Potential physiological and pathological roles for axonal ryanodine receptors

Clinical disability following trauma or disease to the spinal cord often involves the loss of vital white matter elements including axons and glia.Although excessive Cais an established driver of axonal degeneration,therapeutically targeting externally sourced Cato date has had limited success in both basic and clinical studies.Contributing factors that may underlie this limited success include the complexity of the many potential sources of Caentry and the discovery that axons also contain substantial amounts of stored Cathat if inappropriately released could contribute to axonal demise.Axonal Castorage is largely accomplished by the axoplasmic reticulum that is part of a continuous network of the endoplasmic reticulum that provides a major sink and source of intracellular Cafrom the tips of dendrites to axonal terminals.This“neuron-within-a-neuron”is positioned to rapidly respond to diverse external and internal stimuli by amplifying cytosolic Calevels and generating short and long distance regenerative Cawaves through Cainduced Carelease.This review provides a glimpse into the molecular machinery that has been implicated in regulating ryanodine receptor mediated Carelease in axons and how dysregulation and/or overstimulation of these internodal axonal signaling nanocomplexes may directly contribute to Ca-dependent axonal demise.Neuronal ryanodine receptors expressed in dendrites,soma,and axonal terminals have been implicated in synaptic transmission and synaptic plasticity,but a physiological role for internodal localized ryanodine receptors remains largely obscure.Plausible physiological roles for internodal ryanodine receptors and such an elaborate internodal binary membrane signaling network in axons will also be discussed.

Expression and reconstitution of the bioluminescent Ca2+reporter aequorin in human embryonic stem cells,and exploration of the presence of functional IP3 and ryanodine receptors during the early stages of their differentiation into cardiomyocytes

In order to develop a novel method of visualizing possible Ca2＋ signaling during the early differentiation of hESCs into cardi- omyocytes and avoid some of the inherent problems associated with using fluorescent reporters, we expressed the biolumines- cent Ca2＋ reporter, apo-aequorin, in HES2 cells and then reconstituted active holo-aequorin by incubation withf-coelenterazine. The temporal nature of the Ca2＋ signals generated by the holo-f-aequorin-expressing HES2 cells during the earliest stages of differentiation into cardiomyocytes was then investigated. Our data show that no endogenous Ca2＋ transients （generated by re- lease from intracellular stores） were detected in 1-12-day-old cardiospheres but transients were generated in cardiospheres following stimulation with KC1 or CaC12, indicating that holo-f-aequorin was functional in these cells. Furthermore, following the addition of exogenous ATP, an inositol trisphosphate receptor （IP3R） agonist, small Ca2＋transients were generated from day 1 onward. That ATP was inducing Ca2＋ release from functional IP3Rs was demonstrated by treatment with 2-APB, a known IP3R antagonist. In contrast, following treatment with caffeine, a ryanodine receptor （RyR） agonist, a minima/Ca2＋ response was observed at day 8 of differentiation only. Thus, our data indicate that unlike RyRs, IP3Rs are present and continually functional at these early stages of cardiomyocyte differentiation.

Dynamic Coupling between Ryanodine Receptors and its Role in Cacium Release

The calcium release channels/ryanodine receptors (RyRs) usually form 2-D regular lattice in the endoplasmic/sarcoplasmic reticulum membranes. Several inter-RyR coupling

Function of the CaMKII-ryanodine receptor signaling pathway in rabbits with left ventricular hypertrophy and triggered ventricular arrhythmia

