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1.
FEBS Lett ; 594(3): 497-508, 2020 02.
Artigo em Inglês | MEDLINE | ID: mdl-31626714

RESUMO

The voltage-gated sodium channels (VGSCs) are aberrantly expressed in a variety of tumors and play an important role in tumor growth and metastasis. Here, we show that VGSCs auxiliary ß3 subunit, encoded by the SCN3B gene, promotes proliferation and suppresses apoptosis in HepG2 cells by promoting p53 degradation. ß3 significantly increases HepG2 cell proliferation, promotes tumor growth in mouse xenograft models, and suppresses senescence and apoptosis. We found that ß3 knockdown stabilizes p53 protein, leading to potentiation of p53-induced cell cycle arrest, senescence, and apoptosis. Mechanistic studies revealed that ß3 could bind to p53, promoting p53 ubiquitination and degradation by stabilizing the p53/MDM2 complex. Our results suggest that ß3 is a novel negative regulator of p53 and a potential oncogenic factor.


Assuntos
Carcinogênese , Carcinoma Hepatocelular/patologia , Neoplasias Hepáticas/patologia , Proteólise , Proteína Supressora de Tumor p53/metabolismo , Subunidade beta-3 do Canal de Sódio Disparado por Voltagem/metabolismo , Carcinoma Hepatocelular/metabolismo , Ciclo Celular , Proliferação de Células , Senescência Celular , Técnicas de Silenciamento de Genes , Células Hep G2 , Humanos , Neoplasias Hepáticas/metabolismo , Proteínas Proto-Oncogênicas c-mdm2/metabolismo , Ubiquitinação , Subunidade beta-3 do Canal de Sódio Disparado por Voltagem/deficiência , Subunidade beta-3 do Canal de Sódio Disparado por Voltagem/genética
2.
Audiol Neurootol ; 23(3): 135-144, 2018.
Artigo em Inglês | MEDLINE | ID: mdl-30300896

RESUMO

Considering the possibility of a common genetic background of vertigo and epilepsy, we genotyped an affected group of individuals with vertigo and an unaffected group, by studying 26 single-nucleotide polymorphisms (SNPs) in 14 genes which were previously reported to be of particular importance for epilepsy. Significant differences were found between the patients and the control group (χ2 = 38.3, df = 3, p = 1.6 × 10-7) for the frequencies of haplotypes consist ing of 2 SNPs located in chromosome 11 (rs1939012 and rs1783901 within genes MMP8 and SCN3B, respectively). The haplotype rs1939012:C-rs1783901:A, consisting of the minor-frequency alleles was found to be associated with a higher risk of vertigo (OR = 5.0143, 95% CI = 1.6991-14.7980, p = 0.0035). In contrast, the haplotype rs1939012:T-rs1783901:A showed a significant association with a decreased risk of the disease (OR = 0.0597, 95% CI = 0.0136-0.2620, p = 0.0002). Our results suggest that the SNPs rs1939012 and rs1783901 may play a potential role of gene regulation and/or epistasis in a complex etiology of vertigo.


Assuntos
Epilepsia/genética , Metaloproteinase 8 da Matriz/genética , Vertigem/genética , Subunidade beta-3 do Canal de Sódio Disparado por Voltagem/genética , Adulto , Idoso , Estudos de Casos e Controles , Epistasia Genética , Feminino , Regulação da Expressão Gênica , Frequência do Gene , Predisposição Genética para Doença , Genótipo , Haplótipos , Humanos , Masculino , Pessoa de Meia-Idade , Polônia , Polimorfismo de Nucleotídeo Único , Adulto Jovem
3.
J Am Heart Assoc ; 5(11)2016 11 02.
Artigo em Inglês | MEDLINE | ID: mdl-27806967

