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1.
Nat Med ; 25(6): 911-919, 2019 06.
Artigo em Inglês | MEDLINE | ID: mdl-31160820

RESUMO

It is estimated that 350 million individuals worldwide suffer from rare diseases, which are predominantly caused by mutation in a single gene1. The current molecular diagnostic rate is estimated at 50%, with whole-exome sequencing (WES) among the most successful approaches2-5. For patients in whom WES is uninformative, RNA sequencing (RNA-seq) has shown diagnostic utility in specific tissues and diseases6-8. This includes muscle biopsies from patients with undiagnosed rare muscle disorders6,9, and cultured fibroblasts from patients with mitochondrial disorders7. However, for many individuals, biopsies are not performed for clinical care, and tissues are difficult to access. We sought to assess the utility of RNA-seq from blood as a diagnostic tool for rare diseases of different pathophysiologies. We generated whole-blood RNA-seq from 94 individuals with undiagnosed rare diseases spanning 16 diverse disease categories. We developed a robust approach to compare data from these individuals with large sets of RNA-seq data for controls (n = 1,594 unrelated controls and n = 49 family members) and demonstrated the impacts of expression, splicing, gene and variant filtering strategies on disease gene identification. Across our cohort, we observed that RNA-seq yields a 7.5% diagnostic rate, and an additional 16.7% with improved candidate gene resolution.


Assuntos
Doenças Raras/genética , Ceramidase Ácida/genética , Estudos de Casos e Controles , Criança , Pré-Escolar , Estudos de Coortes , Feminino , Variação Genética , Humanos , Masculino , Modelos Genéticos , Mutação , Oxirredutases atuantes sobre Doadores de Grupo CH-CH/genética , Canais de Potássio/genética , RNA/sangue , RNA/genética , Processamento de RNA/genética , Doenças Raras/sangue , Análise de Sequência de RNA , Sequenciamento Completo do Exoma
2.
Int J Mol Sci ; 20(9)2019 Apr 30.
Artigo em Inglês | MEDLINE | ID: mdl-31052176

RESUMO

Sodium and potassium are two alkali cations abundant in the biosphere. Potassium is essential for plants and its concentration must be maintained at approximately 150 mM in the plant cell cytoplasm including under circumstances where its concentration is much lower in soil. On the other hand, sodium must be extruded from the plant or accumulated either in the vacuole or in specific plant structures. Maintaining a high intracellular K+/Na+ ratio under adverse environmental conditions or in the presence of salt is essential to maintain cellular homeostasis and to avoid toxicity. The baker's yeast, Saccharomyces cerevisiae, has been used to identify and characterize participants in potassium and sodium homeostasis in plants for many years. Its utility resides in the fact that the electric gradient across the membrane and the vacuoles is similar to plants. Most plant proteins can be expressed in yeast and are functional in this unicellular model system, which allows for productive structure-function studies for ion transporting proteins. Moreover, yeast can also be used as a high-throughput platform for the identification of genes that confer stress tolerance and for the study of protein-protein interactions. In this review, we summarize advances regarding potassium and sodium transport that have been discovered using the yeast model system, the state-of-the-art of the available techniques and the future directions and opportunities in this field.


Assuntos
Proteínas de Transporte de Cátions/metabolismo , Proteínas de Plantas/metabolismo , Canais de Potássio/metabolismo , Saccharomyces cerevisiae/genética , Canais de Sódio/metabolismo , Técnicas do Sistema de Duplo-Híbrido , Proteínas de Transporte de Cátions/genética , Proteínas de Plantas/genética , Canais de Potássio/genética , Saccharomyces cerevisiae/metabolismo , Canais de Sódio/genética
3.
Ecotoxicol Environ Saf ; 179: 31-39, 2019 Sep 15.
Artigo em Inglês | MEDLINE | ID: mdl-31022653

RESUMO

To analyze the differences between high- and low-accumulation plants in cesium (Cs) uptake and its related mechanism, Brassica juncea (a hyperaccumulation plant for Cs) and Vicia faba (a low-accumulation plant for Cs) were selected as comparative experimental materials. The contributions to Cs uptake of a K-transporter-mediated high-affinity transport system and a K-channel-mediated low-affinity transport system in the two plants were compared and analyzed. The difference between the two plants in the mechanism of Cs uptake was further analyzed using transcription sequence technology. The results show that the transfer characteristics of Cs in the two plants had a similar distribution relationship with K. The contribution rate of the K-channel pathway to Cs uptake was 32.00% in the V. faba seedling roots, which was significantly higher than for B. juncea (9.81%) (P < 0.01); the contribution rate of the K-transporter pathway to Cs uptake of the B. juncea seedlings was 32.08%, which was significantly higher than that of the V. faba seedlings (17.13%)(P < 0.05). Other uptake pathways also mediated the uptake of Cs by roots in B. juncea and V. faba (contribution rate: 54.92-60.09% and 42.18-59.73%, respectively). The transcriptome sequencing results confirmed that Cs-induced treatment significantly inhibited the expression of the K-transporter protein and K-channel protein-related genes in the V. faba roots, but it had no significant effect on the expression of related genes in the B. juncea roots. Thus, one reason for the significant difference between the two plant in the accumulation of Cs is that Cs inhibited the expression of related transporter protein genes in the V. faba roots.


