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1.
Phys Chem Chem Phys ; 21(45): 25290-25301, 2019 Dec 07.
Artigo em Inglês | MEDLINE | ID: mdl-31701097

RESUMO

The voltage-gated potassium channel Kv4.3 plays a vital role in shaping the timing, frequency, and backpropagation of electrical signals in the brain and heart by generating fast transient currents at subthreshold membrane potentials in repetitive firing neurons. To achieve its physiological function, Kv4.3 is assisted by auxiliary ß-subunits that become integral parts of the native A-type potassium channels, among which there are the Kv channel-interacting proteins (KChIPs). KChIPs are a family of cytosolic proteins that, when coexpressed with Kv4, lead to higher current density, modulation of channel inactivation and faster recovery from inactivation, while the loss of KChIP function may lead to severe pathological states. Recently, the structural basis of the KChIP1-Kv4.3 interaction was reported by using two similar X-ray crystallographic structures, which supported a crucial role for KChIP1 in enhancing the stability of the Kv4.3 tetrameric assembly, thus helping the trafficking of the channel to the plasma membrane. Here, we investigate through fully atomistic simulations the structure and stability of the human Kv4.3 tetramerization (T1) domain in complex with KChIP1 upon specific mutations located in the first and second interfaces of the complex, as compared to the wild-type (WT). Our results nicely complement the available structural and biophysical information collected so far on these complex variants. In particular, the degree of structural deviations and energetic instability, from small to substantial, observed in these variants with respect to the WT model seems to parallel well the level of channel dysfunction known from electrophysiology data. Our simulations provide an octameric structure of the WT KChIP1-Kv4.3 assembly very similar to the known crystal structures, and, at the same time, highlight the importance of a previously overlooked site of interaction between KChIP1 and the Kv4.3 T1 domain.


Assuntos
Simulação por Computador , Proteínas Interatuantes com Canais de Kv/química , Canais de Potássio Shal/química , Cristalografia por Raios X , Humanos , Proteínas Interatuantes com Canais de Kv/genética , Modelos Moleculares , Mutação , Canais de Potássio Shal/genética
2.
Int J Mol Sci ; 20(17)2019 Aug 21.
Artigo em Inglês | MEDLINE | ID: mdl-31438481

RESUMO

A-type K+ channels contribute to regulating the propagation and frequency of action potentials in smooth muscle cells (SMCs). The present study (i) identified the molecular components of A-type K+ channels in rat vas deferens SMs (VDSMs) and (ii) showed the long-term, genomic effects of testosterone on their expression in VDSMs. Transcripts of the A-type K+ channel α subunit, Kv4.3L and its regulatory ß subunits, KChIP3, NCS1, and DPP6-S were predominantly expressed in rat VDSMs over the other related subtypes (Kv4.2, KChIP1, KChIP2, KChIP4, and DPP10). A-type K+ current (IA) density in VDSM cells (VDSMCs) was decreased by castration without changes in IA kinetics, and decreased IA density was compensated for by an oral treatment with 17α-methyltestosterone (MET). Correspondingly, in the VDSMs of castrated rats, Kv4.3L and KChIP3 were down-regulated at both the transcript and protein expression levels. Changes in Kv4.3L and KChIP3 expression levels were compensated for by the treatment with MET. These results suggest that testosterone level changes in testosterone disorders and growth processes control the functional expression of A-type K+ channels in VDSMCs.


Assuntos
Castração/efeitos adversos , Regulação para Baixo , Proteínas Interatuantes com Canais de Kv/genética , Proteínas Interatuantes com Canais de Kv/metabolismo , Ducto Deferente/metabolismo , Animais , Western Blotting , Eletrofisiologia , Masculino , Metiltestosterona/farmacologia , Músculo Liso/efeitos dos fármacos , Músculo Liso/metabolismo , Ratos , Ratos Wistar , Testosterona/metabolismo , Ducto Deferente/efeitos dos fármacos
3.
Nucleic Acids Res ; 47(17): 9087-9103, 2019 09 26.
Artigo em Inglês | MEDLINE | ID: mdl-31400114

RESUMO

Most human cancers acquire mutations causing defects in the p53 signaling pathway. The tumor suppressor p53 becomes activated in response to genotoxic stress and is essential for arresting the cell cycle to facilitate DNA repair or to initiate apoptosis. p53-induced cell cycle-arrest is mediated by expression of the CDK inhibitor p21WAF1/Cip1, which prevents phosphorylation and inactivation of the pocket proteins RB, p130, and p107. In a hypophosphorylated state, pocket proteins bind to E2F factors forming RB-E2F and DREAM transcriptional repressor complexes. Here, we analyze the influence of RB and DREAM on p53-induced gene repression and cell-cycle arrest. We show that abrogation of DREAM function by knockout of the DREAM component LIN37 results in a reduced repression of cell-cycle genes. We identify the genes repressed by the p53-DREAM pathway and describe a set of genes that is downregulated by p53 independent of LIN37/DREAM. Most strikingly, p53-dependent repression of cell-cycle genes is completely abrogated in LIN37-/-;RB-/- cells leading to a loss of the G1/S checkpoint. Taken together, we show that DREAM and RB are key factors in the p53 signaling pathway to downregulate a large number of cell-cycle genes and to arrest the cell cycle at the G1/S transition.


