Structural Determinants of Skeletal Muscle Ryanodine Receptor Gating

Ryanodine receptor type 1 (RyR1) releases Ca2+ from intracellular stores upon nerve impulse to trigger skeletal muscle contraction. Effector binding at the cytoplasmic domain tightly controls gating of the pore domain of RyR1 to release Ca2+. However, the molecular mechanism that links effector binding to channel gating is unknown due to lack of structural data. Here, we used a combination of computational and electrophysiological methods and cryo-EM densities to generate structural models of the open and closed states of RyR1. Using our structural models, we identified an interface between the pore-lining helix (Tyr-4912–Glu-4948) and a linker helix (Val-4830–Val-4841) that lies parallel to the cytoplasmic membrane leaflet. To test the hypothesis that this interface controls RyR1 gating, we designed mutations in the linker helix to stabilize either the open (V4830W and T4840W) or closed (H4832W and G4834W) state and validated them using single channel experiments. To further confirm this interface, we designed mutations in the pore-lining helix to stabilize the closed state (Q4947N, Q4947T, and Q4947S), which we also validated using single channel experiments. The channel conductance and selectivity of the mutations that we designed in the linker and pore-lining helices were indistinguishable from those of WT RyR1, demonstrating our ability to modulate RyR1 gating without affecting ion permeation. Our integrated computational and experimental approach significantly advances the understanding of the structure and function of an unusually large ion channel

Channel Gating Dependence on Pore Lining Helix Glycine Residues in Skeletal Muscle Ryanodine Receptor

Type 1 ryanodine receptors (RyR1s) release Ca2+ from the sarcoplasmic reticulum to initiate skeletal muscle contraction. The role of RyR1-G4934 and -G4941 in the pore-lining helix in channel gating and ion permeation was probed by replacing them with amino acid residues of increasing side chain volume. RyR1-G4934A, -G4941A, and -G4941V mutant channels exhibited a caffeine-induced Ca2+ release response in HEK293 cells and bound the RyR-specific ligand [3H]ryanodine. In single channel recordings, significant differences in the number of channel events and mean open and close times were observed between WT and RyR1-G4934A and -G4941A. RyR1-G4934A had reduced K+ conductance and ion selectivity compared with WT. Mutations further increasing the side chain volume at these positions (G4934V and G4941I) resulted in reduced caffeine-induced Ca2+ release in HEK293 cells, low [3H]ryanodine binding levels, and channels that were not regulated by Ca2+ and did not conduct Ca2+ in single channel measurements. Computational predictions of the thermodynamic impact of mutations on protein stability indicated that although the G4934A mutation was tolerated, the G4934V mutation decreased protein stability by introducing clashes with neighboring amino acid residues. In similar fashion, the G4941A mutation did not introduce clashes, whereas the G4941I mutation resulted in intersubunit clashes among the mutated isoleucines. Co-expression of RyR1-WT with RyR1-G4934V or -G4941I partially restored the WT phenotype, which suggested lessening of amino acid clashes in heterotetrameric channel complexes. The results indicate that both glycines are important for RyR1 channel function by providing flexibility and minimizing amino acid clashes

Pore Dynamics and Conductance of RyR1 Transmembrane Domain

AbstractRyanodine receptors (RyR) are calcium release channels, playing a major role in the regulation of muscular contraction. Mutations in skeletal muscle RyR (RyR1) are associated with congenital diseases such as malignant hyperthermia and central core disease (CCD). The absence of high-resolution structures of RyR1 has limited our understanding of channel function and disease mechanisms at the molecular level. Previously, we have reported a hypothetical structure of the RyR1 pore-forming region, obtained by homology modeling and supported by mutational scans, electrophysiological measurements, and cryo-electron microscopy. Here, we utilize the expanded model encompassing six transmembrane helices to calculate the RyR1 pore region conductance, to analyze its structural stability, and to hypothesize the mechanism of the Ile4897 CCD-associated mutation. The calculated conductance of the wild-type RyR1 suggests that the proposed pore structure can sustain ion currents measured in single-channel experiments. We observe a stable pore structure on timescales of 0.2 μs, with multiple cations occupying the selectivity filter and cytosolic vestibule, but not the inner chamber. We further suggest that stability of the selectivity filter critically depends on the interactions between the I4897 residue and several hydrophobic residues of the neighboring subunit. Loss of these interactions in the case of polar substitution I4897T results in destabilization of the selectivity filter, a possible cause of the CCD-specific reduced Ca2+ conductance

