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1.
Proc Natl Acad Sci U S A ; 120(4): e2117503120, 2023 Jan 24.
Artigo em Inglês | MEDLINE | ID: mdl-36649401

RESUMO

Resting skeletal muscle generates heat for endothermy in mammals but not amphibians, while both use the same Ca2+-handling proteins and membrane structures to conduct excitation-contraction coupling apart from having different ryanodine receptor (RyR) isoforms for Ca2+ release. The sarcoplasmic reticulum (SR) generates heat following Adenosine triphosphate (ATP) hydrolysis at the Ca2+ pump, which is amplified by increasing RyR1 Ca2+ leak in mammals, subsequently increasing cytoplasmic [Ca2+] ([Ca2+]cyto). For thermogenesis to be functional, rising [Ca2+]cyto must not interfere with cytoplasmic effectors of the sympathetic nervous system (SNS) that likely increase RyR1 Ca2+ leak; nor should it compromise the muscle remaining relaxed. To achieve this, Ca2+ activated, regenerative Ca2+ release that is robust in lower vertebrates needs to be suppressed in mammals. However, it has not been clear whether: i) the RyR1 can be opened by local increases in [Ca2+]cyto; and ii) downstream effectors of the SNS increase RyR Ca2+ leak and subsequently, heat generation. By positioning amphibian and malignant hyperthermia-susceptible human-skinned muscle fibers perpendicularly, we induced abrupt rises in [Ca2+]cyto under identical conditions optimized for activating regenerative Ca2+ release as Ca2+ waves passed through the junction of fibers. Only mammalian fibers showed resistance to rising [Ca2+]cyto, resulting in increased SR Ca2+ load and leak. Fiber heat output was increased by cyclic adenosine monophosphate (cAMP)-induced RyR1 phosphorylation at Ser2844 and Ca2+ leak, indicating likely SNS regulation of thermogenesis. Thermogenesis occurred despite the absence of SR Ca2+ pump regulator sarcolipin. Thus, evolutionary isolation of RyR1 provided increased dynamic range for thermogenesis with sensitivity to cAMP, supporting endothermy.


Assuntos
Músculo Esquelético , Canal de Liberação de Cálcio do Receptor de Rianodina , Animais , Humanos , Cálcio/metabolismo , Músculo Esquelético/metabolismo , Isoformas de Proteínas/metabolismo , Rianodina/metabolismo , Canal de Liberação de Cálcio do Receptor de Rianodina/metabolismo , Retículo Sarcoplasmático/metabolismo , Termogênese , Anfíbios
2.
Biochem Biophys Res Commun ; 633: 96-103, 2022 12 10.
Artigo em Inglês | MEDLINE | ID: mdl-36344175

RESUMO

The hippocampus is a brain region implicated in synaptic plasticity and memory formation; both processes require neuronal Ca2+ signals generated by Ca2+ entry via plasma membrane Ca2+ channels and Ca2+ release from the endoplasmic reticulum (ER). Through Ca2+-induced Ca2+ release, the ER-resident ryanodine receptor (RyR) Ca2+ channels amplify and propagate Ca2+ entry signals, leading to activation of cytoplasmic and nuclear Ca2+-dependent signaling pathways required for synaptic plasticity and memory processes. Earlier reports have shown that mice and rat hippocampus expresses mainly the RyR2 isoform, with lower expression levels of the RyR3 isoform and almost undetectable levels of the RyR1 isoform; both the RyR2 and RyR3 isoforms have central roles in synaptic plasticity and hippocampal-dependent memory processes. Here, we describe that dendritic spines of rat primary hippocampal neurons express the RyR3 channel isoform, which is also expressed in the neuronal body and neurites. In contrast, the RyR2 isoform, which is widely expressed in the neuronal body and neurites of primary hippocampal neurons, is absent from the dendritic spines. We propose that this asymmetric distribution is of relevance for hippocampal neuronal function. We suggest that the RyR3 isoform amplifies activity-generated Ca2+ entry signals at postsynaptic dendritic spines, from where they propagate to the dendrite and activate primarily RyR2-mediated Ca2+ release, leading to Ca2+ signal propagation into the soma and the nucleus where they activate the expression of genes that mediate synaptic plasticity and memory.


