RESUMEN
Intracellular Ca2+ leak from cardiac ryanodine receptor (RyR2) is an established mechanism of sudden cardiac death (SCD), whereby dysregulated Ca2+ handling causes ventricular arrhythmias. We previously discovered the RyR2-selective inhibitor ent-(+)-verticilide (ent-1), a 24-membered cyclooligomeric depsipeptide that is the enantiomeric form of a natural product (nat-(-)-verticilide). Here, we examined its 18-membered ring-size oligomer (ent-verticilide B1; "ent-B1") in RyR2 single channel and [3H]ryanodine binding assays, and in Casq2 -/- cardiomyocytes and mice, a gene-targeted model of SCD. ent-B1 inhibited RyR2 single channels and RyR2-mediated spontaneous Ca2+ release in Casq2 -/- cardiomyocytes with sub-micromolar potency. ent-B1 was a partial RyR2 inhibitor, with maximal inhibitory efficacy of less than 50%. ent-B1 was stable in plasma, with a peak plasma concentration of 1460 ng/ml at 10 minutes and half-life of 45 minutes after intraperitoneal administration of 3 mg/kg in mice. In vivo, ent-B1 significantly reduced catecholamine-induced ventricular arrhythmias in Casq2 -/- mice in a dose-dependent manner. Hence, we have identified a novel chemical entity - ent-B1 - that preserves the mechanism of action of a hit compound and shows therapeutic efficacy. These findings strengthen RyR2 as an antiarrhythmic drug target and highlight the potential of investigating the mirror-image isomers of natural products to discover new therapeutics. SIGNIFICANCE STATEMENT: The cardiac ryanodine receptor (RyR2) is an untapped target in the stagnant field of antiarrhythmic drug development. We have confirmed RyR2 as an antiarrhythmic target in a mouse model of sudden cardiac death and shown the therapeutic efficacy of a second enantiomeric natural product.
Asunto(s)
Productos Biológicos , Depsipéptidos , Ratones , Animales , Antiarrítmicos/farmacología , Antiarrítmicos/uso terapéutico , Canal Liberador de Calcio Receptor de Rianodina/genética , Canal Liberador de Calcio Receptor de Rianodina/metabolismo , Arritmias Cardíacas/tratamiento farmacológico , Arritmias Cardíacas/metabolismo , Depsipéptidos/metabolismo , Depsipéptidos/uso terapéutico , Muerte Súbita Cardíaca/etiología , Miocitos Cardíacos/metabolismo , Calcio/metabolismoRESUMEN
Ca 2+ leak from cardiac ryanodine receptor (RyR2) is an established mechanism of sudden cardiac death (SCD), whereby dysregulated Ca 2+ handling causes ventricular arrhythmias. We previously discovered the RyR2-selective inhibitor ent- (+)-verticilide ( ent -1), a 24-membered cyclooligomeric depsipeptide that is the enantiomeric form of a natural product ( nat -(-)-verticilide). Here, we examined its 18-membered ring-size oligomer ( ent -verticilide B1; " ent -B1") in single RyR2 channel assays, [ 3 H]ryanodine binding assays, and in Casq2 -/- cardiomyocytes and mice, a gene-targeted model of SCD. ent -B1 inhibited RyR2 single-channels and [ 3 H]ryanodine binding with low micromolar potency, and RyR2-mediated spontaneous Ca 2+ release in Casq2-/- cardiomyocytes with sub-micromolar potency. ent -B1 was a partial RyR2 inhibitor, with maximal inhibitory efficacy of less than 50%. ent -B1 was stable in plasma, with a peak plasma concentration of 1460 ng/ml at 10 min and half-life of 45 min after intraperitoneal administration of 3 mg/kg in mice. Both 3 mg/kg and 30 mg/kg ent -B1 significantly reduced catecholamine-induced ventricular arrhythmia in Casq2-/- mice. Hence, we have identified a novel chemical entity - ent -B1 - that preserves the mechanism of action of a hit compound and shows therapeutic efficacy. These findings strengthen RyR2 as an antiarrhythmic drug target and highlight the potential of investigating the mirror-image isomers of natural products to discover new therapeutics. Significance statement: The cardiac ryanodine receptor (RyR2) is an untapped target in the stagnant field of antiarrhythmic drug development. We have confirmed RyR2 as an antiarrhythmic target in a mouse model of sudden cardiac death and shown the therapeutic efficacy of a second enantiomeric natural product.
