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1.
J Nat Prod ; 87(1): 104-112, 2024 01 26.
Artigo em Inglês | MEDLINE | ID: mdl-38128916

RESUMO

Calcin is a group ligand with high affinity and specificity for the ryanodine receptors (RyRs). Little is known about the effect of its acidic residues on the spacial structure as well as the interaction with RyRs. We screened the opicalcin1 acidic mutants and investigated the effect of mutation on activity. The results indicated that all acidic mutants maintained the structural features, but their surface charge distribution underwent significant changes. Molecular docking and dynamics simulations were used to analyze the interaction between opicalcin1 mutants and RyRs, which demonstrated that all opicalcin1 mutants effectively bound to the channel domain of RyR1. This stable binding induced a pronounced asymmetry in the structure of the RyR tetramer, exhibiting a high degree of structural dissimilarity. [3H]Ryanodine binding to RyR1 was enhanced in D2A and D15A, which was similar to opicalcin1, but that effect was suppressed in E12A and E29A and reversed for the DE-4A, thereby inhibiting ryanodine binding. Opicalcin1 and DE-4A also exhibited the ability to form stable docking structures with RyR2. Acidic residues play a crucial role in the structure of calcin and its functional interaction with RyRs that is beneficial for the calcin optimization to develop more active peptide lead compounds for RyR-related diseases.


Assuntos
Sinalização do Cálcio , Canal de Liberação de Cálcio do Receptor de Rianodina , Rianodina/metabolismo , Canal de Liberação de Cálcio do Receptor de Rianodina/química , Canal de Liberação de Cálcio do Receptor de Rianodina/genética , Canal de Liberação de Cálcio do Receptor de Rianodina/metabolismo , Simulação de Acoplamento Molecular , Mutação , Cálcio/metabolismo
2.
Cell Mol Life Sci ; 80(8): 202, 2023 Jul 13.
Artigo em Inglês | MEDLINE | ID: mdl-37442828

RESUMO

The epidermal growth factor receptor (EGFR) is one of the main tumor drivers and is an important therapeutic target for many cancers. Calcium is important in EGFR signaling pathways. Sorcin is one of the most important calcium sensor proteins, overexpressed in many tumors, that promotes cell proliferation, migration, invasion, epithelial-to-mesenchymal transition, malignant progression and resistance to chemotherapeutic drugs. The present work elucidates a functional mechanism that links calcium homeostasis to EGFR signaling in cancer. Sorcin and EGFR expression are significantly correlated and associated with reduced overall survival in cancer patients. Mechanistically, Sorcin directly binds EGFR protein in a calcium-dependent fashion and regulates calcium (dys)homeostasis linked to EGF-dependent EGFR signaling. Moreover, Sorcin controls EGFR proteostasis and signaling and increases its phosphorylation, leading to increased EGF-dependent migration and invasion. Of note, silencing of Sorcin cooperates with EGFR inhibitors in the regulation of migration, highlighting calcium signaling pathway as an exploitable target to enhance the effectiveness of EGFR-targeting therapies.


Assuntos
Fator de Crescimento Epidérmico , Neoplasias , Humanos , Fator de Crescimento Epidérmico/farmacologia , Fator de Crescimento Epidérmico/metabolismo , Cálcio , Transdução de Sinais , Receptores ErbB/genética , Receptores ErbB/metabolismo , Linhagem Celular Tumoral , Movimento Celular
4.
J Mol Cell Cardiol ; 167: 118-128, 2022 06.
Artigo em Inglês | MEDLINE | ID: mdl-35413295

RESUMO

Ryanodine receptor 2 (RyR2) is an ion channel in the heart responsible for releasing into the cytosol most of the Ca2+ required for contraction. Proper regulation of RyR2 is critical, as highlighted by the association between channel dysfunction and cardiac arrhythmia. Lower RyR2 expression is also observed in some forms of heart disease; however, there is limited information on the impact of this change on excitation-contraction (e-c) coupling, Ca2+-dependent arrhythmias, and cardiac performance. We used a constitutive knock-out of RyR2 in rabbits (RyR2-KO) to assess the extent to which a stable decrease in RyR2 expression modulates Ca2+ handling in the heart. We found that homozygous knock-out of RyR2 in rabbits is embryonic lethal. Remarkably, heterozygotes (KO+/-) show ~50% loss of RyR2 protein without developing an overt phenotype at the intact animal and whole heart levels. Instead, we found that KO+/- myocytes show (1) remodeling of RyR2 clusters, favoring smaller groups in which channels are more densely arranged; (2) lower Ca2+ spark frequency and amplitude; (3) slower rate of Ca2+ release and mild but significant desynchronization of the Ca2+ transient; and (4) a significant decrease in the basal phosphorylation of S2031, likely due to increased association between RyR2 and PP2A. Our data show that RyR2 deficiency, although remarkable at the molecular and subcellular level, has only a modest impact on global Ca2+ release and is fully compensated at the whole-heart level. This highlights the redundancy of RyR2 protein expression and the plasticity of the e-c coupling apparatus.


