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1.
PLoS One ; 15(8): e0236246, 2020.
Artigo em Inglês | MEDLINE | ID: mdl-32804956

RESUMO

K+ is an essential nutrient for plant growth and is responsible for many important physiological processes. K+ deficiency leads to crop yield losses, and overexpression of K+ transporter genes has been proven to be an effective way to resolve this problem. However, current research on the overexpression of K+ transporter genes is limited to plant sources. TrkH is a bacterial K+ transporter whose function generally depends on the regulation of TrkA. To date, whether TrkH can improve K+ uptake in eukaryotic organisms is still unknown. In this study, a novel MbtrkH gene was cloned from marine microbial metagenomic DNA. Functional complementation and K+-depletion analyses revealed that MbTrkH functions in K+ uptake in the K+-deficient yeast strain CY162. Moreover, K+-depletion assays revealed that MbtrkH overexpression improves plant K+ uptake. K+ hydroponic culture experiments showed that, compared with WT tobacco lines, MbtrkH transgenic tobacco lines had significantly greater fresh weights, dry weights and K+ contents. These results indicate that MbTrkH promotes K+ uptake independently of TrkA in eukaryotes and provide a new strategy for improving K+-use efficiency in plants.


Assuntos
Organismos Aquáticos/genética , Potássio/metabolismo , Saccharomyces cerevisiae/metabolismo , Água do Mar/microbiologia , Tabaco/metabolismo , Clonagem Molecular , Metagenoma , Plantas Geneticamente Modificadas/genética , Plantas Geneticamente Modificadas/metabolismo , Canais de Potássio/genética , Canais de Potássio/metabolismo , Saccharomyces cerevisiae/genética , Tabaco/genética
2.
PLoS One ; 15(7): e0236193, 2020.
Artigo em Inglês | MEDLINE | ID: mdl-32692755

RESUMO

BACKGROUND: Naturally occurring human genetic variants provide a valuable tool to identify drug targets and guide drug prioritization and clinical trial design. Ivabradine is a heart rate lowering drug with protective effects on heart failure despite increasing the risk of atrial fibrillation. In patients with coronary artery disease without heart failure, the drug does not protect against major cardiovascular adverse events prompting questions about the ability of genetics to have predicted those effects. This study evaluates the effect of a variant in HCN4, ivabradine's drug target, on safety and efficacy endpoints. METHODS: We used genetic association testing and Mendelian randomization to predict the effect of ivabradine and heart rate lowering on cardiovascular outcomes. RESULTS: Using data from the UK Biobank and large GWAS consortia, we evaluated the effect of a heart rate-reducing genetic variant at the HCN4 locus encoding ivabradine's drug target. These genetic association analyses showed increases in risk for atrial fibrillation (OR 1.09, 95% CI: 1.06-1.13, P = 9.3 ×10-9) in the UK Biobank. In a cause-specific competing risk model to account for the increased risk of atrial fibrillation, the HCN4 variant reduced incident heart failure in participants that did not develop atrial fibrillation (HR 0.90, 95% CI: 0.83-0.98, P = 0.013). In contrast, the same heart rate reducing HCN4 variant did not prevent a composite endpoint of myocardial infarction or cardiovascular death (OR 0.99, 95% CI: 0.93-1.04, P = 0.61). CONCLUSION: Genetic modelling of ivabradine recapitulates its benefits in heart failure, promotion of atrial fibrillation, and neutral effect on myocardial infarction.


Assuntos
Ivabradina/farmacologia , Modelos Genéticos , Ensaios Clínicos Controlados Aleatórios como Assunto , Adulto , Idoso , Alelos , Doenças Cardiovasculares/fisiopatologia , Determinação de Ponto Final , Feminino , Variação Genética , Estudo de Associação Genômica Ampla , Frequência Cardíaca/efeitos dos fármacos , Frequência Cardíaca/genética , Humanos , Canais Disparados por Nucleotídeos Cíclicos Ativados por Hiperpolarização/genética , Masculino , Análise da Randomização Mendeliana , Pessoa de Meia-Idade , Proteínas Musculares/genética , Canais de Potássio/genética , Fatores de Risco
3.
Am J Physiol Gastrointest Liver Physiol ; 319(2): G142-G150, 2020 08 01.
Artigo em Inglês | MEDLINE | ID: mdl-32567323

