RESUMO
Aims: Catecholaminergic polymorphic ventricular tachycardia (CPVT) is an ion channelopathy characterized by ventricular arrhythmia during exertion or stress. Mutations in RYR2-coded Ryanodine Receptor-2 (RyR2) and CASQ2-coded Calsequestrin-2 (CASQ2) genes underlie CPVT1 and CPVT2, respectively. However, prognostic markers are scarce. We sought to better characterize the phenotypic and genotypic spectrum of CPVT, and utilize molecular modelling to help account for clinical phenotypes. Methods and results: This is a Pediatric and Congenital Electrophysiology Society multicentre, retrospective cohort study of CPVT patients diagnosed at <19 years of age and their first-degree relatives. Genetic testing was undertaken in 194 of 236 subjects (82%) during 3.5 (1.4-5.3) years of follow-up. The majority (60%) had RyR2-associated CPVT1. Variant locations were predicted based on a 3D structural model of RyR2. Specific residues appear to have key structural importance, supported by an association between cardiac arrest and mutations in the intersubunit interface of the N-terminus, and the S4-S5 linker and helices S5 and S6 of the RyR2 C-terminus. In approximately one quarter of symptomatic patients, cardiac events were precipitated by only normal wakeful activities. Conclusion: This large, multicentre study identifies contemporary challenges related to the diagnosis and prognostication of CPVT patients. Structural modelling of RyR2 can improve our understanding severe CPVT phenotypes. Wakeful rest, rather than exertion, often precipitated life-threatening cardiac events.
Assuntos
Calsequestrina/genética , Mutação , Canal de Liberação de Cálcio do Receptor de Rianodina/genética , Taquicardia Ventricular/genética , Adolescente , Criança , Análise Mutacional de DNA , Morte Súbita Cardíaca/epidemiologia , Feminino , Marcadores Genéticos , Predisposição Genética para Doença , Hereditariedade , Humanos , Masculino , Modelos Moleculares , Linhagem , Fenótipo , Prognóstico , Conformação Proteica , Sistema de Registros , Estudos Retrospectivos , Fatores de Risco , Canal de Liberação de Cálcio do Receptor de Rianodina/química , Canal de Liberação de Cálcio do Receptor de Rianodina/metabolismo , Relação Estrutura-Atividade , Taquicardia Ventricular/diagnóstico , Taquicardia Ventricular/mortalidade , Taquicardia Ventricular/fisiopatologiaRESUMO
Many physiological events require transient increases in cytosolic Ca(2+) concentrations. Ryanodine receptors (RyRs) are ion channels that govern the release of Ca(2+) from the endoplasmic and sarcoplasmic reticulum. Mutations in RyRs can lead to severe genetic conditions that affect both cardiac and skeletal muscle, but locating the mutated residues in the full-length channel structure has been difficult. Here we show the 2.5 Å resolution crystal structure of a region spanning three domains of RyR type 1 (RyR1), encompassing amino acid residues 1-559. The domains interact with each other through a predominantly hydrophilic interface. Docking in RyR1 electron microscopy maps unambiguously places the domains in the cytoplasmic portion of the channel, forming a 240-kDa cytoplasmic vestibule around the four-fold symmetry axis. We pinpoint the exact locations of more than 50 disease-associated mutations in full-length RyR1 and RyR2. The mutations can be classified into three groups: those that destabilize the interfaces between the three amino-terminal domains, disturb the folding of individual domains or affect one of six interfaces with other parts of the receptor. We propose a model whereby the opening of a RyR coincides with allosterically coupled motions within the N-terminal domains. This process can be affected by mutations that target various interfaces within and across subunits. The crystal structure provides a framework to understand the many disease-associated mutations in RyRs that have been studied using functional methods, and will be useful for developing new strategies to modulate RyR function in disease states.
Assuntos
Modelos Moleculares , Canal de Liberação de Cálcio do Receptor de Rianodina/química , Canal de Liberação de Cálcio do Receptor de Rianodina/genética , Animais , Mutação/genética , Estrutura Terciária de Proteína , Coelhos , Canal de Liberação de Cálcio do Receptor de Rianodina/metabolismoRESUMO
CPVT (catecholaminergic polymorphic ventricular tachycardia) is an inherited life-threatening arrhythmogenic disorder. CPVT is caused by DADs (delayed after-depolarizations) that are induced by spontaneous Ca2+ release during SR (sarcoplasmic reticulum) Ca2+ overload, a process also known as SOICR (store-overload-induced Ca2+ release). A number of mutations in the cardiac ryanodine receptor RyR2 are linked to CPVT. Many of these CPVT-associated RyR2 mutations enhance the propensity for SOICR and DADs by sensitizing RyR2 to luminal or luminal/cytosolic Ca2+ activation. Recently, a novel CPVT RyR2 mutation, G230C, was found to increase the cytosolic, but not the luminal, Ca2+ sensitivity of single RyR2 channels in lipid bilayers. This observation led to the suggestion of a SOICR-independent disease mechanism for the G230C mutation. However, the cellular impact of this mutation on SOICR is yet to be determined. To this end, we generated stable inducible HEK (human embryonic kidney)-293 cell lines expressing the RyR2 WT (wild-type) and the G230C mutant. Using single-cell Ca2+ imaging, we found that the G230C mutation markedly enhanced the propensity for SOICR and reduced the SOICR threshold. Furthermore, the G230C mutation increased the sensitivity of single RyR2 channels to both luminal and cytosolic Ca2+ activation and the Ca2+-dependent activation of [3H]ryanodine binding. In addition, the G230C mutation decreased the thermal stability of the N-terminal region (amino acids 1-547) of RyR2. These data suggest that the G230C mutation enhances the propensity for SOICR by sensitizing the channel to luminal and cytosolic Ca2+ activation, and that G230C has an intrinsic structural impact on the N-terminal domains of RyR2.
