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1.
2.
Bratisl Lek Listy ; 121(2): 111-116, 2020.
Artigo em Inglês | MEDLINE | ID: mdl-32115962

RESUMO

AIM: We aimed to determine the possible correlation between voltage­gated potassium channels and micro RNAs in breast cancer and metastatic breast cancer cells. METHOD: Kv1.3 and Kv10.1 channels were inhibited by specific siRNAs using a lipofectamine-based transfection in MCF-7 and MDA-MB-231 cells. After transfection, total RNA was isolated, and then miR-126 and miR-126* expressions were observed using RT-PCR. RESULTS: There was a negative correlation between Kv channels and miRNAs according to the characteristics of the breast cancer cells. The inhibition was observed not only in Kv1.3 but also in Kv10.1 in MCF-7 cells, and miR-126 and miR-126* expressions were downregulated compared to the control group (p < 0.001). The inhibition of these channels in MDA-MB-231 cells caused an upregulation of miR-126 and miR-126* expressions (p < 0.001). CONCLUSION: The miR-126 and miR-126* expressions differed according to benign and malign breast cancer cell lines. Furthermore, we found that miR-126/126* may interact with Kv1.3 and Kv10.1 voltage-gated potassium channels. Our study suggests and indicates the relationship between Kv channels and miRNAs in breast cancer cells (Tab. 1, Fig. 2, Ref. 51).


Assuntos
Neoplasias da Mama , Regulação Neoplásica da Expressão Gênica , MicroRNAs , Canais de Potássio de Abertura Dependente da Tensão da Membrana , Neoplasias da Mama/metabolismo , Linhagem Celular Tumoral , Humanos , Células MCF-7 , MicroRNAs/metabolismo
3.
Invest Ophthalmol Vis Sci ; 61(3): 33, 2020 03 09.
Artigo em Inglês | MEDLINE | ID: mdl-32191288

RESUMO

Purpose: To characterize the retinal expression and localization of Kcne2, an ancillary (ß) ion-channel subunit with an important role in fine-tuning cellular excitability. Methods: We analyzed available single-cell transcriptome data from tens of thousands of murine retinal cells for cell-type-specific expression of Kcne2 using state-of-the-art bioinformatics techniques. This evidence at the transcriptome level was complemented with a comprehensive immunohistochemical characterization of mouse retina (C57BL/6, ages 8-12 weeks) employing co-labeling techniques and cell-type-specific antibody markers. We furthermore examined how conserved the Kcne2 localization pattern in the retina was across species by performing immunostaining on zebrafish, cowbird, sheep, mice, and macaque. Results: Kcne2 is distinctly expressed in cone photoreceptors and rod bipolar cells. At a subcellular level, the bulk of Kcne2 immunoreactivity can be observed in the outer plexiform layer. Here, it localizes into cone pedicles and likely the postsynaptic membrane of the rod bipolar cells. Thus, the vast majority of Kcne2 immunoreactivity is observed in a thin band in the outer plexiform layer. In addition to this, faint Kcne2 immunoreactivity can also be observed in cone inner segments and the somata of a small subset of cone ON bipolar cells. Strikingly, the localization of Kcne2 in the outer plexiform layer was preserved among all of the species studied, spanning at least 300 million years of evolution of the vertebrate kingdom. Conclusions: The data we present here suggest an important and specific role for Kcne2 in the highly specialized photoreceptor-bipolar cell synapse.


Assuntos
Regulação da Expressão Gênica/fisiologia , Canais de Potássio de Abertura Dependente da Tensão da Membrana/genética , Células Bipolares da Retina/metabolismo , Células Fotorreceptoras Retinianas Cones/metabolismo , Células Fotorreceptoras Retinianas Bastonetes/metabolismo , Animais , Células CHO , Biologia Computacional , Cricetulus , Imuno-Histoquímica , Macaca mulatta , Camundongos , Camundongos Endogâmicos C57BL , Microscopia Confocal , Ovinos , Aves Canoras , Sinapses , Transfecção , Peixe-Zebra
4.
Nucleic Acids Res ; 48(8): 3999-4012, 2020 05 07.
Artigo em Inglês | MEDLINE | ID: mdl-32201888