Assuntos
Adrenérgicos , Canal de Liberação de Cálcio do Receptor de Rianodina , Animais , Arritmias Cardíacas/metabolismo , Cálcio/metabolismo , Sinalização do Cálcio , Acoplamento Excitação-Contração , Miócitos Cardíacos/metabolismo , Coelhos , Canal de Liberação de Cálcio do Receptor de Rianodina/genética , Canal de Liberação de Cálcio do Receptor de Rianodina/metabolismo , Retículo Sarcoplasmático/metabolismo
5.
J Physiol ; 599(23): 5179-5201, 2021 12.
Artigo em Inglês | MEDLINE | ID: mdl-34676560

RESUMO

Catecholaminergic polymorphic ventricular tachycardia type 1 (CPVT1) is a lethal genetic disease causing arrhythmias and sudden cardiac death in children and young adults and is linked to mutations in the cardiac ryanodine receptor (RyR2). The effects of CPVT1 mutations on RyR2 ion-channel function are often investigated using purified recombinant RyR2 channels homozygous for the mutation. However, CPVT1 patients are heterozygous for the disease, so this approach does not reveal the true changes to RyR2 function across the entire RyR2 population of channels in the heart. We therefore investigated the native cardiac RyR2 single-channel abnormalities in mice heterozygous for the CPVT1 mutation, V2475F(+/-)-RyR2, and applied molecular modelling techniques to investigate the possible structural changes that could initiate any altered function. We observed that increased sensitivity of cardiac V2475F(+/-)-RyR2 channels to both activating and inactivating levels of cytosolic Ca2+ , plus attenuation of Mg2+ inhibition, were the most marked changes. Severity of abnormality was not uniform across all channels, giving rise to multiple sub-populations with differing functional characteristics. For example, 46% of V2475F(+/-)-RyR2 channels exhibited reduced Mg2+ inhibition and 23% were actually activated by Mg2+ . Using homology modelling, we discovered that V2475 is situated at a hinge between two regions of the RyR2 helical domain 1 (HD1). Our model proposes that detrimental functional changes to RyR2 arise because mutation at this critical site reduces the angle between these regions. Our results demonstrate the necessity of characterising the total heterozygous population of CPVT1-mutated channels in order to understand CPVT1 phenotypes in patients. KEY POINTS: RyR2 mutations can cause type-1 catecholaminergic polymorphic ventricular tachycardia (CPVT1), a lethal, autosomal-dominant arrhythmic disease. However, the changes in RyR2 ion-channel function that result from the many different patient mutations are rarely investigated in detail and often only recombinant RyR2, homozygous for the mutation, is studied. As CPVT1 is a heterozygous disease and the tetrameric RyR2 channels expressed in the heart will contain varying numbers of mutated monomers, we have investigated the range of RyR2 single-channel abnormalities found in the hearts of mice heterozygous for the CPVT1 mutation, V2475F(+/-)-RyR2. Specific alterations to ligand regulation of V2475F(+/-)-RyR2 were observed. Multiple sub-populations of channels exhibited varying degrees of abnormality. In particular, an increased sensitivity to activating and inactivating cytosolic [Ca2+ ], and reduced sensitivity to Mg2+ inhibition were evident. Our results provide mechanistic insight into the changes to RyR2 gating that destabilise sarcoplasmic reticulum Ca2+ -release causing life-threatening arrhythmias in V2475F(+/-)-CPVT1 patients.


Assuntos
Canal de Liberação de Cálcio do Receptor de Rianodina , Taquicardia Ventricular , Animais , Cálcio/metabolismo , Sinalização do Cálcio , Humanos , Camundongos , Mutação , Canal de Liberação de Cálcio do Receptor de Rianodina/genética , Canal de Liberação de Cálcio do Receptor de Rianodina/metabolismo , Retículo Sarcoplasmático/metabolismo , Taquicardia Ventricular/genética
6.
Circ Res ; 122(11): 1501-1516, 2018 05 25.
Artigo em Inglês | MEDLINE | ID: mdl-29514831

RESUMO

RATIONALE: In cardiomyocytes, NaV1.5 and Kir2.1 channels interact dynamically as part of membrane bound macromolecular complexes. OBJECTIVE: The objective of this study was to test whether NaV1.5 and Kir2.1 preassemble during early forward trafficking and travel together to common membrane microdomains. METHODS AND RESULTS: In patch-clamp experiments, coexpression of trafficking-deficient mutants Kir2.1Δ314-315 or Kir2.1R44A/R46A with wild-type (WT) NaV1.5WT in heterologous cells reduced inward sodium current compared with NaV1.5WT alone or coexpressed with Kir2.1WT. In cell surface biotinylation experiments, expression of Kir2.1Δ314-315 reduced NaV1.5 channel surface expression. Glycosylation analysis suggested that NaV1.5WT and Kir2.1WT channels associate early in their biosynthetic pathway, and fluorescence recovery after photobleaching experiments demonstrated that coexpression with Kir2.1 increased cytoplasmic mobility of NaV1.5WT, and vice versa, whereas coexpression with Kir2.1Δ314-315 reduced mobility of both channels. Viral gene transfer of Kir2.1Δ314-315 in adult rat ventricular myocytes and human induced pluripotent stem cell-derived cardiomyocytes reduced inward rectifier potassium current and inward sodium current, maximum diastolic potential and action potential depolarization rate, and increased action potential duration. On immunostaining, the AP1 (adaptor protein complex 1) colocalized with NaV1.5WT and Kir2.1WT within areas corresponding to t-tubules and intercalated discs. Like Kir2.1WT, NaV1.5WT coimmunoprecipitated with AP1. Site-directed mutagenesis revealed that NaV1.5WT channels interact with AP1 through the NaV1.5Y1810 residue, suggesting that, like for Kir2.1WT, AP1 can mark NaV1.5 channels for incorporation into clathrin-coated vesicles at the trans-Golgi. Silencing the AP1 ϒ-adaptin subunit in human induced pluripotent stem cell-derived cardiomyocytes reduced inward rectifier potassium current, inward sodium current, and maximum diastolic potential and impaired rate-dependent action potential duration adaptation. CONCLUSIONS: The NaV1.5-Kir2.1 macromolecular complex pre-assembles early in the forward trafficking pathway. Therefore, disruption of Kir2.1 trafficking in cardiomyocytes affects trafficking of NaV1.5, which may have important implications in the mechanisms of arrhythmias in inheritable cardiac diseases.