Assuntos
Cálcio/metabolismo , Mutação Puntual/genética , Canal de Liberação de Cálcio do Receptor de Rianodina/química , Canal de Liberação de Cálcio do Receptor de Rianodina/genética , Taquicardia Ventricular/genética , Taquicardia Ventricular/metabolismo , Regulação para Cima/genética , Animais , Cálcio/química , Células HEK293 , Humanos , Camundongos , Estrutura Terciária de Proteína/genética , Canal de Liberação de Cálcio do Receptor de Rianodina/fisiologiaRESUMO
Ryanodine receptors are large channels that release Ca(2+) from the endoplasmic and sarcoplasmic reticulum. Hundreds of RyR mutations can cause cardiac and skeletal muscle disorders, yet detailed mechanisms explaining their effects have been lacking. Here we compare pseudo-atomic models and propose that channel opening coincides with widening of a cytoplasmic vestibule formed by the N-terminal region, thus altering an interface targeted by 20 disease mutations. We solve crystal structures of several disease mutants that affect intrasubunit domain-domain interfaces. Mutations affecting intrasubunit ionic pairs alter relative domain orientations, and thus couple to surrounding interfaces. Buried disease mutations cause structural changes that also connect to the intersubunit contact area. These results suggest that the intersubunit contact region between N-terminal domains is a prime target for disease mutations, direct or indirect, and we present a model whereby ryanodine receptors and inositol-1,4,5-trisphosphate receptors are activated by altering domain arrangements in the N-terminal region.
Assuntos
Doença/genética , Mutação/genética , Subunidades Proteicas/química , Subunidades Proteicas/genética , Canal de Liberação de Cálcio do Receptor de Rianodina/química , Canal de Liberação de Cálcio do Receptor de Rianodina/genética , Animais , Humanos , Ligantes , Simulação de Acoplamento Molecular , Desnaturação Proteica , Estabilidade Proteica , Estrutura Terciária de Proteína , Coelhos , TemperaturaRESUMO
Ten years after Aurora Biomed (Vancouver, British Columbia, Canada) hosted the inaugural Ion Channel Retreat, this event is recognized as a leading conference for ion channel researchers. Held annually in Vancouver, this meeting consistently provides an outlet for researchers to share their findings while learning about new concepts, methods, and technologies. Researchers use this forum to discuss and debate a spectrum of topics from ion channel research and technology to drug discovery and safety. The Retreat covered key subjects in the ion channel industry, including ion channels as disease targets, transient receptor protein channels as pain and disease targets, ion channels as pain targets, ion channel structure and function, ion channel screening technologies, cardiac safety and toxicology, and cardiac function and pharmacology.
Assuntos
Encefalopatias/metabolismo , Desenho de Fármacos , Inflamação/metabolismo , Ativação do Canal Iônico , Canais Iônicos/química , Canais Iônicos/metabolismo , Nefropatias/metabolismo , Animais , HumanosRESUMO
Ryanodine receptors (RyRs) are large tetrameric calcium (Ca(2+)) release channels found on the sarcoplasmic reticulum that respond to dihydropyridine receptor activity through a direct conformational interaction and/or indirect Ca(2+) sensitivity, propagating sarcoplasmic reticulum luminal Ca(2+) release in the process of excitation-contraction coupling. There are three human RyR subtypes, and several debilitating diseases are linked to heritable mutations in RyR1 and RyR2 including malignant hypothermia, central core disease, catecholaminergic polymorphic ventricular tachycardia (CPVT) and arrhythmogenic right ventricular dysplasia type 2 (ARVD2). Despite the recent appreciation that many disease-associated mutations within the N-terminal RyRABC domains (i.e., residues 1-559) are located in the putative interfaces mediating tetrameric channel assembly, the precise structural and dynamical consequences of the mutations are not well understood. We used solution nuclear magnetic resonance (NMR) spectroscopy and X-ray crystallography to examine the effect of ARVD2-associated (i.e., R176Q) and CPVT-associated [i.e., P164S, R169Q and delta exon 3 (Δ3)] mutations on the structure and dynamics of RyR2A. Our solution NMR data exposed a mobile α-helix, unique to type 2; further, this α2 helix rescues the ß-strand lost in RyR2A Δ3 but remains dynamic in the hot-spot loop (HS-loop) P164S, R169Q and R176Q mutant proteins. Docking of our X-ray crystal/NMR hybrid structure into the RyR1 cryo-electron microscopy map revealed that this RyR2A α2 helix is in close proximity to dense "columns" projecting toward the channel pore. This is in contrast to the HS-loop mutations that cause structural changes largely localized to the intersubunit interface between adjacent ABC domains. Taken together, our data suggest that ARVD2 and CPVT mutations have at least two distinct structural consequences linked to channel dysfunction: perturbation of the HS-loop (i.e., domain A):domain B intersubunit interface and disruption of the communication between the N-terminal region and the channel domain.