RESUMEN
Dantrolene is a neutral hydantoin that is clinically used as a skeletal muscle relaxant to prevent overactivation of the skeletal muscle calcium release channel (RyR1) in response to volatile anesthetics. Dantrolene has aroused considerable recent interest as a lead compound for stabilizing calcium release due to overactive cardiac calcium release channels (RyR2) in heart failure. Previously, we found that dantrolene produces up to a 45% inhibition RyR2 with an IC50 of 160 nM, and that this inhibition requires the physiological association between RyR2 and CaM. In this study, we tested the hypothesis that dantrolene inhibition of RyR2 in the presence of CaM is modulated by RyR2 phosphorylation at S2808 and S2814. Phosphorylation was altered by incubations with either exogenous phosphatase (PP1) or kinases; PKA to phosphorylate S2808 or endogenous CaMKII to phosphorylate S2814. We found that PKA caused selective dissociation of FKBP12.6 from the RyR2 complex and a loss of dantrolene inhibition. Rapamycin-induced FKBP12.6 dissociation from RyR2 also resulted in the loss of dantrolene inhibition. Subsequent incubations of RyR2 with exogenous FKBP12.6 reinstated dantrolene inhibition. These findings indicate that the inhibitory action of dantrolene on RyR2 depends on RyR2 association with FKBP12.6 in addition to CaM as previously found.
Asunto(s)
Dantroleno , Canal Liberador de Calcio Receptor de Rianodina , Canal Liberador de Calcio Receptor de Rianodina/fisiología , Dantroleno/farmacología , Rianodina , Membrana Dobles de Lípidos , Calcio/metabolismoRESUMEN
Sensor-based devices can record pressure or force over time during grasping and therefore offer a more comprehensive approach to quantifying grip strength during sustained contractions. The objectives of this study were to investigate the reliability and concurrent validity of measures of maximal tactile pressures and forces during a sustained grasp task using a TactArray device in people with stroke. Participants with stroke (n = 11) performed three trials of sustained maximal grasp over 8 s. Both hands were tested in within- and between-day sessions, with and without vision. Measures of maximal tactile pressures and forces were measured for the complete (8 s) grasp duration and plateau phase (5 s). Tactile measures are reported using the highest value among three trials, the mean of two trials, and the mean of three trials. Reliability was determined using changes in mean, coefficients of variation, and intraclass correlation coefficients (ICCs). Pearson correlation coefficients were used to evaluate concurrent validity. This study found that measures of reliability assessed by changes in means were good, coefficients of variation were good to acceptable, and ICCs were very good for maximal tactile pressures using the average pressure of the mean of three trials over 8 s in the affected hand with and without vision for within-day sessions and without vision for between-day sessions. In the less affected hand, changes in mean were very good, coefficients of variations were acceptable, and ICCs were good to very good for maximal tactile pressures using the average pressure of the mean of three trials over 8 s and 5 s, respectively, in between-day sessions with and without vision. Maximal tactile pressures had moderate correlations with grip strength. The TactArray device demonstrates satisfactory reliability and concurrent validity for measures of maximal tactile pressures in people with stroke.
Asunto(s)
Accidente Cerebrovascular , Tacto , Humanos , Reproducibilidad de los Resultados , Mano , Fuerza de la ManoRESUMEN
Multivalent ligands of ion channels have proven to be both very rare and highly valuable in yielding unique insights into channel structure and pharmacology. Here, we describe a bivalent peptide from the venom of Xibalbanus tulumensis, a troglobitic arthropod from the enigmatic class Remipedia, that causes persistent calcium release by activation of ion channels involved in muscle contraction. The high-resolution solution structure of φ-Xibalbin3-Xt3a reveals a tandem repeat arrangement of inhibitor-cysteine knot (ICK) domains previously only found in spider venoms. The individual repeats of Xt3a share sequence similarity with a family of scorpion toxins that target ryanodine receptors (RyR). Single-channel electrophysiology and quantification of released Ca2+ stores within skinned muscle fibers confirm Xt3a as a bivalent RyR modulator. Our results reveal convergent evolution of RyR targeting toxins in remipede and scorpion venoms, while the tandem-ICK repeat architecture is an evolutionary innovation that is convergent with toxins from spider venoms.