BACKGROUND:Calcium calmodulin-dependent kinase II(CaMKII) can be more active in patients with left ventricular hypertrophy(LVH),which in turn causes phosphorylation of ryanodine receptors,resulting in inactivation and the instability of intracellular calcium homeostasis.The present study aimed to determine the effect of CaMKII-ryanodine receptor pathway signaling in rabbits with left ventricular hypertrophy and triggered ventricular arrhythmia.METHODS:Forty New Zealand rabbits were randomized into four groups(10 per group):sham group,LVH group,KN-93 group(LVH+KN-93),and ryanodine group(LVH+ryanodine).Rabbits in the LVH,KN-93,and ryanodine groups were used to establish a left ventricular hypertrophy model by the coarctation of the abdominal aorta,while those in the sham group did not undergo the coarctation.After eight weeks,action potentials(APs) were recorded simultaneously in the endocardium and epicardium,and a transmural electrocardiogram(ECG) was also recorded in the rabbit left ventricular wedge model.Drugs were administered to the animals in the KN-93 and ryanodine groups,and the frequency of triggered APs and ventricular tachycardia was recorded after the rabbits were given isoprenaline(1 μmol/L) and high-frequency stimulation.RESULTS:The frequency(animals/group) of triggered APs was 0/10 in the sham group,10/10 in the LVH group,4/10 in the KN-93 group,and 1/10 in the ryanodine group.The frequencies of ventricular tachycardia were 0/10,9/10,3/10,and 1/10,respectively.The frequencies of polymorphic ventricular tachycardia or ventricular fibrillation were 0/10,7/10,2/10,and 1/10,respectively.The frequencies of triggered ventricular arrhythmias in the KN-93 and ryanodine groups were much lower than those in the LVH group(P<0.05).CONCLUSIONS:KN-93 and ryanodine can effectively reduce the occurrence of triggered ventricular arrhythmia in rabbits with LVH.The CaMKII-ryanodine signaling pathway can be used as a new means of treating ventricular arrhythmia.

Molecular characterization of the ryanodine receptor from Adoxophyes orana and its response to lethal and sublethal doses of chlorantraniliprole

The insect ryanodine receptor(RyR)is a novel target of the anthranilic and phthalic insecticides,which have high activity against lepidopteran insects.Several diamide insecticides have been used to control pests in orchards in China.To enhance our understanding of the effects of diamides on RyRs,full-length cDNAs were isolated and characterized from the summer fruit tortrix moth,Adoxophyes orana,which is the most severe pest of stone and pome trees worldwide.In addition,the modulation of AoRyRmRNA expression by diamide insecticides was investigated.The AoRyRmRNA obtained had an open reading frame(ORF)of 15402 bp nucleotides encoding 5113 amino acids,and shared high and low identity with its orthologs in other insects and mammals of 77–92 and 45–47%identity,respectively.One alternative splice site with two exclusive exons was revealed in AoRyR(a/b).The usage of exon was more frequent in eggs and larvae than in pupae and adults.Quantitative real-time reverse transcription PCR(qRT-PCR)showed that AoRyRmRNA was expressed at all developmental stages,especially in eggs,male pupae and male adults.The expression levels of AoRyRmRNA in the whole body were up-regulated markedly after 3 rd instar larvae were treated with chlorantraniliprole at LC_(10),LC_(20)and LC_(50)dosages.The results could provide the basis for further functional studies of Ao Ry R and for the development of new chemicals with selective activity against insects.

Study on the effect of doxorubicin on expressions of genes encoding myocardial sarcoplasmic reticulum Ca^2＋ transport proteins and the effect of taurine on myocardial protection in rabbits

To investigate the effect of doxorubicin(DOX) on gene expression of the myocardial sarcoplasmic reticulum (SR)Ca 2+ transport proteins and the mechanism of taurine(Tau) protecting cardiac muscle cells, 9 rabbits were injected with DOX , 8 rabbits with DOX and Tau, and 9 rabbits with normal saline. Cardiac function , concentration of calcium in cardiomyocytes (Myo[Ca 2+ ] \%i\%), activity of SR Ca 2+ ATPase(SERCA2a), level of SERCA2a mRNA and Ca 2+ released channels(RYR2)mRNA were detected. The left ventricle tissues were observed by electron microscopy. The results showed that cardiac index, left ventricular systolic pressure, activity of SR Ca 2+ ATPase and level of SERCA2a mRNA decreased , while Myo[Ca 2+ ] \%i\% increased in DOX treated rabbits. DOX could not affect the level of RYR2 mRNA. Tau intervention could alleviate the increase of left ventricular diastolic pressure, Myo[Ca 2+ ] \%i\% and the decrease of SERCA2a mRNA induced by doxorubicin. The results suggested that downregulation of SERCA2a gene expression was an important mechanism of DOX induced cardiomyopathy and that Tau could partially improve the heart function by reducing calcium overload and alleviating downregulation of SERCA2a mRNA.