Assuntos
Complexo 1 de Proteínas Adaptadoras/metabolismo , Miócitos Cardíacos/metabolismo , Canal de Sódio Disparado por Voltagem NAV1.5/metabolismo , Canais de Potássio Corretores do Fluxo de Internalização/metabolismo , Sarcolema/metabolismo , Potenciais de Ação , Animais , Corantes , Humanos , Células-Tronco Pluripotentes Induzidas/metabolismo , Masculino , Potenciais da Membrana/fisiologia , Miócitos Cardíacos/fisiologia , Canal de Sódio Disparado por Voltagem NAV1.5/genética , Canais de Potássio/metabolismo , Canais de Potássio Corretores do Fluxo de Internalização/genética , Canais de Potássio de Abertura Dependente da Tensão da Membrana/metabolismo , Transporte Proteico/fisiologia , Ratos , Ratos Sprague-Dawley , Canais de Sódio Disparados por Voltagem/metabolismo
7.
Proc Natl Acad Sci U S A ; 112(13): E1669-77, 2015 Mar 31.
Artigo em Inglês | MEDLINE | ID: mdl-25775566

RESUMO

Current mechanisms of arrhythmogenesis in catecholaminergic polymorphic ventricular tachycardia (CPVT) require spontaneous Ca(2+) release via cardiac ryanodine receptor (RyR2) channels affected by gain-of-function mutations. Hence, hyperactive RyR2 channels eager to release Ca(2+) on their own appear as essential components of this arrhythmogenic scheme. This mechanism, therefore, appears inadequate to explain lethal arrhythmias in patients harboring RyR2 channels destabilized by loss-of-function mutations. We aimed to elucidate arrhythmia mechanisms in a RyR2-linked CPVT mutation (RyR2-A4860G) that depresses channel activity. Recombinant RyR2-A4860G protein was expressed equally as wild type (WT) RyR2, but channel activity was dramatically inhibited, as inferred by [(3)H]ryanodine binding and single channel recordings. Mice heterozygous for the RyR2-A4860G mutation (RyR2-A4860G(+/-)) exhibited basal bradycardia but no cardiac structural alterations; in contrast, no homozygotes were detected at birth, suggesting a lethal phenotype. Sympathetic stimulation elicited malignant arrhythmias in RyR2-A4860G(+/-) hearts, recapitulating the phenotype originally described in a human patient with the same mutation. In isoproterenol-stimulated ventricular myocytes, the RyR2-A4860G mutation decreased the peak of Ca(2+) release during systole, gradually overloading the sarcoplasmic reticulum with Ca(2+). The resultant Ca(2+) overload then randomly caused bursts of prolonged Ca(2+) release, activating electrogenic Na(+)-Ca(2+) exchanger activity and triggering early afterdepolarizations. The RyR2-A4860G mutation reveals novel pathways by which RyR2 channels engage sarcolemmal currents to produce life-threatening arrhythmias.


Assuntos
Arritmias Cardíacas/genética , Mutação , Canal de Liberação de Cálcio do Receptor de Rianodina/metabolismo , Taquicardia Ventricular/genética , Animais , Arritmias Cardíacas/fisiopatologia , Cálcio/metabolismo , Coração/fisiologia , Heterozigoto , Homozigoto , Humanos , Isoproterenol/química , Camundongos , Miocárdio/metabolismo , Miócitos Cardíacos/metabolismo , Retículo Sarcoplasmático/metabolismo
9.
J Physiol ; 592(9): 1957-73, 2014 May 01.
Artigo em Inglês | MEDLINE | ID: mdl-24445321

RESUMO

Phosphorylation of the cardiac ryanodine receptor (RyR2) by protein kinase A (PKA) at Ser-2808 is suggested to mediate the physiological 'fight or flight' response and contribute to heart failure by rendering the sarcoplasmic reticulum (SR) leaky for Ca(2+). In the present study, we examined the potential role of RyR2 phosphorylation at Ser-2808 in the progression of Ca(2+)-dependent cardiomyopathy (CCM) by using mice genetically modified to feature elevated SR Ca(2+) leak while expressing RyR2s that cannot be phosphorylated at this site (S2808A). Surprisingly, rather than alleviating the disease phenotype, constitutive dephosphorylation of Ser-2808 aggravated CCM as manifested by shortened survival, deteriorated in vivo cardiac function, exacerbated SR Ca(2+) leak and mitochondrial injury. Notably, the deteriorations of cardiac function, myocyte Ca(2+) handling, and mitochondria integrity were consistently worse in mice with heterozygous ablation of Ser-2808 than in mice with complete ablation. Wild-type (WT) and CCM myocytes expressing unmutated RyR2s exhibited a high level of baseline phosphorylation at Ser-2808. Exposure of these CCM cells to protein phosphatase 1 caused a transitory increase in Ca(2+) leak attributable to partial dephosphorylation of RyR2 tetramers at Ser-2808 from more fully phosphorylated state. Thus, exacerbated Ca(2+) leak through partially dephosphorylated RyR2s accounts for the prevalence of the disease phenotype in the heterozygous S2808A CCM mice. These results do not support the importance of RyR2 hyperphosphorylation in Ca(2+)-dependent heart disease, and rather suggest roles for the opposite process, the RyR2 dephosphorylation at this residue in physiological and pathophysiological Ca(2+) signalling.