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Canal Liberador de Calcio Receptor de Rianodina , Venenos de Escorpión , Canal Liberador de Calcio Receptor de Rianodina/genética , Canal Liberador de Calcio Receptor de Rianodina/metabolismo , Calcio/metabolismo , Rianodina/farmacología , Secuencia de Aminoácidos , Péptidos/química , Venenos de Escorpión/farmacología , Venenos de Escorpión/químicaRESUMEN
The objective of this paper is to develop a computationally efficient simulation model of Calcium signalling in cardiomyocytes. The model considered here consists of more than two million stiff, nonlinear, and stochastic systems, each of which is composed of 62 state equations. The size of the model, combined with the broad numerical scale, non-continuous stochastic state-transitions, and underlying physiological constraints, presents a significant implementation challenge. The method involves development of specialised algorithms for parallelisation, which include fully-implicit Runge-Kutta integration with both L-stability and step-size control, Newton's root finding method with exception handling, and Conjugate Residual Squared for solving linear systems not of full-rank within available computational precision. Parallelisation of the problem across the systems is employed to allow for practical scaling with computing resources. The results produce sparks and waves akin to those observed in actual laboratory experiments within an acceptable timeframe. Performance measures of the simulation model with respect to accuracy and computation time are also given. The conclusion is that the methodologies utilised in this work are can simulate cardiomyocyte's calcium signalling in a computationally efficient manner with the results produced replicating those in the laboratory. The significance of this paper is that computational models such as the one developed here provide a way to simulate and understand the complex biological interactions operating in organisms. Accurate simulations are extremely computationally intensive and this pursuit is considered as the grand challenge for computational science into the 21st century.
Asunto(s)
Calcio , Miocitos Cardíacos , Señalización del Calcio , Algoritmos , Simulación por ComputadorRESUMEN
Ion currents through potassium channels are gated. Constriction of the ion conduction pathway at the inner helix bundle, the textbook gate of Kir potassium channels, has been shown to be an ineffective permeation control, creating a rift in our understanding of how these channels are gated. Here we present evidence that anionic lipids act as interactive response elements sufficient to gate potassium conduction. We demonstrate the limiting barrier to K+ permeation lies within the ion conduction pathway and show that this gate is operated by the fatty acyl tails of lipids that infiltrate the conduction pathway via fenestrations in the walls of the pore. Acyl tails occupying a surface groove extending from the cytosolic interface to the conduction pathway provide a potential means of relaying cellular signals, mediated by anionic lipid head groups bound at the canonical lipid binding site, to the internal gate.
Asunto(s)
Activación del Canal Iónico , Lípidos de la Membrana/metabolismo , Canales de Potasio de Rectificación Interna/metabolismo , Potasio/metabolismo , Aniones/química , Aniones/metabolismo , Sitios de Unión , Cristalografía por Rayos X , Humanos , Transporte Iónico , Liposomas/química , Liposomas/metabolismo , Lípidos de la Membrana/química , Simulación de Dinámica Molecular , Mutación , Fosfatidilcolinas/química , Fosfatidilcolinas/metabolismo , Fosfatidilserinas/química , Fosfatidilserinas/metabolismo , Canales de Potasio de Rectificación Interna/química , Canales de Potasio de Rectificación Interna/genéticaRESUMEN
RATIONALE: The class Ic antiarrhythmic drug flecainide prevents ventricular tachyarrhythmia in patients with catecholaminergic polymorphic ventricular tachycardia (CPVT), a disease caused by hyperactive RyR2 (cardiac ryanodine receptor) mediated calcium (Ca) release. Although flecainide inhibits single RyR2 channels in vitro, reports have claimed that RyR2 inhibition by flecainide is not relevant for its mechanism of antiarrhythmic action and concluded that sodium channel block alone is responsible for flecainide's efficacy in CPVT. OBJECTIVE: To determine whether RyR2 block independently contributes to flecainide's efficacy for suppressing spontaneous sarcoplasmic reticulum Ca release and for preventing ventricular tachycardia in vivo. METHODS AND RESULTS: We synthesized N-methylated flecainide analogues (QX-flecainide and N-methyl flecainide) and showed that N-methylation reduces flecainide's inhibitory potency on RyR2 channels incorporated into artificial lipid bilayers. N-methylation did not alter flecainide's inhibitory activity on human cardiac sodium channels expressed in HEK293T cells. Antiarrhythmic efficacy was tested utilizing a Casq2 (cardiac calsequestrin) knockout (Casq2-/-) CPVT mouse model. In membrane-permeabilized Casq2-/- cardiomyocytes-lacking intact sarcolemma and devoid of sodium channel contribution-flecainide, but not its analogues, suppressed RyR2-mediated Ca release at clinically relevant concentrations. In voltage-clamped, intact Casq2-/- cardiomyocytes pretreated with tetrodotoxin to inhibit sodium channels and isolate the effect of flecainide on RyR2, flecainide significantly reduced the frequency of spontaneous sarcoplasmic reticulum Ca release, while QX-flecainide and N-methyl flecainide did not. In vivo, flecainide effectively suppressed catecholamine-induced ventricular tachyarrhythmias in Casq2-/- mice, whereas N-methyl flecainide had no significant effect on arrhythmia burden, despite comparable sodium channel block. CONCLUSIONS: Flecainide remains an effective inhibitor of RyR2-mediated arrhythmogenic Ca release even when cardiac sodium channels are blocked. In mice with CPVT, sodium channel block alone did not prevent ventricular tachycardia. Hence, RyR2 channel inhibition likely constitutes the principal mechanism of antiarrhythmic action of flecainide in CPVT.
