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1.
Arterioscler Thromb Vasc Biol ; 40(9): e240-e255, 2020 09.
Artigo em Inglês | MEDLINE | ID: mdl-32698687

RESUMO

OBJECTIVE: To determine if endothelial dysfunction in a mouse model of diet-induced obesity and in obese humans is mediated by the suppression of endothelial Kir (inwardly rectifying K+) channels. Approach and Results: Endothelial dysfunction, observed as reduced dilations to flow, occurred after feeding mice a high-fat, Western diet for 8 weeks. The functional downregulation of endothelial Kir2.1 using dominant-negative Kir2.1 construct resulted in substantial reductions in the response to flow in mesenteric arteries of lean mice, whereas no effect was observed in arteries of obese mice. Overexpressing wild-type-Kir2.1 in endothelium of arteries from obese mice resulted in full recovery of the flow response. Exposing freshly isolated endothelial cells to fluid shear during patch-clamp electrophysiology revealed that the flow-sensitivity of Kir was virtually abolished in cells from obese mice. Atomic force microscopy revealed that the endothelial glycocalyx was stiffer and the thickness of the glycocalyx layer reduced in arteries from obese mice. We also identified that the length of the glycocalyx is critical to the flow-activation of Kir. Overexpressing Kir2.1 in endothelium of arteries from obese mice restored flow- and heparanase-sensitivity, indicating an important role for heparan sulfates in the flow-activation of Kir. Furthermore, the Kir2.1-dependent component of flow-induced vasodilation was lost in the endothelium of resistance arteries of obese humans obtained from biopsies collected during bariatric surgery. CONCLUSIONS: We conclude that obesity-induced impairment of flow-induced vasodilation is attributed to the loss of flow-sensitivity of endothelial Kir channels and propose that the latter is mediated by the biophysical alterations of the glycocalyx.


Assuntos
Células Endoteliais/metabolismo , Endotélio Vascular/metabolismo , Glicocálix/metabolismo , Artérias Mesentéricas/metabolismo , Obesidade/metabolismo , Canais de Potássio Corretores do Fluxo de Internalização/metabolismo , Vasodilatação , Adulto , Animais , Células Cultivadas , Dieta Hiperlipídica , Modelos Animais de Doenças , Endotélio Vascular/fisiopatologia , Feminino , Heparitina Sulfato/metabolismo , Humanos , Masculino , Mecanotransdução Celular , Potenciais da Membrana , Artérias Mesentéricas/fisiopatologia , Camundongos , Pessoa de Meia-Idade , Obesidade/genética , Obesidade/fisiopatologia , Canais de Potássio Corretores do Fluxo de Internalização/genética , Fluxo Sanguíneo Regional
2.
Proc Natl Acad Sci U S A ; 117(28): 16626-16637, 2020 07 14.
Artigo em Inglês | MEDLINE | ID: mdl-32601236

RESUMO

Neuronal activity leads to an increase in local cerebral blood flow (CBF) to allow adequate supply of oxygen and nutrients to active neurons, a process termed neurovascular coupling (NVC). We have previously shown that capillary endothelial cell (cEC) inwardly rectifying K+ (Kir) channels can sense neuronally evoked increases in interstitial K+ and induce rapid and robust dilations of upstream parenchymal arterioles, suggesting a key role of cECs in NVC. The requirements of this signal conduction remain elusive. Here, we utilize mathematical modeling to investigate how small outward currents in stimulated cECs can elicit physiologically relevant spread of vasodilatory signals within the highly interconnected brain microvascular network to increase local CBF. Our model shows that the Kir channel can act as an "on-off" switch in cECs to hyperpolarize the cell membrane as extracellular K+ increases. A local hyperpolarization can be amplified by the voltage-dependent activation of Kir in neighboring cECs. Sufficient Kir density enables robust amplification of the hyperpolarizing stimulus and produces responses that resemble action potentials in excitable cells. This Kir-mediated excitability can remain localized in the stimulated region or regeneratively propagate over significant distances in the microvascular network, thus dramatically increasing the efficacy of K+ for eliciting local hyperemia. Modeling results show how changes in cEC transmembrane current densities and gap junctional resistances can affect K+-mediated NVC and suggest a key role for Kir as a sensor of neuronal activity and an amplifier of retrograde electrical signaling in the cerebral vasculature.


