The R615C mutation in porcine RyR1 affects inter-domain interactions that facilitate channel opening

Several cryo-EM structures of RyRs have been reported, but structural investigations of the pathophysiological conformations are scarce. This is partly due to the difficulty in obtaining large quantities of recombinant RyRs, requiring the majority of investigators to isolate channels from only wild-type (WT) native material^{7,8,11,23,24,25,26}. To overcome this hurdle, we employed a known homolog to the human R614C MH RyR1 mutation, identified in pigs^4,27. The homologous pig R615C mutation provided the original link between MH and RyR1 causing a very similar disease state called porcine stress syndrome²⁸. We thus capitalized on the availability of homozygous R615C pigs to obtain large quantities of R615C pig RyR1 (pRyR1) for cryo-EM studies, as a representative for N-terminal disease hot spot mutations in RyR1 and RyR2. Figure 1a, b shows single-channel recordings of WT and R615C pRyR1 purified from porcine tissue, indicating a clear gain-of-function for R615C with increased maximum open probability (P_o) and enhanced sensitivity to Ca²⁺, aligning with previous reports²⁹.

With the expected functionality of the R615C mutation confirmed, we solved cryo-EM structures of wild-type and R615C pRyR1, under nominally Ca²⁺-free conditions (5 mM EGTA), in the presence of FKBP12.6, which was used as a bait protein for purification (Supplementary Fig. 1). In both cases, one major 3D class was obtained, along with a smaller class with poor quality that was not further considered. Density modification or symmetry expansion and local masking of the major class drastically improved the local resolution throughout the structure (Supplementary Figs. 2–7, supplementary Table 1). Importantly, for WT pRyR1, we can observe unambiguous density for the Arg615 sidechain (Fig. 2). The location of this residue seems critical: Arg615 is situated in a junction between three solenoid domains: the N-terminal solenoid (Nsol, residues 395–630) containing Arg615, the junctional solenoid (JSol, residues 1657–2145), and the bridging solenoid (Bsol, residues 2146–3613). Nearby residues that form potential interactions with Arg615 are Asn1678 in Jsol and Glu2175 in Bsol (Supplementary Fig. 8).

Despite identical experimental conditions, the overall R615C pRyR1 resolution is lower than for WT, potentially indicating a larger inherent heterogeneity. Density modification or symmetry expansion combined with local masking resulted in an improved resolution around the site of the mutation (Supplementary Figs. 3–7). A comparison with WT pRyR1 reveals the allosteric mechanism by which a mutation >130 Å away from the pore can facilitate its opening. Superposing the N-terminal solenoid (Nsol) shows that loss of interactions mediated by Arg615 leads to pivoting movements of the neighboring solenoid regions (Bsol, Jsol) relative to Nsol (Fig. 3a, b, Supplementary Fig. 7a, Movie S1). Locally, this results in shifts ~2–3 Å in the Bsol region near residue Glu2175, but due to the pivoting movement this is amplified further away from the mutation site, e.g., up to ~10 Å in the Bsol region around residue 2457 (Fig. 3a, b).

As the latter region of the Bsol makes contacts with the N-terminal domain A (NTD-A) of a neighboring subunit, the relative movement of the Nsol and Bsol regions results in substantial overall changes in RyR1. The N-terminal disease hot spot domains (NTD-A and NTD-B), located around the central 4-fold symmetry axis, are no longer forming inter-subunit interactions with the N-terminal domains of neighboring subunits, as observed for WT RyR1 (Fig. 3a, b, Movie S1 and S2). This result confirms previous predictions based on crystal structures of wild-type and disease mutant versions of the N-terminal disease hot spot^20,21. The overall result is a widening of the cytosolic cap, with the corner regions moving downward toward the SR membrane (Fig. 3c, d, Movie S2). Corresponding movements in the Jsol region cause a splaying of the C-terminal region, which is directly linked to the S6 inner helices of the pore domain. As a result, the pore is considerably widened, consistent with an open channel based on the position of the S6 helices. The narrowest point in WT pRyR1, found at Ile4935, is now wide enough to support permeation (Supplementary Figs. 8c–10, Supplementary Table 2).

