Biology subjects

Kurebayashi, N.

Publications and source records attributed to Kurebayashi, N..

3 recordsLinked to original sources

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↗