Assuntos
Neurônios/metabolismo , Acoplamento Neurovascular , Canais de Potássio Corretores do Fluxo de Internalização/metabolismo , Potássio/metabolismo , Animais , Encéfalo/irrigação sanguínea , Encéfalo/metabolismo , Circulação Cerebrovascular , Células Endoteliais/química , Células Endoteliais/metabolismo , Masculino , Camundongos , Camundongos Endogâmicos C57BL , Modelos Biológicos , Neurônios/química , Potássio/química , Canais de Potássio Corretores do Fluxo de Internalização/química , Canais de Potássio Corretores do Fluxo de Internalização/genética , Transdução de Sinais , Canais de Cátion TRPV/química , Canais de Cátion TRPV/genética , Canais de Cátion TRPV/metabolismo
3.
Circ Arrhythm Electrophysiol ; 13(8): e008627, 2020 08.
Artigo em Inglês | MEDLINE | ID: mdl-32654514

RESUMO

BACKGROUND: During acute infections, the risk of malignant ventricular arrhythmias is increased, partly because of a higher propensity to develop QTc prolongation. Although it is generally believed that QTc changes almost exclusively result from concomitant treatment with QT-prolonging antimicrobials, direct effects of inflammatory cytokines on ventricular repolarization are increasingly recognized. We hypothesized that systemic inflammation per se can significantly prolong QTc during acute infections, via cytokine-mediated changes in K+ channel expression. METHODS: We evaluated (1) the frequency of QTc prolongation and its association with inflammatory markers, in patients with different types of acute infections, during active disease and remission; (2) the prevalence of acute infections in a cohort of consecutive patients with Torsades de Pointes; (3) the relationship between K+ channel mRNA levels in ventricles and peripheral blood mononuclear cells and their changes in patients with acute infection over time. RESULTS: In patients with acute infections, regardless of concomitant QT-prolonging antimicrobial treatments, QTc was significantly prolonged but rapidly normalized in parallel to CRP (C-reactive protein) and cytokine level reduction. Consistently in the Torsades de Pointes cohort, concomitant acute infections were highly prevalent (30%), despite only a minority (25%) of these cases were treated with QT-prolonging antimicrobials. KCNJ2 K+ channel expression in peripheral blood mononuclear cell, which strongly correlated to that in ventricles, inversely associated to CRP and IL (interleukin)-1 changes in acute infection patients. CONCLUSIONS: During acute infections, systemic inflammation rapidly induces cytokine-mediated ventricular electrical remodeling and significant QTc prolongation, regardless concomitant antimicrobial therapy. Although transient, these changes may significantly increase the risk of life-threatening ventricular arrhythmia in these patients. It is timely and warranted to transpose these findings to the current coronavirus disease 2019 (COVID-19) pandemic, in which both increased amounts of circulating cytokines and cardiac arrhythmias are demonstrated along with a frequent concomitant treatment with several QT-prolonging drugs. Graphic Abstract: A graphic abstract is available for this article.


Assuntos
Doenças Transmissíveis/metabolismo , Citocinas/metabolismo , Parada Cardíaca/metabolismo , Frequência Cardíaca , Ventrículos do Coração/metabolismo , Inflamação/metabolismo , Leucócitos Mononucleares/metabolismo , Canais de Potássio Corretores do Fluxo de Internalização/metabolismo , Torsades de Pointes/metabolismo , Potenciais de Ação , Doença Aguda , Adulto , Idoso , Idoso de 80 Anos ou mais , Anti-Infecciosos/efeitos adversos , Doenças Transmissíveis/tratamento farmacológico , Doenças Transmissíveis/epidemiologia , Doenças Transmissíveis/fisiopatologia , Feminino , Parada Cardíaca/epidemiologia , Parada Cardíaca/fisiopatologia , Frequência Cardíaca/efeitos dos fármacos , Ventrículos do Coração/efeitos dos fármacos , Ventrículos do Coração/fisiopatologia , Humanos , Inflamação/epidemiologia , Inflamação/fisiopatologia , Leucócitos Mononucleares/efeitos dos fármacos , Masculino , Pessoa de Meia-Idade , Canais de Potássio Corretores do Fluxo de Internalização/genética , Prevalência , Fatores de Risco , Transdução de Sinais , Fatores de Tempo , Torsades de Pointes/epidemiologia , Torsades de Pointes/fisiopatologia , Adulto Jovem
4.
Invest Ophthalmol Vis Sci ; 61(5): 38, 2020 05 11.
Artigo em Inglês | MEDLINE | ID: mdl-32437550