This is in contrast to our planar lipid bilayer experiments that indicate the channel should be closed under nominally Ca²⁺-free conditions. We hypothesize that this is because the channel was suspended in a detergent/lipid environment and not within a lipid bilayer. Indeed, CHAPS is known to affect channel opening intrinsically²⁵. This is a general limitation of all RyR1 and RyR2 structures that have been solved in conditions with CHAPS detergent. However, given that both WT and mutant were prepared and solved under identical experimental conditions, this highlights the inherent effect of the mutation to facilitate channel opening. In this regard, the cryo-EM conditions can be considered as an activating condition. For wild-type pRyR1, the major class still represents closed channels, but for R615C pRyR1 the combination of cryo-EM condition and mutation results in mostly open channels. Thus, both the planar lipid bilayer assays and the cryo-EM structures confirm the intrinsic ability of the R615C mutation to facilitate channel opening.

How does the R615C mutation accomplish this? A direct comparison with available open and closed RyR1 structures readily reveals the mechanism of the disease mutation. In WT rabbit RyR1, the addition of Ca²⁺, ATP, and caffeine results in a proportion of open channel structures¹¹. In this case also, the angle between the Nsol and Bsol increases upon channel opening (Supplementary Fig. 8a, b). Thus, interactions between Nsol and Bsol, in part mediated by Arg615, need to be disrupted for channel opening to occur. As the mutation intrinsically affects such interactions, weakening the Nsol–Bsol interface, less energy would be required to rearrange the Nsol and Bsol region, readily allowing the conformational change to occur at lower ligand concentrations.

Of note, the relative angle between the Nsol and Bsol region is larger for the R615C structure compared to the WT rabbit RyR1 structure activated with Ca²⁺, ATP, and caffeine (PDB 5TAL) (Supplementary Fig. 8b), and the mutation thus results in a distinct, pathological conformation. The Bsol region contains the location of the central disease hot spot, and clearly makes interactions with the N-terminal disease hot spot. Although the mutation results in a distinct conformation, we do not observe evidence of a disrupted interaction between the Bsol and the NTD-A, counter to the previous hypothesis that the interaction between the N-terminal and central disease hot spot may get disrupted or ‘unzipped’ in pathological conditions¹⁵.

In order to obtain a full picture of the effect of R615C, we aimed to compare the structures in both open and closed states. We therefore considered using Calmodulin, a well-known regulator of RyRs.

Calmodulin has divergent effects on wild-type and disease mutant RyR1

Calmodulin (CaM) is a ubiquitous Ca²⁺ sensor that has been shown to bind and regulate RyR1. For RyR2, CaM has thus far only been shown to inhibit the channel³⁰, but the effect on RyR1 is divergent, with CaM increasing the Po at low Ca²⁺ concentrations, while inhibiting it at high cytosolic Ca^{2+ 31,32,33,34}. Dantrolene, a small molecule used clinically to treat MH, was previously found to only have an effect in the presence of CaM³⁵, although this effect has been questioned³⁶. We thus investigated the effect of CaM, at low (40 nM) Ca²⁺ concentrations, for WT and R615C pRyR1 in planar lipid bilayers. At such low Ca²⁺ concentrations, the intrinsic Po is too low to reliably detect an effect, but this can be circumvented by adding an activating ligand³². We therefore added 2 mM ATP to obtain a sufficiently high initial P_o. Under these conditions, we find that the addition of CaM increased the P_o of wild-type pRyR1 ~2-fold, thus confirming the stimulatory role of CaM at low Ca²⁺, at least when another activating ligand is present. However, under the same conditions, CaM does not increase the P_o of R615C pig RyR1 and instead displayed a statistically insignificant decrease in P_o (Fig. 4a).

