Biology subjects

Kurebayashi, N.

Publications and source records attributed to Kurebayashi, N..

7 recordsLinked to original sources

bioRxiv2026

Structural basis for Ca2+-dependent, RyR2-selective stabilization by Ryanozole in CPVT

The cardiac ryanodine receptor (RyR2) is a Ca{superscript 2} release channel essential for excitation-contraction coupling. RyR2 mutations cause severe arrhythmogenic disorders, including catecholaminergic polymorphic ventricular tachycardia (CPVT), through gain-of-function (GOF) effects leading to aberrant Ca{superscript 2} release. We have recently developed Ryanozole, a potential therapeutic compound for CPVT, which selectively stabilizes RyR2 in a Ca2+-dependent manner. Here, we define the mechanism of action of Ryanozole by combining high-resolution cryo-electron microscopy, targeted mutagenesis and functional assays. Ryanozole binds to the interface between the Ca{superscript 2}-binding site and the pore-forming S6 helix, interfering with conformational changes required for Ca{superscript 2}-induced channel opening at low Ca2+. We identified key residues for the binding and isoform-specific modulation of Ryanozole. Notably, Ryanozole-bound RyR2 retains its ability to open at high Ca2+ via unique conformational changes. These findings provide a structural basis for CPVT-targeted therapy and redefine the paradigm of small molecule-based regulation of large ion channels.

biochemistry↗

bioRxiv2024

A novel RyR2-selective stabilizer prevents and ameliorates severe arrhythmias in stress-induced ventricular tachycardia

Background and PurposeAberrant activation of the type 2 ryanodine receptor (RyR2) causes lethal arrhythmias, such as catecholaminergic polymorphic ventricular tachycardia (CPVT). Developing drugs that suppress RyR2 hyperactivation may be key to novel arrhythmia treatments. This study evaluated the antiarrhythmic potential of Ryanozole, a recently developed novel RyR2 modulator with high affinity and selectivity, using CPVT mouse models harboring mutant RyR2s. Experimental approachIn vitro effects of Ryanozole were evaluated by ER Ca2+-based assay and [3H]Ryanodine binding assay using RyR2-expressing HEK293 cells. Two lines of mice with different arrhythmia severity, RyR2-R420W and -K4750Q, were employed for in vivo assessments. Intracellular Ca2+ signals were analyzed in isolated cardiomyocytes using Cal520. Antiarrhythmic effects were evaluated by ECG under catecholaminergic challenge in anesthetized mice and during spontaneous arrhythmias in conscious mice. ECG and echocardiographic parameters were evaluated before and after drug administration. Key resultsRyanozole inhibited both wild type and mutant RyR2s with similar IC50 of 15-40 nM. The inhibition was more potent at lower cytosolic Ca2+ concentrations. It suppressed Ca2+ waves and Ca2+ sparks without affecting action potential-evoked Ca2+ transients. Ryanozole effectively prevented adrenaline-induced arrhythmias and rapidly terminated spontaneous arrhythmias during daily activity. Importantly, Ryanozole did not impair cardiac conduction or contractility, unlike conventional antiarrhythmic drugs. Conclusions and implicationsRyanozole strongly suppresses RyR2 under diastolic Ca2+ conditions, thereby preventing the arrhythmogenic trigger of aberrant Ca2+ release. This mechanism likely provides potent antiarrhythmic effects while preserving cardiac function. Ryanozole is a promising therapeutic candidate treating RyR2-mediated arrhythmias, such as CPVT.