RESUMO

Transepithelial K+ absorption requires apical K+ uptake and basolateral K+ exit. In the colon, apical H+-K+-ATPase mediates cellular K+ uptake, and it has been suggested that electroneutral basolateral K+ exit reflects K+-Cl- cotransporter-1 (KCC1) operating in parallel with K+ and Cl- channels. The present study was designed to identify basolateral transporter(s) responsible for K+ exit in rat distal colon. Active K+ absorption was determined by measuring 86Rb+ (K+ surrogate) fluxes across colonic epithelia under voltage-clamp conditions. With zero Cl- in the mucosal solution, net K+ absorption was reduced by 38%, indicating that K+ absorption was partially Cl--dependent. Serosal addition of DIOA (KCC1 inhibitor) or Ba2+ (nonspecific K+ channel blocker) inhibited net K+ absorption by 21% or 61%, respectively, suggesting that both KCC1 and K+ channels contribute to basolateral K+ exit. Clotrimazole and TRAM34 (IK channel blockers) added serosally inhibited net K+ absorption, pointing to the involvement of IK channels in basolateral K+ exit. GaTx2 (CLC2 blocker) added serosally also inhibited net K+ absorption, suggesting that CLC2-mediated Cl- exit accompanies IK channel-mediated K+ exit across the basolateral membrane. Net K+ absorption was not inhibited by serosal addition of either IbTX (BK channel blocker), apamin (SK channel blocker), chromanol 293B (KV7 channel blocker), or CFTRinh172 (CFTR blocker). Immunofluorescence studies confirmed basolateral membrane colocalization of CLC2-like proteins and Na+-K+-ATPase α-subunits. We conclude that active K+ absorption in rat distal colon involves electroneutral basolateral K+ exit, which may reflect IK and CLC2 channels operating in parallel.NEW & NOTEWORTHY This study demonstrates that during active electroneutral K+ absorption in rat distal colon, K+ exit across the basolateral membrane mainly reflects intermediate conductance K+ channels operating in conjunction with chloride channel 2, with a smaller, but significant, contribution from K+-Cl- cotransporter-1 (KCC1) activity.


Assuntos
Canais de Cloreto/metabolismo , Colo/fisiologia , Mucosa Intestinal/metabolismo , Canais de Potássio/metabolismo , Potássio/metabolismo , Animais , Canais de Cloreto/genética , Cloretos/metabolismo , Feminino , Transporte de Íons , Masculino , Técnicas de Patch-Clamp , Canais de Potássio/genética , Transporte Proteico , Ratos , Ratos Sprague-Dawley
4.
Proc Natl Acad Sci U S A ; 117(24): 13757-13766, 2020 06 16.
Artigo em Inglês | MEDLINE | ID: mdl-32467161

RESUMO

Inhaled anesthetics are a chemically diverse collection of hydrophobic molecules that robustly activate TWIK-related K+ channels (TREK-1) and reversibly induce loss of consciousness. For 100 y, anesthetics were speculated to target cellular membranes, yet no plausible mechanism emerged to explain a membrane effect on ion channels. Here we show that inhaled anesthetics (chloroform and isoflurane) activate TREK-1 through disruption of phospholipase D2 (PLD2) localization to lipid rafts and subsequent production of signaling lipid phosphatidic acid (PA). Catalytically dead PLD2 robustly blocks anesthetic TREK-1 currents in whole-cell patch-clamp recordings. Localization of PLD2 renders the TRAAK channel sensitive, a channel that is otherwise anesthetic insensitive. General anesthetics, such as chloroform, isoflurane, diethyl ether, xenon, and propofol, disrupt lipid rafts and activate PLD2. In the whole brain of flies, anesthesia disrupts rafts and PLDnull flies resist anesthesia. Our results establish a membrane-mediated target of inhaled anesthesia and suggest PA helps set thresholds of anesthetic sensitivity in vivo.


Assuntos
Anestésicos Inalatórios/administração & dosagem , Animais , Membrana Celular/efeitos dos fármacos , Membrana Celular/genética , Membrana Celular/metabolismo , Clorofórmio/administração & dosagem , Drosophila/efeitos dos fármacos , Drosophila/genética , Drosophila/metabolismo , Proteínas de Drosophila/genética , Proteínas de Drosophila/metabolismo , Isoflurano/administração & dosagem , Ácidos Fosfatídicos/metabolismo , Fosfolipase D/genética , Fosfolipase D/metabolismo , Canais de Potássio/genética , Canais de Potássio/metabolismo , Canais de Potássio de Domínios Poros em Tandem/genética , Canais de Potássio de Domínios Poros em Tandem/metabolismo
5.
Toxicol Lett ; 327: 19-31, 2020 Jul 01.
Artigo em Inglês | MEDLINE | ID: mdl-32234357