a Single-channel open probability (P_o) of wild type and R615C RyR1 with (filled) and without (open) 1 μM calmodulin (mean ± SD, Mann–Whitney two-tailed t-test, n = 6 with each independent recording corresponding to a newly incorporated channel from a different liposome) at low calcium ([Ca^2+]_free = 40 nM) and with 2 mM ATP. Single-channel recordings were carried out under symmetrical conditions ([cis] = [trans]) with a constant −60 mV holding potential. b Binding of apoCaM (purple) at the periphery of the cytosolic cap, showing contacts of the N-lobe with two regions of the Bsol (interface 1 and interface 2). The C-lobe interacts with CaMBD1 and CaMBD2 segments. c Superposition, based on interface 1 helices, of WT pRyR1 without CaM (colors) on closed pRyR1+apoCaM (white) and open pRyR1+apoCaM (black). The corresponding apoCaM chains are shown in light (closed pRyR1) and dark purple (open pRyR1). The panel shows a detail around the C-lobe, showing the relative movement of the Jsol toward the C-lobe compared to the structure without apoCaM bound. CaMBD2 is only visible in the apoCaM bound structure. The interactions in this area are very similar for open and closed pRyR1 structures with apoCaM bound. d Same superposition as in panel (c), but showing the interfaces with the N-lobe. The interface 2 interaction is different for the open pRyR1 in complex with apoCaM.

In order to understand how apoCaM engages with WT and mutant pRyR1, we solved cryo-EM structures of CaM compIexes at nominally Ca²⁺-free conditions, obtained by adding excess EGTA. For WT pRyR1, this resulted in two main classes that could both be refined and with structures built. The main class, corresponding to 70% of the particles, represents closed channels, whereas 27% of the particles contributed to an open channel class (Supplementary Fig. 11). Similarly, two classes and corresponding structures were obtained for R615C pRyR1 in the presence of apoCaM, with 69% open, and 19% closed channels (Supplementary Fig. 11). Importantly, the cryo-EM condition does not contain ATP, and the results have to be interpreted in the context of the cryo-EM condition. So, although the channels are normally closed in the presence of apoCaM and without any other activating molecules, in the cryo-EM condition we can see a significant fraction of open channels. Of note, despite identical conditions used for WT and R615C pRyR1 in the presence of apoCaM, there is still a larger proportion of open channels for R615C pRyR1, further highlighting the inherent effect of this mutation to facilitate channel opening.

As the bilayer shows that apoCaM stimulates opening of WT, but not R615C pRyR1, apoCaM seems to reduce, but not eliminate the relative differences between the two channels. Since the addition of apoCaM resulted in reliable reconstructions for both open and closed versions of WT and R615C pRyR1, this allowed us to compare the structures in different states, and to analyze the intrinsic effect of apoCaM on their conformations.

Structural effect of calmodulin on WT pig RyR1

A more detailed analysis around the binding site of apoCaM reveals the nature of the two conformations. Starting with the structure of the closed complex, the N-lobe is the best resolved (Supplementary Figs. 12 and 13), and mostly makes contacts with α helices in the N-terminal part of the Bsol region, encoded by residues 2190–2242 (=interface 1) (Fig. 4b). In addition, the N-lobe is close to a short loop (~2595–2600) connecting two helices further downstream in the Bsol region (=interface 2). The C-lobe region is poorly resolved, but can be seen to make interactions with a helical region encoded by residues 3627–3634 (interface 3), previously found to interact with CaM in vitro and dubbed CaMBD2 (=CaM binding domain 2)^22,23,37. Additional interactions include two helices in the Jsol region (=interface 4), including a peptide (residues 1975–1999) previously identified as CaMBD1^38,39.

Comparing structures of WT pRyR1 with and without apoCaM, both in the closed state, it is clear that apoCaM introduces conformational changes: using interface 1 as a reference point, the Jsol region including interface 4 is moving closer to interact with the C-lobe (Fig. 4c). Despite these changes, which propagate into the whole cytosolic shell, there is no discernable effect on the channel pore.