Assuntos
Césio/metabolismo , Transporte de Íons/genética , Mostardeira/metabolismo , Potássio/metabolismo , Transcriptoma , Vicia faba/metabolismo , Perfilação da Expressão Gênica , Mostardeira/efeitos dos fármacos , Mostardeira/genética , Raízes de Plantas/efeitos dos fármacos , Raízes de Plantas/genética , Raízes de Plantas/metabolismo , Canais de Potássio/genética , Plântula/efeitos dos fármacos , Plântula/genética , Plântula/metabolismo , Vicia faba/efeitos dos fármacos , Vicia faba/genética
4.
Int J Mol Sci ; 20(5)2019 Mar 02.
Artigo em Inglês | MEDLINE | ID: mdl-30832374

RESUMO

HKT1 and SOS1 are two key Na⁺ transporters that modulate salt tolerance in plants. Although much is known about the respective functions of HKT1 and SOS1 under salt conditions, few studies have examined the effects of HKT1 and SOS1 mutations on the expression of other important Na⁺ and K⁺ transporter genes. This study investigated the physiological parameters and expression profiles of AtHKT1;1, AtSOS1, AtHAK5, AtAKT1, AtSKOR, AtNHX1, and AtAVP1 in wild-type (WT) and athkt1;1 and atsos1 mutants of Arabidopsis thaliana under 25 mM NaCl. We found that AtSOS1 mutation induced a significant decrease in transcripts of AtHKT1;1 (by 56⁻62% at 6⁻24 h), AtSKOR (by 36⁻78% at 6⁻24 h), and AtAKT1 (by 31⁻53% at 6⁻24 h) in the roots compared with WT. This led to an increase in Na⁺ accumulation in the roots, a decrease in K⁺ uptake and transportation, and finally resulted in suppression of plant growth. AtHKT1;1 loss induced a 39⁻76% (6⁻24 h) decrease and a 27⁻32% (6⁻24 h) increase in transcripts of AtSKOR and AtHAK5, respectively, in the roots compared with WT. At the same time, 25 mM NaCl decreased the net selective transport capacity for K⁺ over Na⁺ by 92% in the athkt1;1 roots compared with the WT roots. Consequently, Na⁺ was loaded into the xylem and delivered to the shoots, whereas K⁺ transport was restricted. The results indicate that AtHKT1;1 and AtSOS1 not only mediate Na⁺ transport but also control ion uptake and the spatial distribution of Na⁺ and K⁺ by cooperatively regulating the expression levels of relevant Na⁺ and K⁺ transporter genes, ultimately regulating plant growth under salt stress.


Assuntos
Proteínas de Arabidopsis/genética , Arabidopsis/genética , Proteínas de Transporte de Cátions/genética , Homeostase , Estresse Salino , Trocadores de Sódio-Hidrogênio/genética , Simportadores/genética , Arabidopsis/metabolismo , Proteínas de Arabidopsis/metabolismo , Proteínas de Transporte de Cátions/metabolismo , Regulação da Expressão Gênica de Plantas , Mutação , Canais de Potássio/genética , Canais de Potássio/metabolismo , Superfamília Shaker de Canais de Potássio/genética , Superfamília Shaker de Canais de Potássio/metabolismo , Trocadores de Sódio-Hidrogênio/metabolismo , Simportadores/metabolismo
5.
Biosens Bioelectron ; 133: 141-146, 2019 May 15.
Artigo em Inglês | MEDLINE | ID: mdl-30925363

RESUMO

Natural ion channels on cell membrane can gate the transport of ions and molecules by the conformational alteration of transmembrane proteins to regulate the normal physiological activities of cells. Inspired by the similarity of the conformation change under specific stimuli, here we introduce an ion channel gating model on a single nanoelectrode by anchoring DNA-gated switches on the very nanotip of gold nanoelectrode to mimic the response-to-stimulus behaviors of ion channels on bio-membranes. The surface-tethered DNA ion channels can be switched on by the Watson-Crick base pairing, which can alter the conformation of the tethered DNA from lying state to upright state. And these conformational alterations of the anchored DNA switches can effectively gate the transport of potassium ferricyanide onto the electrode interface. By continuously initiating the gates with DNA of different concentrations, we achieved the stepping gating of ion channels on a single nanoelectrode. Further, we demonstrated that the ion gating system on nanoelectrode showed excellent sensing performance. For example, the response kinetic was very fast with the signal saturation time of ~1 min, the reproducibility of the OFF/ON switch was robust enough to sustain for two cycles, and simultaneously, the specificity was high enough to distinguish complementary DNA and noncomplementary DNA. When used for label-free DNA detection, the limit of detection can be as low as 10 pM. This study provides a promising avenue to achieve label free and real-time detection of multiple biomolecules.