RESUMO

BACKGROUND: Ganglionated plexus have been developed as additional ablation targets to improve the outcome of atrial fibrillation (AF) besides pulmonary vein isolation. Recent studies implicated an intimate relationship between neuronal sodium channel Nav1.8 (encoded by SCN10A) and AF. The underlying mechanism between Nav1.8 and AF remains unclear. This study aimed to determine the role of Nav1.8 in cardiac electrophysiology in an acute AF model and explore possible therapeutic targets. METHODS AND RESULTS: Immunohistochemical study was used on canine cardiac ganglionated plexus. Both Nav1.5 and Nav1.8 were expressed in ganglionated plexus with canonical neuronal markers. Sixteen canines were randomly administered either saline or the Nav1.8 blocker A-803467. Electrophysiological study was compared between the 2 groups before and after 6-hour rapid atrial pacing. Compared with the control group, administration of A-803467 decreased the incidence of AF (87.5% versus 25.0%, P<0.05), shortened AF duration, and prolonged AF cycle length. A-803467 also significantly suppressed the decrease in the effective refractory period and the increase in effective refractory period dispersion and cumulative window of vulnerability caused by rapid atrial pacing in all recording sites. Patch clamp study was performed under 100 nmol/L A-803467 in TSA201 cells cotransfected with SCN10A-WT, SCN5A-WT, and SCN3B-WT. INa,P was reduced by 45.34% at -35 mV, and INa,L by 68.57% at -20 mV. Evident fast inactivation, slow recovery, and use-dependent block were also discovered after applying the drug. CONCLUSIONS: Our study demonstrates that Nav1.8 could exert its effect on electrophysiological characteristics through cardiac ganglionated plexus. It indicates that Nav1.8 is a novel target in understanding cardiac electrophysiology and SCN10A-related arrhythmias.


Assuntos
Fibrilação Atrial/metabolismo , Coração/inervação , Canal de Sódio Disparado por Voltagem NAV1.8/metabolismo , Neurônios/metabolismo , Doença Aguda , Compostos de Anilina/farmacologia , Animais , Fibrilação Atrial/terapia , Cães , Furanos/farmacologia , Coração/efeitos dos fármacos , Imuno-Histoquímica , Canal de Sódio Disparado por Voltagem NAV1.5/genética , Canal de Sódio Disparado por Voltagem NAV1.8/genética , Técnicas de Patch-Clamp , Fatores de Tempo , Bloqueadores do Canal de Sódio Disparado por Voltagem/farmacologia , Subunidade beta-3 do Canal de Sódio Disparado por Voltagem/genética
4.
BMC Cardiovasc Disord ; 16: 1, 2016 Jan 04.
Artigo em Inglês | MEDLINE | ID: mdl-26728597

RESUMO

BACKGROUND: In the initiation and maintenance of arrhythmia, inflammatory processes play an important role. IL-2 is a pro-inflammatory factor which is associated with the morbidity of arrhythmias, however, how IL-2 affects the cardiac electrophysiology is still unknown. METHODS: In the present study, we observed the effect of IL-2 by qRT-PCR on the transcription of ion channel genes including SCN2A, SCN3A, SCN4A, SCN5A, SCN9A, SCN10A, SCN1B, SCN2B, SCN3B, KCNN1, KCNJ5, KCNE1, KCNE2, KCNE3, KCND3, KCNQ1, KCNA5, KCNH2 and CACNA1C. Western blot assays and electrophysiological studies were performed to demonstrate the effect of IL-2 on the translation of SCN3B/scn3b and sodium currents. RESULTS: The results showed that transcriptional level of SCN3B was up-regulated significantly in Hela cells (3.28-fold, p = 0.022 compared with the control group). Consistent results were verified in HL-1 cells (3.73-fold, p = 0.012 compared with the control group). The result of electrophysiological studies showed that sodium current density increased significantly in cells which treated by IL-2 and the effect of IL-2 on sodium currents was independent of SCN3B (1.4 folds, p = 0.023). Western blot analysis showed IL-2 lead to the significantly increasing of p53 and scn3b (2.1 folds, p = 0.021 for p53; 3.1 folds, p = 0.023 for scn3b) in HL-1 cells. Consistent results were showed in HEK293 cells using qRT-PCR analysis (1.43 folds for P53, p = 0.022; 1.57 folds for SCN3B, p = 0.05). CONCLUSIONS: The present study suggested that IL-2, may play role in the arrhythmia by regulating the expression of SCN3B and sodium current density.