Assuntos
Cálcio/metabolismo , Cardiomiopatias/genética , Cardiomiopatias/metabolismo , Canal de Liberação de Cálcio do Receptor de Rianodina/genética , Canal de Liberação de Cálcio do Receptor de Rianodina/metabolismo , Serina/genética , Animais , Feminino , Masculino , Camundongos , Camundongos Knockout , Camundongos Transgênicos , Fosforilação/fisiologia
10.
Int J Biol Macromol ; 260(Pt 1): 129424, 2024 Mar.
Artigo em Inglês | MEDLINE | ID: mdl-38219929

RESUMO

Calcins are a group of scorpion toxin peptides specifically binding to ryanodine receptors (RyRs) with high affinity, and have the ability to activate and stabilize RyR in a long-lasting subconductance state. Five newly calcins synthesized compounds exhibit typical structural characteristics of a specific family through chemical synthesis and virtual analysis. As the calcins from the same species, Petersiicalcin1 and Petersiicalcin2, Jendekicalcin2 and Jendekicalcin3, have only one residue difference. Both Petersiicalcin1 and Petersiicalcin2 exhibited different affinities in stimulating [3H]ryanodine binding, but the residue mutation resulted in a 2.7 folds difference. Other calcins also exhibited a stimulatory effect on [3H]ryanodine binding to RyR1, however, their affinities were significantly lower than that of Petersiiicalcin1 and Petersiiicalcin2. The channel domain of RyR1 was found to be capable of binding with the basic residues of these calcins, which also exhibited interactions with the S6 helices on RyR1. Dynamic simulations were conducted for Petersiicalcin1 and Petersiicalcin2, which demonstrated their ability to form a highly stable conformation and resulting in an asymmetric tetramer structure of RyR1. The discovery of five newly calcins further enriches the diversity of the natural calcin family, which provides more native peptides for the structure-function analysis between calcin and RyRs.


Assuntos
Peptídeos , Canal de Liberação de Cálcio do Receptor de Rianodina , Canal de Liberação de Cálcio do Receptor de Rianodina/genética , Canal de Liberação de Cálcio do Receptor de Rianodina/química , Canal de Liberação de Cálcio do Receptor de Rianodina/metabolismo , Sequência de Aminoácidos , Rianodina/metabolismo , Rianodina/farmacologia , Peptídeos/química , Estrutura Secundária de Proteína , Cálcio/metabolismo , Músculo Esquelético
11.
J Gen Physiol ; 156(4)2024 Apr 01.
Artigo em Inglês | MEDLINE | ID: mdl-38385988

RESUMO

We have previously demonstrated that type II ryanodine receptors (RyR2) tetramers can be rapidly rearranged in response to a phosphorylation cocktail. The cocktail modified downstream targets indiscriminately, making it impossible to determine whether phosphorylation of RyR2 was an essential element of the response. Here, we used the ß-agonist isoproterenol and mice homozygous for one of the following clinically relevant mutations: S2030A, S2808A, S2814A, or S2814D. We measured the length of the dyad using transmission electron microscopy (TEM) and directly visualized RyR2 distribution using dual-tilt electron tomography. We found that the S2814D mutation, by itself, significantly expanded the dyad and reorganized the tetramers, suggesting a direct link between the phosphorylation state of the tetramer and its microarchitecture. S2808A and S2814A mutant mice, as well as wild types, had significant expansions of their dyads in response to isoproterenol, while S2030A mutants did not. In agreement with functional data from these mutants, S2030 and S2808 were necessary for a complete ß-adrenergic response, unlike S2814 mutants. Additionally, all mutants had unique effects on the organization of their tetramer arrays. Lastly, the correlation of structural with functional changes suggests that tetramer-tetramer contacts play an important functional role. We thus conclude that both the size of the dyad and the arrangement of the tetramers are linked to the state of the channel tetramer and can be dynamically altered by a ß-adrenergic receptor agonist.


