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Mobilization of intracellular Ca(2+) stores regulates a multitude of cellular functions, but the role of intracellular Ca(2+) release via the ryanodine receptor (RyR) in the brain remains incompletely understood. We found that nitric oxide (NO) directly activates RyRs, which induce Ca(2+) release from intracellular stores of central neurons, and thereby promote prolonged Ca(2+) signalling in the brain. Reversible S-nitrosylation of type 1 RyR (RyR1) triggers this Ca(2+) release. NO-induced Ca(2+) release (NICR) is evoked by type 1 NO synthase-dependent NO production during neural firing, and is essential for cerebellar synaptic plasticity. NO production has also been implicated in pathological conditions including ischaemic brain injury, and our results suggest that NICR is involved in NO-induced neuronal cell death. These findings suggest that NICR via RyR1 plays a regulatory role in the physiological and pathophysiological functions of the brain.

Variants in the skeletal muscle ryanodine receptor 1 gene (RYR1) result in a spectrum of RYR1-related disorders. Presentation during infancy is typical and ranges from delayed motor milestones and proximal muscle weakness to severe respiratory impairment and ophthalmoplegia. We aimed to elucidate correlations between genotype, protein structure and clinical phenotype in this rare disease population. Genetic and clinical data from 47 affected individuals were analyzed and variants mapped to the cryo-EM RyR1 structure. Comparisons of clinical severity, motor and respiratory function and symptomatology were made according to the mode of inheritance and affected RyR1 structural domain(s). Overall, 49 RYR1 variants were identified in 47 cases (dominant/de novo, n = 35; recessive, n = 12). Three variants were previously unreported. In recessive cases, facial weakness, neonatal hypotonia, ophthalmoplegia/paresis, ptosis, and scapular winging were more frequently observed than in dominant/de novo cases (all, p < 0.05). Both dominant/de novo and recessive cases exhibited core myopathy histopathology. Clinically severe cases were typically recessive or had variants localized to the RyR1 cytosolic shell domain. Motor deficits were most apparent in the MFM-32 standing and transfers dimension, [median (IQR) 85.4 (18.8)% of maximum score] and recessive cases exhibited significantly greater overall motor function impairment compared to dominant/de novo cases [79.7 (18.8)% vs. 87.5 (17.7)% of maximum score, p = 0.03]. Variant mapping revealed patterns of clinical severity across RyR1 domains, including a structural plane of interest within the RyR1 cytosolic shell, in which 84% of variants affected the bridging solenoid. We have corroborated genotype-phenotype correlations and identified RyR1 regions that may be especially sensitive to structural modification.

Ryanodine receptors (RyRs) are large intracellular Ca2+ release channels primarily found in muscle and nerve cells and also present at low levels in pancreatic islet endocrine cells. This review examines the role of RyRs in islet cell function, focusing on calcium signaling and hormone secretion, while addressing the ongoing debate regarding their significance due to their limited expression. We explore conflicting experimental results and their potential causes, synthesizing current knowledge on RyR isoforms in islet cells, particularly in beta and delta cells. The review discusses how RyR-mediated calcium-induced calcium release enhances, rather than drives, glucose-stimulated insulin secretion. We examine the phosphorylation-dependent regulation of beta-cell RyRs, the concept of “leaky ryanodine receptors”, and the roles of RyRs in endoplasmic reticulum stress, apoptosis, store-operated calcium entry, and beta-cell electrical activity. The relationship between RyR dysfunction and the development of impaired insulin secretion in diabetes is assessed, noting their limited role in human diabetes pathogenesis given the disease’s polygenic nature. We highlight the established role of RyR-mediated CICR in the mechanism of action of common type 2 diabetes treatments, such as glucagon-like peptide-1, which enhances insulin secretion. By integrating findings from electrophysiological, molecular, and clinical studies, this review provides a balanced perspective on RyRs in islet cell physiology and pathology, emphasizing their significance in both normal insulin secretion and current diabetes therapies.