Assuntos
Espinhas Dendríticas , Canal de Liberação de Cálcio do Receptor de Rianodina , Animais , Ratos , Cálcio/metabolismo , Espinhas Dendríticas/metabolismo , Retículo Endoplasmático/metabolismo , Hipocampo/metabolismo , Isoformas de Proteínas/genética , Isoformas de Proteínas/metabolismo , Rianodina/metabolismo , Canal de Liberação de Cálcio do Receptor de Rianodina/metabolismo
4.
J Agric Food Chem ; 70(39): 12651-12662, 2022 Oct 05.
Artigo em Inglês | MEDLINE | ID: mdl-36134897

RESUMO

To develop potent and environment-friendly insecticides, novel N-pyridylpyrazole amide derivatives containing a phthalimide were designed and synthesized. The preliminary bioassay results showed that most of the target compounds exhibited good insecticidal activities. For oriental armyworm (Mythimna separata), compounds E5, E29, E30, and E33 displayed higher than 90% lethal rates at 25 mg L-1. In particular, compound E33 displayed 60% mortality at a lower concentration of 6.25 mg L-1. Besides, compound E33 also showed a 30% lethal rate at 5 mg L-1 against diamondback moth (DBM) (Plutella xylostella). Molecular docking between the most active compound E33 and DBM ryanodine receptor (RyR), comparative molecular field analysis (CoMFA), and density functional theory (DFT) calculations were conducted and discussed. Furthermore, according to vitro studies using a calcium imaging technique, compound E33 was a potent novel lead targeting insect RyR.


Assuntos
Inseticidas , Mariposas , Amidas , Animais , Cálcio/metabolismo , Inseticidas/farmacologia , Larva/metabolismo , Simulação de Acoplamento Molecular , Mariposas/metabolismo , Ftalimidas , Rianodina , Canal de Liberação de Cálcio do Receptor de Rianodina/metabolismo , Relação Estrutura-Atividade
5.
Sci Rep ; 12(1): 16113, 2022 09 27.
Artigo em Inglês | MEDLINE | ID: mdl-36167878

RESUMO

Ca2+ signaling is altered substantially in many cancers. The ryanodine receptors (RYRs) are among the key ion channels in Ca2+ signaling. This study aimed to establish the mutational profile of RYR in cancers and investigate the correlation between RYR alterations and cancer phenotypes. The somatic mutation and clinical data of 11,000 cancer patients across 33 cancer types was downloaded from The Cancer Genome Atlas (TCGA) database. Subsequent data processing was performed with corresponding packages of the R software. Mutational profile was analyzed and its correlation with tumor mutational burden (TMB), patient prognosis, age and smoking status was analyzed and compared. All three RYR isoforms exhibited random mutational distribution without hotspot mutations when all cancers were analyzed together. The number of mutations in RYR2 (2388 mutations) far overweight that of RYR1 (1439 mutations) and RYR3 (1573 mutations). Linear correlation was observed between cumulative TMB and cumulative number of mutations for all RYR isoforms. Patients with RYR mutations exhibited significantly higher TMB than those without RYR mutations for most cancer types. Strong correlation was also revealed in the average number of mutations per person between pairs of RYR isoforms. No stratification of patient overall survival (OS) by mutational status was found for all three RYR isoforms when all cancers were analyzed together, however, significant stratification of OS by RYR mutations was revealed in several individual cancers, most strikingly in LUAD (P = 0.0067, RYR1), BLCA (P = 0.00071, RYR2), LUSC (P = 0.036, RYR2) and KIRC (P = 0.0042, RYR3). Furthermore, RYR mutations were correlated with higher age, higher smoking history grading and higher number of pack years. Characteristic mutation profile of RYRs in cancers has been revealed for the first time. RYR mutations were correlated with TMB, age, smoking status and capable of stratifying the prognosis of patients in several cancer types.


Assuntos
Neoplasias , Canal de Liberação de Cálcio do Receptor de Rianodina , Cálcio/metabolismo , Sinalização do Cálcio , Humanos , Mutação , Neoplasias/genética , Isoformas de Proteínas/metabolismo , Rianodina , Canal de Liberação de Cálcio do Receptor de Rianodina/genética , Canal de Liberação de Cálcio do Receptor de Rianodina/metabolismo
6.
J Gen Physiol ; 154(9)2022 09 05.
Artigo em Inglês | MEDLINE | ID: mdl-35713932

RESUMO

Flecainide, a cardiac class 1C blocker of the surface membrane sodium channel (NaV1.5), has also been reported to reduce cardiac ryanodine receptor (RyR2)-mediated sarcoplasmic reticulum (SR) Ca2+ release. It has been introduced as a clinical antiarrhythmic agent for catecholaminergic polymorphic ventricular tachycardia (CPVT), a condition most commonly associated with gain-of-function RyR2 mutations. Current debate concerns both cellular mechanisms of its antiarrhythmic action and molecular mechanisms of its RyR2 actions. At the cellular level, it targets NaV1.5, RyR2, Na+/Ca2+ exchange (NCX), and additional proteins involved in excitation-contraction (EC) coupling and potentially contribute to the CPVT phenotype. This Viewpoint primarily addresses the various direct molecular actions of flecainide on isolated RyR2 channels in artificial lipid bilayers. Such studies demonstrate different, multifarious, flecainide binding sites on RyR2, with voltage-dependent binding in the channel pore or voltage-independent binding at distant peripheral sites. In contrast to its single NaV1.5 pore binding site, flecainide may bind to at least four separate inhibitory sites on RyR2 and one activation site. None of these binding sites have been specifically located in the linear RyR2 sequence or high-resolution structure. Furthermore, it is not clear which of the inhibitory sites contribute to flecainide's reduction of spontaneous Ca2+ release in cellular studies. A confounding observation is that flecainide binding to voltage-dependent inhibition sites reduces cation fluxes in a direction opposite to physiological Ca2+ flow from SR lumen to cytosol. This may suggest that, rather than directly blocking Ca2+ efflux, flecainide can reduce Ca2+ efflux by blocking counter currents through the pore which otherwise limit SR membrane potential change during systolic Ca2+ efflux. In summary, the antiarrhythmic effects of flecainide in CPVT seem to involve multiple components of EC coupling and multiple actions on RyR2. Their clarification may identify novel specific drug targets and facilitate flecainide's clinical utilization in CPVT.