In the open structure with apoCaM, interface 1 appears nearly identical to the closed structure with apoCaM bound. Using interface 1 as a reference point, there is no visible difference in the contacts with the C-lobe comparing open and closed structures with apoCaM bound (Fig. 4c, Movie S3). However, interface 2 with the N-lobe is altered, including shifts up to ~6 Å in the contacting region (Fig. 4d, Movie S3). This results in an overall bending of the Bsol region in the open versus closed structure, with positional shifts up to 10 Å in the Bsol region far from the apoCaM binding site (Fig. 5a, b). This has very large effects on RyR1: as the Bsol interacts with multiple regions, including the SPRY domains of a neighboring subunit, its bending results in motions within the Jsol, Csol, and ultimately the CTD and pore-forming region. There is an overall downward and outward motion of the cytosolic shell, and inter-subunit interactions in the N-terminal region are disrupted, similar to the movements seen for RyRs upon binding activating ligands¹¹ (Fig. 5c, d). ApoCaM can thus promote channel opening by bending of the Bsol region, induced by the N-lobe.

Of note, a previous structure of pig RyR2 in complex with apoCaM (PDB 6JI8) has not shown this bending of the Bsol region²³ (Supplementary Fig. 13). As the main regions that interact with apoCaM appear conserved between pRyR1 and pRyR2 (Supplementary Fig. 13), the structural differences may rather be explained by various sequence differences scattered throughout the Bsol region of pRyR1 and pRyR2, affecting the interactions allosterically.

Structural effect of apo-calmodulin on R615C pig RyR1

The interfaces between apoCaM and pRyR1 are conserved between WT and R615C. The C-lobe forms interactions with CaMBD1 and CaMBD2, and these do not differ between the open and closed structures of R615C pRyR1:apoCaM. The N-lobe can interact with the Bsol region in two different modes, with the open state complex involving a bending of the Bsol region, induced by movement of interface 2, just like in the WT pRyR1:apoCaM complexes. Thus, at nominally zero Ca²⁺ levels, the interactions of apoCaM with R615C pRyR1 are nearly indistinguishable from apoCaM interacting with WT pRyR1.

How then does the addition of CaM at low Ca²⁺ promote opening of WT, but not R615C pRyR1, as observed in the bilayer experiments? It is clear that there are two relatively stable conformations of the Bsol region and in the way the Bsol region interacts with the N-lobe at interface 2 (Fig. 6a, b; Movie S4). One of these two conformations, corresponding to a closed pRyR1, is already observed for WT pRyR1 in the absence of apoCaM. However, for R615C pRyR1, the main conformation of the Bsol region without apoCaM bound is different from either of the two conformations with apoCaM. Instead, it can be best described as a conformation that is ‘intermediate’ between the two when considering the degree of bending of the Bsol region. Thus, upon binding, apoCaM pushes the R615C pRyR1 into the same two conformations observed for WT pRyR1, with one corresponding to an open, and the other to a closed channel. Thus, the original major conformation of the Bsol in R615C pRyR1 without apoCaM bound no longer exists in the presence of apoCaM.

As the ‘intermediate’ conformation for the R615C pRyR1 without apoCaM corresponds to an open channel, the addition of apoCaM thus results in a redistribution of such open channels to a mixture of both open and closed channels. This would imply that apoCaM partially inhibits opening of R615C, but in the bilayer data (Fig. 4a) this effect is not statistically significant.

The distinct state of R615C pRyR1 without apoCaM, different from both open and closed R615C pRyR1:apoCaM, is also visible throughout the structure. The different degree of bending of the Bsol region affects its contacts, including the SPRY domains of a neighboring subunit (Fig. 6c). Through links with the Jsol and Csol regions, this results in changes in the position of the ‘Thumb and forefinger’ (TaF) domain, which interacts directly with the CTD (Fig. 7a). Despite this, the position of the inner helix S6 and the width of the pore are very similar for R615C pRyR1 and open R615C pRyR1:apoCaM (Fig. 7a, b). In conclusion, binding of apoCaM to R615C pRyR1 thus disrupts the overall original conformation induced by the mutation.