Assuntos
Interleucina-2/farmacologia , Potenciais da Membrana/efeitos dos fármacos , Miócitos Cardíacos/efeitos dos fármacos , Canais de Potássio de Abertura Dependente da Tensão da Membrana/efeitos dos fármacos , Sódio/metabolismo , Subunidade beta-3 do Canal de Sódio Disparado por Voltagem/efeitos dos fármacos , Canais de Cálcio/efeitos dos fármacos , Canais de Cálcio/genética , Regulação da Expressão Gênica , Células HEK293 , Células HeLa , Humanos , Miócitos Cardíacos/metabolismo , Técnicas de Patch-Clamp , Canais de Potássio de Abertura Dependente da Tensão da Membrana/genética , RNA Mensageiro/efeitos dos fármacos , RNA Mensageiro/metabolismo , Reação em Cadeia da Polimerase Via Transcriptase Reversa , Subunidade beta-3 do Canal de Sódio Disparado por Voltagem/genética , Canais de Sódio Disparados por Voltagem/efeitos dos fármacos , Canais de Sódio Disparados por Voltagem/genética
5.
J Mol Cell Cardiol ; 74: 297-306, 2014 Sep.
Artigo em Inglês | MEDLINE | ID: mdl-24956219

RESUMO

Our in vitro studies revealed that a transcription factor, Forkhead box protein O1 (FoxO1), negatively regulates the expression of NaV1.5, a main α subunit of the cardiac Na(+) channel, by altering the promoter activity of SCN5a in HL-1 cardiomyocytes. The in vivo role of FoxO1 in the regulation of cardiac NaV1.5 expression remains unknown. The present study aimed to define the role of FoxO1 in the regulation of NaV1.5 expression and cardiac Na(+) channel activity in mouse ventricular cardiomyocytes and assess the cardiac electrophysiological phenotype of mice with cardiac FoxO1 deletion. Tamoxifen-induced and cardiac-specific FoxO1 deletion was confirmed by polymerase chain reaction (PCR). Cardiac FoxO1 deletion failed to result in either cardiac functional changes or hypertrophy as assessed by echocardiography and individual ventricular cell capacitances, respectively. Western blotting showed that FoxO1 was significantly decreased while NaV1.5 protein level was significantly increased in mouse hearts with FoxO1 deletion. Reverse transcription-PCR (RT-PCR) revealed that FoxO1 deletion led to an increase in NaV1.5 and Na(+) channel subunit ß3 mRNA, but not ß1, 2, and 4, or connexin 43. Whole patch-clamp recordings demonstrated that cardiac Na(+) currents were significantly augmented by FoxO1 deletion without affecting the steady-state activation and inactivation, leading to accelerated depolarization of action potentials in mouse ventricular cardiomyocytes. Electrocardiogram recordings showed that the QRS complex was significantly shortened and the P wave amplitude was significantly increased in conscious and unrestrained mice with cardiac FoxO1 deletion. NaV1.5 expression was decreased in the peri-infarct (border-zone) of mice with myocardial infarction and FoxO1 accumulated in the cardiomyocyte nuclei of chronic ischemic human hearts. Our findings indicate that FoxO1 plays an important role in the regulation of NaV1.5 and ß3 subunit expressions as well as Na(+) channel activity in the heart and that FoxO1 is involved in the modulation of NaV1.5 expression in ischemic heart disease.


Assuntos
Fatores de Transcrição Forkhead/genética , Ventrículos do Coração/metabolismo , Infarto do Miocárdio/genética , Miócitos Cardíacos/metabolismo , Canal de Sódio Disparado por Voltagem NAV1.5/genética , Subunidade beta-3 do Canal de Sódio Disparado por Voltagem/genética , Potenciais de Ação/fisiologia , Animais , Núcleo Celular/metabolismo , Núcleo Celular/patologia , Conexina 43/genética , Conexina 43/metabolismo , Eletrocardiografia , Proteína Forkhead Box O1 , Fatores de Transcrição Forkhead/deficiência , Regulação da Expressão Gênica , Ventrículos do Coração/patologia , Humanos , Camundongos , Camundongos Knockout , Infarto do Miocárdio/metabolismo , Infarto do Miocárdio/patologia , Miócitos Cardíacos/patologia , Canal de Sódio Disparado por Voltagem NAV1.5/metabolismo , Técnicas de Patch-Clamp , Cultura Primária de Células , Transdução de Sinais , Subunidade beta-3 do Canal de Sódio Disparado por Voltagem/metabolismo
6.
Proc Natl Acad Sci U S A ; 111(18): 6816-21, 2014 May 06.
Artigo em Inglês | MEDLINE | ID: mdl-24753596