pharmacology and toxicology↗

bioRxiv2023

Screening for novel RyR2 inhibitors by ER Ca2+ monitoring

Type 2 ryanodine receptor (RyR2) is a Ca2+ release channel on the endoplasmic/sarcoplasmic reticulum (ER/SR) that plays a central role in the excitation-contraction coupling in the heart. Hyperactivity of RyR2 has been linked to ventricular arrhythmias in patients with catecholaminergic polymorphic ventricular tachycardia (CPVT) and heart failure, where spontaneous Ca2+ release via hyperactivated RyR2 depolarizes diastolic membrane potential to induce triggered activity. In such cases, drugs that suppress RyR2 activity are expected to prevent the arrhythmias, but there is no clinically available RyR2 inhibitors at present. In this study, we searched for RyR2 inhibitors from a well-characterized compound library using a recently developed ER Ca2+-based assay, where the inhibition of RyR2 activity was detected by the increase in ER Ca2+ signals from R-CEPIA1er, a genetically encoded ER Ca2+ indicator, in RyR2-expressing HEK293 cells. By screening 1535 compounds in the library, we identified three compounds (chloroxylenol, methyl orsellinate and riluzole) that greatly increased the ER Ca2+ signal. All of the three compounds suppressed spontaneous Ca2+ oscillations in RyR2-expressing HEK293 cells and correspondingly reduced the Ca2+-dependent [3H]ryanodine binding activity. In cardiomyocytes from RyR2-mutant mice, the three compounds effectively suppressed abnormal Ca2+ waves without substantial effects on the action-potential-induced Ca2+ transients. These results confirm that ER Ca2+-based screening is useful for identifying modulators of ER Ca2+ release channels and suggest that RyR2 inhibitors have potential to be developed as a new category of antiarrhythmic drugs. Significance statementWe successfully identified three compounds having RyR2 inhibitory action from a well-characterized compound library using an ER Ca2+-based assay, and demonstrated that these compounds suppressed arrhythmogenic Ca2+ wave generation without substantially affecting physiological action-potential induced Ca2+ transients in cardiomyocytes. This study will facilitate the development of RyR2 specific inhibitors as a potential new class of drugs for life-threatening arrhythmias induced by hyperactivation of RyR2.

pharmacology and toxicology↗

bioRxiv2022

High-throughput platform for depolarization-induced Ca2+ release for skeletal muscle disease mutation validation and drug discovery

In skeletal muscle excitation-contraction (E-C) coupling, depolarization of the plasma membrane triggers Ca2+ release from the sarcoplasmic reticulum (SR), referred to as depolarization-induced Ca2+ release (DICR). DICR occurs via the type 1 ryanodine receptor (RyR1), which physically interacts with the dihydropyridine receptor Cav1.1 subunit in specific machinery formed with additional essential components including {beta}1a, Stac3 adaptor protein and junctophilins. Exome sequencing has accelerated the discovery of many novel mutations in genes encoding DICR machinery in various skeletal muscle diseases. However, functional validation is time-consuming because it must be performed in a skeletal muscle environment. In this study, we established a platform of the reconstituted DICR in HEK293 cells. The essential components were effectively transduced into HEK293 cells expressing RyR1 using baculovirus vectors, and Ca2+ release was quantitatively measured with R-CEPIA1er, a fluorescent ER Ca2+ indicator, without contaminant of extracellular Ca2+ influx. In these cells, [K+]-dependent Ca2+ release was triggered by chemical depolarization with the aid of inward rectifying potassium channel, indicating a successful reconstitution of DICR. Using the platform, we evaluated several Cav1.1 mutations that are implicated in malignant hyperthermia and myopathy. We also tested several RyR1 inhibitors; whereas dantrolene and Cpd1 inhibited DICR, procaine had no effect. Furthermore, twitch potentiators such as perchlorate and thiocyanate shifted the voltage dependence of DICR to more negative potentials without affecting Ca2+-induced Ca2+ release. These results well reproduced the findings with the muscle fibers and the cultured myotubes. Since the procedure is simple and reproducible, the reconstituted DICR platform will be highly useful for validation of mutations and drug discovery for skeletal muscle diseases. SummaryMutations in essential components for depolarization-induced Ca2+ release (DICR) are implicated into various skeletal muscle diseases. Murayama et al. establish a reconstituted DICR platform in nonmuscle cells for evaluation of disease-causing mutations and drug discovery.

physiology↗

bioRxiv2021

Enhanced CICR activity reduces ER Ca2+ level in cells expressing CPVT-linked mutant RyR2