Asunto(s)
Antiarrítmicos/farmacología , Bloqueadores de los Canales de Calcio/farmacología , Flecainida/farmacología , Frecuencia Cardíaca/efectos de los fármacos , Miocitos Cardíacos/efectos de los fármacos , Canal Liberador de Calcio Receptor de Rianodina/efectos de los fármacos , Retículo Sarcoplasmático/efectos de los fármacos , Taquicardia Ventricular/prevención & control , Potenciales de Acción , Animales , Señalización del Calcio , Calsecuestrina/genética , Calsecuestrina/metabolismo , Modelos Animales de Enfermedad , Femenino , Células HEK293 , Humanos , Masculino , Ratones Noqueados , Miocitos Cardíacos/metabolismo , Fosforilación , Canal Liberador de Calcio Receptor de Rianodina/metabolismo , Retículo Sarcoplasmático/metabolismo , Oveja Doméstica , Taquicardia Ventricular/genética , Taquicardia Ventricular/metabolismo , Taquicardia Ventricular/fisiopatología , Bloqueadores del Canal de Sodio Activado por Voltaje/farmacologíaRESUMEN
The canonical mechanistic model explaining potassium channel gating is of a conformational change that alternately dilates and constricts a collar-like intracellular entrance to the pore. It is based on the premise that K+ ions maintain a complete hydration shell while passing between the transmembrane cavity and cytosol, which must be accommodated. To put the canonical model to the test, we locked the conformation of a Kir K+ channel to prevent widening of the narrow collar. Unexpectedly, conduction was unimpaired in the locked channels. In parallel, we employed all-atom molecular dynamics to simulate K+ ions moving along the conduction pathway between the lower cavity and cytosol. During simulations, the constriction did not significantly widen. Instead, transient loss of some water molecules facilitated K+ permeation through the collar. The low free energy barrier to partial dehydration in the absence of conformational change indicates Kir channels are not gated by the canonical mechanism.
Asunto(s)
Canales de Potasio Rectificados Internamente Asociados a la Proteína G/metabolismo , Potasio/metabolismo , Citosol/química , Citosol/metabolismo , Conductividad Eléctrica , Impedancia Eléctrica , Canales de Potasio Rectificados Internamente Asociados a la Proteína G/química , Canales de Potasio Rectificados Internamente Asociados a la Proteína G/genética , Humanos , Transporte Iónico , Iones/química , Iones/metabolismo , Simulación de Dinámica Molecular , Potasio/química , Conformación Proteica , Agua/metabolismoRESUMEN
TRP channels of the transient receptor potential ion channel superfamily are involved in a wide variety of mechanosensory processes, including touch sensation, pain, blood pressure regulation, bone loading and detection of cerebrospinal fluid flow. However, in many instances it is unclear whether TRP channels are the primary transducers of mechanical force in these processes. In this study, we tested stretch activation of eleven TRP channels from six mammalian subfamilies. We found that these TRP channels were insensitive to short membrane stretches in cellular systems. Furthermore, we purified TRPC6 and demonstrated its insensitivity to stretch in liposomes, an artificial bilayer system free from cellular components. Additionally, we demonstrated that, when expressed in C. elegans neurons, mouse TRPC6 restores the mechanoresponse of a touch insensitive mutant but requires diacylglycerol for activation. These results strongly suggest that the mammalian members of the TRP ion channel family are insensitive to tension induced by cell membrane stretching and, thus, are more likely to be activated by cytoplasmic tethers or downstream components and to act as amplifiers of cellular mechanosensory signaling cascades.