Remodeling of the ryanodine receptor isoform 1 channel regulates the sweet and umami taste perception of Rattus norvegicus

Sweet and umami tastes are elicited by sweet and umami receptors on the tongue and palate epithelium,respectively.However,the molecular machinery allowing the taste reaction remains incompletely understood.Through a phosphoproteomic approach,we identified the key proteins that trigger taste mechanisms based on phosphorylation cascades.Ryanodine receptor isoform 1(RYR1)was further verified by sensory and behavioral assays.We propose a model of RYR1-mediated sweet/umami signaling in which the RYR1 channel,which mediates Ca^(2+)release from the endoplasmic reticulum,is closed by dephosphorylation in bud tissue after sweet/umami treatment.The alteration in Ca^(2+)content in the cytosol induces transient membrane depolarization and generates a cell current for taste signal transduction.We demonstrate that RYR1 is a new channel involved in the regulation of sweet/umami signal transduction and propose a“metabolic clock”notion based on sweet/umami sensing.Our study provides a valuable foundation for a system-level understanding of the taste perception mechanism.

INTRACELLULAR SOLITARY PULSE CALCIUM WAVES IN FROG SYMPATHETIC NEURONS

In certain extracellular environments, there would appear a kind of solitary pulse calcium waves in Rana pipiens sympathetic neurons, propagating inwards along the radial direction from the plasma membrane. To gain a deeper insight into the waves, a model describing intracellular calcium waves in frog sympathetic neurons was established. In the piecewise linear approximation, the present model is identical to the Sneyd model. Thus, with (Sneyd's) method, analytical expressions for the wave speed and profiles of 1-D solitary pulse wave were obtained. A wave speed of (21.5 μm/s) was deduced, which agrees rather well with experimental data.

The current status of malignant hyperthermia

Malignant hyperthermia(MH) is a rare and life-threatening pharmacogenetic disorder triggered by volatile anesthetics, the depolarizing muscle relaxant succinylcholine, and rarely by strenuous exercise or environmental heat. The exact prevalence of MH is unknown, and it varies from 1:16 000 in Denmark to 1:100 000 in New York State. The underlying mechanism of MH is excessive calcium release from the sarcoplasmic reticulum(SR),leading to uncontrolled skeletal muscle hyper-metabolism. Genetic mutations in ryanodine receptor type 1(RYR1)and CACNA1 S have been identified in approximately 50% to 86% and 1% of MH-susceptible(MHS) individuals,respectively. Classic clinical symptoms of MH include hypercarbia, sinus tachycardia, masseter spasm,hyperthermia, acidosis, muscle rigidity, hyperkalemia, myoglobinuria, and etc. There are two types of testing for MH: a genetic test and a contracture test. Contracture testing is still being considered as the gold standard for MH diagnosis. Dantrolene is the only available drug approved for the treatment of MH through suppressing the calcium release from SR. Since clinical symptoms of MH are highly variable, it can be difficult to establish a diagnosis of MH. Nevertheless, prompt diagnosis and treatments are crucial to avoid a fatal outcome. Therefore, it is very important for anesthesiologists to raise awareness and understand the characteristics of MH. This review summarizes epidemiology, clinical symptoms, diagnosis and treatments of MH and any new developments.

Arrhythmogenic mechanisms in ryanodine receptor channelopathies

Ryanodine receptors(Ry Rs) are the calcium release channels of sarcoplasmic reticulum(SR) that provide the majority of calcium ions(Ca2+) necessary to induce contraction of cardiac and skeletal muscle cells.In their intracellular environment,Ry R channels are regulated by a variety of cytosolic and luminal factors so that their output signal(Ca2+) induces finely-graded cell contraction without igniting cellular processes that may lead to aberrant electrical activity(ventricular arrhythmias) or cellular remodeling.The importance of Ry R dysfunction has been recently highlighted with the demonstration that point mutations in RYR2,the gene encoding for the cardiac isoform of the Ry R(Ry R2),are associated with catecholaminergic polymorphic ventricular tachycardia(CPVT),an arrhythmogenic syndrome characterized by the development of adrenergically-mediated ventricular tachycardia in individuals with an apparently normal heart.Here we summarize the state of the field in regards to the main arrhythmogenic mechanisms triggered by Ry R2 channels harboring mutations linked to CPVT.Most CPVT mutations characterized to date endow Ry R2 channels with a gain of function,resulting in hyperactive channels that release Ca2+ spontaneously,especially during diastole.The spontaneous Ca2+ release is extruded by the electrogenic Na+/Ca2+ exchanger,which depolarizes the external membrane(delayed afterdepolarization or DAD) and may trigger untimely action potentials.However,a rare set of CPVT mutations yield Ry R2 channels that are intrinsically hypo-active and hypo-responsive to stimuli,and it is unclear whether these channels release Ca2+ spontaneously during diastole.We discuss novel cellular mechanisms that appear more suitable to explain ventricular arrhythmias due to Ry R2 loss-of-function mutations.