Assuntos
Pontos de Checagem do Ciclo Celular/genética , Regulação da Expressão Gênica , Proteínas Interatuantes com Canais de Kv/metabolismo , Proteínas Repressoras/metabolismo , Proteína do Retinoblastoma/genética , Transativadores/fisiologia , Proteína Supressora de Tumor p53/metabolismo , Animais , Células Cultivadas , Proteína Substrato Associada a Crk/genética , Inibidor de Quinase Dependente de Ciclina p21/genética , Inibidor de Quinase Dependente de Ciclina p21/metabolismo , Fatores de Transcrição E2F/genética , Fatores de Transcrição E2F/metabolismo , Fibroblastos/metabolismo , Genes cdc , Células HCT116 , Humanos , Proteínas Interatuantes com Canais de Kv/genética , Camundongos , Proteínas Repressoras/genética , Proteína do Retinoblastoma/metabolismo , Proteína p107 Retinoblastoma-Like/genética , Transativadores/genética , Transativadores/metabolismo , Proteína Supressora de Tumor p53/genética
4.
Pediatr Cardiol ; 40(7): 1325-1330, 2019 Oct.
Artigo em Inglês | MEDLINE | ID: mdl-31346662

RESUMO

Spatiotemporal gene expression during cardiac development is a highly regulated process. Activation of key signaling pathways involved in electrophysiological programming, such as Notch and Wnt signaling, occurs in early cardiovascular development and these pathways are reactivated during pathologic remodeling. Direct targets of these signaling pathways have also been associated with inherited arrhythmias such as Brugada syndrome and arrhythmogenic cardiomyopathy. In addition, evidence is emerging from animal models that reactivation of Notch and Wnt signaling during cardiac pathology may predispose to acquired arrhythmias, underscoring the importance of elucidating the transcriptional and epigenetic effects on cardiac gene regulation. Here, we highlight specific examples where gene expression dictates electrophysiological properties in both normal and diseased hearts.


Assuntos
Arritmias Cardíacas/genética , Eletrofisiologia , Epigênese Genética , Animais , Fatores de Transcrição Hélice-Alça-Hélice Básicos/genética , Humanos , Proteínas Interatuantes com Canais de Kv/genética , Receptores Notch , Proteínas Repressoras/genética , Transdução de Sinais , Via de Sinalização Wnt
5.
J Biol Chem ; 294(10): 3683-3695, 2019 03 08.
Artigo em Inglês | MEDLINE | ID: mdl-30622142

RESUMO

The Kv4 family of A-type voltage-gated K+ channels regulates the excitability in hippocampal pyramidal neuron dendrites and are key determinants of dendritic integration, spike timing-dependent plasticity, long-term potentiation, and learning. Kv4.2 channel expression is down-regulated following hippocampal seizures and in epilepsy, suggesting A-type currents as therapeutic targets. In addition to pore-forming Kv4 subunits, modulatory auxiliary subunits called K+ channel-interacting proteins (KChIPs) modulate Kv4 expression and activity and are required to recapitulate native hippocampal A-type currents in heterologous expression systems. KChIP mRNAs contain multiple start sites and alternative exons that generate considerable N-terminal variation and functional diversity in shaping Kv4 currents. As members of the EF-hand domain-containing neuronal Ca2+ sensor protein family, KChIP auxiliary proteins may convey Ca2+ sensitivity upon Kv4 channels; however, to what degree intracellular Ca2+ regulates KChIP-Kv4.2 complexes is unclear. To answer this question, we expressed KChIP2 with Kv4.2 in HEK293T cells, and, with whole-cell patch-clamp electrophysiology, measured an ∼1.5-fold increase in Kv4.2 current density in the presence of elevated intracellular Ca2+ Intriguingly, the Ca2+ regulation of Kv4 current was specific to KChIP2b and KChIP2c splice isoforms that lack a putative polybasic domain that is present in longer KChIP2a1 and KChIP2a isoforms. Site-directed acidification of the basic residues within the polybasic motif of KChIP2a1 rescued Ca2+-mediated regulation of Kv4 current density. These results support divergent Ca2+ regulation of Kv4 channels mediated by alternative splicing of KChIP2 isoforms. They suggest that distinct KChIP-Kv4 interactions may differentially control excitability and function of hippocampal dendrites.


Assuntos
Processamento Alternativo , Cálcio/metabolismo , Proteínas Interatuantes com Canais de Kv/química , Proteínas Interatuantes com Canais de Kv/metabolismo , Canais de Potássio Shal/metabolismo , Motivos de Aminoácidos , Sequência de Aminoácidos , Dendritos/metabolismo , Fenômenos Eletrofisiológicos , Células HEK293 , Hipocampo/citologia , Humanos , Interações Hidrofóbicas e Hidrofílicas , Espaço Intracelular/metabolismo , Cinética , Proteínas Interatuantes com Canais de Kv/genética , Domínios Proteicos , Isoformas de Proteínas/química , Isoformas de Proteínas/genética , Isoformas de Proteínas/metabolismo
6.
Oncogene ; 38(7): 1080-1092, 2019 02.
Artigo em Inglês | MEDLINE | ID: mdl-30206359

RESUMO

Overexpression of the oncogene MYBL2 (B-Myb) is associated with increased cell proliferation and serves as a marker of poor prognosis in cancer. However, the mechanism by which B-Myb alters the cell cycle is not fully understood. In proliferating cells, B-Myb interacts with the MuvB core complex including LIN9, LIN37, LIN52, RBBP4, and LIN54, forming the MMB (Myb-MuvB) complex, and promotes transcription of genes required for mitosis. Alternatively, the MuvB core interacts with Rb-like protein p130 and E2F4-DP1 to form the DREAM complex that mediates global repression of cell cycle genes in G0/G1, including a subset of MMB target genes. Here, we show that overexpression of B-Myb disrupts the DREAM complex in human cells, and this activity depends on the intact MuvB-binding domain in B-Myb. Furthermore, we found that B-Myb regulates the protein expression levels of the MuvB core subunit LIN52, a key adapter for assembly of both the DREAM and MMB complexes, by a mechanism that requires S28 phosphorylation site in LIN52. Given that high expression of B-Myb correlates with global loss of repression of DREAM target genes in breast and ovarian cancer, our findings offer mechanistic insights for aggressiveness of cancers with MYBL2 amplification, and establish the rationale for targeting B-Myb to restore cell cycle control.