Assuntos
Flecainida , Taquicardia Ventricular , Antiarrítmicos/farmacologia , Cálcio/metabolismo , Flecainida/metabolismo , Flecainida/farmacologia , Humanos , Miócitos Cardíacos/metabolismo , Rianodina/metabolismo , Rianodina/farmacologia , Canal de Liberação de Cálcio do Receptor de Rianodina/metabolismo , Sódio/metabolismo , Taquicardia Ventricular/genética , Taquicardia Ventricular/metabolismo
7.
Br J Pharmacol ; 179(21): 4941-4957, 2022 11.
Artigo em Inglês | MEDLINE | ID: mdl-35703154

RESUMO

BACKGROUND AND PURPOSE: Statins, inhibitors of HMG-CoA reductase, are mainstay treatment for hypercholesterolaemia. However, muscle pain and weakness prevent many patients from benefiting from their cardioprotective effects. We previously demonstrated that simvastatin activates skeletal ryanodine receptors (RyR1), an effect that could be important in initiating myopathy. Using a range of structurally diverse statin analogues, we examined structural features associated with RyR1 activation, aiming to identify statins lacking this property. EXPERIMENTAL APPROACH: Compounds were screened for RyR1 activity utilising [3 H]ryanodine binding. Mechanistic insight into RyR1 activity was studied by incorporating RyR1 channels from sheep, mouse or rabbit skeletal muscle into bilayers. KEY RESULTS: All UK-prescribed statins activated RyR1 at nanomolar concentrations. Cerivastatin, withdrawn from the market due to life-threatening muscle-related side effects, was more effective than currently-prescribed statins and possessed the unique ability to open RyR1 channels independently of cytosolic Ca2+ . We synthesised the one essential structural moiety that all statins must possess for HMG-CoA reductase inhibition, the R-3,5-dihydroxypentanoic acid unit, and it did not activate RyR1. We also identified five analogues retaining potent HMG-CoA reductase inhibition that inhibited RyR1 and four that lacked the ability to modulate RyR1. CONCLUSION AND IMPLICATIONS: That cerivastatin activates RyR1 most strongly supports the hypothesis that RyR1 activation is implicated in statin-induced myopathy. Demonstrating that statin regulation of RyR1 and HMG-CoA reductase are separable effects will allow the role of RyR1 in statin-induced myopathy to be further elucidated by the tool compounds we have identified, allowing development of effective cardioprotective statins with improved patient tolerance.


Assuntos
Inibidores de Hidroximetilglutaril-CoA Redutases , Doenças Musculares , Acil Coenzima A , Animais , Inibidores de Hidroximetilglutaril-CoA Redutases/farmacologia , Camundongos , Músculo Esquelético , Doenças Musculares/induzido quimicamente , Doenças Musculares/tratamento farmacológico , Coelhos , Rianodina/farmacologia , Canal de Liberação de Cálcio do Receptor de Rianodina , Ovinos , Sinvastatina/farmacologia
8.
J Cardiovasc Pharmacol ; 79(5): 749-757, 2022 05 01.
Artigo em Inglês | MEDLINE | ID: mdl-35239284

RESUMO

ABSTRACT: Propofol, a general anesthetic administered intravenously, may cause pain at the injection site. The pain is in part due to irritation of vascular endothelial cells. We here investigated the effects of propofol on Ca2+ transport and pain mediator release in human umbilical vein endothelial cells (EA.hy926). Propofol mobilized Ca2+ from cyclopiazonic acid (CPA)-dischargeable pool but did not cause Ca2+ release from the lysosomal Ca2+ stores. Propofol-elicited Ca2+ release was suppressed by 100 µM ryanodine, suggesting the participation of ryanodine receptor channels. Propofol did not affect ATP-triggered Ca2+ release but abolished the Ca2+ influx triggered by ATP; in addition, propofol also suppressed store-operated Ca2+ entry elicited by CPA. Ca2+ clearance during CPA-induced Ca2+ discharge was unaffected by a low Na+ (50 mM) extracellular solution, but strongly suppressed by 5 mM La3+ (an inhibitor of plasmalemmal Ca2+ pump), suggesting Ca2+ extrusion was predominantly through the plasmalemmal Ca2+ pump. Propofol mimicked the effect of La3+ in suppressing Ca2+ clearance. Propofol also stimulated release of pain mediators, namely, reactive oxygen species and bradykinin. Our data suggest propofol elicited Ca2+ release and repressed Ca2+ clearance, causing a sustained cytosolic [Ca2+]i elevation. The latter may cause reactive oxygen species and bradykinin release, resulting in pain.