Assuntos
Técnicas Biossensoriais , DNA/isolamento & purificação , Ativação do Canal Iônico/genética , DNA/química , DNA/genética , Cinética , Hibridização de Ácido Nucleico , Canais de Potássio/química , Canais de Potássio/genética
6.
Clin Chim Acta ; 493: 87-91, 2019 Jun.
Artigo em Inglês | MEDLINE | ID: mdl-30825425

RESUMO

Progressive myoclonic epilepsies (PME) are a clinically and genetically heterogeneous group of rare diseases characterized by myoclonic seizures, tonic-clonic seizures, and neurological deterioration. Here, we genetically analyzed a Chinese patient affected by infantile-onset progressive myoclonic epilepsy. We applied next-generation whole exome capture sequencing with Sanger direct sequencing to the proband and her unaffected parents. Two compound heterozygous mutations were identified in the KCTD7 gene. The first mutation [c. 434A > G(p.Q145R)] was inherited from her father, while the second [c.631C > T(p.R211X)] was inherited from her mother. The two were co-segregated with disease phenotype in the family. To our knowledge, this is the first report of KCTD7 mutations causing PME in the Chinese population, with c. 434A > G in particular being a novel mutation. Our findings supported the important role of KCTD7 in PME and broadened the gene's mutation spectrum. Thus, this study contributes to genetic diagnoses and counselling of families with PME.


Assuntos
Mutação , Epilepsias Mioclônicas Progressivas/genética , Canais de Potássio/genética , Sequenciamento Completo do Exoma , Pré-Escolar , Biologia Computacional , DNA/genética , Feminino , Humanos , Linhagem
7.
Proc Natl Acad Sci U S A ; 116(9): 3811-3816, 2019 02 26.
Artigo em Inglês | MEDLINE | ID: mdl-30755524

RESUMO

The ability to detect environmental cold serves as an important survival tool. The sodium channels NaV1.8 and NaV1.9, as well as the TRP channel Trpm8, have been shown to contribute to cold sensation in mice. Surprisingly, transcriptional profiling shows that NaV1.8/NaV1.9 and Trpm8 are expressed in nonoverlapping neuronal populations. Here we have used in vivo GCaMP3 imaging to identify cold-sensing populations of sensory neurons in live mice. We find that ∼80% of neurons responsive to cold down to 1 °C do not express NaV1.8, and that the genetic deletion of NaV1.8 does not affect the relative number, distribution, or maximal response of cold-sensitive neurons. Furthermore, the deletion of NaV1.8 had no observable effect on transient cold-induced (≥5 °C) behaviors in mice, as measured by the cold-plantar, cold-plate (5 and 10 °C), or acetone tests. In contrast, nocifensive-like behavior to extreme cold-plate stimulation (-5 °C) was completely absent in mice lacking NaV1.8. Fluorescence-activated cell sorting (FACS) and subsequent microarray analysis of sensory neurons activated at 4 °C identified an enriched repertoire of ion channels, which include the Trp channel Trpm8 and potassium channel Kcnk9, that are potentially required for cold sensing above freezing temperatures in mouse DRG neurons. These data demonstrate the complexity of cold-sensing mechanisms in mouse sensory neurons, revealing a principal role for NaV1.8-negative neurons in sensing both innocuous and acute noxious cooling down to 1 °C, while NaV1.8-positive neurons are likely responsible for the transduction of prolonged extreme cold temperatures, where tissue damage causes pan-nociceptor activation.


Assuntos
Canal de Sódio Disparado por Voltagem NAV1.8/genética , Canais de Potássio/genética , Células Receptoras Sensoriais/fisiologia , Canais de Cátion TRPM/genética , Animais , Temperatura Baixa , Gânglios Espinais/diagnóstico por imagem , Gânglios Espinais/metabolismo , Gânglios Espinais/fisiologia , Camundongos , Nociceptores/metabolismo , Nociceptores/fisiologia , Células Receptoras Sensoriais/metabolismo , Sensação Térmica/genética
8.
Epileptic Disord ; 21(1): 48-54, 2019 Feb 01.
Artigo em Inglês | MEDLINE | ID: mdl-30782581