Assuntos
Neoplasias da Mama/metabolismo , Proteínas de Ciclo Celular/biossíntese , Ciclo Celular , Regulação Neoplásica da Expressão Gênica , Proteínas Interatuantes com Canais de Kv/metabolismo , Complexos Multiproteicos/metabolismo , Proteínas de Neoplasias/metabolismo , Neoplasias Ovarianas/metabolismo , Proteínas Repressoras/metabolismo , Transativadores/biossíntese , Neoplasias da Mama/genética , Proteínas de Ciclo Celular/genética , Linhagem Celular Tumoral , Feminino , Humanos , Proteínas Interatuantes com Canais de Kv/genética , Complexos Multiproteicos/genética , Proteínas de Neoplasias/genética , Neoplasias Ovarianas/genética , Neoplasias Ovarianas/patologia , Proteínas Repressoras/genética , Transativadores/genética
7.
Nat Commun ; 9(1): 4866, 2018 11 19.
Artigo em Inglês | MEDLINE | ID: mdl-30451831

RESUMO

Chromosome 17q gains are almost invariably present in high-risk neuroblastoma cases. Here, we perform an integrative epigenomics search for dosage-sensitive transcription factors on 17q marked by H3K27ac defined super-enhancers and identify TBX2 as top candidate gene. We show that TBX2 is a constituent of the recently established core regulatory circuitry in neuroblastoma with features of a cell identity transcription factor, driving proliferation through activation of p21-DREAM repressed FOXM1 target genes. Combined MYCN/TBX2 knockdown enforces cell growth arrest suggesting that TBX2 enhances MYCN sustained activation of FOXM1 targets. Targeting transcriptional addiction by combined CDK7 and BET bromodomain inhibition shows synergistic effects on cell viability with strong repressive effects on CRC gene expression and p53 pathway response as well as several genes implicated in transcriptional regulation. In conclusion, we provide insight into the role of the TBX2 CRC gene in transcriptional dependency of neuroblastoma cells warranting clinical trials using BET and CDK7 inhibitors.


Assuntos
Neoplasias Encefálicas/genética , Proteína Forkhead Box M1/genética , Regulação Neoplásica da Expressão Gênica , Proteínas Interatuantes com Canais de Kv/genética , Proteína Proto-Oncogênica N-Myc/genética , Neuroblastoma/genética , Proteínas Repressoras/genética , Proteínas com Domínio T/genética , Antineoplásicos/farmacologia , Azepinas/farmacologia , Neoplasias Encefálicas/tratamento farmacológico , Neoplasias Encefálicas/metabolismo , Neoplasias Encefálicas/patologia , Linhagem Celular Tumoral , Sobrevivência Celular/efeitos dos fármacos , Quinases Ciclina-Dependentes/genética , Quinases Ciclina-Dependentes/metabolismo , Variações do Número de Cópias de DNA , Epigênese Genética , Proteína Forkhead Box M1/metabolismo , Células HEK293 , Histonas/genética , Histonas/metabolismo , Humanos , Proteínas Interatuantes com Canais de Kv/metabolismo , Proteína Proto-Oncogênica N-Myc/metabolismo , Neuroblastoma/tratamento farmacológico , Neuroblastoma/metabolismo , Neuroblastoma/patologia , Organoides/efeitos dos fármacos , Organoides/metabolismo , Organoides/patologia , Panobinostat/farmacologia , Fenilenodiaminas/farmacologia , Pirimidinas/farmacologia , Proteínas Repressoras/metabolismo , Transdução de Sinais , Proteínas com Domínio T/metabolismo , Triazóis/farmacologia , Proteína Supressora de Tumor p53/genética , Proteína Supressora de Tumor p53/metabolismo
8.
Elife ; 72018 10 01.
Artigo em Inglês | MEDLINE | ID: mdl-30272559

RESUMO

Regulated mucin secretion from specialized goblet cells by exogenous agonist-dependent (stimulated) and -independent (baseline) manner is essential for the function of the epithelial lining. Over extended periods, baseline release of mucin can exceed quantities released by stimulated secretion, yet its regulation remains poorly characterized. We have discovered that ryanodine receptor-dependent intracellular Ca2+ oscillations effect the dissociation of the Ca2+-binding protein, KChIP3, encoded by KCNIP3 gene, from mature mucin-filled secretory granules, allowing for their exocytosis. Increased Ca2+ oscillations, or depleting KChIP3, lead to mucin hypersecretion in a human differentiated colonic cell line, an effect reproduced in the colon of Kcnip3-/- mice. Conversely, overexpressing KChIP3 or abrogating its Ca2+-sensing ability, increases KChIP3 association with granules, and inhibits baseline secretion. KChIP3 therefore emerges as the high-affinity Ca2+ sensor that negatively regulates baseline mucin secretion. We suggest KChIP3 marks mature, primed mucin granules, and functions as a Ca2+ oscillation-dependent brake to control baseline secretion. Editorial note: This article has been through an editorial process in which the authors decide how to respond to the issues raised during peer review. The Reviewing Editor's assessment is that all the issues have been addressed (see decision letter).