Assuntos
Propofol , Canal de Liberação de Cálcio do Receptor de Rianodina , Trifosfato de Adenosina , Bradicinina/farmacologia , Cálcio/metabolismo , Células Endoteliais/metabolismo , Humanos , Dor , Propofol/farmacologia , Espécies Reativas de Oxigênio , Rianodina/farmacologia
9.
Semin Thorac Cardiovasc Surg ; 34(1): 123-132, 2022.
Artigo em Inglês | MEDLINE | ID: mdl-33460764

RESUMO

The impairment of intracellular calcium homeostasis plays an essential role during ischemia-reperfusion injury. Calcium release from sarcoplasmic reticulum which is triggered by myocardial ischemia is mainly mediated by ryanodine receptors. Dantrolene sodium is a ryanodine receptor antagonist. The objective of the present study was to evaluate the in-vivo impact of dantrolene sodium on myocardial ischemia-reperfusion injury in swine models. An in vivo, experimental trial comparing 10 experimental animals which received dantrolene sodium with 9 control swine models was conducted. Their left anterior descending coronary artery was temporarily occluded for 75 minutes via a vessel tourniquet, which was then released. Myocardial reperfusion was allowed for 24 hours. Dantrolene was administered at the onset of the reperfusion period and levels of troponin, creatine phosphokinase and creatine kinase myocardial band between the two groups were compared. Additionally, various other hemodynamic parameters and left ventricular morphology and function were examined. There were significantly lower values of troponin, creatine phosphokinase and creatine kinase myocardial band in the dantrolene group indicating less ischemia-reperfusion injury. Moreover, the postischemic cardiac index was also greater in the dantrolene group, whereas viable myocardium was also better preserved. In conclusion, the in vivo cardioprotective role of dantrolene sodium against ischemia-reperfusion injury in swine models was indicated in this study. Therefore, dantrolene sodium administration could be a promising treatment against ischemia-reperfusion injury in humans. However, large randomized clinical studies should be firstly carried out to prove this hypothesis.


Assuntos
Isquemia Miocárdica , Traumatismo por Reperfusão Miocárdica , Animais , Cálcio/metabolismo , Creatina Quinase/metabolismo , Dantroleno/farmacologia , Dantroleno/uso terapêutico , Homeostase , Traumatismo por Reperfusão Miocárdica/prevenção & controle , Miocárdio/metabolismo , Rianodina , Canal de Liberação de Cálcio do Receptor de Rianodina , Suínos , Resultado do Tratamento , Troponina
10.
J Neurotrauma ; 39(3-4): 311-319, 2022 02.
Artigo em Inglês | MEDLINE | ID: mdl-34913747

RESUMO

Ryanodine receptors (RyRs) mediate calcium release from calcium stores and have been implicated in axonal degeneration. Here, we use an intravital imaging approach to determine axonal fate after spinal cord injury (SCI) in real-time and assess the efficacy of ryanodine receptor inhibition as a potential therapeutic approach to prevent intra-axonal calcium-mediated axonal degeneration. Adult 6-8 week old Thy1YFP transgenic mice that express YFP in axons, as well as triple transgenic Avil-Cre:Ai9:Ai95 mice that express the genetically-encoded calcium indicator GCaMP6f in tdTomato positive axons, were used to visualize axons and calcium changes in axons, respectively. Mice received a mild SCI at the T12 level of the spinal cord. Ryanodine, a RyR antagonist, was given at a concentration of 50 µM intrathecally within 15 min of SCI or delayed 3 h after injury and compared with vehicle-treated mice. RyR inhibition within 15 min of SCI significantly reduced axonal spheroid formation from 1 h to 24 h after SCI and increased axonal survival compared with vehicle controls. Delayed ryanodine treatment increased axonal survival and reduced intra-axonal calcium levels at 24 h after SCI but had no effect on axonal spheroid formation. Together, our results support a role for RyR in secondary axonal degeneration.