Assuntos
Canal de Liberação de Cálcio do Receptor de Rianodina , Animais , Camundongos , Isoproterenol/farmacologia , Mutação , Fosforilação , Canal de Liberação de Cálcio do Receptor de Rianodina/química
12.
J Mol Cell Cardiol ; 61: 102-10, 2013 Aug.
Artigo em Inglês | MEDLINE | ID: mdl-23541953

RESUMO

AIMS: Mutations in CAV3-encoding caveolin-3 (Cav3) have been implicated in type 9 long QT syndrome (LQT9) and sudden infant death syndrome (SIDS). When co-expressed with SCN5A-encoded cardiac sodium channels these mutations increased late sodium current (INa) but the mechanism was unclear. The present study was designed to address the mechanism by which the LQT9-causing mutant Cav3-F97C affects the function of caveolar SCN5A. METHODS AND RESULTS: HEK-293 cells expressing SCN5A and LQT9 mutation Cav3-F97C resulted in a 2-fold increase in late INa compared to Cav3-WT. This increase was reversed by the neural nitric oxide synthase (nNOS) inhibitor L-NMMA. Based on these findings, we hypothesized that an nNOS complex mediated the effect of Cav3 on SCN5A. A SCN5A macromolecular complex was established in HEK-293 cells by transiently expressing SCN5A, α1-syntrophin (SNTA1), nNOS, and Cav3. Compared with Cav3-WT, Cav3-F97C produced significantly larger peak INa amplitudes, and showed 3.3-fold increase in the late INa associated with increased S-nitrosylation of SCN5A. L-NMMA reversed both the Cav3-F97C induced increase in late and peak INa and decreased S-nitrosylation of SCN5A. Overexpression of Cav3-F97C in adult rat cardiomyocytes caused a significant increase in late INa compared to Cav3-WT, and prolonged the action potential duration (APD90) in a nNOS-dependent manner. CONCLUSIONS: Cav3 is identified as an important negative regulator for cardiac late INa via nNOS dependent direct S-nitrosylation of SCN5A. This provides a molecular mechanism for how Cav3 mutations increase late INa to cause LQT9. This article is part of a Special Issue entitled "Na(+) Regulation in Cardiac Myocytes".


Assuntos
Caveolina 3/fisiologia , Canal de Sódio Disparado por Voltagem NAV1.5/metabolismo , Óxido Nítrico Sintase Tipo I/metabolismo , S-Nitrosotióis/metabolismo , Animais , Células HEK293 , Humanos , Síndrome do QT Longo/genética , Potenciais da Membrana , Mutação de Sentido Incorreto , Miócitos Cardíacos/fisiologia , Óxido Nítrico/metabolismo , Processamento de Proteína Pós-Traducional , Ratos , Sódio/metabolismo
13.
Am J Physiol Heart Circ Physiol ; 304(7): H994-H1001, 2013 Apr 01.
Artigo em Inglês | MEDLINE | ID: mdl-23376825

RESUMO

SCN5A and SNTA1 are reported susceptible genes for long QT syndrome (LQTS). This study was designed to elucidate a plausible pathogenic arrhythmia mechanism for the combined novel mutations R800L-SCN5A and A261V-SNTA1 on cardiac sodium channels. A Caucasian family with syncope and marginally prolonged QT interval was screened for LQTS-susceptibility genes and found to harbor the R800L mutation in SCN5A and A261V mutation in SNTA1, and those with both mutations had the strongest clinical phenotype. The mutations were engineered into the most common splice variant of human SCN5A and SNTA1 cDNA, respectively, and sodium current (INa) was characterized in human embryonic kidney 293 cells cotransfected with neuronal nitric oxide synthase (nNOS) and the cardiac isoform of the plasma membrane Ca-ATPase (PMCA4b). Peak INa densities were unchanged for wild-type (WT) and for mutant channels containing R800L-SCN5A, A261V-SNTA1, or R800L-SCN5A plus A261V-SNTA1. However, late INa for either single mutant was moderately increased two- to threefold compared with WT. The combined mutations of R800L-SCN5A plus A261V-SNTA1 significantly enhanced the INa late/peak ratio by 5.6-fold compared with WT. The time constants of current decay of combined mutant channel were markedly increased. The gain-of-function effect could be blocked by the N(G)-monomethyl-l-arginine, a nNOS inhibitor. We conclude that novel mutations in SCN5A and SNTA1 jointly exert a nNOS-dependent gain-of-function on SCN5A channels, which may consequently prolong the action potential duration and lead to LQTS phenotype.


Assuntos
Potenciais de Ação/genética , Proteínas de Ligação ao Cálcio/genética , Síndrome do QT Longo/genética , Proteínas de Membrana/genética , Proteínas Musculares/genética , Mutação de Sentido Incorreto , Canal de Sódio Disparado por Voltagem NAV1.5/genética , Adulto , Idoso , Proteínas de Ligação ao Cálcio/metabolismo , Criança , Feminino , Predisposição Genética para Doença , Células HEK293 , Humanos , Síndrome do QT Longo/diagnóstico , Masculino , Proteínas de Membrana/metabolismo , Proteínas Musculares/metabolismo , Canal de Sódio Disparado por Voltagem NAV1.5/metabolismo , Óxido Nítrico Sintase/antagonistas & inibidores , Óxido Nítrico Sintase/metabolismo , Linhagem , Fenótipo , ATPases Transportadoras de Cálcio da Membrana Plasmática/metabolismo , Sódio/metabolismo , Síncope/genética , ômega-N-Metilarginina/farmacologia
14.
Sci Adv ; 9(21): eadf4936, 2023 05 24.
Artigo em Inglês | MEDLINE | ID: mdl-37224245

RESUMO

Calcins are peptides from scorpion venom with the unique ability to cross cell membranes, gaining access to intracellular targets. Ryanodine Receptors (RyR) are intracellular ion channels that control release of Ca2+ from the endoplasmic and sarcoplasmic reticulum. Calcins target RyRs and induce long-lived subconductance states, whereby single-channel currents are decreased. We used cryo-electron microscopy to reveal the binding and structural effects of imperacalcin, showing that it opens the channel pore and causes large asymmetry throughout the cytosolic assembly of the tetrameric RyR. This also creates multiple extended ion conduction pathways beyond the transmembrane region, resulting in subconductance. Phosphorylation of imperacalcin by protein kinase A prevents its binding to RyR through direct steric hindrance, showing how posttranslational modifications made by the host organism can determine the fate of a natural toxin. The structure provides a direct template for developing calcin analogs that result in full channel block, with potential to treat RyR-related disorders.