RESUMO

In eukaryotic cells, with the exception of the specialized genomes of mitochondria and plastids, all genetic information is sequestered within the nucleus. This arrangement imposes constraints on how the information can be tailored for different cellular regions, particularly in cells with complex morphologies like neurons. Although messenger RNAs (mRNAs), and the proteins that they encode, can be differentially sorted between cellular regions, the information itself does not change. RNA editing by adenosine deamination can alter the genome's blueprint by recoding mRNAs; however, this process too is thought to be restricted to the nucleus. In this work, we show that ADAR2 (adenosine deaminase that acts on RNA), an RNA editing enzyme, is expressed outside of the nucleus in squid neurons. Furthermore, purified axoplasm exhibits adenosine-to-inosine activity and can specifically edit adenosines in a known substrate. Finally, a transcriptome-wide analysis of RNA editing reveals that tens of thousands of editing sites (>70% of all sites) are edited more extensively in the squid giant axon than in its cell bodies. These results indicate that within a neuron RNA editing can recode genetic information in a region-specific manner.


Assuntos
Adenosina Desaminase/metabolismo , Neurônios/enzimologia , Edição de RNA , Adenosina/metabolismo , Animais , Axônios/enzimologia , Citoplasma/enzimologia , Decapodiformes/enzimologia , Células HEK293 , Humanos , Inosina/metabolismo , Canais de Potássio de Abertura Dependente da Tensão da Membrana/genética , Canais de Potássio de Abertura Dependente da Tensão da Membrana/metabolismo , Sinapses/enzimologia
5.
Nat Commun ; 11(1): 676, 2020 02 03.
Artigo em Inglês | MEDLINE | ID: mdl-32015334

RESUMO

In voltage-gated potassium (KV) channels, the voltage-sensing domain (VSD) undergoes sequential activation from the resting state to the intermediate state and activated state to trigger pore opening via electro-mechanical (E-M) coupling. However, the spatial and temporal details underlying E-M coupling remain elusive. Here, utilizing KV7.1's unique two open states, we report a two-stage E-M coupling mechanism in voltage-dependent gating of KV7.1 as triggered by VSD activations to the intermediate and then activated state. When the S4 segment transitions to the intermediate state, the hand-like C-terminus of the VSD-pore linker (S4-S5L) interacts with the pore in the same subunit. When S4 then proceeds to the fully-activated state, the elbow-like hinge between S4 and S4-S5L engages with the pore of the neighboring subunit to activate conductance. This two-stage hand-and-elbow gating mechanism elucidates distinct tissue-specific modulations, pharmacology, and disease pathogenesis of KV7.1, and likely applies to numerous domain-swapped KV channels.


Assuntos
Canais de Potássio de Abertura Dependente da Tensão da Membrana/química , Canais de Potássio de Abertura Dependente da Tensão da Membrana/fisiologia , Humanos , Ativação do Canal Iônico/fisiologia , Canal de Potássio KCNQ1/química , Canal de Potássio KCNQ1/fisiologia , Simulação de Acoplamento Molecular , Oócitos/metabolismo , Canais de Potássio , Conformação Proteica
6.
Proc Natl Acad Sci U S A ; 117(7): 3575-3582, 2020 02 18.
Artigo em Inglês | MEDLINE | ID: mdl-32024761

RESUMO

Excitability-a threshold-governed transient in transmembrane voltage-is a fundamental physiological process that controls the function of the heart, endocrine, muscles, and neuronal tissues. The 1950s Hodgkin and Huxley explicit formulation provides a mathematical framework for understanding excitability, as the consequence of the properties of voltage-gated sodium and potassium channels. The Hodgkin-Huxley model is more sensitive to parametric variations of protein densities and kinetics than biological systems whose excitability is apparently more robust. It is generally assumed that the model's sensitivity reflects missing functional relations between its parameters or other components present in biological systems. Here we experimentally assembled excitable membranes using the dynamic clamp and voltage-gated potassium ionic channels (Kv1.3) expressed in Xenopus oocytes. We take advantage of a theoretically derived phase diagram, where the phenomenon of excitability is reduced to two dimensions defined as combinations of the Hodgkin-Huxley model parameters, to examine functional relations in the parameter space. Moreover, we demonstrate activity dependence and hysteretic dynamics over the phase diagram due to the impacts of complex slow inactivation kinetics. The results suggest that maintenance of excitability amid parametric variation is a low-dimensional, physiologically tenable control process. In the context of model construction, the results point to a potentially significant gap between high-dimensional models that capture the full measure of complexity displayed by ion channel function and the lower dimensionality that captures physiological function.