RESUMO

Traditional Chinese Medicines (TCMs)-containing aconitine are popular and indispensable home remedies in Asia for thousands of years due to its excellent pharmaceutical effects. Accumulating evidence has identified that repeated-dose of aconitine could cause polymorphic ventricular arrhythmias. However, underlying molecular mechanisms are still not fully understood. Hence, the present study firstly investigated the potential role of Notch1 signaling in aconitine-induced cardiotoxicity, aiming to elaborate possible molecular mechanisms involved in aconitine triggered ventricular arrhythmias. Our results showed that aconitine increased Notch1 signaling and downstream KDM5A expression in human and rat cardiomyocytes at non-detectable cytotoxic doses. Furthermore, aconitine promoted the formation of a new regulatory complex containing NICD and KDM5A in a CK2αHI regime, which then targeted to HCN4 promoter and induced re-expression of HCN4 in mature cardiomyocytes. Ultimately, HCN4-mediated If current contributed to aconitine-caused alterations in beating rate of rat cardiomyocytes. All changes aforementioned were significantly ameliorated by Notch1 inhibitor, suggesting that Notch1-mediated epigenetic regulation of HCN4 contributes to aconitine-induced ventricular myocardial dysrhythmia. Thus, our findings provide a novel toxic mechanism and position Notch1/NICD/KDM5A/HCN4 toxicity pathway as a potential target for the treatments of repeated-dose of medicine containing aconitine induced ventricular arrhythmias.


Assuntos
Aconitina/farmacologia , Arritmias Cardíacas/induzido quimicamente , Ventrículos do Coração/efeitos dos fármacos , Canais Disparados por Nucleotídeos Cíclicos Ativados por Hiperpolarização/metabolismo , Canais de Potássio/metabolismo , Receptor Notch1/metabolismo , Animais , Animais Recém-Nascidos , Linhagem Celular , Sobrevivência Celular/efeitos dos fármacos , Regulação da Expressão Gênica/efeitos dos fármacos , Histonas , Humanos , Canais Disparados por Nucleotídeos Cíclicos Ativados por Hiperpolarização/genética , Mitocôndrias/efeitos dos fármacos , Mitocôndrias/metabolismo , Proteínas Musculares/genética , Proteínas Musculares/metabolismo , Miócitos Cardíacos/efeitos dos fármacos , Canais de Potássio/genética , Ratos , Receptor Notch1/genética , Superóxidos/metabolismo
6.
Nat Commun ; 11(1): 1419, 2020 03 17.
Artigo em Inglês | MEDLINE | ID: mdl-32184399

RESUMO

Hyperpolarization-activated cyclic nucleotide-gated (HCN) channels are essential for rhythmic activity in the heart and brain, and mutations in HCN channels are linked to heart arrhythmia and epilepsy. HCN channels belong to the family of voltage-gated K+ (Kv) channels. However, why HCN channels are activated by hyperpolarization whereas Kv channels are activated by depolarization is not clear. Here we reverse the voltage dependence of HCN channels by mutating only two residues located at the interface between the voltage sensor and the pore domain such that the channels now open upon depolarization instead of hyperpolarization. Our data indicate that what determines whether HCN channels open by hyperpolarizations or depolarizations are small differences in the energies of the closed and open states, due to different interactions between the voltage sensor and the pore in the different channels.


Assuntos
Canais Disparados por Nucleotídeos Cíclicos Ativados por Hiperpolarização/metabolismo , Canais de Potássio/metabolismo , Motivos de Aminoácidos , Sequência de Aminoácidos , Animais , Humanos , Canais Disparados por Nucleotídeos Cíclicos Ativados por Hiperpolarização/química , Canais Disparados por Nucleotídeos Cíclicos Ativados por Hiperpolarização/genética , Mutação , Canais de Potássio/química , Canais de Potássio/genética , Ouriços-do-Mar , Alinhamento de Sequência
7.
Proc Natl Acad Sci U S A ; 117(13): 7171-7175, 2020 03 31.
Artigo em Inglês | MEDLINE | ID: mdl-32188782

RESUMO

Transmembrane allosteric coupling is a feature of many critical biological signaling events. Here we test whether transmembrane allosteric coupling controls the potassium binding affinity of the prototypical potassium channel KcsA in the context of C-type inactivation. Activation of KcsA is initiated by proton binding to the pH gate upon an intracellular drop in pH. Numerous studies have suggested that this proton binding also prompts a conformational switch, leading to a loss of affinity for potassium ions at the selectivity filter and therefore to channel inactivation. We tested this mechanism for inactivation using a KcsA mutant (H25R/E118A) that exhibits an open pH gate across a broad range of pH values. We present solid-state NMR measurements of this open mutant at neutral pH to probe the affinity for potassium at the selectivity filter. The potassium binding affinity in the selectivity filter of this mutant, 81 mM, is about four orders of magnitude weaker than that of wild-type KcsA at neutral pH and is comparable to the value for wild-type KcsA at low pH (pH ≈ 3.5). This result strongly supports our assertion that the open pH gate allosterically affects the potassium binding affinity of the selectivity filter. In this mutant, the protonation state of a glutamate residue (E120) in the pH sensor is sensitive to potassium binding, suggesting that this mutant also has flexibility in the activation gate and is subject to transmembrane allostery.