Asunto(s)
Canal Catiónico TRPC6/química , Animales , Células CHO , Caenorhabditis elegans/metabolismo , Cricetulus , Electrofisiología , Células HEK293 , Células HeLa , Humanos , Mecanotransducción Celular/fisiología , Neuronas/metabolismo , Proteolípidos/químicaRESUMEN
Objective: To investigate the reliability and the concurrent validity of maximal tactile pressures and forces of a sustained grasp task using a TactArray device in healthy adults.Methods: Healthy participants (n = 18, mean age: 62.2 ± 9.9 years) performed three repeat trials of sustained maximal grasp over 8 seconds. Both hands were tested in within-day and between-day sessions, with vision and without vision. Measures of maximal tactile pressures and forces were measured for the complete grasp duration (8s) and for the plateau phase (5s). Measures of maximal tactile pressures and forces were reported using the highest value among three repeat trials, the mean of two repeat trials, and the mean of three repeat trials. Reliability was determined using changes in mean, coefficients of variation and intraclass correlation coefficients (ICCs). Pearson correlation coefficients were used to evaluate concurrent validity.Results: Changes in mean were very good, coefficients of variation were good to acceptable and ICCs were very good to good for maximal tactile pressures using the highest value among three repeat trials and the mean of three repeat trials for the complete grasp duration (8s) and for the plateau phase (5s) in the dominant hand with and without vision and in the non-dominant hand without vision for within-day and between-day sessions. Maximal tactile pressures had moderate to large correlations with grip strength.Conclusion: The TactArray device demonstrates satisfactory reliability for maximal tactile pressures during a sustained grasp for within-day and between-day testing sessions in both hands. Validity was satisfactory with grip strength in both hands.
Asunto(s)
Fenómenos Biomecánicos/fisiología , Retroalimentación Sensorial/fisiología , Mano/fisiología , Actividad Motora/fisiología , Desempeño Psicomotor/fisiología , Anciano , Femenino , Lateralidad Funcional/fisiología , Humanos , Masculino , Persona de Mediana Edad , Reproducibilidad de los ResultadosRESUMEN
BACKGROUND: Circulating SN (secretoneurin) concentrations are increased in patients with myocardial dysfunction and predict poor outcome. Because SN inhibits CaMKIIδ (Ca2+/calmodulin-dependent protein kinase IIδ) activity, we hypothesized that upregulation of SN in patients protects against cardiomyocyte mechanisms of arrhythmia. METHODS: Circulating levels of SN and other biomarkers were assessed in patients with catecholaminergic polymorphic ventricular tachycardia (CPVT; n=8) and in resuscitated patients after ventricular arrhythmia-induced cardiac arrest (n=155). In vivo effects of SN were investigated in CPVT mice (RyR2 [ryanodine receptor 2]-R2474S) using adeno-associated virus-9-induced overexpression. Interactions between SN and CaMKIIδ were mapped using pull-down experiments, mutagenesis, ELISA, and structural homology modeling. Ex vivo actions were tested in Langendorff hearts and effects on Ca2+ homeostasis examined by fluorescence (fluo-4) and patch-clamp recordings in isolated cardiomyocytes. RESULTS: SN levels were elevated in patients with CPVT and following ventricular arrhythmia-induced cardiac arrest. In contrast to NT-proBNP (N-terminal pro-B-type natriuretic peptide) and hs-TnT (high-sensitivity troponin T), circulating SN levels declined after resuscitation, as the risk of a new arrhythmia waned. Myocardial pro-SN expression was also increased in CPVT mice, and further adeno-associated virus-9-induced overexpression of SN attenuated arrhythmic induction during stress testing with isoproterenol. Mechanistic studies mapped SN binding to the substrate binding site in the catalytic region of CaMKIIδ. Accordingly, SN attenuated isoproterenol induced autophosphorylation of Thr287-CaMKIIδ in Langendorff hearts and inhibited CaMKIIδ-dependent RyR phosphorylation. In line with CaMKIIδ and RyR inhibition, SN treatment decreased Ca2+ spark frequency and dimensions in cardiomyocytes during isoproterenol challenge, and reduced the incidence of Ca2+ waves, delayed afterdepolarizations, and spontaneous action potentials. SN treatment also lowered the incidence of early afterdepolarizations during isoproterenol; an effect paralleled by reduced magnitude of L-type Ca2+ current. CONCLUSIONS: SN production is upregulated in conditions with cardiomyocyte Ca2+ dysregulation and offers compensatory protection against cardiomyocyte mechanisms of arrhythmia, which may underlie its putative use as a biomarker in at-risk patients.