Assuntos
Modelos Biológicos , Xenopus/metabolismo , Animais , Cinética , Potenciais da Membrana , Oócitos/química , Oócitos/metabolismo , Canais de Potássio de Abertura Dependente da Tensão da Membrana/química , Canais de Potássio de Abertura Dependente da Tensão da Membrana/metabolismo , Canais de Sódio Disparados por Voltagem/química , Canais de Sódio Disparados por Voltagem/metabolismo
8.
Cell Mol Life Sci ; 77(13): 2473-2482, 2020 Jul.
Artigo em Inglês | MEDLINE | ID: mdl-31894358

RESUMO

Voltage-gated K+ (Kv) channel opening repolarizes excitable cells by allowing K+ efflux. Over the last two decades, multiple Kv functions in the nervous system have been found to be unrelated to or beyond the immediate control of excitability, such as shaping action potential contours or regulation of inter-spike frequency. These functions include neuronal exocytosis and neurite formation, neuronal cell death, regulation of astrocyte Ca2+, glial cell and glioma proliferation. Some of these functions have been shown to be independent of K+ conduction, that is, they suggest the non-canonical functions of Kv channels. In this review, we focus on neuronal or glial plasmalemmal Kv channel functions which are unrelated to shaping action potentials or immediate control of excitability. Similar functions in other cell types will be discussed to some extent in appropriate contexts.


Assuntos
Neuroglia/metabolismo , Neurônios/metabolismo , Canais de Potássio de Abertura Dependente da Tensão da Membrana/fisiologia , Potenciais de Ação , Apoptose , Astrócitos/metabolismo , Cálcio/metabolismo , Movimento Celular , Proliferação de Células , Exocitose , Glioma/patologia , Neuritos/fisiologia , Neuroglia/citologia
9.
Eur J Paediatr Neurol ; 24: 105-116, 2020 Jan.
Artigo em Inglês | MEDLINE | ID: mdl-31932120

RESUMO

Next-generation sequencing has enhanced discovery of many disease-associated genes in previously unexplained epilepsies, mainly in developmental and epileptic encephalopathies and familial epilepsies. We now classify these disorders according to the underlying molecular pathways, which encompass a diverse array of cellular and sub-cellular compartments/signalling processes including voltage-gated ion-channel defects. With the aim to develop and increase the use of precision medicine therapies, understanding the pathogenic mechanisms and consequences of disease-causing variants has gained major relevance in clinical care. The super-family of voltage-gated potassium channels is the largest and most diverse family among the ion channels, encompassing approximately 80 genes. Key potassium channelopathies include those affecting the KV, KCa and Kir families, a significant proportion of which have been implicated in neurological disease. As for other ion channel disorders, different pathogenic variants within any individual voltage-gated potassium channel gene tend to affect channel protein function differently, causing heterogeneous clinical phenotypes. The focus of this review is to summarise recent clinical developments regarding the key voltage-gated potassium (KV) family-related epilepsies, which now encompasses approximately 12 established disease-associated genes, from the KCNA-, KCNB-, KCNC-, KCND-, KCNV-, KCNQ- and KCNH-subfamilies.


Assuntos
Epilepsia/genética , Canais de Potássio de Abertura Dependente da Tensão da Membrana/genética , Humanos
10.
Am J Physiol Cell Physiol ; 318(2): C346-C359, 2020 02 01.
Artigo em Inglês | MEDLINE | ID: mdl-31693400