Assuntos
Axônios/patologia , Cálcio/metabolismo , Canal de Liberação de Cálcio do Receptor de Rianodina , Rianodina/antagonistas & inibidores , Traumatismos da Medula Espinal , Medula Espinal/efeitos dos fármacos , Animais , Modelos Animais de Doenças , Microscopia Intravital , Camundongos , Camundongos Transgênicos , Traumatismos da Medula Espinal/complicações , Traumatismos da Medula Espinal/fisiopatologia
12.
Nat Commun ; 12(1): 6815, 2021 11 24.
Artigo em Inglês | MEDLINE | ID: mdl-34819493

RESUMO

Bistable motoneurons of the spinal cord exhibit warmth-activated plateau potential driven by Na+ and triggered by a brief excitation. The thermoregulating molecular mechanisms of bistability and their role in motor functions remain unknown. Here, we identify thermosensitive Na+-permeable Trpm5 channels as the main molecular players for bistability in mouse motoneurons. Pharmacological, genetic or computational inhibition of Trpm5 occlude bistable-related properties (slow afterdepolarization, windup, plateau potentials) and reduce spinal locomotor outputs while central pattern generators for locomotion operate normally. At cellular level, Trpm5 is activated by a ryanodine-mediated Ca2+ release and turned off by Ca2+ reuptake through the sarco/endoplasmic reticulum Ca2+-ATPase (SERCA) pump. Mice in which Trpm5 is genetically silenced in most lumbar motoneurons develop hindlimb paresis and show difficulties in executing high-demanding locomotor tasks. Overall, by encoding bistability in motoneurons, Trpm5 appears indispensable for producing a postural tone in hindlimbs and amplifying the locomotor output.


Assuntos
Locomoção/fisiologia , Neurônios Motores/metabolismo , Paresia/fisiopatologia , Medula Espinal/fisiologia , Canais de Cátion TRPM/metabolismo , Potenciais de Ação/efeitos dos fármacos , Potenciais de Ação/fisiologia , Animais , Animais Recém-Nascidos , Simulação por Computador , Modelos Animais de Doenças , Feminino , Inativação Gênica , Células HEK293 , Membro Posterior/fisiologia , Humanos , Locomoção/efeitos dos fármacos , Masculino , Camundongos , Neurônios Motores/efeitos dos fármacos , Paresia/genética , Técnicas de Patch-Clamp , Proteínas Recombinantes/genética , Proteínas Recombinantes/metabolismo , Rianodina/metabolismo , ATPases Transportadoras de Cálcio do Retículo Sarcoplasmático/antagonistas & inibidores , ATPases Transportadoras de Cálcio do Retículo Sarcoplasmático/metabolismo , Medula Espinal/citologia , Canais de Cátion TRPM/antagonistas & inibidores , Canais de Cátion TRPM/genética
13.
Fish Physiol Biochem ; 47(6): 1969-1982, 2021 Dec.
Artigo em Inglês | MEDLINE | ID: mdl-34668117

RESUMO

This study investigated the dependence of contraction from extracellular Ca2+, the presence of a functional sarcoplasmic reticulum (SR), and the effects of ß-adrenergic stimulation using isometric cardiac muscle preparations. Moreover, the expression of Ca2+-handling proteins such as SR-Ca2+-ATPase (SERCA), phospholamban (PLN), and Na+/Ca2+ exchanger (NCX) were also evaluated in the ventricular tissue of adult African sharptooth catfish, Clarias gariepinus, a facultative air-breathing fish. In summary, we observed that (1) contractility was strongly regulated by extracellular Ca2+; (2) inhibition of SR Ca2+-release by application of ryanodine reduced steady-state force production; (3) ventricular myocardium exhibited clear post-rest decay, even in the presence of ryanodine, indicating a decrease in SR Ca2+ content and NCX as the main pathway for Ca2+ extrusion; (4) a positive force-frequency relationship was observed above 60 bpm (1.0 Hz); (5) ventricular tissue was responsive to ß-adrenergic stimulation, which caused significant increases in twitch force, kept a linear force-frequency relationship from 12 to 96 bpm (0.2 to Hz), and improved the cardiac pumping capacity (CPC); and (6) African catfish myocardium exhibited similar expression patterns of NCX, SERCA, and PLN, corroborating our findings that both mechanisms for Ca2+ transport across the SR and sarcolemma contribute to Ca2+ activator. In conclusion, this fish species displays great physiological plasticity of E-C coupling, able to improve the ability to maintain cardiac performance under physiological conditions to ecological and/or adverse environmental conditions, such as hypoxic air-breathing activity.