RESUMO

Several recent studies have reported potassium sodium-activated channel subfamily T member 1 (KCNT1) mutations in epilepsy patients on quinidine therapy. The efficacy and safety of quinidine for epilepsy treatment, however, remains controversial. We herein report the cases of four patients with KCNT1 mutations treated with quinidine. A reduction in seizures of more than 50% after quinidine treatment was observed in one patient with epilepsy of infancy with migrating focal seizures (EIMFS), whereas two patients with EIMFS and one with focal epilepsy did not achieve apparent seizure reduction. The relationship between quinidine dose and serum quinidine concentration was inconsistent, particularly at high quinidine doses. One patient with EIMFS developed ventricular tachycardia the day after an increase in quinidine dose from 114 to 126 mg/kg/day. The serum trough quinidine concentration and the corrected QT interval (QTc) before arrhythmia onset were 2.4 µg/ml and 420 ms, respectively, and peak serum quinidine concentration after arrhythmia onset was 9.4 µg/ml. Another patient with EIMFS showed aberrant intraventricular conduction with a quinidine dose of 74.5 mg/kg/day and a serum trough concentration of 3.2 µg/ml. Given that serum quinidine levels may elevate sharply after a dose increase, careful monitoring of electrocardiographs and serum concentrations is required. Based on a review of previous reports and our experience with this case, quinidine should be considered as a promising drug for patients with EIMFS harbouring KCNT1 mutations, however, its efficacy remains controversial due to the limited number of cases, and more information on optimal serum concentrations and appropriate titration methods is required.


Assuntos
Anticonvulsivantes/farmacologia , Arritmias Cardíacas/induzido quimicamente , Epilepsias Parciais/tratamento farmacológico , Proteínas do Tecido Nervoso/genética , Canais de Potássio/genética , Quinidina/farmacologia , Anticonvulsivantes/administração & dosagem , Anticonvulsivantes/efeitos adversos , Anticonvulsivantes/sangue , Criança , Pré-Escolar , Monitoramento de Medicamentos , Eletrocardiografia , Feminino , Humanos , Lactente , Masculino , Quinidina/administração & dosagem , Quinidina/efeitos adversos , Quinidina/sangue
9.
Int J Mol Sci ; 20(3)2019 Feb 09.
Artigo em Inglês | MEDLINE | ID: mdl-30744118

RESUMO

Ion channels are transmembrane proteins that conduct specific ions across biological membranes. Ion channels are present at the onset of many cellular processes, and their malfunction triggers severe pathologies. Potassium channels (KChs) share a highly conserved signature that is necessary to conduct K⁺ through the pore region. To be functional, KChs require an exquisite regulation of their subcellular location and abundance. A wide repertoire of signatures facilitates the proper targeting of the channel, fine-tuning the balance that determines traffic and location. These signature motifs can be part of the secondary or tertiary structure of the protein and are spread throughout the entire sequence. Furthermore, the association of the pore-forming subunits with different ancillary proteins forms functional complexes. These partners can modulate traffic and activity by adding their own signatures as well as by exposing or masking the existing ones. Post-translational modifications (PTMs) add a further dimension to traffic regulation. Therefore, the fate of a KCh is not fully dependent on a gene sequence but on the balance of many other factors regulating traffic. In this review, we assemble recent evidence contributing to our understanding of the spatial expression of KChs in mammalian cells. We compile specific signatures, PTMs, and associations that govern the destination of a functional channel.


Assuntos
Ativação do Canal Iônico , Canais de Potássio/metabolismo , Animais , Transporte Biológico , Membrana Celular/química , Membrana Celular/metabolismo , Humanos , Espaço Intracelular/metabolismo , Organelas/metabolismo , Potássio/metabolismo , Canais de Potássio/química , Canais de Potássio/genética , Ligação Proteica , Domínios e Motivos de Interação entre Proteínas , Multimerização Proteica , Processamento de Proteína Pós-Traducional , Transdução de Sinais
10.
Seizure ; 66: 26-30, 2019 Mar.
Artigo em Inglês | MEDLINE | ID: mdl-30776697

RESUMO

PURPOSE: This study aimed to investigate the genetic etiology of epilepsy in a cohort of Chinese children. METHODS: Targeted next-generation sequencing (NGS) was performed for 120 patients with unexplained epilepsy, including 71 patients with early-onset epileptic encephalopathies, and 16 patients with Dravet syndrome (including three patients with a Dravet-like phenotype) but without SCN1A pathogenic variants. RESULTS: Pathogenic variants of 14 genes were discovered in 22 patients (18%). A de novo KCND3 pathogenic variant (c.1174G > A, p.Val392Ile) was identified in a boy with refractory epilepsy, psychomotor regression, attention deficit, and visual decline. Pathogenic variants in other coding genes were excluded via whole exome sequencing. This KCND3 variant was previously confirmed to be pathogenic by Giudicessi, et al. However, the clinical profile was different: sudden death at 20 years old without any medical history of neurological disorders, nor with any diseases typically caused by KCND3 pathogenic variants such as Brugada syndrome, spinocerebellar ataxia type 19/22 or ataxia accompanied by epilepsy. This indicates that we have identified a new KCND3 phenotype. In addition, we also uncovered a GRIN1 pathogenic variant and a novel HCN1 pathogenic variant in the Dravet cohort. CONCLUSION: Our study highlights the significant utility of NGS panels in the genetic diagnosis of pediatric epilepsy. Our findings indicate that KCND3 pathogenic variants may be responsible for a wider phenotypic spectrum than previously thought, by including childhood epileptic encephalopathy. Furthermore, this study provides evidence that GRIN1 and HCN1 are candidate genes for Dravet and Dravet-like phenotypes.