Assuntos
Proteínas de Bactérias/metabolismo , Canais de Potássio/metabolismo , Proteínas de Bactérias/genética , Concentração de Íons de Hidrogênio , Espectroscopia de Ressonância Magnética , Mutação , Potássio/metabolismo , Canais de Potássio/genética , Conformação Proteica
8.
Artigo em Inglês | MEDLINE | ID: mdl-32114250

RESUMO

Potassium homeostasis is essential for pollen development and pollen-pistil interactions during the sexual reproduction of flowering plants. Here, we described the role of a Shaker K+ channel, OsAKT1.2, in rice pollen germination and growth. OsAKT1.2 is specifically expressed in the tricellular pollen, mature pollen grains and growing pollen tubes. Using CRISPR gene editing, we found that knockout lines did not differ from wildtype in vegetative growth, but showed decreased pollen germination rate both in the germination medium and in vivo. OsAKT1.2-GFP fusion protein was localized in the plasma membrane and enriched at the pollen tube tip. OsAKT1.2 could complement the yeast strain which is deficient in K+ intake. These findings suggest that OsAKT1.2 is associated with pollen germination and tube elongation in rice.


Assuntos
Germinação/genética , Oryza/fisiologia , Proteínas de Plantas/genética , Pólen/crescimento & desenvolvimento , Canais de Potássio/genética , Sequência de Bases , Oryza/genética , Proteínas de Plantas/metabolismo
9.
Nat Commun ; 11(1): 480, 2020 01 24.
Artigo em Inglês | MEDLINE | ID: mdl-31980599

RESUMO

Mutations in the actively expressed, maternal allele of the imprinted KCNK9 gene cause Birk-Barel intellectual disability syndrome (BBIDS). Using a BBIDS mouse model, we identify here a partial rescue of the BBIDS-like behavioral and neuronal phenotypes mediated via residual expression from the paternal Kcnk9 (Kcnk9pat) allele. We further demonstrate that the second-generation HDAC inhibitor CI-994 induces enhanced expression from the paternally silenced Kcnk9 allele and leads to a full rescue of the behavioral phenotype suggesting CI-994 as a promising molecule for BBIDS therapy. Thus, these findings suggest a potential approach to improve cognitive dysfunction in a mouse model of an imprinting disorder.


Assuntos
Anormalidades Craniofaciais/genética , Anormalidades Craniofaciais/metabolismo , Histonas/metabolismo , Deficiência Intelectual/genética , Deficiência Intelectual/metabolismo , Hipotonia Muscular/genética , Hipotonia Muscular/metabolismo , Canais de Potássio/genética , Animais , Comportamento Animal , Encéfalo/metabolismo , Anormalidades Craniofaciais/tratamento farmacológico , Modelos Animais de Doenças , Feminino , Técnicas de Silenciamento de Genes , Impressão Genômica , Inibidores de Histona Desacetilases/farmacologia , Humanos , Deficiência Intelectual/tratamento farmacológico , Locus Cerúleo/metabolismo , Masculino , Camundongos , Camundongos Endogâmicos C57BL , Camundongos Knockout , Hipotonia Muscular/tratamento farmacológico , Mutação , Fenótipo , Fenilenodiaminas/farmacologia , Canais de Potássio/deficiência , Canais de Potássio/metabolismo , Regulação para Cima/efeitos dos fármacos
10.
DNA Cell Biol ; 39(2): 289-298, 2020 Feb.
Artigo em Inglês | MEDLINE | ID: mdl-31916853

RESUMO

TBX3 reprograms cardiac myocytes into cells that possess sinoatrial node phenotype, but no specific funny current (If) was detected. We explore whether overexpression of TBX3 alone or combined with HCN2 can reprogram human-induced pluripotent stem cells-derived cardiomyocytes (hiPSC-CMs) into pacemaker-like cells. HiPSC-CMs were transfected with TBX3 and/or HCN2 in this study. Expression analysis showed that overexpression of TBX3 induces a reduced reduction expression profile of working cardiomyocytes into that of pacemaker cells. Reverse transcription-quantitative polymerase chain reaction (RT-qPCR) and electrophysiological analyses showed a reduced expression of connexins subunits (CX40, CX43), the sodium current (SCN5A, INa), the inward rectified potassium channels (Kir2.1, IK1), and an increased expression of connexins subunits (CX30.2, CX45). No If was detected. The reduction of IK1 resulted in a more depolarized maximum diastolic potential together with an expression of If (generated by HCN2), which they work in synergy to generate spontaneous diastolic depolarization that was the most typical characteristic of pacemaker cells. In conclusion, overexpression of TBX3 and HCN2 could reprogram hiPSC-CMs into pacemaker-like cells. The ability to enable diastolic depolarization formation provides a new strategy for the construction of a biological pacemaker.