Asunto(s)
Paro Cardíaco/metabolismo , Neuropéptidos/metabolismo , Secretogranina II/metabolismo , Taquicardia Ventricular/metabolismo , Animales , Biomarcadores/metabolismo , Calcio/metabolismo , Señalización del Calcio , Proteína Quinasa Tipo 2 Dependiente de Calcio Calmodulina/metabolismo , Paro Cardíaco/fisiopatología , Humanos , Ratones , Miocitos Cardíacos/metabolismo , Péptido Natriurético Encefálico/metabolismo , Técnicas de Placa-Clamp , Fragmentos de Péptidos/metabolismo , Fosforilación , Canal Liberador de Calcio Receptor de Rianodina/metabolismo , Taquicardia Ventricular/fisiopatología , Troponina T/metabolismo , Regulación hacia ArribaRESUMEN
Calmodulin (CaM) is a Ca-binding protein that binds to, and can directly inhibit cardiac ryanodine receptor calcium release channels (RyR2). Animal studies have shown that RyR2 hyperphosphorylation reduces CaM binding to RyR2 in failing hearts, but data are lacking on how CaM regulates human RyR2 and how this regulation is affected by RyR2 phosphorylation. Physiological concentrations of CaM (100â¯nM) inhibited the diastolic activity of RyR2 isolated from failing human hearts by ~50% but had no effect on RyR2 from healthy human hearts. Using FRET between donor-FKBP12.6 and acceptor-CaM bound to RyR2, we determined that CaM binds to RyR2 from healthy human heart with a Kdâ¯=â¯121⯱â¯14â¯nM. Ex-vivo phosphorylation/dephosphorylation experiments suggested that the divergent CaM regulation of healthy and failing human RyR2 was caused by differences in RyR2 phosphorylation by protein kinase A and Ca-CaM-dependent kinase II. Ca2+-spark measurements in murine cardiomyocytes harbouring RyR2 phosphomimetic or phosphoablated mutants at S2814 and S2808 suggest that phosphorylation of residues corresponding to either human RyR2-S2808 or S2814 is both necessary and sufficient for RyR2 regulation by CaM. Our results challenge the current concept that CaM universally functions as a canonical inhibitor of RyR2 across species. Rather, CaM's biological action on human RyR2 appears to be more nuanced, with inhibitory activity only on phosphorylated RyR2 channels, which occurs during exercise or in patients with heart failure.
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Calmodulina/metabolismo , Insuficiencia Cardíaca/metabolismo , Miocitos Cardíacos/metabolismo , Canal Liberador de Calcio Receptor de Rianodina/metabolismo , Proteína Quinasa Tipo 2 Dependiente de Calcio Calmodulina/metabolismo , Proteínas Quinasas Dependientes de AMP Cíclico/metabolismo , Insuficiencia Cardíaca/patología , Humanos , Miocitos Cardíacos/patología , Fosforilación , Unión ProteicaRESUMEN
Ryanodine receptors (RyRs) are the Ca2+ release channels in the sarcoplasmic reticulum in striated muscle which play an important role in excitation-contraction coupling and cardiac pacemaking. Single channel recordings have revealed a wealth of information about ligand regulation of RyRs from mammalian skeletal and cardiac muscle (RyR1 and RyR2, respectively). RyR subunit has a Ca2+ activation site located in the luminal and cytoplasmic domains of the RyR. These sites synergistically feed into a common gating mechanism for channel activation by luminal and cytoplasmic Ca2+. RyRs also possess two inhibitory sites in their cytoplasmic domains with Ca2+ affinities of the order of 1 µM and 1 mM. Magnesium competes with Ca2+ at these sites to inhibit RyRs and this plays an important role in modulating their Ca2+-dependent activity in muscle. This review focuses on how these sites lead to RyR modulation by Ca2+ and Mg2+ and how these mechanisms control Ca2+ release in excitation-contraction coupling and cardiac pacemaking.
RESUMEN
In human pancreatic islets, the neurotransmitter γ-aminobutyric acid (GABA) is an extracellular signaling molecule synthesized by and released from the insulin-secreting ß cells. The effective, physiological GABA concentration range within human islets is unknown. Here we use native GABAA receptors in human islet ß cells as biological sensors and reveal that 100-1000nM GABA elicit the maximal opening frequency of the single-channels. In saturating GABA, the channels desensitized and stopped working. GABA modulated insulin exocytosis and glucose-stimulated insulin secretion. GABAA receptor currents were enhanced by the benzodiazepine diazepam, the anesthetic propofol and the incretin glucagon-like peptide-1 (GLP-1) but not affected by the hypnotic zolpidem. In type 2 diabetes (T2D) islets, single-channel analysis revealed higher GABA affinity of the receptors. The findings reveal unique GABAA receptors signaling in human islets ß cells that is GABA concentration-dependent, differentially regulated by drugs, modulates insulin secretion and is altered in T2D.