Type 2 ryanodine receptor (RyR2) is a cardiac Ca2+ release channel in the endoplasmic reticulum (ER). Mutations in RyR2 are linked to catecholaminergic polymorphic ventricular tachycardia (CPVT), which is considered to be associated with enhanced spontaneous Ca2+ release. This spontaneous Ca2+ release tends to occur when ER Ca2+ ([Ca2+]ER) reaches a certain threshold level, and CPVT mutations are reported to lower this threshold. There are two explanations for this lowered threshold: the mutations increase sensitivity to luminal Ca2+ or they enhance cytosolic [Ca2+] ([Ca2+]cyt)-induced Ca2+ release (CICR) activity. However, no quantitative analysis of this issue has been performed so far. Here, we quantitatively explored how the change in CICR activity of RyR2 affects the threshold [Ca2+]ER experimentally and by model-based simulation. Wild-type (WT) and CPVT-linked mutant RyR2s were expressed in HEK293 cells. [Ca2+]cyt and [Ca2+]ER measurements with Ca2+ indicators revealed that CPVT RyR2 cells showed higher oscillation frequency and lower threshold [Ca2+]ER in a mutation-specific manner compared with WT cells. The CICR activity of mutant RyR2s was assessed by Ca2+-dependent [3H]ryanodine binding and parameter analysis. CICR activity at resting [Ca2+]cyt, A7.0, was higher in CPVT mutants than in WT and a strong inverse correlation was found between threshold [Ca2+]ER and A7.0. Interestingly, lowering RyR2 expression increased threshold [Ca2+]ER, suggesting that the threshold [Ca2+]ER depends on net Ca2+ release rate via RyR2, a product of A7.0 for each mutant and the density of RyR2 molecules. A model-based simulation successfully reproduced the [Ca2+]cyt and [Ca2+]ER changes. Interestingly, the CICR activity associated with specific mutations correlated well with the age of onset of the disease in CPVT patients carrying the mutations. Our data suggest that the reduction in threshold [Ca2+]ER for spontaneous Ca2+ release by CPVT mutation is explained by enhanced CICR activity without considering a change in the [Ca2+]ER sensitivity of RyR2. SummaryCPVT-linked RyR2 mutations are prone to induce spontaneous Ca2+ release from ER, which is strongly associated with arrhythmias. Kurebayashi et al. quantitatively explore how the changes in CICR activity by RyR2 mutations affect spontaneous Ca2+ experimentally and by model simulation.

physiology↗

bioRxiv2020

Gating mechanism of cardiac ryanodine receptor 2 upon calcium ion binding

Cardiac ryanodine receptor (RyR2) is a large Ca2+ release channel in the sarcoplasmic reticulum and indispensable for excitation-contraction coupling in the heart. RyR2 is activated by Ca2+ and RyR2 mutations are implicated in severe arrhythmogenic diseases. Yet, the structural basis underlying channel opening and how mutations affect the channel remain unknown. Here, we addressed gating mechanism of RyR2 by combining high-resolution structures determined by cryo-electron microscopy with quantitative functional analysis of channels carrying various mutations in specific residues. We demonstrated two fundamental mechanisms for channel gating: interactions close to the channel pore stabilize the channel to prevent hyperactivity and a series of interactions in the surrounding regions is necessary for channel opening upon Ca2+ binding. Mutations at the residues involved in the former and the latter mechanisms cause gain-of-function and loss-of-function, respectively. Our results reveal gating mechanisms of the RyR2 channel and alterations by pathogenic mutations at the atomic level.

biochemistry↗

bioRxiv2020

A novel saline-soluble, rapidly-metabolized RyR1 inhibitor rescues volatile anesthesia-induced death and environmental heat stroke in a mouse model relevant to malignant hyperthermia

Mutations in the type 1 ryanodine receptor (RyR1), a Ca2+ release channel in skeletal muscle, hyperactivate the channel to cause malignant hyperthermia (MH) and are implicated in severe heat stroke. Dantrolene, the only approved drug for MH, has the disadvantages of having very poor water solubility and long plasma half-life. We show here that a novel RyR1-selective inhibitor, 6,7-(methylenedioxy)-1-octyl-4-quinolone-3-carboxylic acid (Compound 1, Cpd1), effectively prevents and treats MH and heat stroke in several mouse models relevant to MH. Cpd1 reduced resting intracellular Ca2+, inhibited halothane- and isoflurane-induced Ca2+ release, suppressed caffeine-induced contracture in skeletal muscle, reduced sarcolemmal cation influx, and prevented or reversed the fulminant MH crisis induced by isoflurane anesthesia and rescued animals from heat stroke caused by environmental heat stress. Notably, Cpd1 has great advantages of better water solubility and rapid clearance in vivo over dantrolene. Cpd1 has the potential to be a promising new candidate for effective treatment of patients carrying RyR1 mutations.

pharmacology and toxicology↗