Assuntos
Diferenciação Celular/genética , Canais Disparados por Nucleotídeos Cíclicos Ativados por Hiperpolarização/genética , Células-Tronco Pluripotentes Induzidas/metabolismo , Canais de Potássio/genética , Proteínas com Domínio T/genética , Potenciais de Ação/fisiologia , Relógios Biológicos/genética , Humanos , Miócitos Cardíacos/metabolismo
11.
Nat Commun ; 11(1): 547, 2020 Jan 28.
Artigo em Inglês | MEDLINE | ID: mdl-31992706

RESUMO

TrkH is a bacterial ion channel implicated in K+ uptake and pH regulation. TrkH assembles with its regulatory protein, TrkA, which closes the channel when bound to ADP and opens it when bound to ATP. However, it is unknown how nucleotides control the gating of TrkH through TrkA. Here we report the structures of the TrkH-TrkA complex in the presence of ADP or ATP. TrkA forms a tetrameric ring when bound to ADP and constrains TrkH to a closed conformation. The TrkA ring splits into two TrkA dimers in the presence of ATP and releases the constraints on TrkH, resulting in an open channel conformation. Functional studies show that both the tetramer-to-dimer conversion of TrkA and the loss of constraints on TrkH are required for channel gating. In addition, deletion of TrkA in Escherichia coli depolarizes the cell, suggesting that the TrkH-TrkA complex couples changes in intracellular nucleotides to membrane potential.


Assuntos
Transportadores de Cassetes de Ligação de ATP/química , Transportadores de Cassetes de Ligação de ATP/metabolismo , Proteínas de Escherichia coli/química , Proteínas de Escherichia coli/metabolismo , Escherichia coli/metabolismo , Potenciais da Membrana/fisiologia , Canais de Potássio/química , Canais de Potássio/metabolismo , Transportadores de Cassetes de Ligação de ATP/genética , Difosfato de Adenosina , Trifosfato de Adenosina/metabolismo , Proteínas de Bactérias/química , Proteínas de Bactérias/metabolismo , Transporte Biológico/fisiologia , Escherichia coli/genética , Proteínas de Escherichia coli/genética , Modelos Moleculares , Mutagênese , Potássio/metabolismo , Canais de Potássio/genética , Conformação Proteica , Domínios e Motivos de Interação entre Proteínas , Deleção de Sequência , Vibrio parahaemolyticus/genética , Difração de Raios X
12.
J Hum Genet ; 65(4): 363-369, 2020 Apr.
Artigo em Inglês | MEDLINE | ID: mdl-31907387

RESUMO

Spinocerebellar ataxia (SCA) is a genetically heterogeneous disease characterized by cerebellar ataxia. Many causative genes have been identified to date, the most common etiology being the abnormal expansion of repeat sequences, and the mutation of ion channel genes also play an important role in the development of SCA. Some of them encode calcium and potassium channels. However, due to limited reports about potassium genes in SCA, we screened 192 Japanese individuals with dominantly inherited SCA who had no abnormal repeat expansions of causative genes for potassium channel mutations (KCNC3 for SCA13 and KCND3 for SCA19/SCA22) by target sequencing. As a result, two variants were identified from two patients: c.1973G>A, p.R658Q and c.1018G>A, p.V340M for KCNC3, and no pathogenic variant was identified for KCND3. The newly identified p.V340M exists in the extracellular domain, and p.R658Q exists in the intracellular domain on the C-terminal side, although most of the reported KCNC3 mutations are present at the transmembrane site. Adult-onset and slowly progressive cerebellar ataxia are the main clinical features of SCA13 and SCA19 caused by potassium channel mutations, which was similar in our cases. SCA13 caused by KCNC3 mutations may present with deep sensory loss and cognitive impairment in addition to cerebellar ataxia. In this study, mild deep sensory loss was observed in one case. SCA caused by potassium channel gene mutations is extremely rare, and more cases should be accumulated in the future to elucidate its pathogenesis due to channel dysfunction.


Assuntos
Disfunção Cognitiva/genética , Mutação , Canais de Potássio/genética , Ataxias Espinocerebelares/genética , Adulto , Grupo com Ancestrais do Continente Asiático , Disfunção Cognitiva/diagnóstico por imagem , Feminino , Testes Genéticos , Humanos , Japão , Imagem por Ressonância Magnética , Masculino , Pessoa de Meia-Idade , Ataxias Espinocerebelares/diagnóstico por imagem
13.
Artigo em Inglês | MEDLINE | ID: mdl-31977249