Assuntos
Canal de Liberação de Cálcio do Receptor de Rianodina , Venenos de Escorpião , Fosforilação , Microscopia Crioeletrônica , Proteínas Quinases Dependentes de AMP Cíclico , Venenos de Escorpião/farmacologia
15.
Mater Today Bio ; 23: 100859, 2023 Dec.
Artigo em Inglês | MEDLINE | ID: mdl-38033368

RESUMO

Background: Reducing Ca2+ content in the sarcoplasmic reticulum (SR) through ryanodine receptors (RyRs) by calcin is a potential intervention strategy for the SR Ca2+ overload triggered by ß-adrenergic stress in acute heart diseases. Methods: OpiCal-PEG-PLGA nanomicelles were prepared by thin film dispersion, of which the antagonistic effects were observed using an acute heart failure model induced by epinephrine and caffeine in mice. In addition, cardiac targeting, self-stability as well as biotoxicity were determined. Results: The synthesized OpiCa1-PEG-PLGA nanomicelles were elliptical with a particle size of 72.26 nm, a PDI value of 0.3, and a molecular weight of 10.39 kDa. The nanomicelles showed a significant antagonistic effect with 100 % survival rate to the death induced by epinephrine and caffeine, which was supported by echocardiography with significantly recovered heart rate, ejection fraction and left ventricular fractional shortening rate. The FITC labeled nanomicelles had a strong membrance penetrating capacity within 2 h and cardiac targeting within 12 h that was further confirmed by immunohistochemistry with a self-prepared OpiCa1 polyclonal antibody. Meanwhile, the nanomicelles can keep better stability and dispersibility in vitro at 4 °C rather than 20 °C or 37 °C, while maintain a low but stable plasma OpiCa1 concentration in vivo within 72 h. Finally, no obvious biotoxicities were observed by CCK-8, flow cytometry, H&E staining and blood biochemical examinations. Conclusion: Our study also provide a novel nanodelivery pathway for targeting RyRs and antagonizing the SR Ca2+ disordered heart diseases by actively releasing SR Ca2+ through RyRs with calcin.

16.
Physiol Genomics ; 43(9): 461-6, 2011 May 13.
Artigo em Inglês | MEDLINE | ID: mdl-21385947

RESUMO

The common polymorphism SCN5A-S1103Y (∼13% allelic frequency in African Americans) is a risk factor for arrhythmia, sudden unexplained death (SUD), and sudden infant death syndrome. Prompted by a case of autopsy-negative SUD in a 23-year-old African American man who collapsed while playing football, we hypothesized that S1103Y interacted with other SCN5A variants to pathologically modify sodium current (I(Na)). Mutational analysis of arrhythmia-associated genes in the victim revealed the variants SCN5A-R680H and SCN5A-S1103Y. These variants were made both separately and in the same cDNA construct of the alternative splice variant backgrounds (SCN5A-Q1077del and Q1077) and expressed in HEK293 cells. In the most abundant SCN5A-Q1077del, late I(Na) for S1103Y alone was not significantly different from wild type (WT). However, late I(Na) for R680H, R680H+S1103Y (coexpressed), and R680H/S1103Y (on the same cDNA) was increased 2.1-, 3.4-, and 3.6-fold, respectively, compared with WT. Intracellular acidosis (pH 6.7) increased late I(Na) for S1103Y, R680H, R680H+S1103Y, and R680H/S1103Y by 2.2-, 2.4-, 5.0-, and 5.5-fold, respectively, compared with WT at pH 6.7. Expression in the less abundant SCN5A-Q1077 showed no increased late I(Na). This is the initial report of a functional interaction for the common polymorphism S1103Y with another mutation in the major transcript Q1077del of SCN5A. The "double hit" and environmental factor of acidosis may have converged to cause arrhythmic sudden death in this case.


Assuntos
Negro ou Afro-Americano/genética , Mutação , Polimorfismo Genético , Canais de Sódio/genética , Arritmias Cardíacas/genética , Sequência de Bases , Análise Mutacional de DNA , Morte Súbita/etnologia , Frequência do Gene , Células HEK293 , Humanos , Masculino , Dados de Sequência Molecular , Canal de Sódio Disparado por Voltagem NAV1.5 , Sódio/metabolismo , Canais de Sódio/metabolismo
17.
Am J Physiol Heart Circ Physiol ; 297(4): H1446-52, 2009 Oct.
Artigo em Inglês | MEDLINE | ID: mdl-19666841