RESUMO

Diabetic corneal endothelial keratopathy is an intractable ocular complication characterized by corneal edema and endothelial decompensation, which seriously threaten vision. It has been suggested that diabetes is associated with pyroptosis, a type of programmed cell death via the activation of inflammation. Long noncoding RNA KCNQ1OT1 is commonly associated with various pathophysiological mechanisms of diabetic complications, including diabetic cardiomyopathy and diabetic retinopathy. However, whether KCNQ1OT1 is capable of regulating pyroptosis and participates in the pathogenesis of diabetic corneal endothelial keratopathy remains unknown. The aim of this study was to investigate the mechanisms of KCNQ1OT1 in diabetic corneal endothelial keratopathy. Here, we reveal that KCNQ1OT1 and pyroptosis can be triggered in diabetic human and rat corneal endothelium, along with the high glucose-treated corneal endothelial cells. However, miR-214 expression was substantially decreased in vivo and in experiments with cultured cells. LDH assay was also used to verify the existence of pyroptosis in high glucose-treated cells. Bioinformatics prediction and luciferase assays showed that KCNQ1OT1 may function as a competing endogenous RNA binding miR-214 to regulate the expression of caspase-1. To further analyze the KCNQ1OT1-mediated mechanism, miR-214 mimic and inhibitor were introduced into the high glucose-treated corneal endothelial cells. The results showed that upregulation of miR-214 attenuated pyroptosis; conversely, knockdown of miR-214 promoted it. In addition, KCNQ1OT1 knockdown by a small interfering RNA decreased pyroptosis factors expressions but enhanced miR-214 expression in corneal endothelial cells. To understand the signaling mechanisms underlying the prepyroptotic properties of KCNQ1OT1, si-KCNQ1OT1 was cotransfected with or without miR-214 inhibitor. The results showed that pyroptosis was repressed after silencing KCNQ1OT1 but was reversed by cotransfection with miR-214 inhibitor, suggesting that KCNQ1OT1 mediated pyroptosis induced by high glucose via targeting miR-214. Therefore, the KCNQ1OT1/miR-214/caspase-1 signaling pathway represents a new mechanism of diabetic corneal endothelial keratopathy progression, and KCNQ1OT1 could potentially be a novel therapeutic target.


Assuntos
Complicações do Diabetes/genética , Cardiomiopatias Diabéticas/genética , Células Endoteliais/metabolismo , Epitélio Posterior/metabolismo , Piroptose/genética , Animais , Estudos de Casos e Controles , Caspase 1/genética , Linhagem Celular , Complicações do Diabetes/metabolismo , Diabetes Mellitus/genética , Diabetes Mellitus/metabolismo , Cardiomiopatias Diabéticas/metabolismo , Feminino , Humanos , Masculino , MicroRNAs/genética , Pessoa de Meia-Idade , Canais de Potássio de Abertura Dependente da Tensão da Membrana/genética , RNA Longo não Codificante/genética , RNA Interferente Pequeno/genética , Ratos , Ratos Sprague-Dawley , Regulação para Cima/genética
11.
Mol Pharmacol ; 97(2): 132-144, 2020 02.
Artigo em Inglês | MEDLINE | ID: mdl-31722973

RESUMO

The pairing of KCNQ1 and KCNE1 subunits together mediates the cardiac slow delayed rectifier current (I Ks ), which is partly responsible for cardiomyocyte repolarization and physiologic shortening of the cardiac action potential. Mefenamic acid, a nonsteroidal anti-inflammatory drug, has been identified as an I Ks activator. Here, we provide a biophysical and pharmacological characterization of mefenamic acid's effect on I Ks Using whole-cell patch clamp, we show that mefenamic acid enhances I Ks activity in both a dose- and stoichiometry-dependent fashion by changing the slowly activating and deactivating I Ks current into an almost linear current with instantaneous onset and slowed tail current decay, sensitive to the I Ks blocker (3R,4S)-(+)-N-[3-hydroxy-2,2-dimethyl-6-(4,4,4-trifluorobutoxy) chroman-4-yl]-N-methylmethanesulfonamide (HMR1556). Both single channels, which reveal no change in the maximum conductance, and whole-cell studies, which reveal a dramatically altered conductance-voltage relationship despite increasingly longer interpulse intervals, suggest mefenamic acid decreases the voltage sensitivity of the I Ks channel and shifts channel gating kinetics toward more negative potentials. Modeling studies revealed that changes in voltage sensor activation kinetics are sufficient to reproduce the dose and frequency dependence of mefenamic acid action on I Ks channels. Mutational analysis showed that mefenamic acid's effect on I Ks required residue K41 and potentially other surrounding residues on the extracellular surface of KCNE1, and explains why the KCNQ1 channel alone is insensitive to up to 1 mM mefenamic acid. Given that mefenamic acid can enhance all I Ks channel complexes containing different ratios of KCNQ1 to KCNE1, it may provide a promising therapeutic approach to treating life-threatening cardiac arrhythmia syndromes. SIGNIFICANCE STATEMENT: The channels which generate the cardiac slow delayed rectifier K+ current (I Ks ) are composed of KCNQ1 and KCNE1 subunits. Due to the critical role played by I Ks in heartbeat regulation, enhancing I Ks current has been identified as a promising therapeutic strategy to treat various heart rhythm diseases. Most I Ks activators, unfortunately, only work on KCNQ1 alone and not the physiologically relevant I Ks channel. We have demonstrated that mefenamic acid can enhance I Ks in a dose- and stoichiometry-dependent fashion, regulated by its interactions with KCNE1.