Assuntos
Sinalização do Cálcio , Cálcio/metabolismo , Colo/metabolismo , Proteínas Interatuantes com Canais de Kv/metabolismo , Mucina-5AC/metabolismo , Animais , Células Caliciformes/metabolismo , Células HEK293 , Células HT29 , Humanos , Proteínas Interatuantes com Canais de Kv/genética , Camundongos Endogâmicos C57BL , Camundongos Knockout , Microscopia Confocal , Mucina-5AC/genética , Vesículas Secretórias/metabolismo
9.
Arch Endocrinol Metab ; 62(2): 205-211, 2018.
Artigo em Inglês | MEDLINE | ID: mdl-29641740

RESUMO

OBJECTIVE: The transcriptional repressor DREAM is involved in thyroid-specific gene expression, thyroid enlargement and nodular development, but its clinical utility is still uncertain. In this study we aimed to investigate whether DREAM mRNA levels differ in different thyroid tumors and how this possible difference would allow the use of DREAM gene expression as molecular marker for diagnostic and/or prognosis purpose. MATERIALS AND METHODS: We quantified DREAM gene mRNA levels and investigated its mutational status, relating its expression and genetic changes to diagnostic and prognostic features of 200 thyroid tumors, being 101 malignant [99 papillary thyroid carcinomas (PTC) and 2 anaplastic thyroid carcinomas] and 99 benign thyroid lesions [49 goiter and 50 follicular adenomas (FA)]. RESULTS: Levels of mRNA of DREAM gene were higher in benign (0.7909 ± 0.6274 AU) than in malignant (0.3373 ± 0.6274 AU) thyroid lesions (p < 0.0001). DREAM gene expression was able to identify malignancy with 66.7% sensitivity, 85.4% specificity, 84.2% positive predictive value (PPV), 68.7% negative predictive value (NPV), and 75.3% accuracy. DREAM mRNA levels were also useful distinguishing the follicular lesions FA and FVPTC with 70.2% sensitivity, 73.5% specificity, 78.5% PPV, 64.1% NPV, and 71.6% accuracy. However, DREAM gene expression was neither associated with clinical features of tumor aggressiveness, nor with recurrence or survival. Six different genetic changes in non-coding regions of DREAM gene were also found, not related to DREAM gene expression or tumor features. CONCLUSION: We suggest that DREAM gene expression may help diagnose thyroid nodules, identifying malignancy and characterizing follicular-patterned thyroid lesions; however, it is not useful as a prognostic marker.


Assuntos
Biomarcadores Tumorais/genética , Proteínas Interatuantes com Canais de Kv/genética , RNA Mensageiro/genética , Elementos Reguladores de Transcrição/genética , Proteínas Repressoras/genética , Neoplasias da Glândula Tireoide/diagnóstico , Biomarcadores Tumorais/metabolismo , Feminino , Humanos , Proteínas Interatuantes com Canais de Kv/metabolismo , Masculino , Pessoa de Meia-Idade , Estadiamento de Neoplasias , Prognóstico , RNA Mensageiro/metabolismo , Reação em Cadeia da Polimerase em Tempo Real , Proteínas Repressoras/metabolismo , Sensibilidade e Especificidade , Neoplasias da Glândula Tireoide/genética , Neoplasias da Glândula Tireoide/metabolismo
10.
Int J Mol Sci ; 19(4)2018 Apr 13.
Artigo em Inglês | MEDLINE | ID: mdl-29652865

RESUMO

Calsenilin modulates A-type potassium channels, regulates presenilin-mediated γ-secretase activity, and represses prodynorphin and c-fos genes expression. RhoA is involved in various cellular functions including proliferation, differentiation, migration, transcription, and regulation of the actin cytoskeleton. Although recent studies demonstrate that calsenilin can directly interact with RhoA and that RhoA inactivation is essential for neuritogenesis, it is uncertain whether there is a link between calsenilin and RhoA-regulated neuritogenesis. Here, we investigated the role of calsenilin in RhoA-regulated neuritogenesis using in vitro and in vivo systems. We found that calsenilin induced RhoA inactivation, which accompanied RhoA phosphorylation and the reduced phosphorylation levels of LIM kinase (LIMK) and cofilin. Interestingly, PC12 cells overexpressing either full-length (FL) or the caspase 3-derived C-terminal fragment (CTF) of calsenilin significantly inactivated RhoA through its interaction with RhoA and p190 Rho GTPase-activating protein (p190RhoGAP). In addition, cells expressing FL and the CTF of calsenilin had increased neurite outgrowth compared to cells expressing the N-terminal fragment (NTF) of calsenilin or vector alone. Moreover, Tat-C3 and Y27632 treatment significantly increased the percentage of neurite-bearing cells, neurite length, and the number of neurites in cells. Finally, calsenilin deficiency in the brains of calsenilin-knockout mice significantly interfered with RhoA inactivation. These findings suggest that calsenilin contributes to neuritogenesis through RhoA inactivation.


Assuntos
Proteínas Interatuantes com Canais de Kv/genética , Proteínas Interatuantes com Canais de Kv/metabolismo , Crescimento Neuronal , Proteína rhoA de Ligação ao GTP/metabolismo , Animais , Técnicas de Inativação de Genes , Células HEK293 , Humanos , Proteínas Interatuantes com Canais de Kv/química , Camundongos , Células PC12 , Fosforilação , Ratos , Transdução de Sinais
11.
Endocr J ; 65(5): 537-545, 2018 May 28.
Artigo em Inglês | MEDLINE | ID: mdl-29491224