RESUMO

The SCN5A-encoded cardiac sodium channel underlies excitability in the heart, and dysfunction of sodium current (I(Na)) can cause fatal ventricular arrhythmia in maladies such as long QT syndrome, Brugada syndrome (BrS), and sudden infant death syndrome (SIDS). The gene GPD1L encodes the glycerol phosphate dehydrogenase 1-like protein with homology to glycerol phosphate dehydrogenase (GPD1), but the function for this enzyme is unknown. Mutations in GPD1L have been associated with BrS and SIDS and decrease I(Na) through an unknown mechanism. Using a heterologous expression system, we show that GPD1L associated with SCN5A and that the BrS- and SIDS-related mutations in GPD1L caused a loss of enzymatic function resulting in glycerol-3-phosphate PKC-dependent phosphorylation of SCN5A at serine 1503 (S1503) through a GPD1L-dependent pathway. The direct phosphorylation of S1503 markedly decreased I(Na). These results show a function for GPD1L in cell physiology and a mechanism linking mutations in GPD1L to sudden cardiac arrest. Because the enzymatic step catalyzed by GPD1L depends upon nicotinamide adenine dinucleotide, this GPD1L pathway links the metabolic state of the cell to I(Na) and excitability and may be important more generally in cardiac ischemia and heart failure.


Assuntos
Glicerolfosfato Desidrogenase/metabolismo , Proteínas Musculares/metabolismo , Miocárdio/metabolismo , Proteína Quinase C/metabolismo , Canais de Sódio/metabolismo , Sódio/metabolismo , Síndrome de Brugada/genética , Síndrome de Brugada/metabolismo , Linhagem Celular , Morte Súbita Cardíaca/etiologia , Glicerolfosfato Desidrogenase/genética , Glicerofosfatos/metabolismo , Humanos , Recém-Nascido , Potenciais da Membrana , Proteínas Musculares/genética , Mutação , Canal de Sódio Disparado por Voltagem NAV1.5 , Oxirredução , Fosforilação , Proteína Quinase C/antagonistas & inibidores , Inibidores de Proteínas Quinases/farmacologia , Serina , Canais de Sódio/genética , Morte Súbita do Lactente/genética , Transfecção , Subunidade beta-1 do Canal de Sódio Disparado por Voltagem
18.
JCI Insight ; 52019 03 05.
Artigo em Inglês | MEDLINE | ID: mdl-30835254

RESUMO

Hypertrophic cardiomyopathy (HCM) is triggered mainly by mutations in genes encoding sarcomeric proteins, but a significant proportion of patients lack a genetic diagnosis. We identified a novel mutation in the ryanodine receptor 2, RyR2-P1124L, in a patient from a genotype-negative HCM cohort. The aim of this study was to determine whether RyR2-P1124L triggers functional and structural alterations in isolated RyR2 channels and whole hearts. We found that P1124L induces significant conformational changes in the SPRY2 domain of RyR2. Recombinant RyR2-P1124L channels displayed a cytosolic loss-of-function phenotype, which contrasted with a higher sensitivity to luminal [Ca2+], indicating a luminal gain-of-function. Homozygous mice for RyR2-P1124L showed mild cardiac hypertrophy, similar to the human patient. This phenotype, evident at 1 yr of age, was accompanied by an increase in the expression of calmodulin (CaM). P1124L mice also showed higher susceptibility to arrhythmia at 8 mo of age, before the onset of hypertrophy. RyR2-P1124L has a distinct cytosolic loss-of-function and a luminal gain-of-function phenotype. This bifunctionally-divergent behavior triggers arrhythmias and structural cardiac remodeling, and involves overexpression of calmodulin as a potential hypertrophic mediator. This study is relevant to continue elucidating the possible causes of genotype-negative HCM and the role of RyR2 in cardiac hypertrophy.


Assuntos
Arritmias Cardíacas/genética , Cardiomegalia/genética , Cardiomegalia/metabolismo , Predisposição Genética para Doença/genética , Mutação , Canal de Liberação de Cálcio do Receptor de Rianodina/genética , Adolescente , Animais , Arritmias Cardíacas/metabolismo , Calmodulina/metabolismo , Cardiomegalia/patologia , Ecocardiografia , Feminino , Células HEK293 , Coração/fisiopatologia , Humanos , Peptídeos e Proteínas de Sinalização Intracelular , Masculino , Proteínas de Membrana/metabolismo , Camundongos , Camundongos Endogâmicos C57BL , Camundongos Knockout , Modelos Moleculares , Fenótipo , Conformação Proteica , Domínios Proteicos , Proteínas Serina-Treonina Quinases , Análise de Sequência de Proteína
19.
Circulation ; 116(20): 2253-9, 2007 Nov 13.
Artigo em Inglês | MEDLINE | ID: mdl-17967976