Assuntos
Antiarrítmicos/farmacologia , Ativação do Canal Iônico/efeitos dos fármacos , Ácido Mefenâmico/farmacologia , Canais de Potássio de Abertura Dependente da Tensão da Membrana/metabolismo , Potássio/metabolismo , Animais , Antiarrítmicos/uso terapêutico , Arritmias Cardíacas/tratamento farmacológico , Arritmias Cardíacas/fisiopatologia , Relação Dose-Resposta a Droga , Fibroblastos , Células HEK293 , Frequência Cardíaca/fisiologia , Humanos , Canal de Potássio KCNQ1/química , Canal de Potássio KCNQ1/genética , Canal de Potássio KCNQ1/metabolismo , Potenciais da Membrana/efeitos dos fármacos , Camundongos , Mutagênese Sítio-Dirigida , Mutação , Miocárdio/metabolismo , Técnicas de Patch-Clamp , Canais de Potássio de Abertura Dependente da Tensão da Membrana/agonistas , Canais de Potássio de Abertura Dependente da Tensão da Membrana/química , Canais de Potássio de Abertura Dependente da Tensão da Membrana/genética , Proteínas Recombinantes/química , Proteínas Recombinantes/genética , Proteínas Recombinantes/metabolismo , Transdução de Sinais/efeitos dos fármacos
12.
Life Sci ; 241: 117161, 2020 Jan 15.
Artigo em Inglês | MEDLINE | ID: mdl-31837329

RESUMO

AIMS: Acute myeloid leukemia (AML) is an aggressive cancer that invariably produces drug resistance after treatment. The aim is to explore the role of lncRNA potassium voltage-gated channel subfamily Q member 1 overlapping transcript 1 (KCNQ1OT1) and associated novel mechanisms in the progression and chemoresistance of AML. MAIN METHODS: The expression of KCNQ1OT1, miR-193a-3p, and Tspan3 was measured by qRT-PCR. The values of IC50 for adriamycin (ADR) and the ability of proliferation were analyzed by CCK-8 assay. Cell migration and invasion were assessed by transwell assay. Cell apoptosis was monitored by flow cytometry assay. The expression of Tspan3, MRP1, P-gp and LRP at the protein level was quantified by western blot. The relationship between miR-193a-3p and KCNQ1OT1 or Tspan3 was predicted by bioinformatics tool Diana and verified by dual-luciferase reporter assay, RIP assay or RNA pull-down assay. KEY FINDINGS: KCNQ1OT1 and Tspan3 were up-regulated, while miR-193a-3p was down-regulated in ADR resistant AML samples and cells. KCNQ1OT1 knockdown reduced ADR resistance, inhibited proliferation, migration and invasion but promoted apoptosis of ADR resistant AML cells, miR-193a-3p inhibition reversed these effects. MiR-193a-3p was a target of KCNQ1OT1 and combined with Tspan3 3' untranslated region (3' UTR). Enrichment of miR-193a-3p decreased ADR resistance, inhibited proliferation, migration and invasion and stimulated apoptosis in ADR resistant AML cells, but Tspan3 overexpression overturned these impacts. SIGNIFICANCE: KCNQ1OT1 aggravates AML progression and chemoresistance to ADR by inducing Tspan3 expression via adsorbing miR-193a-3p in ADR resistant AML cells, providing a theoretical basis for the treatment of AML with chemoresistance.