Identification of the ryanodine receptor mutation I4743M and its contribution to diamide insecticide resistance in Spodoptera exigua (Lepidoptera: Noctuidae)

Insect ryanodine receptors(RyRs)are the targets of diamide insecticides.Two point mutations G4946E and I4790M(numbering according to Plutella xylostella,PxRyR)in the transmembrane domain of the insect RyRs associated with diamide resistance have so far been identified in three lepidopteran pests,P.xylostella,Tuta absoluta and Chilo suppressalis.In this study,we identified one of the known RyR target site resistance mutations(I4790M)in a field-collected population of Spodoptera exigua.The field-collected WF population of S.exigua exhibited 154 fold resistance to chlorantraniliprole when compared with the susceptible WH-S strain.Sequencing the transmembrane domains of S.exigua RyR(SeRyR)revealed that the resistant WF strain was homozygous for the 14743M mutation(corresponding to I4790M in PxRyR),whereas the G4900E allele(corresponding to G4946E of PxRyR)was not detected.The 4743M allele was introgressed into the susceptible WH-S strain by crossing WF with WH-S,followed by three rounds of backcrossing with WH-S.The introgressed strain 4743M was homozygous for the mutant 4743M allele and shared about 94%of its genetic background with that of the recipient WH-S strain.Compared with WH-S,the near-isogenic 4743M strain showed moderate levels of resistance to chlorantraniliprole(21 fold),cyantraniliprole(25 fold)and flubendiamide(22 fold),suggesting that the I4743M mutation confers medium levels of resistance to all three diamides.Genetic analysis showed diamide resistance in the 4743M strain was inherited as an autosomal and recessive trait.Results from this study have direct implications for the design of appropriate resistance monitoring and management practices to sustainably control S.exigua.

Cardiac ryanodine receptor gene(hRyR2) mutation underlying catecholaminergic polymorphic ventricular tachycardia in a Chinese adolescent presenting with sudden cardiac arrest and cardiac syncope

Sudden cardiac death （SCD） in children and adolescents is uncommon and yet it is devastating for both victim＇s family and the society. Recently, it was increasingly recognized that SCD in young patients with structurally normal heart may be caused by inheritable primary electrical diseases due to the malfunction of cardiac ion channels, a disease entity known as the ion channelopathies. Catecholaminergic polymorphic ventricular tachycardia （CPVT） is a specific form of ion channelopathy which can cause cardiac syncope or SCD in young patients by producing catecholamine-induced bi-directional ventricular tachycardia （BiVT）, polymorphic VT and ventricular fibrillation （VF） during physical exertion or emotion. We reported here an index case of CPVT caused by cardiac ryanodine receptor gene （hRyR2） mutation which presented as cardiac syncope and sudden cardiac arrest in a Chinese adolescent female.

Design, Synthesis, Biological Evaluation and SARs of Novel N-Substituted Sulfoximfnes as Potential Ryanodine Receptor Modulators

The sulfoximine group has been evaluated as a pharmacophore. Introducing the sulfoximine structure into medicinal compounds as exciting motifs has brought opportunities in drug discovery. In order to develop new ryanodine receptor （RyR） modulators, a series of phthalamides containing sulfoximine derivatives were designed, synthesized, and evaluated against oriental armyworm and diamondback moth for their insecticidal activities. These studies helped to elucidate the electronic and structural requirements around the sulfoximine motif for insecticidal activity. All new structures were synthesized and characterized by ^1H NMR, ^13C NMR, HRMS and bioassay and a preliminary structure-activity relationship （SAR） was discussed. The biological assessment indicated that most title compounds showed good to excellent larvicidal activities. Compounds la, le, and If gave excellent insecticidal activity against oriental armyworm, which showed 100% larvicidal activity at 0.5 mg/L. All compounds showed 100% larvicidal activity at 0.1 mg/L against diamondback moth. In particular, the larvicidal activities of le, If, and lh at 0.0001 mg/L were 50%, 20%, and 40%, respectively, reaching an activity as high as that of the commercial flubendiamide （40%, 0.0001mg/L）. Therefore, Ia, Ie, If and Ih could be considered as new lead structures for the development of new ryanodine receptor （RyR） modulators.