Assuntos
Adrenérgicos/farmacologia , Cálcio , Peixes-Gato , Contração Miocárdica , Retículo Sarcoplasmático , Animais , Cálcio/metabolismo , Peixes-Gato/metabolismo , Rianodina , Retículo Sarcoplasmático/metabolismo , ATPases Transportadoras de Cálcio do Retículo Sarcoplasmático/metabolismo , Trocador de Sódio e Cálcio
14.
Pestic Biochem Physiol ; 178: 104921, 2021 Oct.
Artigo em Inglês | MEDLINE | ID: mdl-34446197

RESUMO

Ryanodine receptors (RyRs) are the targets of diamide insecticides, which have been identified and characterized in a dozen insect pests of Lepidoptera, Hemiptera, Diptera and Coleoptera, but limited attention has been paid to the RyR in parasitoid natural enemies. Without this knowledge, it will hinder our effective and efficient application using both parasitoid natural enemies and diamide insecticides simultaneously in the integrated pest management (IPM). In this study, the full-length cDNA of RyR was cloned from Encarsia formosa (EfRyR), a parasitic wasp used worldwide for the biological control of whitefly. Its expression profile was examined in various tissues of E. formosa adults. The toxicities of four diamide insecticides to E. formosa were measured, and then the expression profile of EfRyR after 12 h and 24 h exposure to the LC50 dosages of diamide insecticides was investigated. The results showed that the full-length cDNA of EfRyR was 16, 778 bp including a 15, 345 bp open reading frame, and two alternative splice (AS) sites. Comparing to its expression in the abdomen, EfRyR was highly expressed in the head (11.9-fold) and the thorax (3.7-fold). The toxicities of four dimide insecticides against E. formosa from low to high were chlorantraniliprole (LC50 = 367.84 mg L-1), cyantraniliprole (221.72 mg L-1), cyclaniliprole (51.77 mg L-1), and tetrachlorantraniliprole (8.35 mg L-1). The expressions of EfRyR and its variants with AS were significantly increased after E. formosa adults were exposed to different diamide insecticides. This study improves our understanding of the RyR in parasitoid wasps and provides useful information on IPM by using E. formosa.


Assuntos
Diamida , Inseticidas , Animais , Diamida/toxicidade , Inseticidas/toxicidade , Rianodina , Canal de Liberação de Cálcio do Receptor de Rianodina/genética , Taiwan
15.
J Physiol ; 599(19): 4415-4426, 2021 10.
Artigo em Inglês | MEDLINE | ID: mdl-34411300

RESUMO

Ryanodine receptors (RyRs) are intracellular Ca2+ release channels ubiquitously expressed in various cell types. RyRs were extensively studied in striated muscle cells due to their crucial role in muscle contraction. In contrast, the role of RyRs in Ca2+ signalling and functions in non-excitable cells, such as T lymphocytes, remains poorly understood. Expression of different isoforms of RyRs was shown in primary T cells and T cell lines. In T cells, RyRs co-localize with the plasmalemmal store-operated Ca2+ channels of the Orai family and endoplasmic reticulum Ca2+ sensing Stim family proteins and are activated by store-operated Ca2+ entry and pyridine nucleotide metabolites, the intracellular second messengers generated upon stimulation of T cell receptors. Experimental data indicate that together with d-myo-inositol 1,4,5-trisphosphate receptors, RyRs regulate intercellular Ca2+ dynamics by controlling Ca2+ concentration within the lumen of the endoplasmic reticulum and, consequently, store-operated Ca2+ entry. Gain-of-function mutations, genetic deletion or pharmacological inhibition of RyRs alters T cell Ca2+ signalling and effector functions. The picture emerging from the collective data shows that RyRs are the essential regulators of T cell Ca2+ signalling and can be potentially used as molecular targets for immunomodulation or T cell-based diagnostics of the disorders associated with RyRs dysregulation.


Assuntos
Cálcio , Canal de Liberação de Cálcio do Receptor de Rianodina , Cálcio/metabolismo , Sinalização do Cálcio , Receptores de Inositol 1,4,5-Trifosfato/metabolismo , Receptores de Antígenos de Linfócitos T , Rianodina , Canal de Liberação de Cálcio do Receptor de Rianodina/metabolismo , Linfócitos T
16.
J Biol Chem ; 297(1): 100808, 2021 07.
Artigo em Inglês | MEDLINE | ID: mdl-34022226

RESUMO

Ryanodine receptors (RyRs) are ion channels that mediate the release of Ca2+ from the sarcoplasmic reticulum/endoplasmic reticulum, mutations of which are implicated in a number of human diseases. The adjacent C-terminal domains (CTDs) of cardiac RyR (RyR2) interact with each other to form a ring-like tetrameric structure with the intersubunit interface undergoing dynamic changes during channel gating. This mobile CTD intersubunit interface harbors many disease-associated mutations. However, the mechanisms of action of these mutations and the role of CTD in channel function are not well understood. Here, we assessed the impact of CTD disease-associated mutations P4902S, P4902L, E4950K, and G4955E on Ca2+- and caffeine-mediated activation of RyR2. The G4955E mutation dramatically increased both the Ca2+-independent basal activity and Ca2+-dependent activation of [3H]ryanodine binding to RyR2. The P4902S and E4950K mutations also increased Ca2+ activation but had no effect on the basal activity of RyR2. All four disease mutations increased caffeine-mediated activation of RyR2 and reduced the threshold for activation and termination of spontaneous Ca2+ release. G4955D dramatically increased the basal activity of RyR2, whereas G4955K mutation markedly suppressed channel activity. Similarly, substitution of P4902 with a negatively charged residue (P4902D), but not a positively charged residue (P4902K), also dramatically increased the basal activity of RyR2. These data suggest that electrostatic interactions are involved in stabilizing the CTD intersubunit interface and that the G4955E disease mutation disrupts this interface, and thus the stability of the closed state. Our studies shed new insights into the mechanisms of action of RyR2 CTD disease mutations.