[³H]S107 binding to SR vesicles.

<p>SR vesicles with low and high B_max of [³H]ryanodine binding were obtained as described in <a href="http://www.plosone.org/article/info:doi/10.1371/journal.pone.0054208#s2" target="_blank">Materials and Methods</a>. B_max of [³H]ryanodine binding was obtained as shown in <a href="http://www.plosone.org/article/info:doi/10.1371/journal.pone.0054208#pone.0054208.s002" target="_blank">Fig. S2A</a>. Shown are the amounts of [³H]ryanodine specifically bound to SR vesicles after 5 h at 24°C. Specific [³H]S107 binding to SR vesicles was determined at 44 µM [³H]S107 and indicated free Ca²⁺ concentration as described in <a href="http://www.plosone.org/article/info:doi/10.1371/journal.pone.0054208#pone.0054208.s002" target="_blank">Fig. S2B</a>. Shown are the amounts of S107 specifically bound to SR vesicles after 9 h incubation at 24°C. Data are the mean ± SD of 3–6 determinations.</p

Stabilization of the Skeletal Muscle Ryanodine Receptor Ion Channel-FKBP12 Complex by the 1,4-Benzothiazepine Derivative S107

<div><p>Activation of the skeletal muscle ryanodine receptor (RyR1) complex results in the rapid release of Ca²⁺ from the sarcoplasmic reticulum and muscle contraction. Dissociation of the small FK506 binding protein 12 subunit (FKBP12) increases RyR1 activity and impairs muscle function. The 1,4-benzothiazepine derivative JTV519, and the more specific derivative S107 <b>(</b>2,3,4,5,-tetrahydro-7-methoxy-4-methyl-1,4-benzothiazepine), are thought to improve skeletal muscle function by stabilizing the RyR1-FKBP12 complex. Here, we report a high degree of nonspecific and specific low affinity [³H]S107 binding to SR vesicles. SR vesicles enriched in RyR1 bound ∼48 [³H]S107 per RyR1 tetramer with EC₅₀ ∼52 µM and Hillslope ∼2. The effects of S107 and FKBP12 on RyR1 were examined under conditions that altered the redox state of RyR1. S107 increased FKBP12 binding to RyR1 in SR vesicles in the presence of reduced glutathione and the NO-donor NOC12, with no effect in the presence of oxidized glutathione. Addition of 0.15 µM FKBP12 to SR vesicles prevented FKBP12 dissociation; however, in the presence of oxidized glutathione and NOC12, FKBP12 dissociation was observed in skeletal muscle homogenates that contained 0.43 µM myoplasmic FKBP12 and was attenuated by S107. In single channel measurements with FKBP12-depleted RyR1s, in the absence and presence of NOC12, S107 augmented the FKBP12-mediated decrease in channel activity. The data suggest that S107 can reverse the harmful effects of redox active species on SR Ca²⁺ release in skeletal muscle by binding to RyR1 low affinity sites.</p> </div

[³H]S107 binding to SR vesicles.