RESUMO

Copy number variation (CNV) has emerged as another important genetic marker in addition to SNP for understanding etiology of complex disease. Kv channel interacting protein 1 (KCNIP1) is a Ca2+-dependent transcriptional modulator that contributes to the regulation of insulin secretion. Previous genome-wide CNV assay identified the KCNIP1 gene encompassing a CNV region, however, its further effect and risk rate on type 2 diabetes (T2D) have rarely been addressed, especially in Chinese population. The current study aims to detect and excavate genetic distribution profile of KCNIP1 CNV in Chinese T2D and control populations, and further to investigate the associations with clinical characteristics. Divergent patterns of the KCNIP1 CNV were identified (p < 0.01), in which the copy number gain was predominant in T2D, while the copy number normal accounted for the most in control group. Consistently, the individuals with copy number gain showed significant risk on T2D (OR = 4.550, p < 0.01). The KCNIP1 copy numbers presented significantly positive correlations with fasting plasma glucose and glycated hemoglobin in T2D. For OGTT test, the T2D patients with copy number gain had remarkably elevated glucose contents (60, 120, 180-min, p < 0.05 or p < 0.01) and diminished insulin levels (60, 120-min, p < 0.05) than those with copy number loss and normal, which suggested that the KCNIP1 CNV was correlated with the glucose and insulin action. This is the first CNV association study of the KCNIP1 gene in Chinese population, and these data indicated that KCNIP1 might function as a T2D-susceptibility gene whose dysregulation alters insulin production.


Assuntos
Variações do Número de Cópias de DNA , Diabetes Mellitus Tipo 2/genética , Predisposição Genética para Doença , Proteínas Interatuantes com Canais de Kv/genética , Polimorfismo de Nucleotídeo Único , Idoso , China , Feminino , Humanos , Masculino , Pessoa de Meia-Idade
12.
Proc Natl Acad Sci U S A ; 115(15): E3559-E3568, 2018 04 10.
Artigo em Inglês | MEDLINE | ID: mdl-29581270

RESUMO

A de novo mutation in the KCND2 gene, which encodes the Kv4.2 K+ channel, was identified in twin boys with intractable, infant-onset epilepsy and autism. Kv4.2 channels undergo closed-state inactivation (CSI), a mechanism by which channels inactivate without opening during subthreshold depolarizations. CSI dynamically modulates neuronal excitability and action potential back propagation in response to excitatory synaptic input, controlling Ca2+ influx into dendrites and regulating spike timing-dependent plasticity. Here, we show that the V404M mutation specifically affects the mechanism of CSI, enhancing the inactivation of channels that have not opened while dramatically impairing the inactivation of channels that have opened. The mutation gives rise to these opposing effects by increasing the stability of the inactivated state and in parallel, profoundly slowing the closure of open channels, which according to our data, is required for CSI. The larger volume of methionine compared with valine is a major factor underlying altered inactivation gating. Our results suggest that V404M increases the strength of the physical interaction between the pore gate and the voltage sensor regardless of whether the gate is open or closed. Furthermore, in contrast to previous proposals, our data strongly suggest that physical coupling between the voltage sensor and the pore gate is maintained in the inactivated state. The state-dependent effects of V404M on CSI are expected to disturb the regulation of neuronal excitability and the induction of spike timing-dependent plasticity. Our results strongly support a role for altered CSI gating in the etiology of epilepsy and autism in the affected twins.


Assuntos
Transtorno Autístico/genética , Epilepsia/genética , Canais de Potássio Shal/genética , Animais , Transtorno Autístico/metabolismo , Epilepsia/metabolismo , Feminino , Humanos , Proteínas Interatuantes com Canais de Kv/genética , Proteínas Interatuantes com Canais de Kv/metabolismo , Potenciais da Membrana/fisiologia , Mutação , Oócitos/fisiologia , Técnicas de Patch-Clamp/métodos , Polimorfismo Genético , Proteínas Repressoras/genética , Proteínas Repressoras/metabolismo , Canais de Potássio Shal/metabolismo , Transfecção , Xenopus laevis
13.
PLoS One ; 13(1): e0191911, 2018.
Artigo em Inglês | MEDLINE | ID: mdl-29385176

RESUMO

Kv4.2 channels mediate a subthreshold-activating somatodendritic A-type current (ISA) in hippocampal neurons. We examined the role of accessory Kv channel interacting protein (KChIP) binding in somatodendritic surface expression and activity-dependent decrease in the availability of Kv4.2 channels. For this purpose we transfected cultured hippocampal neurons with cDNA coding for Kv4.2 wild-type (wt) or KChIP binding-deficient Kv4.2 mutants. All channels were equipped with an externally accessible hemagglutinin (HA)-tag and an EGFP-tag, which was attached to the C-terminal end. Combined analyses of EGFP self-fluorescence, surface HA immunostaining and patch-clamp recordings demonstrated similar dendritic trafficking and functional surface expression for Kv4.2[wt]HA,EGFP and the KChIP binding-deficient Kv4.2[A14K]HA,EGFP. Coexpression of exogenous KChIP2 augmented the surface expression of Kv4.2[wt]HA,EGFP but not Kv4.2[A14K]HA,EGFP. Notably, activity-dependent decrease in availability was more pronounced in Kv4.2[wt]HA,EGFP + KChIP2 coexpressing than in Kv4.2[A14K]HA,EGFP + KChIP2 coexpressing neurons. Our results do not support the notion that accessory KChIP binding is a prerequisite for dendritic trafficking and functional surface expression of Kv4.2 channels, however, accessory KChIP binding may play a potential role in Kv4.2 modulation during intrinsic plasticity processes.