The type 2 ryanodine receptor (RyR2) controls the release of calcium ions from the sarcoplasmic reticulum in cardiac cells—the initiating step in cardiac muscle contraction. Mutations in RyR2 are associated with cardiac diseases. Peng et al. used single-particle electron cryomicroscopy to determine the structure of RyR2 from porcine heart at 4.4-Å resolution with the calcium channel closed and at 4.2-Å resolution with the calcium channel open. The structures reveal how interdomain motions result in a conformational change in the cytoplasmic region of RyR2 that is transduced by a central domain to cause motions that open or close the channel. Science , this issue p. 301 Motion of the cytoplasmic region of a key intracellular Ca 2+ channel is transduced by a central domain to gate the channel domain. RyR2 is a high-conductance intracellular calcium (Ca 2+ ) channel that controls the release of Ca 2+ from the sarco(endo)plasmic reticulum of a variety of cells. Here, we report the structures of RyR2 from porcine heart in both the open and closed states at near-atomic resolutions determined using single-particle electron cryomicroscopy. Structural comparison reveals a breathing motion of the overall cytoplasmic region resulted from the interdomain movements of amino-terminal domains (NTDs), Helical domains, and Handle domains, whereas almost no intradomain shifts are observed in these armadillo repeats–containing domains. Outward rotations of the Central domains, which integrate the conformational changes of the cytoplasmic region, lead to the dilation of the cytoplasmic gate through coupled motions. Our structural and mutational characterizations provide important insights into the gating and disease mechanism of RyRs.

The resistance of pests to common insecticides is a global issue that threatens food production worldwide. Diamide insecticides target insect ryanodine receptors (RyRs), causing uncontrolled calcium release from the sarcoplasmic and endoplasmic reticulum. Despite their high potency and species selectivity, several resistance mutations have emerged. Using a chimeric RyR (chiRyR) approach and cryo-electron microscopy (cryo-EM), we investigate how insect RyRs engage two different diamide insecticides from separate families: flubendiamide, a phthalic acid derivative, and tetraniliprole, an anthranilic compound. Both compounds target the same site in the transmembrane region of the RyR, albeit with different poses, and promote channel opening through coupling with the pore-forming domain. To explore the resistance mechanisms, we also solve two cryo-EM structures of chiRyR carrying the two most common resistance mutations, I4790M and G4946E, both alone and in complex with the diamide insecticide chlorantraniliprole. The resistance mutations perturb the local structure, directly reducing the binding affinity and altering the binding pose. Our findings elucidate the mode of action of different diamide insecticides, reveal the molecular mechanism of resistance mutations, and provide important clues for the development of novel pesticides that can bypass the resistance mutations. Pesticide resistance poses a serious threat to global food security. In this study, the authors show the mode of action of various diamide insecticides and elucidate the molecular mechanisms underlying resistance mutations.

The type 2 ryanodine receptor (RyR2) is responsible for releasing Ca2+ from the sarcoplasmic reticulum of cardiomyocytes, subsequently leading to muscle contraction. Here, we report 4 cryo-electron microscopy (cryo-EM) structures of porcine RyR2 bound to distinct modulators that, together with our published structures, provide mechanistic insight into RyR2 regulation. Ca2+ alone induces a contraction of the central domain that facilitates the dilation of the S6 bundle but is insufficient to open the pore. The small-molecule agonist PCB95 helps Ca2+ to overcome the barrier for opening. FKBP12.6 induces a relaxation of the central domain that decouples it from the S6 bundle, stabilizing RyR2 in a closed state even in the presence of Ca2+ and PCB95. Although the channel is open when PCB95 is replaced by caffeine and adenosine 5′-triphosphate (ATP), neither of the modulators alone can sufficiently counter the antagonistic effect to open the channel. Our study marks an important step toward mechanistic understanding of the sophisticated regulation of this key channel whose aberrant activity engenders life-threatening cardiac disorders.

Background Pathogenic variations affecting the ryanodine receptor 1 ( RYR1 ) gene may result in a variety of neuromuscular disorders, collectively known as RYR1 -related myopathies. Considered the most common form of congenital myopathy, individuals with RYR1 -related myopathies may experience skeletal muscle weakness and fatigue, as well as reduced functional capacity. This study examined the exercise capacity in individuals with RYR1 -related myopathies during a cardiopulmonary exercise test. Methods Ambulatory individuals (32 adults, 16 children) with genetically confirmed RYR1 -related myopathies performed exercise testing on a cycle ergometer and a six-minute walk test at baseline and month six (pre-intervention phase) of a randomized controlled trial (NCT02362425). Outcomes at peak exercise were compared to expected values among the adult and pediatric populations, while longitudinal changes were assessed after six months. Correlations between peak exercise outcomes and the six-minute walk test distance were also examined. Results The peak outcomes of oxygen uptake, work rate and heart rate at baseline were lower (all p  < 0.001) than expected in both adults and children. Peak oxygen uptake expressed as percent predicted was 62 ± 20% and 49 ± 24% in adults and children, respectively. No changes were observed across six months for peak exercise outcomes in either group. A moderately strong positive correlation was observed for peak work rate and six-minute walk test distance among adults (r s = 0.75, p  < 0.001) and children (r s = 0.64, p  = 0.008). Conclusion Exercise capacity is diminished in adults and children with RYR1 -related myopathies yet remains stable over six months. The six-minute walk test distance had a direct relationship to peak exercise work rate in adults and children. Exercise capacity testing may be informative for individualizing exercise regimens for persons with RYR1 -related myopathies. This study was registered with www.clinicaltrials.gov (NCT02362425) on February 12, 2015.