RESUMO

The brainstem nucleus locus coeruleus (LC) is the primary source of norepinephrine (NE) to the mammalian neocortex. It is believed to operate as a homogeneous syncytium of transmitter-specific cells that regulate brain function and behavior via an extensive network of axonal projections and global transmitter-mediated modulatory influences on a diverse assembly of neural targets within the CNS. The data presented here challenge this longstanding notion and argue instead for segregated operation of the LC-NE system with respect to the functions of the circuits within its efferent domain. Anatomical, molecular, and electrophysiological approaches were used in conjunction with a rat model to show that LC cells innervating discrete cortical regions are biochemically and electrophysiologically distinct from one another so as to elicit greater release of norepinephrine in prefrontal versus motor cortex. These findings challenge the consensus view of LC as a relatively homogeneous modulator of forebrain activity and have important implications for understanding the impact of the system on the generation and maintenance of adaptive and maladaptive behaviors.


Assuntos
Locus Cerúleo/anatomia & histologia , Locus Cerúleo/fisiologia , Córtex Motor/anatomia & histologia , Córtex Motor/fisiologia , Córtex Pré-Frontal/anatomia & histologia , Córtex Pré-Frontal/fisiologia , Animais , Comportamento Animal/fisiologia , Vias Eferentes/anatomia & histologia , Vias Eferentes/fisiologia , Masculino , Norepinefrina/fisiologia , Análise de Componente Principal , RNA Mensageiro/genética , RNA Mensageiro/metabolismo , Ratos , Ratos Sprague-Dawley , Receptores de AMPA/genética , Receptores de N-Metil-D-Aspartato/genética , Tirosina 3-Mono-Oxigenase/genética , Proteínas Vesiculares de Transporte de Monoamina/genética , Subunidade beta-3 do Canal de Sódio Disparado por Voltagem/genética , Ácido alfa-Amino-3-hidroxi-5-metil-4-isoxazol Propiônico/metabolismo
7.
C R Biol ; 337(2): 73-7, 2014 Feb.
Artigo em Inglês | MEDLINE | ID: mdl-24581800

RESUMO

The voltage-gated sodium channel (VGSC) is a complex, which is composed of one pore-forming α subunit and at least one ß subunit. Up to now, five ß subunits are known: ß1/ß1A, ß1B, ß2, ß3, and ß4, encoded by four genes (SCN1B∼SCN4B). It is critical to have a deep understanding of the interaction between ß1 and ß3 subunits, two subunits which frequently appear in many diseases concurrently. In this study, we had screened out the new template of ß1 subunit for homology modelling, which shares higher similarity to ß3. Docking studies of the ß1 and ß3 homology model were conducted, and likely ß1 and ß3 binding loci were investigated. The results revealed that ß1-ß3 is more likely to form a di-polymer than ß1-ß1 based on molecular interaction analysis, including potential energy analysis, Van der Waals (VDW) energy analysis and electrostatic energy analysis, and in addition, consideration of the hydrogen bonds and hydrophobic contacts that are involved. Based on these analyses, the residues His122 and Lys140 of ß1 and Glu 66, Asn 131, Asp 118, Glu 120, Glu133, Asn135, Ser 137 of ß3 were predicted to play a functional role.


Assuntos
Subunidade beta-3 do Canal de Sódio Disparado por Voltagem/genética , Animais , Sítios de Ligação , Sistemas de Liberação de Medicamentos , Ligação de Hidrogênio , Camundongos , Modelos Moleculares , Ligação Proteica , Subunidade beta-1 do Canal de Sódio Disparado por Voltagem/genética
9.
FASEB J ; 27(2): 568-80, 2013 Feb.
Artigo em Inglês | MEDLINE | ID: mdl-23118027