RESUMO

Tandem pore domain acid-sensitive K+ (TASK) channels are present in cardiac tissue; however, their contribution to cardiac pathophysiology is not well understood. Here, we investigate the role of TASK-1 and TASK-3 in the pathogenesis of cardiac dysfunction using both human tissue and mouse models of genetic TASK channel loss of function. Compared with normal human cardiac tissue, TASK-1 gene expression is reduced in association with either cardiac hypertrophy alone or combined cardiac hypertrophy and heart failure. In a pressure overload cardiomyopathy model, TASK-1 global knockout (TASK-1 KO) mice have both reduced cardiac hypertrophy and preserved cardiac function compared with wild-type mice. In contrast to the TASK-1 KO mouse pressure overload response, TASK-3 global knockout (TASK-3 KO) mice develop cardiac hypertrophy and a delayed onset of cardiac dysfunction compared with wild-type mice. The cardioprotective effects observed in TASK-1 KO mice are associated with pressure overload-induced augmentation of AKT phosphorylation and peroxisome proliferator-activated receptor-γ coactivator-1α (PGC-1α) expression, with consequent augmentation of cardiac energetics and fatty acid oxidation. The protective effects of TASK-1 loss of function are associated with an enhancement of physiologic hypertrophic signaling and preserved metabolic functions. These findings may provide a rationale for TASK-1 channel inhibition in the treatment of cardiac dysfunction.NEW & NOTEWORTHY The role of tandem pore domain acid-sensitive K+ (TASK) channels in cardiac function is not well understood. This study demonstrates that TASK channel gene expression is associated with the onset of human cardiac hypertrophy and heart failure. TASK-1 and TASK-3 strongly affect the development of pressure overload cardiomyopathies in genetic models of TASK-1 and TASK-3 loss of function. The effects of TASK-1 loss of function were associated with enhanced AKT phosphorylation and expression of peroxisome proliferator-activated receptor-γ coactivator-1 (PGC-1) transcription factor. These data suggest that TASK channels influence the development of cardiac hypertrophy and dysfunction in response to injury.


Assuntos
Cardiomegalia/metabolismo , Cardiomiopatias/metabolismo , Miocárdio/metabolismo , Proteínas do Tecido Nervoso/metabolismo , Canais de Potássio de Domínios Poros em Tandem/metabolismo , Canais de Potássio/metabolismo , Remodelação Ventricular/fisiologia , Animais , Cardiomegalia/genética , Cardiomiopatias/genética , Humanos , Camundongos , Camundongos Knockout , Miócitos Cardíacos/metabolismo , Proteínas do Tecido Nervoso/genética , Coativador 1-alfa do Receptor gama Ativado por Proliferador de Peroxissomo/genética , Coativador 1-alfa do Receptor gama Ativado por Proliferador de Peroxissomo/metabolismo , Fosforilação , Canais de Potássio/genética , Canais de Potássio de Domínios Poros em Tandem/genética , Proteínas Proto-Oncogênicas c-akt/metabolismo
14.
J Nanobiotechnology ; 18(1): 21, 2020 Jan 28.
Artigo em Inglês | MEDLINE | ID: mdl-31992314

RESUMO

BACKGROUND: Carbon nanoparticles (CNPs) have been reported to boost plant growth, while the mechanism that CNPs enhanced potassium uptake for plant growth has not been reported so far. RESULTS: In this study, the function that CNPs promoted potassium uptake in BY-2 cells was established and the potassium accumulated in cells had a significant correlation with the fresh biomass of BY-2 cells. The K+ accumulation in cells increased with the increasing concentration of CNPs. The K+ influx reached high level after treatment with CNPs and was significantly higher than that of the control group and the negative group treated with K+ channels blocker, tetraethylammonium chloride (TEA+). The K+ accumulation was not reduced in the presence of CNPs inhibitors. In the presence of potassium channel blocker TEA+ or CNPs inhibitors, the NKT1 gene expression was changed compared with the control group. The CNPs were found to preferentially transport K+ than other cations determined by rectification of ion current assay (RIC) in a conical nanocapillary. CONCLUSIONS: These results indicated that CNPs upregulated potassium gene expression to enhance K+ accumulation in BY-2 cells. Moreover, it was speculated that the CNPs simulated protein of ion channels via bulk of carboxyl for K+ permeating. These findings will provide support for improving plant growth by carbon nanoparticles.


Assuntos
Carbono/química , Nanopartículas/química , Nanopartículas/metabolismo , Canais de Potássio/genética , Canais de Potássio/metabolismo , Potássio/metabolismo , Aminoácidos/análise , Aminoácidos/metabolismo , Permeabilidade da Membrana Celular , Regulação da Expressão Gênica/efeitos dos fármacos , Melhoramento Genético , Humanos , Potenciais da Membrana , Bloqueadores dos Canais de Potássio/química , Bloqueadores dos Canais de Potássio/metabolismo , Tetraetilamônio/química , Tetraetilamônio/metabolismo , Regulação para Cima/efeitos dos fármacos
15.
Cell Mol Life Sci ; 77(5): 903-918, 2020 Mar.
Artigo em Inglês | MEDLINE | ID: mdl-31312877