Assuntos
Doxorrubicina/farmacologia , Resistencia a Medicamentos Antineoplásicos/genética , Regulação Neoplásica da Expressão Gênica/efeitos dos fármacos , Leucemia Mieloide Aguda/patologia , MicroRNAs/genética , Tetraspaninas/metabolismo , Antibióticos Antineoplásicos/farmacologia , Estudos de Casos e Controles , Movimento Celular , Proliferação de Células , Progressão da Doença , Humanos , Leucemia Mieloide Aguda/tratamento farmacológico , Leucemia Mieloide Aguda/genética , Leucemia Mieloide Aguda/metabolismo , Canais de Potássio de Abertura Dependente da Tensão da Membrana/genética , Tetraspaninas/genética , Células Tumorais Cultivadas
13.
Am J Physiol Lung Cell Mol Physiol ; 318(1): L10-L26, 2020 01 01.
Artigo em Inglês | MEDLINE | ID: mdl-31553627

RESUMO

Downregulated expression of K+ channels and decreased K+ currents in pulmonary artery smooth muscle cells (PASMC) have been implicated in the development of sustained pulmonary vasoconstriction and vascular remodeling in patients with idiopathic pulmonary arterial hypertension (IPAH). However, it is unclear exactly how K+ channels are downregulated in IPAH-PASMC. MicroRNAs (miRNAs) are small non-coding RNAs that are capable of posttranscriptionally regulating gene expression by binding to the 3'-untranslated regions of their targeted mRNAs. Here, we report that specific miRNAs are responsible for the decreased K+ channel expression and function in IPAH-PASMC. We identified 3 miRNAs (miR-29b, miR-138, and miR-222) that were highly expressed in IPAH-PASMC in comparison to normal PASMC (>2.5-fold difference). Selectively upregulated miRNAs are correlated with the decreased expression and attenuated activity of K+ channels. Overexpression of miR-29b, miR-138, or miR-222 in normal PASMC significantly decreased whole cell K+ currents and downregulated voltage-gated K+ channel 1.5 (KV1.5/KCNA5) in normal PASMC. Inhibition of miR-29b in IPAH-PASMC completely recovered K+ channel function and KV1.5 expression, while miR-138 and miR-222 had a partial or no effect. Luciferase assays further revealed that KV1.5 is a direct target of miR-29b. Additionally, overexpression of miR-29b in normal PASMC decreased large-conductance Ca2+-activated K+ (BKCa) channel currents and downregulated BKCa channel ß1 subunit (BKCaß1 or KCNMB1) expression, while inhibition of miR-29b in IPAH-PASMC increased BKCa channel activity and BKCaß1 levels. These data indicate upregulated miR-29b contributes at least partially to the attenuated function and expression of KV and BKCa channels in PASMC from patients with IPAH.


Assuntos
Regulação para Baixo/genética , Hipertensão Pulmonar Primária Familiar/genética , MicroRNAs/genética , Canais de Potássio de Abertura Dependente da Tensão da Membrana/genética , Adolescente , Adulto , Células Cultivadas , Hipertensão Pulmonar Primária Familiar/metabolismo , Feminino , Humanos , Masculino , Potenciais da Membrana/genética , Pessoa de Meia-Idade , Músculo Liso Vascular/metabolismo , Miócitos de Músculo Liso/metabolismo , Artéria Pulmonar/metabolismo , RNA Mensageiro/genética , Regulação para Cima/genética , Vasoconstrição/genética , Adulto Jovem
14.
Am J Physiol Heart Circ Physiol ; 318(2): H212-H222, 2020 02 01.
Artigo em Inglês | MEDLINE | ID: mdl-31834838

RESUMO

The cardiac potassium IKs current is carried by a channel complex formed from α-subunits encoded by KCNQ1 and ß-subunits encoded by KCNE1. Deleterious mutations in either gene are associated with hereditary long QT syndrome. Interactions between the transmembrane domains of the α- and ß-subunits determine the activation kinetics of IKs. A physical and functional interaction between COOH termini of the proteins has also been identified that impacts deactivation rate and voltage dependence of activation. We sought to explore the specific physical interactions between the COOH termini of the subunits that confer such control. Hydrogen/deuterium exchange coupled to mass spectrometry narrowed down the region of interaction to KCNQ1 residues 352-374 and KCNE1 residues 70-81, and provided evidence of secondary structure within these segments. Key mutations of residues in these regions tended to shift voltage dependence of activation toward more depolarizing voltages. Double-mutant cycle analysis then revealed energetic coupling between KCNQ1-I368 and KCNE1-D76 during channel activation. Our results suggest that the proximal COOH-terminal regions of KCNQ1 and KCNE1 participate in a physical and functional interaction during channel opening that is sensitive to perturbation and may explain the clustering of long QT mutations in the region.NEW & NOTEWORTHY Interacting ion channel subunits KCNQ1 and KCNE1 have received intense investigation due to their critical importance to human cardiovascular health. This work uses physical (hydrogen/deuterium exchange with mass spectrometry) and functional (double-mutant cycle analyses) studies to elucidate precise and important areas of interaction between the two proteins in an area that has eluded structural definition of the complex. It highlights the importance of pathogenic mutations in these regions.