Assuntos
Epilepsias Mioclônicas/genética , Mutação/genética , Canais de Potássio Shal/genética , Análise de Variância , Pré-Escolar , China , Estudos de Coortes , Análise Mutacional de DNA , Epilepsias Mioclônicas/diagnóstico por imagem , Feminino , Frequência do Gene , Humanos , Canais Disparados por Nucleotídeos Cíclicos Ativados por Hiperpolarização/genética , Lactente , Imagem por Ressonância Magnética , Masculino , Proteínas do Tecido Nervoso/genética , Canais de Potássio/genética , Receptores de N-Metil-D-Aspartato/genética
11.
Neuron ; 101(2): 193-195, 2019 01 16.
Artigo em Inglês | MEDLINE | ID: mdl-30653930

RESUMO

In this issue of Neuron, Royal et al. (2018) find that a mutant form of the TRESK ion channel linked to migraine undergoes alternative translation to produce an inhibitory protein that blocks TREK channels, leading to neuronal hyperexcitability and migraine in rodents.


Assuntos
Transtornos de Enxaqueca , Canais de Potássio/genética , Humanos , Mutação , Neurônios
12.
Nat Commun ; 10(1): 123, 2019 01 10.
Artigo em Inglês | MEDLINE | ID: mdl-30631074

RESUMO

Spontaneous activity shifts at constant experimental conditions represent a widespread regulatory mechanism in ion channels. The molecular origins of these modal gating shifts are poorly understood. In the K+ channel KcsA, a multitude of fast activity shifts that emulate the native modal gating behaviour can be triggered by point-mutations in the hydrogen bonding network that controls the selectivity filter. Using solid-state NMR and molecular dynamics simulations in a variety of KcsA mutants, here we show that modal gating shifts in K+ channels are associated with important changes in the channel dynamics that strongly perturb the selectivity filter equilibrium conformation. Furthermore, our study reveals a drastically different motional and conformational selectivity filter landscape in a mutant that mimics voltage-gated K+ channels, which provides a foundation for an improved understanding of eukaryotic K+ channels. Altogether, our results provide a high-resolution perspective on some of the complex functional behaviour of K+ channels.


Assuntos
Proteínas de Bactérias/metabolismo , Ativação do Canal Iônico/fisiologia , Canais de Potássio/metabolismo , Potássio/metabolismo , Sequência de Aminoácidos , Proteínas de Bactérias/química , Proteínas de Bactérias/genética , Ligações de Hidrogênio , Ativação do Canal Iônico/genética , Simulação de Dinâmica Molecular , Mutação , Canais de Potássio/química , Canais de Potássio/genética , Conformação Proteica , Homologia de Sequência de Aminoácidos
13.
Molecules ; 24(1)2019 Jan 05.
Artigo em Inglês | MEDLINE | ID: mdl-30621310

RESUMO

Sustained pacemaker function is a challenge in biological pacemaker engineering. Human cardiomyocyte progenitor cells (CMPCs) have exhibited extended survival in the heart after transplantation. We studied whether lentivirally transduced CMPCs that express the pacemaker current If (encoded by HCN4) can be used as functional gene delivery vehicle in biological pacing. Human CMPCs were isolated from fetal hearts using magnetic beads coated with Sca-1 antibody, cultured in nondifferentiating conditions, and transduced with a green fluorescent protein (GFP)- or HCN4-GFP-expressing lentivirus. A patch-clamp analysis showed a large hyperpolarization-activated, time-dependent inward current (-20 pA/pF at -140 mV, n = 14) with properties typical of If in HCN4-GFP-expressing CMPCs. Gap-junctional coupling between CMPCs and neonatal rat ventricular myocytes (NRVMs) was demonstrated by efficient dye transfer and changes in spontaneous beating activity. In organ explant cultures, the number of preparations showing spontaneous beating activity increased from 6.3% in CMPC/GFP-injected preparations to 68.2% in CMPC/HCN4-GFP-injected preparations (P < 0.05). Furthermore, in CMPC/HCN4-GFP-injected preparations, isoproterenol induced a significant reduction in cycle lengths from 648 ± 169 to 392 ± 71 ms (P < 0.05). In sum, CMPCs expressing HCN4-GFP functionally couple to NRVMs and induce physiologically controlled pacemaker activity and may therefore provide an attractive delivery platform for sustained pacemaker function.