Comparison of WT and R615C RyR1 in different states

Since the complexes with apoCaM resulted in open and closed state structures for both WT and R615C pRyR1, we can perform a more detailed analysis of the effect of the mutation within each state. Comparison of the closed WT and R615C pRyR1, both in the presence of apoCaM, shows that the mutation still results in conformational differences. There is a relative tilt between Nsol and Bsol, resulting in shifts ~2–7 Å across the entire Bsol region (Fig. 8a, b). As the Bsol interacts with the NTD-A of a neighboring subunit, this results in relative movements of the N-terminal domains (Fig. 8c), and downward movements of the cytosolic cap overall (Fig. 8d, e, Movie S5). Although the pore is closed, it is ~0.8 Å wider for closed R615C, as measured by the position of the Cα atom at Ile4935 (Movie S5, Supplementary Table 2). Comparing closed channels, the R615C mutation thus still results in a pathological conformation, with partial movements generally linked to channel opening. Indeed, the planar lipid bilayer shows that the P_o of R615C remains higher than WT even in the presence of apoCaM. ApoCaM can dampen, but not fully prevent a pathological conformation induced by the mutation, which remains more sensitive to activating ligands than WT.

a Superposition, based on the Nsol, of closed WT pRyR1 + apoCaM (colors) and closed R615C pRyR1 + apoCaM dark gray), showing the relative movement of the Bsol and the NTD-A′ of a neighboring subunit. Despite apoCaM binding, the R615C pRyR1 still displays a pathological conformation. b Same view as in panel (a), but showing only the closed WT pRyR1 + apoCaM, with vector trajectories of alpha carbons with displacement >2 Å going from closed Wt pRyR1+apoCaM structure to closed R615C pRyR1+apoCaM. c Overall superposition of closed WT pRyR1+apoCaM (colors) and closed R615C pRyR1+apoCaM (dark gray), showing the changes in the NTD between different subunits. This is more readily visualized in Movie S5. d Same superposition as in panel (c), showing two subunits in a side view. This highlights the overall different conformational state of the closed WT pRyR1+apoCaM and closed R615C pRyR1+apoCaM. e Same view as in panel (d), but showing only the closed WT pRyR1+apoCaM, with vector trajectories indicating shifts >2 Å going from closed WT pRyR1+apoCaM to closed R615C pRyR1+apoCaM. The Repeat34 domain is not shown as it could not be placed reliably in the closed R615C pRyR1+apoCaM map.

Comparing the open structures in complex with apoCaM, the differences between WT and R615C pRyR1 are much smaller. Relative to the Nsol, the movements in the Bsol are dampened to 1 Å around residue 2443 (Fig. 9a, b). Similarly, the relative positions of the inner helices within the pore are almost identical, and there is little additional movement in the N-terminal region (Fig. 9c; Movie S6). Figure 9d compares the relative tilt of the Bsol region across all structures presented in this study, showing the relative tilt angle in the following increasing order: WT (no CaM) = closed WT + apoCaM < closed R615C + apoCaM < open WT + apoCaM < open R615C + apoCaM = R615C (no CaM). Since tilting of Bsol relative to Nsol is generally involved with channel opening, its interface, close to the hinge point, presents an attractive allosteric pocket for novel molecules with therapeutic potential.

Species-specific differences

As previous cryo-EM studies have focused on rabbit RyR1, we compared potential structural differences between rabbit and pig RyR1 (Supplementary Fig. 14). Not surprisingly, the structures align very well, with the differences only found in regions that are generally more flexible. This includes the Repeat34 domain, which is poorly resolved in all RyR cryo-EM reconstructions. Minor differences were also observed for the N-terminal domains (2–4 Å r.m.s.d.), SPRY domains (3–5 Å r.m.s.d.), and repeat 12 domain (3–6 Å r.m.s.d.) (Supplementary Fig. 14).