Assuntos
Hipocampo/metabolismo , Proteínas Interatuantes com Canais de Kv/metabolismo , Canais de Potássio Shal/metabolismo , Potenciais de Ação , Animais , Células Cultivadas , Dendritos/metabolismo , Epitopos/genética , Epitopos/metabolismo , Hipocampo/citologia , Humanos , Imuno-Histoquímica , Proteínas Interatuantes com Canais de Kv/genética , Proteínas Mutantes/química , Proteínas Mutantes/genética , Proteínas Mutantes/metabolismo , Neurônios/metabolismo , Técnicas de Patch-Clamp , Ligação Proteica , Ratos , Ratos Wistar , Proteínas Recombinantes de Fusão/química , Proteínas Recombinantes de Fusão/genética , Proteínas Recombinantes de Fusão/metabolismo , Canais de Potássio Shal/química , Canais de Potássio Shal/genética , Transfecção
14.
J Neurosci ; 38(7): 1756-1773, 2018 02 14.
Artigo em Inglês | MEDLINE | ID: mdl-29335353

RESUMO

Potassium voltage-gated channel interacting protein 3 (KChIP3), also termed downstream regulatory element antagonist modulator (DREAM) and calsenilin, is a multifunctional protein belonging to the neuronal calcium sensor (NCS) family. Recent studies revealed the expression of KChIP3 in dorsal root ganglion (DRG) neurons, suggesting the potential role of KChIP3 in peripheral sensory processing. Herein, we show that KChIP3 colocalizes with transient receptor potential ion channel V1 (TRPV1), a critical molecule involved in peripheral sensitization during inflammatory pain. Furthermore, the N-terminal 31-50 fragment of KChIP3 is capable of binding both the intracellular N and C termini of TRPV1, which substantially decreases the surface localization of TRPV1 and the subsequent Ca2+ influx through the channel. Importantly, intrathecal administration of the transmembrane peptide transactivator of transcription (TAT)-31-50 remarkably reduces Ca2+ influx via TRPV1 in DRG neurons and alleviates thermal hyperalgesia and gait alterations in a complete Freund's adjuvant-induced inflammatory pain model in male rats. Moreover, intraplantar injection of TAT-31-50 attenuated the capsaicin-evoked spontaneous pain behavior and thermal hyperalgesia, which further strengthened the regulatory role of TAT-31-50 on TRPV1 channel. In addition, TAT-31-50 could also alleviate inflammatory thermal hyperalgesia in kcnip3-/- rats generated in our study, suggesting that the analgesic effect mediated by TAT-31-50 is independent of endogenous KChIP3. Our study reveals a novel peripheral mechanism for the analgesic function of KChIP3 and provides a potential analgesic agent, TAT-31-50, for the treatment of inflammatory pain.SIGNIFICANCE STATEMENT Inflammatory pain arising from inflamed or injured tissues significantly compromises the quality of life in patients. This study aims to elucidate the role of peripheral potassium channel interacting protein 3 (KChIP3) in inflammatory pain. Direct interaction of the KChIP3 N-terminal 31-50 fragment with transient receptor potential ion channel V1 (TRPV1) was demonstrated. The KChIP3-TRPV1 interaction reduces the surface localization of TRPV1 and thus alleviates heat hyperalgesia and gait alterations induced by peripheral inflammation. Furthermore, the transmembrane transactivator of transcription (TAT)-31-50 peptide showed analgesic effects on inflammatory hyperalgesia independently of endogenous KChIP3. This work reveals a novel mechanism of peripheral KChIP3 in inflammatory hyperalgesia that is distinct from its classical role as a transcriptional repressor in pain modulation.


Assuntos
Hiperalgesia/fisiopatologia , Inflamação/fisiopatologia , Proteínas Interatuantes com Canais de Kv/metabolismo , Canais de Cátion TRPV/metabolismo , Animais , Sinalização do Cálcio , Repressão Epigenética , Adjuvante de Freund , Marcha , Gânglios Espinais/efeitos dos fármacos , Técnicas de Inativação de Genes , Hiperalgesia/induzido quimicamente , Inflamação/induzido quimicamente , Injeções Espinhais , Proteínas Interatuantes com Canais de Kv/genética , Masculino , Medição da Dor/efeitos dos fármacos , Fragmentos de Peptídeos/metabolismo , Ligação Proteica , Ratos , Canais de Cátion TRPV/efeitos dos fármacos
15.
Mol Neurobiol ; 55(1): 1-12, 2018 01.
Artigo em Inglês | MEDLINE | ID: mdl-28840473

RESUMO

Amyotrophic lateral sclerosis (ALS) is a neurodegenerative disease of unknown origin and characterized by a relentless loss of motor neurons that causes a progressive muscle weakness until death. Among the several pathogenic mechanisms that have been related to ALS, a dysregulation of calcium-buffering proteins in motor neurons of the brain and spinal cord can make these neurons more vulnerable to disease progression. Downstream regulatory element antagonist modulator (DREAM) is a neuronal calcium-binding protein that plays multiple roles in the nucleus and cytosol. The main aim of this study was focused on the characterization of DREAM and glial fibrillary acid protein (GFAP) in the brain and spinal cord tissues from transgenic SOD1G93A mice and ALS patients to unravel its potential role under neurodegenerative conditions. The DREAM and GFAP levels in the spinal cord and different brain areas from transgenic SOD1G93A mice and ALS patients were analyzed by Western blot and immunohistochemistry. Our findings suggest that the calcium-dependent excitotoxicity progressively enhanced in the CNS in ALS could modulate the multifunctional nature of DREAM, strengthening its apoptotic way of action in both motor neurons and astrocytes, which could act as an additional factor to increase neuronal damage. The direct crosstalk between astrocytes and motor neurons can become vulnerable under neurodegenerative conditions, and DREAM could act as an additional switch to enhance motor neuron loss. Together, these findings could pave the way to further study the molecular targets of DREAM to find novel therapeutic strategies to fight ALS.