Assuntos
Citoplasma/metabolismo , Canal de Potássio KCNQ1/química , Canal de Potássio KCNQ1/metabolismo , Canais de Potássio de Abertura Dependente da Tensão da Membrana/química , Canais de Potássio de Abertura Dependente da Tensão da Membrana/metabolismo , Animais , Clonagem Molecular , Cricetinae , Deutério/metabolismo , Fenômenos Eletrofisiológicos , Células HEK293 , Humanos , Hidrogênio/metabolismo , Canal de Potássio KCNQ1/genética , Síndrome do QT Longo/genética , Síndrome do QT Longo/metabolismo , Mutação , Plasmídeos/genética , Canais de Potássio de Abertura Dependente da Tensão da Membrana/genética
15.
Eur J Pharmacol ; 866: 172828, 2020 Jan 05.
Artigo em Inglês | MEDLINE | ID: mdl-31790651

RESUMO

The venoarteriolar reflex is a local mechanism that induces vasoconstriction during venous congestion in various tissues, including skin. This response is thought to play a critical role in minimizing capillary damage or edema resulting from overperfusion, though factors that modulate this response remain largely unknown. Here, we hypothesized that nitric oxide synthase (NOS), cyclooxygenase (COX), and Ca2+-activated, ATP-sensitive, and voltage-gated K+ channels (KCa, KATP, and KV channels, respectively) modulate the venoarteriolar reflex in human skin. Cutaneous blood flow (laser-Doppler flowmetry) was monitored during a 3-min pre-occlusion baseline and following a 3-min venous occlusion of 45 mmHg, the latter maneuver was used to induce the venoarteriolar reflex. The venoarteriolar reflex was assessed at the following forearm skin sites: Experiment 1 (n = 11): 1) lactated Ringer solution (Control), 2) 10 mM Nω-nitro-L-arginine (NOS inhibitor), 3) 10 mM ketorolac (COX inhibitor), and 4) combined NOS + COX inhibition; Experiment 2 (n = 15): 1) lactated Ringer solution (Control), 2) 50 mM tetraethylammonium (KCa channel blocker), 3) 5 mM glybenclamide (KATP channel blocker), and 4) 10 mM 4-aminopyridine (KV channel blocker). Separate and combined NOS and COX inhibition as well as KATP channel blocker had no effect on venoarteriolar reflex. Conversely, venoarteriolar reflex was attenuated by KCa channel blockade (36-38%) and augmented by KV channel blockade (38-55%). We showed that KCa and KV channels modulate the venoarteriolar reflex with minimum roles of NOS, COX, and KATP channels in human non-glabrous forearm skin in vivo. Thus, cutaneous venoarteriolar reflex changes could reflect altered K+ channel function.


Assuntos
Arteríolas/fisiologia , Canais de Potássio Cálcio-Ativados/metabolismo , Canais de Potássio de Abertura Dependente da Tensão da Membrana/metabolismo , Pele/irrigação sanguínea , Pele/metabolismo , Veias/fisiologia , Adulto , Humanos , Canais KATP/metabolismo , Masculino , Óxido Nítrico Sintase/metabolismo , Prostaglandina-Endoperóxido Sintases/metabolismo , Fluxo Sanguíneo Regional
17.
Cell ; 180(2): 340-347.e9, 2020 01 23.
Artigo em Inglês | MEDLINE | ID: mdl-31883792