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Células Secretoras de Insulina/metabolismo , Receptores de GABA-A/metabolismo , Diabetes Mellitus Tipo 2/metabolismo , Exocitosis/efectos de los fármacos , Humanos , Insulina/metabolismo , Células Secretoras de Insulina/efectos de los fármacos , Activación del Canal Iónico/efectos de los fármacos , Cinética , Modelos Biológicos , Subunidades de Proteína/metabolismo , Ácido gamma-Aminobutírico/farmacologíaAsunto(s)
Calmodulina/genética , Ventrículos Cardíacos , Canal Liberador de Calcio Receptor de Rianodina/metabolismo , Taquicardia Ventricular , Ventrículos Cardíacos/metabolismo , Ventrículos Cardíacos/patología , Humanos , Técnicas In Vitro , Mutación , Fosforilación , Taquicardia Ventricular/genéticaRESUMEN
Neural and agonist-induced contractions of proximal (i.e. upper half adjacent to the cervix) and distal mouse vaginal smooth muscle strips were investigated. We hypothesised that nerve-mediated vaginal contractions arise through activity of cholinergic nerves. Nerve activation by bursts of electrical field stimulation (EFS) caused a primary transient contraction often accompanied by a secondary transient contraction, both larger in proximal than distal tissues (i.e. primary: 7-fold larger; secondary: 3-fold larger). Our hypothesis was supported as we found that cholinergic nerves mediated the primary transient contraction in both proximal and distal vaginal strips, as EFS responses were enhanced by neostigmine an anticholinesterase, massively inhibited by the competitive muscarinic receptor antagonist atropine and not affected by the non-selective α-adrenergic receptor antagonist phentolamine. Primary transient contractions were halved in amplitude by the L-type Ca2+ channel blocker nifedipine and markedly inhibited by the sarco-endoplasmic reticulum calcium ATPase (SERCA) inhibitor cyclopiazonic acid (CPA). Resultant secondary transient contractions were abolished by nifedipine. Notably, the selective α1-adrenergic receptor agonist phenylephrine caused tonic contracture in distal but not proximal strips. Low-frequency EFS often initiated recurrent transient contractions similar to those elicited by CCh. Immunohistochemical studies demonstrated innervation of the smooth muscle. Findings of enhanced proximal cholinergic nerve-induced transient contractions, evidence that maintained nerve stimulation could cause recurrent contractions and the finding of distal phenylephrine-mediated tonic contraction have implications on insemination.
Asunto(s)
Contracción Muscular , Músculo Liso/fisiología , Vagina/fisiología , Acetilcolina/farmacología , Animales , Atropina/farmacología , Sistema Nervioso Autónomo/efectos de los fármacos , Estimulación Eléctrica/métodos , Femenino , Ratones , Contracción Muscular/efectos de los fármacos , Músculo Liso/efectos de los fármacos , Fenilefrina/farmacología , Vagina/efectos de los fármacosRESUMEN
BACKGROUND: Halogenated anesthetics activate cardiac ryanodine receptor 2-mediated sarcoplasmic reticulum Ca release, leading to sarcoplasmic reticulum Ca depletion, reduced cardiac function, and providing cell protection against ischemia-reperfusion injury. Anesthetic activation of ryanodine receptor 2 is poorly defined, leaving aspects of the protective mechanism uncertain. METHODS: Ryanodine receptor 2 from the sheep heart was incorporated into artificial lipid bilayers, and their gating properties were measured in response to five halogenated anesthetics. RESULTS: Each anesthetic rapidly and reversibly activated ryanodine receptor 2, but only from the cytoplasmic side. Relative activation levels were as follows: halothane (approximately 4-fold; n = 8), desflurane and enflurane (approximately 3-fold,n = 9), and isoflurane and sevoflurane (approximately 1.5-fold, n = 7, 10). Half-activating concentrations (Ka) were in the range 1.3 to 2.1 mM (1.4 to 2.6 minimum alveolar concentration [MAC]) with the exception of isoflurane (5.3 mM, 6.6 minimum alveolar concentration). Dantrolene (10 µM with 100 nM calmodulin) inhibited ryanodine receptor 2 by 40% but did not alter the Ka for halothane activation. Halothane potentiated luminal and cytoplasmic Ca activation of ryanodine receptor 2 but had no effect on Mg inhibition. Halothane activated ryanodine receptor 2 in the absence and presence (2 mM) of adenosine triphosphate (ATP). Adenosine, a competitive antagonist to ATP activation of ryanodine receptor 2, did not antagonize halothane activation in the absence of ATP. CONCLUSIONS: At clinical concentrations (1 MAC), halothane desflurane and enflurane activated ryanodine receptor 2, whereas isoflurane and sevoflurane were ineffective. Dantrolene inhibition of ryanodine receptor 2 substantially negated the activating effects of anesthetics. Halothane acted independently of the adenine nucleotide-binding site on ryanodine receptor 2. The previously observed adenosine antagonism of halothane activation of sarcoplasmic reticulum Ca release was due to competition between adenosine and ATP, rather than between halothane and ATP.