Assuntos
Ativação do Canal Iônico , Mutação/genética , Canal de Liberação de Cálcio do Receptor de Rianodina/química , Canal de Liberação de Cálcio do Receptor de Rianodina/genética , Animais , Cafeína/farmacologia , Cálcio/metabolismo , Análise Mutacional de DNA , Células HEK293 , Humanos , Ativação do Canal Iônico/efeitos dos fármacos , Camundongos , Ligação Proteica/efeitos dos fármacos , Domínios Proteicos , Subunidades Proteicas/química , Subunidades Proteicas/metabolismo , Rianodina/metabolismo , Trítio/metabolismo
17.
Pestic Biochem Physiol ; 174: 104798, 2021 May.
Artigo em Inglês | MEDLINE | ID: mdl-33838722

RESUMO

The ryanodine receptor (RyR) is an intracellular calcium channel critical to the regulation of insect muscle contraction and the target site of diamide insecticides such as chlorantraniliprole, cyantraniliprole and flubendiamide. To-date, diamides are the only known class of synthetic molecules with high potency against insect RyRs. Target-based screening of an informer library led to discovery of a novel class of RyR activators, pyrrole-2-carboxamides. Efforts to optimize receptor activity resulted in analogs with potency comparable to that of commercial diamides when tested against RyR of the fruit fly, Drosophila melanogaster. Surprisingly, testing of pyrrole-2-carboxamides in whole-insect screens showed poor insecticidal activity, which is partially attributed to differential selectivity among insect receptors and rapid detoxification. Among various lepidopteran species field resistance to diamide insecticides has been well documented and in many cases has been attributed to a single point mutation, G4946E, of the RyR gene. As with diamide insecticides, the G4946E mutation confers greatly reduced sensitivity to pyrrole-2-carboxamides. This, coupled with findings from radioligand binding studies, indicates a shared binding domain between anthranilic diamides and pyrrole-2-carboxamides.


Assuntos
Inseticidas , Mariposas , Animais , Drosophila melanogaster/metabolismo , Proteínas de Insetos/genética , Proteínas de Insetos/metabolismo , Resistência a Inseticidas , Inseticidas/toxicidade , Mariposas/metabolismo , Pirróis/toxicidade , Rianodina , Canal de Liberação de Cálcio do Receptor de Rianodina/genética , ortoaminobenzoatos/toxicidade
18.
Biosci Rep ; 41(4)2021 04 30.
Artigo em Inglês | MEDLINE | ID: mdl-33825858

RESUMO

Mutations in cardiac ryanodine receptor (RyR2) are linked to catecholaminergic polymorphic ventricular tachycardia (CPVT). Most CPVT RyR2 mutations characterized are gain-of-function (GOF), indicating enhanced RyR2 function as a major cause of CPVT. Loss-of-function (LOF) RyR2 mutations have also been identified and are linked to a distinct entity of cardiac arrhythmia termed RyR2 Ca2+ release deficiency syndrome (CRDS). Exercise stress testing (EST) is routinely used to diagnose CPVT, but it is ineffective for CRDS. There is currently no effective diagnostic tool for CRDS in humans. An alternative strategy to assess the risk for CRDS is to directly determine the functional impact of the associated RyR2 mutations. To this end, we have functionally screened 18 RyR2 mutations that are associated with idiopathic ventricular fibrillation (IVF) or sudden death. We found two additional RyR2 LOF mutations E4146K and G4935R. The E4146K mutation markedly suppressed caffeine activation of RyR2 and abolished store overload induced Ca2+ release (SOICR) in human embryonic kidney 293 (HEK293) cells. E4146K also severely reduced cytosolic Ca2+ activation and abolished luminal Ca2+ activation of single RyR2 channels. The G4935R mutation completely abolished caffeine activation of and [3H]ryanodine binding to RyR2. Co-expression studies showed that the G4935R mutation exerted dominant negative impact on the RyR2 wildtype (WT) channel. Interestingly, the RyR2-G4935R mutant carrier had a negative EST, and the E4146K carrier had a family history of sudden death during sleep, which are different from phenotypes of typical CPVT. Thus, our data further support the link between RyR2 LOF and a new entity of cardiac arrhythmias distinct from CPVT.