RESUMO

The ß subunits of voltage-gated sodium (Na(v)) channels possess an extracellular immunoglobulin (Ig) domain that is related to the L1 family of cell-adhesion molecules (CAMs). Here we show that in HEK293 cells, secretion of the free Ig domain of the ß3 subunit is reduced significantly when it is coexpressed with the full-length ß3 and ß1 subunits but not with the ß2 subunit. Using immunoprecipitation, we show that the ß3 subunit can mediate trans homophilic-binding via its Ig domain and that the ß3-Ig domain can associate heterophilically with the ß1 subunit. Evolutionary tracing analysis and structural modeling identified a cluster of surface-localized amino acids fully conserved between the Ig domains of all known ß3 and ß1 sequences. A notable feature of this conserved surface cluster is the presence of two adjacent cysteine residues that previously we have suggested may form a disulfide bond. We now confirm the presence of the disulfide bond in ß3 using mass spectrometry, and we show that its integrity is essential for the association of the full-length, membrane-anchored ß3 subunit with itself. However, selective reduction of this surface disulfide bond did not inhibit homophilic binding of the purified ß3-Ig domain in free solution. Hence, the disulfide bond itself is unlikely to be part of the homophilic binding site. Rather, we suggest that its integrity ensures the Ig domain of the membrane-tethered ß3 subunit adopts the correct orientation for productive association to occur in vivo.


Assuntos
Subunidade beta-3 do Canal de Sódio Disparado por Voltagem/química , Sequência de Aminoácidos , Sítios de Ligação , Dissulfetos/química , Evolução Molecular , Células HEK293 , Humanos , Modelos Moleculares , Dados de Sequência Molecular , Domínios e Motivos de Interação entre Proteínas , Multimerização Proteica , Subunidades Proteicas , Proteínas Recombinantes/química , Proteínas Recombinantes/genética , Proteínas Recombinantes/metabolismo , Espectrometria de Massas por Ionização por Electrospray , Espectrometria de Massas em Tandem , Subunidade beta-1 do Canal de Sódio Disparado por Voltagem/química , Subunidade beta-1 do Canal de Sódio Disparado por Voltagem/genética , Subunidade beta-1 do Canal de Sódio Disparado por Voltagem/metabolismo , Subunidade beta-3 do Canal de Sódio Disparado por Voltagem/genética , Subunidade beta-3 do Canal de Sódio Disparado por Voltagem/metabolismo
10.
Circ J ; 77(4): 959-67, 2013.
Artigo em Inglês | MEDLINE | ID: mdl-23257389

RESUMO

BACKGROUND: Brugada syndrome (BrS) is characterized by specific alterations on ECG in the right precordial leads and associated with ventricular arrhythmia that may manifest as syncope or sudden cardiac death. The major causes of BrS are mutations in SCN5A for a large subunit of the sodium channel, Nav1.5, but a mutation in SCN3B for a small subunit of sodium channel, Navß3, has been recently reported in an American patient. METHODS AND RESULTS: A total of 181 unrelated BrS patients, 178 Japanese and 3 Koreans, who had no mutations in SCN5A, were examined for mutations in SCN3B by direct sequencing of all exons and adjacent introns. A mutation, Val110Ile, was identified in 3 of 178 (1.7%) Japanese patients, but was not found in 480 Japanese controls. The SCN3B mutation impaired the cytoplasmic trafficking of Nav1.5, the cell surface expression of which was decreased in transfected cells. Whole-cell patch clamp recordings of the transfected cells revealed that the sodium currents were significantly reduced by the SCN3B mutation. CONCLUSIONS: The Val110Ile mutation of SCN3B is a relatively common cause of SCN5A-negative BrS in Japan, which has a reduced sodium current because of the loss of cell surface expression of Nav1.5.


Assuntos
Síndrome de Brugada/genética , Síndrome de Brugada/metabolismo , Mutação de Sentido Incorreto , Canal de Sódio Disparado por Voltagem NAV1.5/metabolismo , Subunidade beta-3 do Canal de Sódio Disparado por Voltagem/genética , Adolescente , Adulto , Idoso , Substituição de Aminoácidos , Animais , Povo Asiático , Linhagem Celular , Criança , Feminino , Humanos , Japão , Masculino , Pessoa de Meia-Idade , Canal de Sódio Disparado por Voltagem NAV1.5/genética , Transporte Proteico/genética , Subunidade beta-3 do Canal de Sódio Disparado por Voltagem/metabolismo
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