RESUMO

MicroRNAs (miRs) contribute to different aspects of cardiovascular pathology, among others cardiac hypertrophy and atrial fibrillation. The aim of our study was to evaluate the impact of miR-221/222 on cardiac electrical remodeling. Cardiac miR expression was analyzed in a mouse model with altered electrocardiography parameters and severe heart hypertrophy. Next generation sequencing revealed 14 differentially expressed miRs in hypertrophic hearts, with miR-221 and -222 being the strongest regulated miR-cluster. This increase was restricted to cardiomyocytes and not observed in cardiac fibroblasts. Additionally, we evaluated the change of miR-221/222 in vivo in two models of pharmacologically induced heart hypertrophy (angiotensin II, isoprenaline), thereby demonstrating a stimulus-induced increase in miR-221/222 in vivo by angiotensin II but not by isoprenaline. Whole transcriptome analysis by RNA-seq and qRT-PCR validation revealed an enriched number of downregulated mRNAs coding for proteins located in the T-tubule, which are also predicted targets for miR-221/222. Among those, mRNAs were the L-type Ca2+ channel subunits as well as potassium channel subunits. We confirmed that both miRs target the 3'-untranslated regions of Cacna1c and Kcnj5. Furthermore, enhanced expression of these miRs reduced L-type Ca2+ channel and Kcnj5 channel abundance and function, which was analyzed by whole-cell patch clamp recordings or Western blot and flux measurements, respectively. miR-221 and -222 contribute to the regulation of L-type Ca2+ channels as well as Kcnj5 channels and, therefore, potentially contribute to disturbed cardiac excitation generation and propagation. Future studies will have to evaluate the pathophysiological and clinical relevance of aberrant miR-221/222 expression for electrical remodeling.


Assuntos
Canais de Cálcio Tipo L/metabolismo , Canais de Potássio Corretores do Fluxo de Internalização Acoplados a Proteínas G/metabolismo , MicroRNAs/genética , Canais de Potássio/metabolismo , Animais , Canais de Cálcio Tipo L/genética , Cardiomegalia/genética , Cardiomegalia/patologia , Linhagem Celular , Canais de Potássio Corretores do Fluxo de Internalização Acoplados a Proteínas G/genética , Camundongos , Camundongos Knockout , Miócitos Cardíacos/citologia , Técnicas de Patch-Clamp , Canais de Potássio/genética
16.
Ann Neurol ; 87(1): 139-153, 2020 01.
Artigo em Inglês | MEDLINE | ID: mdl-31658403

RESUMO

OBJECTIVE: The TMEM175/GAK/DGKQ locus is the 3rd strongest risk locus in genome-wide association studies of Parkinson disease (PD). We aimed to identify the specific disease-associated variants in this locus, and their potential implications. METHODS: Full sequencing of TMEM175/GAK/DGKQ followed by genotyping of specific associated variants was performed in PD (n = 1,575) and rapid eye movement sleep behavior disorder (RBD) patients (n = 533) and in controls (n = 1,583). Adjusted regression models and a meta-analysis were performed. Association between variants and glucocerebrosidase (GCase) activity was analyzed in 715 individuals with available data. Homology modeling, molecular dynamics simulations, and lysosomal localization experiments were performed on TMEM175 variants to determine their potential effects on structure and function. RESULTS: Two coding variants, TMEM175 p.M393T (odds ratio [OR] = 1.37, p = 0.0003) and p.Q65P (OR = 0.72, p = 0.005), were associated with PD, and p.M393T was also associated with RBD (OR = 1.59, p = 0.001). TMEM175 p.M393T was associated with reduced GCase activity. Homology modeling and normal mode analysis demonstrated that TMEM175 p.M393T creates a polar side-chain in the hydrophobic core of the transmembrane, which could destabilize the domain and thus impair either its assembly, maturation, or trafficking. Molecular dynamics simulations demonstrated that the p.Q65P variant may increase stability and ion conductance of the transmembrane protein, and lysosomal localization was not affected by these variants. INTERPRETATION: Coding variants in TMEM175 are likely to be responsible for the association in the TMEM175/GAK/DGKQ locus, which could be mediated by affecting GCase activity. ANN NEUROL 2020;87:139-153.


Assuntos
Canais de Potássio/genética , Sinucleinopatias/genética , Adulto , Idoso , Estudos de Casos e Controles , Feminino , Predisposição Genética para Doença/genética , Genótipo , Glucosilceramidase/metabolismo , Humanos , Lisossomos/metabolismo , Masculino , Pessoa de Meia-Idade , Modelos Moleculares , Simulação de Dinâmica Molecular , Doença de Parkinson/genética , Doença de Parkinson/fisiopatologia , Polimorfismo de Nucleotídeo Único/genética , Canais de Potássio/fisiologia , Transtorno do Comportamento do Sono REM/genética , Transtorno do Comportamento do Sono REM/fisiopatologia , Sinucleinopatias/fisiopatologia
17.
Neurol India ; 67(6): 1469-1471, 2019.
Artigo em Inglês | MEDLINE | ID: mdl-31857537

RESUMO

Objective: To study the DNA methylation profiles in brain tissue of patients with refractory epilepsy due to malformations of cortical development (MCDs). Materials and Methods: Clinical, neuroimaging, and pathology characteristics were defined for 13 patients who underwent resective surgery for epilepsy. Methylation analysis was performed using Illumina® 450k Methylation Microarray. Data analysis was completed, and pathway identification was done using the R/Bioconductor package. Results: Genes associated with Ephrin-Reelin pathway, potassium channels, and glutathione metabolism were differentially methylated in the MCD group when compared with patients who had no evidence of MCD. Conclusions: Our preliminary data reveal that epigenetic pathways may have a role in the pathobiogenesis of MCDs.