<p>(A) Time course of total, nonspecific and specific [³H]S107 binding to SR vesicles incubated with 0.2 µM [³H]S107 in 0.25 M KCl, 20 mM imidazole, pH 7.0, 50 µM free Ca²⁺ and protease inhibitors for 1 to 9 h at 24°C. Nonspecific binding was determined by measuring [³H]S107 binding to SR vesicles heat-inactivated for 10 min at 95°C. Data are the mean ± SD of 3 experiments. (B) Total, nonspecific and specific [³H]S107 binding to SR vesicles incubated with 0.1 µM [³H]S107 and 0.1 to 100 µM S107 as above for 7 h at 24°C. Nonspecific binding was determined as in A. Specific binding curve was obtained using four parameter logistic equation shown in <a href="http://www.plosone.org/article/info:doi/10.1371/journal.pone.0054208#s2" target="_blank">Materials and Methods</a>.</p

Effects of NOC12 and S107 on [³H]ryanodine binding to RyR1.

<p>(A) Dependence of [³H]ryanodine binding on NOC12 concentration. SR vesicles not treated with FK506 were incubated for 5 h at 24°C in 0.25 M KCl, 20 mM imidazole, pH 7.0, 7 µM free Ca²⁺, protease inhibitors and the indicated concentrations of NOC12 in the presence (•) and absence (○) of 44 µM S107. Data are the mean ± SEM of 4 experiments. *p<0.05 compared to vesicles without S107. (B) Dependence of [³H]ryanodine binding to RyR1 on S107 concentration. SR vesicles were incubated as in A in the absence (○) and presence of 50 µM NOC12 (•) and the indicated concentrations of S107. Data are the mean ± SEM of 4 experiments. *p<0.05 compared to vesicles with 50 µM NOC12 and without S107. (C) Specific [³H]ryanodine binding to SR vesicles containing (−FK506) and depleted (+FK506) of FKBP12. [³H]Ryanodine binding was determined in the presence of 50 µM NOC12 and the absence and presence of 44 µM S107. Data are the mean ± SEM of 8 experiments. *p<0.05 compared to vesicles treated with FK506 and incubated in the absence of S107, ^#p<0.05 compared to vesicles not treated with FK506 and incubated in the absence of S107.</p

Effect of S107 on the stability of FKBP12-RyR1 complex in skeletal muscle homogenates.

<p>(A and B) Skeletal muscle homogenates were incubated without (control) or with 5 mM GSH, 5 mM GSSG or 0.10 mM NOC12 in the absence or presence of 44 µM S107 for 20 h at 24°C. Unbound FKBP12 was removed by centrifugation and the amounts of RyR1 and FKBP12 were detected using anti-RyR1 and anti-FKBP12 antibodies. Homogenates incubated without glutathione and NOC12 served as control. Data are the mean ± SEM of 8 experiments. *p<0.05 compared to control homogenates without S107. ^#p<0.05 compared to homogenates incubated with NOC12 in the absence of S107.</p

FKBP12 dissociation from SR vesicles in the presence of NOC12.

<p>(A–C) SR vesicles not treated with FK506 were incubated for 5 h at 24°C with or without 0.10 mM NOC12 in the absence and presence of 44 µM S107 in 0.25 M KCl, 20 mM imidazole, pH 7.0, 7 µM free Ca²⁺ and protease inhibitors. S-nitrosylation was stopped by centrifugation. Resuspended samples were separated on 8–20% (FKBP12) and 3–12% (RyR1 and Cys-SNO) gradient SDS-PAGE gels and transferred to nitrocellulose membranes to detect S-nitrosylation of RyR1, and FKBP12 and RyR1 proteins. Data are the mean ± SEM of 4 determinations. *p<0.05 compared to control samples (B) and samples with NOC12 and S107 (C). (D and E) SR membranes were incubated with and without 44 µM S107 and 0.1 mM NOC12 at 24°C for 90 min, solubilized, and immunoprecipitated as described in Methods. Immunoblots of RyR1 and FKBP12 are shown. Data are the mean ± SEM of 4 experiments. ^*p<0.05 compared to control samples and samples incubated with NOC12 and S107.</p