Assuntos
Esclerose Amiotrófica Lateral/metabolismo , Astrócitos/metabolismo , Proteínas Interatuantes com Canais de Kv/metabolismo , Neurônios Motores/metabolismo , Proteínas Repressoras/metabolismo , Medula Espinal/metabolismo , Idoso , Esclerose Amiotrófica Lateral/genética , Esclerose Amiotrófica Lateral/patologia , Animais , Astrócitos/patologia , Modelos Animais de Doenças , Progressão da Doença , Humanos , Proteínas Interatuantes com Canais de Kv/genética , Masculino , Camundongos , Camundongos Transgênicos , Pessoa de Meia-Idade , Neurônios Motores/patologia , Proteínas Repressoras/genética , Medula Espinal/patologia , Regulação para Cima
16.
Am J Physiol Heart Circ Physiol ; 314(1): H68-H81, 2018 01 01.
Artigo em Inglês | MEDLINE | ID: mdl-28939651

RESUMO

Notch receptor signaling is active during cardiac development and silenced in myocytes after birth. Conversely, outward K+ Kv currents progressively appear in postnatal myocytes leading to shortening of the action potential (AP) and acquisition of the mature electrical phenotype. In the present study, we tested the possibility that Notch signaling modulates the electrical behavior of cardiomyocytes by interfering with Kv currents. For this purpose, the effects of Notch receptor activity on electrophysiological properties of myocytes were evaluated using transgenic mice with inducible expression of the Notch1 intracellular domain (NICD), the functional fragment of the activated Notch receptor, and in neonatal myocytes after inhibition of the Notch transduction pathway. By patch clamp, NICD-overexpressing cells presented prolonged AP duration and reduced upstroke amplitude, properties that were coupled with reduced rapidly activating Kv and fast Na+ currents, compared with cells obtained from wild-type mice. In cultured neonatal myocytes, inhibition of the proteolitic release of NICD with a γ-secretase antagonist increased transcript levels of the Kv channel-interacting proteins 2 (KChIP2) and enhanced the density of Kv currents. Collectively, these results indicate that Notch signaling represents an important regulator of the electrophysiological behavior of developing and adult myocytes by repressing, at least in part, repolarizing Kv currents. NEW & NOTEWORTHY We investigated the effects of Notch receptor signaling on the electrical properties of cardiomyocytes. Our results indicate that the Notch transduction pathway interferes with outward K+ Kv currents, critical determinants of the electrical repolarization of myocytes.


Assuntos
Miócitos Cardíacos/metabolismo , Canais de Potássio de Abertura Dependente da Tensão da Membrana/metabolismo , Potássio/metabolismo , Receptor Notch1/metabolismo , Transdução de Sinais , Animais , Células Cultivadas , Feminino , Cinética , Proteínas Interatuantes com Canais de Kv/genética , Proteínas Interatuantes com Canais de Kv/metabolismo , Masculino , Potenciais da Membrana , Camundongos Endogâmicos C57BL , Camundongos Transgênicos , Canais de Potássio de Abertura Dependente da Tensão da Membrana/genética , Receptor Notch1/genética , Sódio/metabolismo
17.
J Cell Physiol ; 233(5): 4317-4326, 2018 05.
Artigo em Inglês | MEDLINE | ID: mdl-29139549

RESUMO

Supplementation of 100% oxygen is a very common intervention in intensive care units (ICU) and critical care centers for patients with dysfunctional lung and lung disorders. Although there is advantage in delivering sufficient levels of oxygen, hyperoxia is reported to be directly associated with increasing in-hospital deaths. Our previous studies reported ventricular and electrical remodeling in hyperoxia treated mouse hearts, and in this article, for the first time, we are investigating the effects of hyperoxia on atrial electrophysiology using whole-cell patch-clamp electrophysiology experiments along with assessment of Kv1.5, Kv4.2, and KChIP2 transcripts and protein profiles using real-time quantitative RT-PCR and Western blotting. Our data showed that induction of hyperoxia for 3 days in mice showed larger outward potassium currents with shorter action potential durations (APD). This increase in current densities is due to significant increase in ultrarapid delayed rectifier outward K+ currents (IKur ) and rapidly activating, rapidly inactivating transient outward K+ current (Ito ) densities. We also observed a significant increase in both transcripts and protein levels of Kv1.5 and KChIP2 in hyperoxia treated atrial cardiomyocytes, whereas no significant change was observed in Kv4.2 transcripts or protein. The data presented here further support our previous findings that hyperoxia induces not only ventricular remodeling, but also atrial electrical remodeling.


Assuntos
Proteínas Interatuantes com Canais de Kv/genética , Canal de Potássio Kv1.6/genética , Pneumopatias/terapia , Oxigênio/efeitos adversos , Canais de Potássio Shal/genética , Potenciais de Ação/efeitos dos fármacos , Animais , Regulação da Expressão Gênica , Átrios do Coração/fisiopatologia , Mortalidade Hospitalar , Humanos , Hiperóxia/etiologia , Hiperóxia/fisiopatologia , Unidades de Terapia Intensiva , Pulmão/metabolismo , Pulmão/fisiopatologia , Pneumopatias/complicações , Pneumopatias/mortalidade , Pneumopatias/fisiopatologia , Camundongos , Miócitos Cardíacos/efeitos dos fármacos , Miócitos Cardíacos/patologia , Técnicas de Patch-Clamp , Potássio/metabolismo
18.
PLoS One ; 12(11): e0188678, 2017.
Artigo em Inglês | MEDLINE | ID: mdl-29176790