The cardiac ryanodine receptor (RyR2) constitutes the molecular basis of the process of calcium-induced calcium release where activation of RyR2s can be locally regenerative. Here, we present purely optical data of RyR2 distribution with sub-molecular resolution by applying 3D MINFLUX microscopy. Using single-domain antibodies and DNA-PAINT we determine the location of individual RyR2 subunits with high precision (~3 nm) and resolve the 3D orientations of RyR2s in-situ. We measured labeling efficiencies of ~50%, implying RyR2 tetramer detection probability approaching 95%. In HEK293 cells, RyR2 expression was dense, with some clusters containing several hundred RyR2s. Ventricular myocytes from mice contained large clusters containing many tens of close-packed RyR2s, resolving apparent discrepancies between electron microscopy and previous super-resolution microscopy data. The methodology developed here reveals the full 3D morphological complexity of RyR2 channels and is applicable to other multi-subunit complexes in a variety of cell types. Cardiac ryanodine receptors (RyR2) are critical for heart contraction. Here, the authors use 3D MINFLUX microscopy to image receptor subunits and RyR2 orientation with nanometre resolution, thereby providing a molecular view of the organisation and clustering of these cardiac muscle receptors.

The ryanodine receptor (RyR) is a critical drug target, yet dantrolene (DAN) remains the only FDA-approved inhibitor, limited by hepatotoxicity and unsuitable for chronic use. To guide improved inhibitor development, we determine high-resolution crystal structures of the RyR Repeat12 (R12) domain bound to DAN, its analog azumolene (AZU), and adenine nucleotides (AMP-PCP or ADP). DAN/AZU and nucleotides bind cooperatively to a pseudosymmetric cleft, with key interactions involving Trp880 and Trp994. Binding induces a clamshell-like closure of the R12 domain. Isothermal titration calorimetry (ITC) reveals higher affinity in the presence of nucleotides and lower affinity for RyR2 due to nearby substitutions. Structural comparison with cryo-EM data suggests that DAN/AZU binding allosterically influences RyR gating and functional regulation. Structure-based screening identifies a potent compound targeting the same site but with a distinct binding mode. Our findings highlight the power of domain-focused crystallography in guiding RyR inhibitor discovery and overcoming cryo-EM resolution limitations. This study reports crystal structures of the ryanodine receptor domain bound to dantrolene and azumolene. The work reveals cooperative nucleotide binding and provides structural insights to guide the design of potent inhibitors targeting the receptor.

Human induced pluripotent stem cells (hiPSCs) offer a unique opportunity for disease modeling. However, it is not invariably successful to recapitulate the disease phenotype because of the immaturity of hiPSC-derived cardiomyocytes (hiPSC-CMs). The purpose of this study was to establish and analyze iPSC-based model of catecholaminergic polymorphic ventricular tachycardia (CPVT), which is characterized by adrenergically mediated lethal arrhythmias, more precisely using electrical pacing that could promote the development of new pharmacotherapies. We generated hiPSCs from a 37-year-old CPVT patient and differentiated them into cardiomyocytes. Under spontaneous beating conditions, no significant difference was found in the timing irregularity of spontaneous Ca2+ transients between control- and CPVT-hiPSC-CMs. Using Ca2+ imaging at 1 Hz electrical field stimulation, isoproterenol induced an abnormal diastolic Ca2+ increase more frequently in CPVT- than in control-hiPSC-CMs (control 12% vs. CPVT 43%, p<0.05). Action potential recordings of spontaneous beating hiPSC-CMs revealed no significant difference in the frequency of delayed afterdepolarizations (DADs) between control and CPVT cells. After isoproterenol application with pacing at 1 Hz, 87.5% of CPVT-hiPSC-CMs developed DADs, compared to 30% of control-hiPSC-CMs (p<0.05). Pre-incubation with 10 μM S107, which stabilizes the closed state of the ryanodine receptor 2, significantly decreased the percentage of CPVT-hiPSC-CMs presenting DADs to 25% (p<0.05). We recapitulated the electrophysiological features of CPVT-derived hiPSC-CMs using electrical pacing. The development of DADs in the presence of isoproterenol was significantly suppressed by S107. Our model provides a promising platform to study disease mechanisms and screen drugs.