Asunto(s)
Enflurano/farmacología , Halotano/farmacología , Isoflurano/análogos & derivados , Isoflurano/farmacología , Éteres Metílicos/farmacología , Canal Liberador de Calcio Receptor de Rianodina/efectos de los fármacos , Anestésicos por Inhalación/farmacología , Animales , Técnicas de Cultivo de Célula , Desflurano , Corazón , Sevoflurano , OvinosRESUMEN
Cardiac output and rhythm depend on the release and the take-up of calcium from the sarcoplasmic reticulum (SR). Excessive diastolic calcium leak from the SR due to dysfunctional calcium release channels (RyR2) contributes to the formation of delayed after-depolarizations, which underlie the fatal arrhythmias that occur in heart failure and inherited syndromes. Calmodulin (CaM) is a calcium-binding protein that regulates target proteins and acts as a calcium sensor. CaM is comprised of two calcium-binding EF-hand domains and a flexible linker. CaM is an accessory protein that partially inhibits RyR2 channel activity. CaM is critical for normal cardiac function, and altered CaM binding and efficacy may contribute to defects in SR calcium release. The present paper reviews CaM binding to RyR2 and how it regulates RyR2 channel activity. It then goes on to review how mutations in the CaM amino acid sequence give rise to inherited syndromes such as Catecholaminergic Polymorphic Ventricular Tachychardia (CPVT) and long QT syndrome (LQTS). In addition, the role of reduced CaM binding to RyR2 that results from RyR2 phosphorylation or from oxidation of either RyR2 or CaM contributes to the progression of heart failure is reviewed. Finally, this manuscript reviews recent evidence that CaM binding to RyR2 is required for the inhibitory action of a pharmaceutical agent (dantrolene) on RyR2. Dantrolene is a clinically used muscle relaxant that has recently been found to exert antiarrhythmic effects against SR Ca2+ overload arrhythmias.
Asunto(s)
Antiarrítmicos/uso terapéutico , Calmodulina/fisiología , Dantroleno/uso terapéutico , Progresión de la Enfermedad , Insuficiencia Cardíaca/tratamiento farmacológico , Canal Liberador de Calcio Receptor de Rianodina/fisiología , Animales , Arritmias Cardíacas/tratamiento farmacológico , Arritmias Cardíacas/metabolismo , Arritmias Cardíacas/patología , Insuficiencia Cardíaca/metabolismo , Insuficiencia Cardíaca/patología , HumanosRESUMEN
Dantrolene is the first line therapy of malignant hyperthermia. Animal studies suggest that dantrolene also protects against heart failure and arrhythmias caused by spontaneous Ca(2+) release. Although dantrolene inhibits Ca(2+) release from the sarcoplasmic reticulum of skeletal and cardiac muscle preparations, its mechanism of action has remained controversial, because dantrolene does not inhibit single ryanodine receptor (RyR) Ca(2+) release channels in lipid bilayers. Here we test the hypothesis that calmodulin (CaM), a physiologic RyR binding partner that is lost during incorporation into lipid bilayers, is required for dantrolene inhibition of RyR channels. In single channel recordings (100 nM cytoplasmic [Ca(2+)] + 2 mM ATP), dantrolene caused inhibition of RyR1 (rabbit skeletal muscle) and RyR2 (sheep) with a maximal inhibition of Po (Emax) to 52 ± 4% of control only after adding physiologic [CaM] = 100 nM. Dantrolene inhibited RyR2 with an IC50 of 0.16 ± 0.03 µM. Mutant N98S-CaM facilitated dantrolene inhibition with an IC50 = 5.9 ± 0.3 nM. In mouse cardiomyocytes, dantrolene had no effect on cardiac Ca(2+) release in the absence of CaM, but reduced Ca(2+) wave frequency (IC50 = 0.42 ± 0.18 µM, Emax = 47 ± 4%) and amplitude (IC50 = 0.19 ± 0.04 µM, Emax = 66 ± 4%) in the presence of 100 nM CaM. We conclude that CaM is essential for dantrolene inhibition of RyR1 and RyR2. Its absence explains why dantrolene inhibition of single RyR channels has not been previously observed.