Assuntos
Técnicas de Transferência de Genes , Ventrículos do Coração/transplante , Canais Disparados por Nucleotídeos Cíclicos Ativados por Hiperpolarização/genética , Proteínas Musculares/genética , Miócitos Cardíacos/transplante , Canais de Potássio/genética , Células-Tronco/citologia , Animais , Terapia Genética/métodos , Proteínas de Fluorescência Verde/química , Ventrículos do Coração/patologia , Humanos , Canais Disparados por Nucleotídeos Cíclicos Ativados por Hiperpolarização/uso terapêutico , Proteínas Musculares/uso terapêutico , Técnicas de Patch-Clamp , Canais de Potássio/uso terapêutico , Ratos , Transplante de Células-Tronco
14.
Gen Comp Endocrinol ; 277: 49-55, 2019 06 01.
Artigo em Inglês | MEDLINE | ID: mdl-30633873

RESUMO

The caudal neurosecretory system (CNSS) is a part of stress response system, a neuroendocrine structure unique to fish. To gain a better understanding of the physiological roles of CNSS in fluid homeostasis, we characterized the tissue distribution of urotensin I (UI) expression in European flounder (Platichthys flesus), analyzed the effect chronic exposure to seawater (SW) or freshwater (FW), transfer from SW to FW, and reverse transfer on mRNA levels of UI, L-type Ca2+ channels and Ca-activated K+ channels transcripts in CNSS. The tissue distribution demonstrated that the CNSS is dominant sites of UI expression, and UI mRNA level in fore brain appeared greater than other non-CNSS tissues. There were no consistent differences in CNSS UI expression or urophysis UI content between SW- and FW-adapted fish in July and September. After transfer from SW to FW, at 8 h CNSS UI expression was significantly increased, but urophysis UI content was no significantly changes. At 24 h transfer from SW to FW, expression of CNSS UI was no apparent change and urophysis UI content was reduced. At 8 h and 24 h after transfer from FW to SW UI expression and urophysis UI content was no significantly effect. The expression of bursting dependent L-type Ca2+ channels and Ca-activated K+ channels in SW-adapted fish significantly decreased compared to those in FW-adapted. However, there were no differences in transfer from SW to FW or from FW to SW at 8 h and 24 h. Thus, these results suggest CNSS UI acts as a modulator in response to osmotic stress and plays important roles in the body fluid homeostasis.


Assuntos
Linguado/genética , Regulação da Expressão Gênica , Sistemas Neurossecretores/metabolismo , Osmose , Urotensinas/genética , Animais , Canais de Cálcio Tipo L/genética , Canais de Cálcio Tipo L/metabolismo , Linguado/sangue , Água Doce , Canais de Potássio/genética , Canais de Potássio/metabolismo , RNA Mensageiro/genética , RNA Mensageiro/metabolismo , Salinidade , Água do Mar , Distribuição Tecidual , Urotensinas/metabolismo
15.
Neuron ; 101(2): 232-245.e6, 2019 01 16.
Artigo em Inglês | MEDLINE | ID: mdl-30573346

RESUMO

It is often unclear why some genetic mutations to a given gene contribute to neurological disorders and others do not. For instance, two mutations have previously been found to produce a dominant negative for TRESK, a two-pore-domain K+ channel implicated in migraine: TRESK-MT, a 2-bp frameshift mutation, and TRESK-C110R. Both mutants inhibit TRESK, but only TRESK-MT increases sensory neuron excitability and is linked to migraine. Here, we identify a new mechanism, termed frameshift mutation-induced alternative translation initiation (fsATI), that may explain why only TRESK-MT is associated with migraine. fsATI leads to the production of a second protein fragment, TRESK-MT2, which co-assembles with and inhibits TREK1 and TREK2, two other two-pore-domain K+ channels, to increase trigeminal sensory neuron excitability, leading to a migraine-like phenotype in rodents. These findings identify TREK1 and TREK2 as potential molecular targets in migraine and suggest that fsATI should be considered as a distinct class of mutations.