Assuntos
Cardiomiopatias/genética , Éxons , Mutação , Canal de Liberação de Cálcio do Receptor de Rianodina/química , Canal de Liberação de Cálcio do Receptor de Rianodina/genética , Sequência de Aminoácidos , Animais , Camundongos , Dados de Sequência Molecular , Estrutura Secundária de Proteína , Estrutura Terciária de Proteína , Alinhamento de Sequência/métodosRESUMO
Ryanodine receptors (RyRs) are calcium release channels located in the membrane of the endoplasmic and sarcoplasmic reticulum and play a major role in muscle excitation-contraction coupling. The cardiac isoform (RyR2) is the target for >150 mutations that cause catecholaminergic polymorphic ventricular tachycardia (CPVT) and other conditions. Here, we present the crystal structure of the N-terminal region of RyR2 (1-547), an area encompassing 29 distinct disease mutations. The protein folds up in three individual domains, which are held together via a central chloride anion that shields repulsive positive charges. Several disease mutant versions of the construct drastically destabilize the protein. The R420Q disease mutant causes CPVT and ablates chloride binding. The mutation results in reorientations of the first two domains relative to the third domain. These conformational changes likely activate the channel by destabilizing intersubunit interactions that are disrupted upon channel opening.
Assuntos
Canal de Liberação de Cálcio do Receptor de Rianodina/química , Animais , Sítios de Ligação , Brometos/química , Cristalografia por Raios X , Ligação de Hidrogênio , Camundongos , Modelos Moleculares , Mutação de Sentido Incorreto , Cloreto de Potássio/química , Compostos de Potássio/química , Estabilidade Proteica , Estrutura Secundária de Proteína , Estrutura Terciária de Proteína , Canal de Liberação de Cálcio do Receptor de Rianodina/genética , Taquicardia Ventricular/genéticaRESUMO
Ryanodine receptors are ion channels that allow for the release of Ca(2+) from the endoplasmic or sarcoplasmic reticulum. They are expressed in many different cell types but are best known for their predominance in skeletal and cardiac myocytes, where they are directly involved in excitation-contraction coupling. With molecular weights exceeding 2 MDa, Ryanodine Receptors are the largest ion channels known to date and present major challenges for structural biology. Since their discovery in the 1980s, significant progress has been made in understanding their behaviour through multiple structural methods. Cryo-electron microscopy reconstructions of intact channels depict a mushroom-shaped structure with a large cytoplasmic region that presents many binding sites for regulatory molecules. This region undergoes significant motions during opening and closing of the channel, demonstrating that the Ryanodine Receptor is a bona fide allosteric protein. High-resolution structures through X-ray crystallography and NMR currently cover â¼11% of the entire protein. The combination of high- and low-resolution methods allows us to build pseudo-atomic models. Here we present an overview of the electron microscopy, NMR, and crystallographic analyses of this membrane protein giant.
Assuntos
Conformação Proteica , Canal de Liberação de Cálcio do Receptor de Rianodina/química , Animais , Sítios de Ligação , Microscopia Crioeletrônica , Cristalografia por Raios X , Humanos , Receptores de Inositol 1,4,5-Trifosfato/química , Receptores de Inositol 1,4,5-Trifosfato/metabolismo , Ativação do Canal Iônico , Modelos Moleculares , Ressonância Magnética Nuclear Biomolecular , Ligação Proteica , Isoformas de Proteínas/química , Isoformas de Proteínas/genética , Isoformas de Proteínas/metabolismo , Canal de Liberação de Cálcio do Receptor de Rianodina/genética , Canal de Liberação de Cálcio do Receptor de Rianodina/metabolismoRESUMO
Mutations in the cardiac Ryanodine Receptor (RYR2) are linked to triggered arrhythmias. Removal of exon 3 results in a severe form of catecholaminergic polymorphic ventricular tachycardia (CPVT). This exon encodes secondary structure elements that are crucial for folding of the N-terminal domain (NTD), raising the question of why the deletion is neither lethal nor confers a loss of function. We determined the 2.3 Å crystal structure of the NTD lacking exon 3. The removal causes a structural rescue whereby a flexible loop inserts itself into the ß trefoil domain and increases thermal stability. The exon 3 deletion is not tolerated in the corresponding RYR1 domain. The rescue shows a novel mechanism by which RYR2 channels can adjust their Ca²âº release properties through altering the structure of the NTD. Despite the rescue, the deletion affects interfaces with other RYR2 domains. We propose that relative movement of the NTD is allosterically coupled to the pore region.