Assuntos
Encéfalo/metabolismo , Metilação de DNA , Epilepsia/genética , Malformações do Desenvolvimento Cortical/genética , Encéfalo/diagnóstico por imagem , Encéfalo/cirurgia , Moléculas de Adesão Celular Neuronais/genética , Moléculas de Adesão Celular Neuronais/metabolismo , Criança , Pré-Escolar , Epilepsia/etiologia , Epilepsia/cirurgia , Proteínas da Matriz Extracelular/genética , Proteínas da Matriz Extracelular/metabolismo , Feminino , Humanos , Lactente , Masculino , Malformações do Desenvolvimento Cortical/complicações , Malformações do Desenvolvimento Cortical/cirurgia , Proteínas do Tecido Nervoso/genética , Proteínas do Tecido Nervoso/metabolismo , Projetos Piloto , Canais de Potássio/genética , Canais de Potássio/metabolismo , Serina Endopeptidases/genética , Serina Endopeptidases/metabolismo , Transdução de Sinais/genética
18.
Elife ; 82019 12 23.
Artigo em Inglês | MEDLINE | ID: mdl-31868587

RESUMO

RCK domains regulate the activity of K+ channels and transporters in eukaryotic and prokaryotic organisms by responding to ions or nucleotides. The mechanisms of RCK activation by Ca2+ in the eukaryotic BK and bacterial MthK K+ channels are well understood. However, the molecular details of activation in nucleotide-dependent RCK domains are not clear. Through a functional and structural analysis of the mechanism of ATP activation in KtrA, a RCK domain from the B. subtilis KtrAB cation channel, we have found that activation by nucleotide requires binding of cations to an intra-dimer interface site in the RCK dimer. In particular, divalent cations are coordinated by the γ-phosphates of bound-ATP, tethering the two subunits and stabilizing the active state conformation. Strikingly, the binding site residues are highly conserved in many different nucleotide-dependent RCK domains, indicating that divalent cations are a general cofactor in the regulatory mechanism of many nucleotide-dependent RCK domains.


Assuntos
Proteínas de Bactérias/química , Proteínas de Transporte de Cátions/química , Nucleotídeos/química , Conformação Proteica , Trifosfato de Adenosina/química , Bacillus subtilis/química , Bacillus subtilis/genética , Proteínas de Bactérias/genética , Proteínas de Bactérias/ultraestrutura , Sítios de Ligação/genética , Cálcio/metabolismo , Proteínas de Transporte de Cátions/genética , Proteínas de Transporte de Cátions/ultraestrutura , Cátions/química , Cristalografia por Raios X , Canal de Potássio Kv1.6/química , Canal de Potássio Kv1.6/ultraestrutura , Nucleotídeos/genética , Potássio/química , Potássio/metabolismo , Canais de Potássio/química , Canais de Potássio/genética , Canais de Potássio/ultraestrutura , Domínios Proteicos/genética , Estrutura Terciária de Proteína , Proteínas Ribossômicas
19.
Nat Commun ; 10(1): 5366, 2019 11 26.
Artigo em Inglês | MEDLINE | ID: mdl-31772184

RESUMO

Potassium channels are presumed to have two allosterically coupled gates, the activation gate and the selectivity filter gate, that control channel opening, closing, and inactivation. However, the molecular mechanism of how these gates regulate K+ ion flow through the channel remains poorly understood. An activation process, occurring at the selectivity filter, has been recently proposed for several potassium channels. Here, we use X-ray crystallography and extensive molecular dynamics simulations, to study ion permeation through a potassium channel MthK, for various opening levels of both gates. We find that the channel conductance is controlled at the selectivity filter, whose conformation depends on the activation gate. The crosstalk between the gates is mediated through a collective motion of channel helices, involving hydrophobic contacts between an isoleucine and a conserved threonine in the selectivity filter. We propose a gating model of selectivity filter-activated potassium channels, including pharmacologically relevant two-pore domain (K2P) and big potassium (BK) channels.


Assuntos
Proteínas Arqueais/química , Proteínas Arqueais/metabolismo , Ativação do Canal Iônico , Canais de Potássio/química , Canais de Potássio/metabolismo , Proteínas Arqueais/genética , Cristalografia por Raios X , Escherichia coli/genética , Interações Hidrofóbicas e Hidrofílicas , Ativação do Canal Iônico/fisiologia , Isoleucina/química , Methanobacteriaceae/química , Simulação de Dinâmica Molecular , Mutação , Potássio/metabolismo , Canais de Potássio/genética , Conformação Proteica , Treonina/química
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