Assuntos
Potenciais de Ação/genética , Transtornos de Enxaqueca/patologia , Mutação/genética , Neurônios/fisiologia , Canais de Potássio de Domínios Poros em Tandem/genética , Potenciais de Ação/efeitos dos fármacos , Animais , Células Cultivadas , Modelos Animais de Doenças , Feminino , Expressão Gênica/genética , Células HEK293 , Humanos , Masculino , Camundongos , Camundongos Endogâmicos C57BL , Camundongos Transgênicos , Transtornos de Enxaqueca/induzido quimicamente , Transtornos de Enxaqueca/genética , Transtornos de Enxaqueca/fisiopatologia , Modelos Biológicos , Modelos Moleculares , Neurotransmissores/toxicidade , Óxido Nítrico/toxicidade , Oócitos , Canais de Potássio/genética , Canais de Potássio de Domínios Poros em Tandem/metabolismo , Ratos , Ratos Sprague-Dawley , Xenopus
16.
Proc Natl Acad Sci U S A ; 115(40): E9459-E9468, 2018 10 02.
Artigo em Inglês | MEDLINE | ID: mdl-30224462

RESUMO

A primary goal of sleep research is to understand the molecular basis of sleep. Although some sleep/wake-promoting circuits and secreted substances have been identified, the detailed molecular mechanisms underlying the regulation of sleep duration have been elusive. Here, to address these mechanisms, we developed a simple computational model of a cortical neuron with five channels and a pump, which recapitulates the cortical electrophysiological characteristics of slow-wave sleep (SWS) and wakefulness. Comprehensive bifurcation and detailed mathematical analyses predicted that leak K+ channels play a role in generating the electrophysiological characteristics of SWS, leading to a hypothesis that leak K+ channels play a role in the regulation of sleep duration. To test this hypothesis experimentally, we comprehensively generated and analyzed 14 KO mice, and found that impairment of the leak K+ channel (Kcnk9) decreased sleep duration. Based on these results, we hypothesize that leak K+ channels regulate sleep duration in mammals.


Assuntos
Ondas Encefálicas/fisiologia , Canais de Potássio/metabolismo , Fases do Sono/fisiologia , Animais , Camundongos , Camundongos Knockout , Canais de Potássio/genética
17.
Cell Physiol Biochem ; 49(1): 65-77, 2018.
Artigo em Inglês | MEDLINE | ID: mdl-30134221

RESUMO

BACKGROUND/AIMS: Cardiac arrhythmias are triggered by environmental stimuli that may modulate expression of cardiac ion channels. Underlying epigenetic regulation of cardiac electrophysiology remains incompletely understood. Histone deacetylases (HDACs) control gene expression and cardiac integrity. We hypothesized that class I/II HDACs transcriptionally regulate ion channel expression and determine action potential duration (APD) in cardiac myocytes. METHODS: Global class I/II HDAC inhibition was achieved by administration of trichostatin A (TSA). HDAC-mediated effects on K+ channel expression and electrophysiological function were evaluated in murine atrial cardiomyocytes (HL-1 cells) using real-time PCR, Western blot, and patch clamp analyses. Electrical tachypacing was employed to recapitulate arrhythmia-related effects on ion channel remodeling in the absence and presence of HDAC inhibition. RESULTS: Global HDAC inhibition increased histone acetylation and prolonged APD90 in atrial cardiomyocytes compared to untreated control cells. Transcript levels of voltage-gated or inwardly rectifying K+ channels Kcnq1, Kcnj3 and Kcnj5 were significantly reduced, whereas Kcnk2, Kcnj2 and Kcnd3 mRNAs were upregulated. Ion channel remodeling was similarly observed at protein level. Short-term tachypacing did not induce significant transcriptional K+ channel remodeling. CONCLUSION: The present findings link class I/II HDAC activity to regulation of ion channel expression and action potential duration in atrial cardiomyocytes. Clinical implications for HDAC-based antiarrhythmic therapy and cardiac safety of HDAC inhibitors require further investigation.


Assuntos
Potenciais de Ação/efeitos dos fármacos , Canais de Potássio Corretores do Fluxo de Internalização Acoplados a Proteínas G/metabolismo , Inibidores de Histona Desacetilases/farmacologia , Histona Desacetilases/metabolismo , Canais de Potássio de Domínios Poros em Tandem/metabolismo , Canais de Potássio/metabolismo , Animais , Linhagem Celular , Canais de Potássio Corretores do Fluxo de Internalização Acoplados a Proteínas G/genética , Inibidores de Histona Desacetilases/química , Histona Desacetilases/química , Ácidos Hidroxâmicos/química , Ácidos Hidroxâmicos/farmacologia , Camundongos , Miócitos Cardíacos/citologia , Miócitos Cardíacos/efeitos dos fármacos , Miócitos Cardíacos/metabolismo , Técnicas de Patch-Clamp , Canais de Potássio/genética , Canais de Potássio de Domínios Poros em Tandem/genética , Transcrição Genética/efeitos dos fármacos , Regulação para Cima/efeitos dos fármacos
18.
Genetics ; 209(3): 637-650, 2018 07.
Artigo em Inglês | MEDLINE | ID: mdl-29967058

