving subregion 6. The model was created using an RyR1 cryo-EM map (EMDB 1607)35 with interactions as shown by Yin et al55.

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Domain architecture of the RyR. One-dimensional view of the RyR1 sequence. Each colored square represents an individual domain with the type of predicted or known fold indicated. The gray portions correspond to divergent regions (DR1-3). TM: transmembrane. Vertical black lines indicate positions of suspected disease-causing mutations in RyR1.

Opening and closing. A) Top view of a superposition of RyR1 in presumed closed (gray, EMDB 1606) and open (red, EMDB 1607) states. The red portion in the central rim indicates this portion moves upwards during opening, whereas the gray portions in the clamps indicate this area moves downward toward the ER/SR membrane. B) Side View. Arrows indicate relative movements during opening, which can generally be described as a tilting motion. C) Side view, clipped through the surface at the center to show movements within the central rim. D) Top view, clipped through the surface at the location of the inner branches, which connect to the pore forming region, showing their radially outward movement. Arrows indicate the relative movement during opening.

Figure 5 High-resolution structures of RyR and IP3R domains. A) N-terminal 3 domains (A,B,C) of RyR1. Black sticks represent positions for known or suspected disease-causing mutations in RyR1. Mutations in 28

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flexible loop regions are not shown. B) N-terminal three domains of RyR2, showing a close-up around the three-domain boundary. A chloride ion, coordinated by three Arg residues, is shown as a white sphere. C) N-terminal domains of IP3R1. Shown is a superposition of the structures in the presence (colors) and absence (light gray) of IP3 (sticks). D) The phosphorylation domain of RyR1. A flexible loop containing the PKA target residue S2843 is indicated in dotted lines. The corresponding loop in the RyR2 phosphodomain structure contains the controversial S2808 and S2814, along with multiple additional phosphorylation sites85. Black sticks represent positions of suspected disease-causing mutations.

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Pseudo-atomic models. A,B) Top and side views of RyR1 crystal structures docked in an RyR1 cryo-EM map (EMDB 1607)35. The domains are colored according to figure 5. Positions for disease mutations are indicated in black. The largest cluster is found at the intersubunit interface. C,D) Top and side view for the IP3R1-ABC crystal structure docked into the IP3R1 cryo-EM map (EMDB 5278)45.

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Allostery in the N-terminal region. A) Superposition of the RyR1ABC crystal structure docked into closed (EMDB 1606) and open (EMDB1607) RyR1 cryo-EM maps. Blue: closed state positions; red: open state positions. Shown is a side view, showing the N-terminal region from only 2 subunits for clarity. B) Superposition of wild type RyR1ABC (colors) and R45C (white). A salt bridge is present between R45 in domain A and D447 in domain C (rabbit RyR1 numbering). The MH mutation R45C results in a reorientation of domain C relative to domain A (arrows). Similar domain-domain reorientations are observed for two other salt bridge disease mutations. C) Superposition of IP3R1ABC with (colors) and without (white) bound IP3. IP3 binding induces a reorientation of domain B relative to domains A and C. IP3 is shown in stick representation.

Figure 8 Model for the N-terminal region as a gating ring in RyRs and IP3Rs. Opening of the channel involves breaking intersubunit interactions between domains A and B, a process requiring energy delivered from a trigger (e.g. Ca2+ binding). Mutations at the interface directly weaken the contacts and thus facilitate channel opening. Salt bridge mutations within one subunit can cause domain misalignment, resulting in incorrect presentation of the domains at the intersubunit interface. This indirectly weakens the intersubunit interface, resulting in facilitated channel opening. Domain misalignment can also be triggered by binding of IP3 to the IP3R-ABC domains.

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β-strand swapping in the RyR2 N-terminal domain. A) Hybrid model (NMR/X-ray) of RyR2 domain A (PDB: 2MC2). A flexible loop, invisible in the crystal structure, was shown to be a mobile α helix via NMR (exon 4, red). Exon 3, a 35 residue segment found deleted in some families, is shown in green. B) Crystal structure of RyR2 domain A Δexon 3 (PDB: 3QR5). Exon 4 undergoes a helix-to-strand transition and prevents unfolding of the N-terminal domain.

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RyR modulators. An overview of small molecule and protein modulators, providing either stimulatory (+) or inhibitory (-) input signals. Some of these (e.g. Ca2+ and CaM) fall in both categories, as their input is concentration dependent. In addition to these, several monovalent cations and anions have been shown to affect RyR activity and [3H] ryanodine binding178.

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EF hand regulators and FKBPs. A) Crystal structure of Ca2+/CaM bound to RyR1 CaMBD2. The N-lobe is shown in blue, and the C-lobe in purple. Calcium ions are shown by white spheres. The CaMBD2 segment is shown in red, with two aromatic anchor residues indicated. B) NMR structure of an S100A1 dimer bound to two RyR1 CaMBD2 peptides. The two S100A1 subunits are shown in light blue and gold. Trp3620 is involved in binding both S100A1 and the Ca2+/C-lobe, indicating that both proteins compete for the site. C) Superposition of RyR1 cryo-EM maps without (EMDB: 5014, solid gray) and with FKBP12 (EMDB: 1606, black mesh). The major difference density readily fits FKBP12, which can also dock to this site via 6-dimensional searches using ADP_EM179. Notice that the FKBP12-bound map has the clamps upward compared to the FKBP12-free map, likely explaining how FKBP12 stabilizes the closed state. Also superposed are difference densities obtained for apo-CaM (blue) and Ca2+/CaM (red) bound to RyR (Data obtained by Drs. Z. Liu and T. Wagenknecht, Wadsworth Centre). D) Close-up of the FKBP12 crystal structure (magenta), as docked in closed-state RyR1 with FKBP12 bound (EMDB 1606), shown here with a closed RyR1 map without FKBP bound (EMDB 5014). The black sticks represent rapamycin, a highaffinity FKBP12 ligand, superposed from a FKBP12-rapamycin crystal structure. Rapamycin clashes with RyR1 at the FKBP12-RyR1 interface, and thus disrupts their binding.

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ACCEPTED MANUSCRIPT Highlights * Ryanodine Receptors (RyRs) are Ca2+ release channels in the SR/ER membrane

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* Cryo-EM studies have shown mushroom-like shapes * Crystal structures cover 15% of the sequence and can be docked into cryo-EM maps

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* RyR opening results in tilting of the cytosolic cap and altered subunit interactions

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* Gating of RyRs is influenced by disease-causing mutations and auxiliary proteins

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