RESUMO

BACKGROUND: Autopsy-negative sudden unexplained death, including sudden infant death syndrome, can be caused by cardiac channelopathies such as Brugada syndrome (BrS). Type 1 BrS, caused by mutations in the SCN5A-encoded sodium channel, accounts for approximately 20% of BrS. Recently, a novel mutation in the glycerol-3-phosphate dehydrogenase 1-like gene (GPD1-L) disrupted trafficking of SCN5A in a multigenerational family with BrS. We hypothesized that mutations in GPD1-L may be responsible for some cases of sudden unexplained death/sudden infant death syndrome. METHODS AND RESULTS: Using denaturing high-performance liquid chromatography and direct DNA sequencing, we performed comprehensive open-reading frame/splice site mutational analysis of GPD1-L on genomic DNA extracted from necropsy tissue of 83 unrelated cases of sudden unexplained death (26 females, 57 males; average age, 14.6+/-10.7 years; range, 1 month to 48 years). A putative, sudden unexplained death-associated GPD1-L missense mutation, E83K, was discovered in a 3-month-old white boy. Further mutational analysis was then performed on genomic DNA derived from a population-based cohort of 221 anonymous cases of sudden infant death syndrome (84 females, 137 males; average age, 3+/-2 months; range, 3 days to 12 months), revealing 2 additional mutations, I124V and R273C, in a 5-week-old white girl and a 1-month-old white boy, respectively. All mutations occurred in highly conserved residues and were absent in 600 reference alleles. Compared with wild-type GPD1-L, GPD1-L mutations coexpressed with SCN5A in heterologous HEK cells produced a significantly reduced sodium current (P<0.01). Adenovirus-mediated gene transfer of the E83K-GPD1-L mutation into neonatal mouse myocytes markedly attenuated the sodium current (P<0.01). These decreases in current density are consistent with sodium channel loss-of-function diseases like BrS. CONCLUSIONS: The present study is the first to report mutations in GPD1-L as a pathogenic cause for a small subset of sudden infant death syndrome via a secondary loss-of-function mechanism whereby perturbations in GPD1-L precipitate a marked decrease in the peak sodium current and a potentially lethal BrS-like proarrhythmic substrate.


Assuntos
Morte Súbita/etiologia , Glicerolfosfato Desidrogenase/genética , Morte Súbita do Lactente/genética , Desidrogenase do Álcool de Açúcar/genética , Adolescente , Adulto , Linhagem Celular , Criança , Pré-Escolar , Estudos de Coortes , Análise Mutacional de DNA , Morte Súbita/epidemiologia , Feminino , Predisposição Genética para Doença/epidemiologia , Glicerolfosfato Desidrogenase/metabolismo , Humanos , Lactente , Recém-Nascido , Rim/citologia , Masculino , Pessoa de Meia-Idade , Mutação , Morte Súbita do Lactente/epidemiologia , Desidrogenase do Álcool de Açúcar/metabolismo
20.
Circ Arrhythm Electrophysiol ; 11(3): e005659, 2018 03.
Artigo em Inglês | MEDLINE | ID: mdl-29540372

RESUMO

BACKGROUND: The mechanisms underlying spontaneous atrial fibrillation (AF) associated with atrial ischemia/infarction are incompletely elucidated. Here, we investigate the mechanisms underlying spontaneous AF in an ovine model of left atrial myocardial infarction (LAMI). METHODS AND RESULTS: LAMI was created by ligating the atrial branch of the left anterior descending coronary artery. ECG loop recorders were implanted to monitor AF episodes. In 7 sheep, dantrolene-a ryanodine receptor blocker-was administered in vivo during the 8-day observation period (LAMI-D, 2.5 mg/kg, IV, BID). LAMI animals experienced numerous spontaneous AF episodes during the 8-day monitoring period that were suppressed by dantrolene (LAMI, 26.1±5.1; sham, 4.3±1.1; LAMI-D, 2.8±0.8; mean±SEM episodes per sheep, P<0.01). Optical mapping showed spontaneous focal discharges (SFDs) originating from the ischemic/normal-zone border. SFDs were calcium driven, rate dependent, and enhanced by isoproterenol (0.03 µmol/L, from 210±87 to 3816±1450, SFDs per sheep) but suppressed by dantrolene (to 55.8±32.8, SFDs per sheep, mean±SEM). SFDs initiated AF-maintaining reentrant rotors anchored by marked conduction delays at the ischemic/normal-zone border. NOS1 (NO synthase-1) protein expression decreased in ischemic zone myocytes, whereas NADPH (nicotinamide adenine dinucleotide phosphate, reduced form) oxidase and xanthine oxidase enzyme activities and reactive oxygen species (DCF [6-carboxy-2',7'-dichlorodihydrofluorescein diacetate]-fluorescence) increased. CaM (calmodulin) aberrantly increased [3H]ryanodine binding to cardiac RyR2 (ryanodine receptors) in the ischemic zone. Dantrolene restored the physiological binding of CaM to RyR2. CONCLUSIONS: Atrial ischemia causes spontaneous AF episodes in sheep, caused by SFDs that initiate reentry. Nitroso-redox imbalance in the ischemic zone is associated with intense reactive oxygen species production and altered RyR2 responses to CaM. Dantrolene administration normalizes the CaM response, prevents LAMI-related SFDs, and AF initiation. These findings provide novel insights into the mechanisms underlying ischemia-related atrial arrhythmias.


Assuntos
Fibrilação Atrial/complicações , Dantroleno/farmacologia , Isquemia Miocárdica/etiologia , Canal de Liberação de Cálcio do Receptor de Rianodina/metabolismo , Animais , Fibrilação Atrial/metabolismo , Fibrilação Atrial/fisiopatologia , Fibrilação Atrial/terapia , Western Blotting , Sinalização do Cálcio , Modelos Animais de Doenças , Átrios do Coração , Masculino , Relaxantes Musculares Centrais/farmacologia , Isquemia Miocárdica/metabolismo , Isquemia Miocárdica/fisiopatologia , Miócitos Cardíacos/metabolismo , Canal de Liberação de Cálcio do Receptor de Rianodina/efeitos dos fármacos , Retículo Sarcoplasmático/metabolismo , Ovinos
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