RESUMO

Like all species, the model eukaryote Saccharomyces cerevisiae, or Bakers' yeast, concentrates potassium in the cytosol as an electrogenic osmolyte and enzyme cofactor. Yeast are capable of robust growth on a wide variety of potassium concentrations, ranging from 10 µM to 2.5 M, due to the presence of a high-affinity potassium uptake system and a battery of cation exchange transporters. Genetic perturbation of either of these systems retards yeast growth on low or high potassium, respectively. However, these potassium-sensitized yeast are a powerful genetic tool, which has been leveraged for diverse studies. Notably, the potassium-sensitive cells can be transformed with plasmids encoding potassium channels from bacteria, plants, or mammals, and subsequent changes in growth rate have been found to correlate with the activity of the introduced potassium channel. Discoveries arising from the use of this assay over the past three decades have increased our understanding of the structure-function relationships of various potassium channels, the mechanisms underlying the regulation of potassium channel function and trafficking, and the chemical basis of potassium channel modulation. In this article, we provide an overview of the major genetic tools used to study potassium channels in S. cerevisiae, a survey of seminal studies utilizing these tools, and a prospective for the future use of this elegant genetic approach.


Assuntos
Engenharia Genética/métodos , Canais de Potássio/genética , Saccharomyces cerevisiae/crescimento & desenvolvimento , Citosol/metabolismo , Potássio/metabolismo , Canais de Potássio/química , Canais de Potássio/metabolismo , Saccharomyces cerevisiae/genética , Saccharomyces cerevisiae/metabolismo , Proteínas de Saccharomyces cerevisiae/química , Proteínas de Saccharomyces cerevisiae/genética , Proteínas de Saccharomyces cerevisiae/metabolismo
19.
Biochim Biophys Acta Bioenerg ; 1859(9): 676-683, 2018 09.
Artigo em Inglês | MEDLINE | ID: mdl-29981721

RESUMO

In natural variable environments, plants rapidly adjust photosynthesis for optimum balance between photochemistry and photoprotection. These adjustments mainly occur via changes in their proton motive force (pmf). Recent studies based on time resolved analysis of the Electro Chromic Signal (ECS) bandshift of photosynthetic pigments in the model plant Arabidopsis thaliana have suggested an active role of ion fluxes across the thylakoid membranes in the regulation of the pmf. Among the different channels and transporters possibly involved in this phenomenon, we previously identified the TPK3 potassium channel. Plants silenced for TPK3 expression displayed light stress signatures, with reduced Non Photochemical Quenching (NPQ) capacity and sustained anthocyanin accumulation, even at moderate intensities. In this work we re-examined the role of this protein in pmf regulation, starting from the observation that both TPK3 knock-down (TPK3 KD) and WT plants display enhanced anthocyanin accumulation in the light under certain growth conditions, especially in old leaves. We thus compared the pmf features of young "green" (without anthocyanins) and old "red" (with anthocyanins) leaves in both genotypes using a global fit analysis of the ECS. We found that the differences in the ECS profile measured between the two genotypes reflect not only differences in TPK3 expression level, but also a modified photosynthetic activity of stressed red leaves, which are present in a larger amounts in the TPK3 KD plants.


Assuntos
Arabidopsis/metabolismo , Clorofila/metabolismo , Complexos de Proteínas Captadores de Luz/metabolismo , Plantas Geneticamente Modificadas/metabolismo , Canais de Potássio/metabolismo , Força Próton-Motriz , Arabidopsis/genética , Arabidopsis/efeitos da radiação , Luz , Complexos de Proteínas Captadores de Luz/genética , Fotossíntese , Plantas Geneticamente Modificadas/genética , Plantas Geneticamente Modificadas/efeitos da radiação , Canais de Potássio/genética , Tilacoides/metabolismo
20.
Adv Neurobiol ; 20: 63-83, 2018.
Artigo em Inglês | MEDLINE | ID: mdl-29916016

RESUMO

The molecular process of RNA editing allows changes in RNA transcripts that increase genomic diversity. These highly conserved RNA editing events are catalyzed by a group of enzymes known as adenosine deaminases acting on double-stranded RNA (ADARs). ADARs are necessary for normal development, they bind to over thousands of genes, impact millions of editing sites, and target critical components of the central nervous system (CNS) such as glutamate receptors, serotonin receptors, and potassium channels. Dysfunctional ADARs are known to cause alterations in CNS protein products and therefore play a role in chronic or acute neurodegenerative and psychiatric diseases as well as CNS cancer. Here, we review how RNA editing deficiency impacts CNS function and summarize its role during disease pathogenesis.


Assuntos
Doenças Neurodegenerativas/metabolismo , Edição de RNA , Humanos , Doenças Neurodegenerativas/genética , Canais de Potássio/genética , Canais de Potássio/metabolismo , Receptores de Glutamato/genética , Receptores de Glutamato/metabolismo , Receptores de Serotonina/genética , Receptores de Serotonina/metabolismo
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