RESUMO

KCNQ1, also known as Kv7.1, is a voltage-dependent K+ channel that regulates gastric acid secretion, salt and glucose homeostasis, and heart rhythm. Its functional properties are regulated in a tissue-specific manner through co-assembly with beta subunits KCNE1-5. In non-excitable cells, KCNQ1 forms a complex with KCNE3, which suppresses channel closure at negative membrane voltages that otherwise would close it. Pore opening is regulated by the signaling lipid PIP2. Using cryoelectron microscopy (cryo-EM), we show that KCNE3 tucks its single-membrane-spanning helix against KCNQ1, at a location that appears to lock the voltage sensor in its depolarized conformation. Without PIP2, the pore remains closed. Upon addition, PIP2 occupies a site on KCNQ1 within the inner membrane leaflet, which triggers a large conformational change that leads to dilation of the pore's gate. It is likely that this mechanism of PIP2 activation is conserved among Kv7 channels.


Assuntos
Canal de Potássio KCNQ1/metabolismo , Canal de Potássio KCNQ1/ultraestrutura , Microscopia Crioeletrônica , Humanos , Ativação do Canal Iônico/fisiologia , Canal de Potássio KCNQ1/química , Potenciais da Membrana/fisiologia , Técnicas de Patch-Clamp , Fosfatidilinositol 4,5-Difosfato/metabolismo , Canais de Potássio de Abertura Dependente da Tensão da Membrana/química , Canais de Potássio de Abertura Dependente da Tensão da Membrana/metabolismo , Canais de Potássio de Abertura Dependente da Tensão da Membrana/ultraestrutura
18.
Cardiovasc Ther ; 2019: 6032631, 2019.
Artigo em Inglês | MEDLINE | ID: mdl-31772612

RESUMO

Background: LCZ696 (valsartan/sacubitril) therapy significantly reduced mortality in patients with heart failure (HF). Although a clinical trial (PARADISE-MI Trial) has been ongoing to examine the effects of LCZ696 in myocardial infarction (MI) patients, the effects of LCZ696 on remodeling of cardiac electrophysiology in animal models remain largely unclear. Methods: We performed coronary artery ligation to create MI in Sprague-Dawley rats. Echocardiography was performed one week after MI to confirm the development of HF with left ventricular ejection fraction ≤ 40%. MI rats were randomly assigned to receive medical therapy for 4 weeks: LCZ696, enalapril, or vehicle. The sham-operation rats received sham operation without MI creation. In vivo electrophysiological exams were performed under general anesthesia. Western blot analyses were conducted to quantify ion channel proteins. Results: The HF-vehicle group did not show significant changes in LVEF. Both enalapril and LCZ696 therapy significantly improved LVEF. The HF-vehicle group had higher ventricular arrhythmia (VA) inducibility than the sham group. As compared with the HF-vehicle group, LCZ696 therapy significantly reduced VA inducibility, but enalapril therapy did not. Western blot analyses showed significant downregulation of NaV1.5, ERG, KCNE1, and KCNE2 channel proteins in the HF vehicle group compared with the sham group. LCZ696 therapy upregulated protein expression of ERG, KCNE1, and KCNE2. Conclusion: As compared with enalapril therapy, LCZ696 therapy led to improvement of LVEF, reduced VA inducibility, and upregulated expression of K+ channel proteins.


Assuntos
Aminobutiratos/farmacologia , Antiarrítmicos/farmacologia , Arritmias Cardíacas/prevenção & controle , Insuficiência Cardíaca/tratamento farmacológico , Frequência Cardíaca/efeitos dos fármacos , Ventrículos do Coração/efeitos dos fármacos , Infarto do Miocárdio/complicações , Tetrazóis/farmacologia , Função Ventricular Esquerda/efeitos dos fármacos , Animais , Arritmias Cardíacas/etiologia , Arritmias Cardíacas/metabolismo , Arritmias Cardíacas/fisiopatologia , Modelos Animais de Doenças , Canal de Potássio ERG1/metabolismo , Feminino , Insuficiência Cardíaca/etiologia , Insuficiência Cardíaca/metabolismo , Insuficiência Cardíaca/fisiopatologia , Ventrículos do Coração/metabolismo , Ventrículos do Coração/fisiopatologia , Masculino , Canal de Sódio Disparado por Voltagem NAV1.5/metabolismo , Canais de Potássio de Abertura Dependente da Tensão da Membrana/metabolismo , Ratos Sprague-Dawley , Volume Sistólico/efeitos dos fármacos
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