RESUMO

Attention-deficit/hyperactivity disorder (ADHD) is an early onset childhood neurodevelopmental disorder with high heritability. A number of genetic risk factors and environment factors have been implicated in the pathogenesis of ADHD. Genes encoding for subtypes of voltage-dependent K channels (Kv) and accessory proteins to these channels have been identified in genome-wide association studies (GWAS) of ADHD. We conducted a two-stage case-control study to investigate the associations between five key genes (KChIP4, KChIP1, DPP10, FHIT, and KCNC1) and the risk of developing ADHD. In the discovery stage comprising 256 cases and 372 controls, KChIP1 rs1541665 and FHIT rs3772475 were identified; they were further genotyped in the validation stage containing 328cases and 431 controls.KChIP1 rs1541665 showed significant association with a risk of ADHD at both stages, with CC vs TT odds ratio (OR) = 1.961, 95% confidence interval (CI) = 1.366-2.497, in combined analyses (P-FDR = 0.007). Moreover, we also found rs1541665 involvement in ADHD-I subtype (OR (95% CI) = 2.341(1.713, 3.282), and Hyperactive index score (P = 0.005) in combined samples.Intriguingly, gene-environmental interactions analysis consistently revealed the potential interactionsof rs1541665 collaboratingwith maternal stress pregnancy (Pmul = 0.021) and blood lead (Padd = 0.017) to modify ADHD risk. In conclusion, the current study provides evidence that genetic variants of Kv accessory proteins may contribute to the susceptibility of ADHD.Further studies with different ethnicitiesare warranted to produce definitive conclusions.


Assuntos
Transtorno do Deficit de Atenção com Hiperatividade/genética , Predisposição Genética para Doença , Proteínas Interatuantes com Canais de Kv/genética , Polimorfismo de Nucleotídeo Único/genética , Criança , Feminino , Interação Gene-Ambiente , Estudo de Associação Genômica Ampla , Humanos , Masculino , Fatores de Risco , Inquéritos e Questionários
19.
Sheng Li Xue Bao ; 69(5): 703-714, 2017 Oct 25.
Artigo em Chinês | MEDLINE | ID: mdl-29063118

RESUMO

DREAM (downstream regulatory element antagonist modulator), Calsenilin and KChIP3 (potassium channel interacting protein 3) belong to the neuronal calcium sensor (NCS) superfamily, which transduces the intracellular calcium signaling into a variety of activities. They are encoded by the same gene locus, but have distinct subcellular locations. DREAM was first found to interact with DRE (downstream regulatory element) site in the vicinity of the promoter of prodynorphin gene to suppress gene transcription. Calcium can disassemble this interaction by binding reversibly to DREAM protein on its four EF-hand motifs. Apart from having calcium dependent DRE site binding, DREAM can also interact with other transcription factors, such as cAMP responsive element binding protein (CREB), CREB-binding protein (CBP) and cAMP responsive element modulator (CREM), by this concerted actions, DREAM extends the gene pool under its control. DREAM is predominantly expressed in central nervous system with its highest level in cerebellum, and accumulating evidence demonstrated that DREAM might play important roles in pain sensitivity. Novel findings have shown that DREAM is also involved in learning and memory processes, Alzheimer's disease and stroke. This mini-review provides a brief introduction of its discovery history and protein structure properties, focusing on the mechanism of DREAM nuclear translocation and gene transcription regulation functions.


Assuntos
Regulação da Expressão Gênica , Proteínas Interatuantes com Canais de Kv/fisiologia , Proteínas Repressoras/fisiologia , Animais , Sinalização do Cálcio/fisiologia , Humanos , Proteínas Interatuantes com Canais de Kv/genética , Limiar da Dor , Proteínas Repressoras/genética
20.
Virology ; 512: 95-103, 2017 12.
Artigo em Inglês | MEDLINE | ID: mdl-28946006

RESUMO

Human cytomegalovirus (HCMV) encodes a viral cyclin-dependent kinase (v-CDK), the UL97 protein. UL97 phosphorylates Rb, p107 and p130, thereby inactivating all three retinoblastoma (Rb) family members. Rb proteins function through regulating the activity of transcription factors to which they bind. Therefore, we examined whether the UL97-mediated regulation of the Rb tumor suppressors also extended to their binding partners. We observed that UL97 phosphorylates LIN52, a component of p107- and p130-assembled transcriptionally repressive DREAM complexes that control transcription during the G0/G1 phases, and the Rb-associated E2F3 protein that activates transcription through G1 and S phases. Intriguingly, we also identified FoxM1B, a transcriptional regulator during the S and G2 phases, as a UL97 substrate. This survey extends the influence of UL97 beyond simply the Rb proteins themselves to their binding partners, as well as past the G1/S transition into later stages of the cell cycle.


Assuntos
Citomegalovirus/enzimologia , Fosfotransferases (Aceptor do Grupo Álcool)/metabolismo , Proteína do Retinoblastoma/metabolismo , Células Cultivadas , Ciclinas/genética , Ciclinas/metabolismo , Citomegalovirus/metabolismo , Fator de Transcrição E2F3/genética , Fator de Transcrição E2F3/metabolismo , Proteína Forkhead Box M1/genética , Proteína Forkhead Box M1/metabolismo , Fase G1 , Regulação Viral da Expressão Gênica/fisiologia , Humanos , Proteínas Interatuantes com Canais de Kv/genética , Proteínas Interatuantes com Canais de Kv/metabolismo , Mutagênese Sítio-Dirigida , Fosforilação , Fosfotransferases (Aceptor do Grupo Álcool)/genética , Subunidades Proteicas , Proteínas Repressoras/genética , Proteínas Repressoras/metabolismo , Fase de Repouso do Ciclo Celular , Proteína do Retinoblastoma/genética
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