Assuntos
Morte Súbita Cardíaca/etiologia , Mutação com Perda de Função , Canal de Liberação de Cálcio do Receptor de Rianodina/genética , Fibrilação Ventricular/genética , Cálcio/metabolismo , Células HEK293 , Humanos , Rianodina/metabolismo , Canal de Liberação de Cálcio do Receptor de Rianodina/metabolismo , Fibrilação Ventricular/metabolismo , Fibrilação Ventricular/patologia
19.
J Neurosci ; 41(17): 3764-3776, 2021 04 28.
Artigo em Inglês | MEDLINE | ID: mdl-33731449

RESUMO

The axon initial segment (AIS) is a specialized neuronal compartment in which synaptic input is converted into action potential (AP) output. This process is supported by a diverse complement of sodium, potassium, and calcium channels (CaV). Different classes of sodium and potassium channels are scaffolded at specific sites within the AIS, conferring unique functions, but how calcium channels are functionally distributed within the AIS is unclear. Here, we use conventional two-photon laser scanning and diffraction-limited, high-speed spot two-photon imaging to resolve AP-evoked calcium dynamics in the AIS with high spatiotemporal resolution. In mouse layer 5 prefrontal pyramidal neurons, calcium influx was mediated by a mix of CaV2 and CaV3 channels that differentially localized to discrete regions. CaV3 functionally localized to produce nanodomain hotspots of calcium influx that coupled to ryanodine-sensitive stores, whereas CaV2 localized to non-hotspot regions. Thus, different pools of CaVs appear to play distinct roles in AIS function.SIGNIFICANCE STATEMENT The axon initial segment (AIS) is the site where synaptic input is transformed into action potential (AP) output. It achieves this function through a diverse complement of sodium, potassium, and calcium channels (CaV). While the localization and function of sodium channels and potassium channels at the AIS is well described, less is known about the functional distribution of CaVs. We used high-speed two-photon imaging to understand activity-dependent calcium dynamics in the AIS of mouse neocortical pyramidal neurons. Surprisingly, we found that calcium influx occurred in two distinct domains: CaV3 generates hotspot regions of calcium influx coupled to calcium stores, whereas CaV2 channels underlie diffuse calcium influx between hotspots. Therefore, different CaV classes localize to distinct AIS subdomains, possibly regulating distinct cellular processes.


Assuntos
Segmento Inicial do Axônio/fisiologia , Segmento Inicial do Axônio/ultraestrutura , Canais de Cálcio/fisiologia , Sinalização do Cálcio/fisiologia , Potenciais de Ação/fisiologia , Animais , Axônios , Caveolina 2/efeitos dos fármacos , Caveolina 2/fisiologia , Caveolina 3/efeitos dos fármacos , Caveolina 3/fisiologia , Feminino , Masculino , Camundongos , Camundongos Endogâmicos C57BL , Microscopia Confocal , Rianodina/farmacologia , Canal de Liberação de Cálcio do Receptor de Rianodina/efeitos dos fármacos
20.
Adv Exp Med Biol ; 1303: 305-317, 2021.
Artigo em Inglês | MEDLINE | ID: mdl-33788199

RESUMO

Ca2+/calmodulin-dependent protein kinase II (CaMKII) is a multifunctional protein kinase and has been recently recognized to play a vital role in pathological events in the pulmonary system. CaMKII has diverse downstream targets that promote vascular disease, asthma, and cancer, so improved understanding of CaMKII signaling has the potential to lead to new therapies for lung diseases. Multiple studies have demonstrated that CaMKII is involved in redox modulation of ryanodine receptors (RyRs). CaMKII can be directly activated by reactive oxygen species (ROS) which then regulates RyR activity, which is essential for Ca2+-dependent processes in lung diseases. Furthermore, both CaMKII and RyRs participate in the inflammation process. However, their role in the pulmonary physiology in response to ROS is still an ambiguous one. Because CaMKII and RyRs are important in pulmonary biology, cell survival, cell cycle control, and inflammation, it is possible that the relationship between ROS and CaMKII/RyRs signal complex will be necessary for understanding and treating lung diseases. Here, we review roles of CaMKII/RyRs in lung diseases to understand with how CaMKII/RyRs may act as a transduction signal to connect prooxidant conditions into specific downstream pathological effects that are relevant to rare and common forms of pulmonary disease.


Assuntos
Pneumopatias , Canal de Liberação de Cálcio do Receptor de Rianodina , Cálcio/metabolismo , Proteína Quinase Tipo 2 Dependente de Cálcio-Calmodulina/genética , Proteína Quinase Tipo 2 Dependente de Cálcio-Calmodulina/metabolismo , Humanos , Inflamação , Rianodina , Canal de Liberação de Cálcio do Receptor de Rianodina/genética
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