Dissociation of FK506 Binding Protein 12.6 from Ryanodine Receptor Type 2 Is Regulated by cADPR but not β-Adrenergic Stimulation in Mouse Cardiomyocytes

AIMS: β-adrenergic augmentation of Ca2+ sparks and cardiac contractility has been functionally linked to phosphorylation-dependent dissociation of FK506 binding protein 12.

Design,synthesis and insecticidal activities of novel anthranilic diamides containing polyfluoroalkyl pyrazole moiety

In order to discover new molecules with good insecticidal activities, a series of anthranilic diamides containing polyfluoroalkyl pyrazole were designed and synthesized, and their structures were characterized by1 H NMR and HRMS. Bioassays demonstrated that some of the title compound exhibited excellent insecticidal activities. The larvicidal activities of compound 8a, 8c, 8g, 8k and 8l against Mythimna separata Walker were 100% at 0.8 mg/L. The insecticidal activities of compound 8a, 8c,8e, 8g, 8k and 8l against Plutella xylostella Linnaeus were 100% at 0.4 mg/L. Surprisingly compounds 8a and 8c still showed 100% larvicidal activities against Plutella xylostella Linnaeus at 0.08 mg/L comparable to the commercialized Chlorantraniliprole. The LC_（50） of compound 8a and 8c against M. separata is 0.048 and 0.043 mg/L respectively.

Design, synthesis and insecticidal activities of novel 1-substituted-5-(trifluoromethyl)-1H-pyrazole-4-carboxamide derivatives

A series of novel 5-（trifluoromethyl）-1H-pyrazole-4-carboxamide derivatives（6a–6n, 7a, 7b, and 8a-8f）were synthesised by placing the amide bond at the 4-position of the pyrazole ring. These derivatives differed from the structure of chlorantraniliprole analogues with the amide bond at the 5-position of the pyrazole ring. Preliminary bioassay results revealed that a few title compounds exhibited good insecticidal activities against lepidopteran pests, such as Plutella xylostella, Mythimna separate, Heliothis armigera, and Ostrinia nubilalis. Some title compounds also elicited broad-spectrum insecticidal activities against dipterous insects including Culex pipiens pallens after altering the amide position. Similar to pyrazole-5-carboxamide analogues, compounds 6b and 6e showed 100% insecticidal activity against P. xylostella, C. pipiens pallens, and M. separate at concentrations of 200, 2, and 200 mg/m L, respectively.This finding suggested that 5-（trifluoromethyl）-1H-pyrazole-4-carboxamide derivatives are potential alternative insecticides for management of agriculture pests.

The effects of local anesthetics on intracellular Ca2+ release from ryanod ine-sensitive Ca2+ stores in gerbil hippocampal neurons

OBJECTIVE: To examine the effects of procaine and lidocaine on intracellular Ca(2+) release from sarcoplasmic reticulum ryanodine-sensitive Ca(2+) stores. METHODS: The experiment was performed on hippocampal slices from 60-80 g male Mongolian gerbils. Levels of intracellular Ca(2+) concentration in the slices were measured by microfluorometry. The slices were perfused with 50 mmol/L KCl containing medium for 30 seconds. Then, the medium was switched to physiological medium. After 5 min of incubation, the slice was perfused with 20 mmol/L caffeine containing physiology medium for 2 min. Following incubation, the slice was superfused with physiological medium until the end of the experiment. The effects of procaine and lidocanin (100 micro mol/L) on caffeine-evoked Ca(2+) release were evaluated by adding them to the medium after high K(+) medium perfusion. RESULTS: Caffeine induced a marked increase in intracellular Ca(2+) concentration which was then decreased 12% upon the addition of procaine (P

Design,Synthesis,Biological Evaluation and SARs of Anthranilic Diamide Derivatives Containing Pyrrole Moieties

In order to find a new variety of ryanodine receptor(RyR)regulator with greater biological activity,a se-ries of anthranilic diamide derivatives possessing pyrrole structure was designed and synthesized in this study.The pyrrole derivatives were evaluated for their insecticidal activity against Mythimna separata and Plutella xylostella.As indicated by the preliminary biological activities,compounds 12h-12j and 121-12n exhibited a remarkable in-secticidal activity against M.separata at 0.25 mg/L.Compared to control chlorantraniliprole.compound 12i exhibited more excellent insecticidal activity at 0.1 mg/L.Meanwhile,compounds 12c,12h,12i.12j.121,and 12m were selected to test the insecticidal activity against P xylostella,which led to the desirable insecticidal activity at 1x103 mg/L.Notably,compound 121 demonstrated 47%insecticidal activity at 5×10^(-6) mg/L over the control.In addition,the biological mechanism of action of compound 12i was investigated by means of insect clectrophysiology experiment.

Molecular bases of Sorcin-dependent resistance to chemotherapeutic agents

Soluble resistance-related calcium binding protein(Sorcin)is a protein initially labelled“resistance-related”,since it is co-amplified with ABCB1 in multidrug(MD)-resistant cells.While for years Sorcin overproduction was believed to be a by-product of the co-amplification of its gene with the P-glycoprotein gene,many recent studies view Sorcin as an oncoprotein,playing an important role in MD resistance(MDR).Sorcin is one of the most highly expressed calciumbinding proteins,which is overexpressed in many human tumors and MD resistant cancers,and represents a novel MDR marker.Sorcin expression in tumors inversely correlates with patients’response to chemotherapies and overall prognosis.Sorcin is highly expressed in MDR cell lines over their parent cells.Sorcin overexpression by gene transfection increases drug resistance to a variety of chemotherapeutic drugs in many cancer lines.On the other hand,Sorcin silencing leads to reversal of drug resistance in many cell lines.This review describes:(1)the roles of Sorcin in the cell;(2)the studies showing Sorcin overexpression in tumors and cancer cells;(3)the studies showing the effects of Sorcin overexpression and silencing;(4)the molecular effects of Sorcin overexpression;and(5)the structural and genetic bases of Sorcin-dependent MDR.

Distinctive characteristics and functions of multiple mitochondrial Ca^(2+) influx mechanisms

Intracellular Ca2+ is vital for cell physiology.Disruption of Ca2+ homeostasis contributes to human diseases such as heart failure,neuron-degeneration,and diabetes.To ensure an effective intracellular Ca2+ dynamics,various Ca2+ transport proteins localized in different cellular regions have to work in coordination.The central role of mitochondrial Ca2+ transport mechanisms in responding to physiological Ca2+ pulses in cytosol is to take up Ca2+ for regulating energy production and shaping the amplitude and duration of Ca2+ transients in various micro-domains.Since the discovery that isolated mitochondria can take up large quantities of Ca2+ approximately 5 decades ago,extensive studies have been focused on the functional characterization and implication of ion channels that dictate Ca2+ transport across the inner mitochondrial membrane.The mitochondrial Ca2+ uptake sensitive to non-specific inhibitors ruthenium red and Ru360 has long been considered as the activity of mitochondrial Ca2+ uniporter(MCU) .The general consensus is that MCU is dominantly or exclusively responsible for the mitochondrial Ca2+ influx.Since multiple Ca2+ influx mechanisms(e.g.L-,T-,and N-type Ca2+ channel) have their unique functions in the plasma membrane,it is plausible that mitochondrial inner membrane has more than just MCU to decode complex intracellular Ca2+ signaling in various cell types.During the last decade,four molecular identities related to mitochondrial Ca2+ influx mechanisms have been identified.These are mitochondrial ryanodine receptor,mitochondrial uncoupling proteins,LETM1(Ca2+ /H+ exchanger) ,and MCU and its Ca2+ sensing regulatory subunit MICU1.Here,we briefly review recent progress in these and other reported mitochondrial Ca2+ influx pathways and their differences in kinetics,Ca2+ dependence,and pharmacological characteristics.Their potential physiological and pathological implications are also discussed.