RESUMO

Purpose: The purpose of this study was to establish and analyze a cell model of Leber congenital amaurosis type 16 (LCA16), which is caused by mutations in the KCNJ13 gene encoding Kir7.1, an inward-rectifying potassium ion channel. Methods: The two guide RNAs specific to the target sites in the KCNJ13 gene were designed and KCNJ13 knock-out (KO) human-induced pluripotent stem cells (hiPSCs) were generated using the CRISPR/Cas9 system. The KCNJ13-KO hiPSCs were differentiated into retinal pigment epithelial cells (hiPSC-RPEs). The KCNJ13-KO in hiPSC-RPEs was confirmed by immunostaining. Phagocytic activity of hiPSC-RPEs was assessed using the uptake of fluorescently labeled porcine photoreceptor outer segments (POSs). Phagocytosis-related genes in RPE cells were assessed by quantitative polymerase chain reaction. Results: Most of the translated region of the KCNJ13 gene was deleted in the KCNJ13-KO hiPSCs by the CRISPR/Cas9 system, and this confirmed that the Kir7.1 protein was not present in RPE cells induced from the hiPSCs. Expression of RPE marker genes such as BEST1 and CRALBP was retained in the wild-type (WT) and in the KCNJ13-KO hiPSC-RPE cells. However, phagocytic activity and expression of phagocytosis-related genes in the KCNJ13-null hiPSC-RPE cells were significantly reduced compared to those of WT. Conclusions: We succeeded in generating an RPE model of LCA16 using hiPSCs. We suggest that Kir7.1 is required for phagocytosis of POSs by RPE cells and that impaired phagocytosis in the absence of Kir7.1 would be involved in the retinal degeneration found in LCA16.


Assuntos
Deleção de Genes , Células-Tronco Pluripotentes Induzidas/citologia , Fagocitose/fisiologia , Canais de Potássio Corretores do Fluxo de Internalização/genética , Epitélio Pigmentado da Retina/fisiologia , Animais , Western Blotting , Proteína 9 Associada à CRISPR , Diferenciação Celular , Linhagem Celular , Técnicas de Inativação de Genes , Humanos , Amaurose Congênita de Leber/patologia , Microscopia Eletrônica de Transmissão , Microscopia de Fluorescência , Reação em Cadeia da Polimerase em Tempo Real , Segmento Externo das Células Fotorreceptoras da Retina/metabolismo , Epitélio Pigmentado da Retina/ultraestrutura , Suínos
5.
Science ; 368(6494): 993-1001, 2020 05 29.
Artigo em Inglês | MEDLINE | ID: mdl-32467389

RESUMO

Sophisticated devices for remote-controlled medical interventions require an electrogenetic interface that uses digital electronic input to directly program cellular behavior. We present a cofactor-free bioelectronic interface that directly links wireless-powered electrical stimulation of human cells to either synthetic promoter-driven transgene expression or rapid secretion of constitutively expressed protein therapeutics from vesicular stores. Electrogenetic control was achieved by coupling ectopic expression of the L-type voltage-gated channel CaV1.2 and the inwardly rectifying potassium channel Kir2.1 to the desired output through endogenous calcium signaling. Focusing on type 1 diabetes, we engineered electrosensitive human ß cells (Electroß cells). Wireless electrical stimulation of Electroß cells inside a custom-built bioelectronic device provided real-time control of vesicular insulin release; insulin levels peaked within 10 minutes. When subcutaneously implanted, this electrotriggered vesicular release system restored normoglycemia in type 1 diabetic mice.


Assuntos
Diabetes Mellitus Experimental/terapia , Diabetes Mellitus Tipo 1/terapia , Estimulação Elétrica/instrumentação , Secreção de Insulina/genética , Células Secretoras de Insulina/metabolismo , Tecnologia sem Fio/instrumentação , Animais , Biônica , Canais de Cálcio Tipo L/genética , Sinalização do Cálcio , Engenharia Celular , Células HEK293 , Humanos , Masculino , Camundongos , Canais de Potássio Corretores do Fluxo de Internalização/genética , Próteses e Implantes , Transcrição Genética , Transgenes
6.
Nat Rev Endocrinol ; 16(7): 378-393, 2020 07.
Artigo em Inglês | MEDLINE | ID: mdl-32376986

RESUMO

The ATP-sensitive potassium channel (KATP channel) couples blood levels of glucose to insulin secretion from pancreatic ß-cells. KATP channel closure triggers a cascade of events that results in insulin release. Metabolically generated changes in the intracellular concentrations of adenosine nucleotides are integral to this regulation, with ATP and ADP closing the channel and MgATP and MgADP increasing channel activity. Activating mutations in the genes encoding either of the two types of KATP channel subunit (Kir6.2 and SUR1) result in neonatal diabetes mellitus, whereas loss-of-function mutations cause hyperinsulinaemic hypoglycaemia of infancy. Sulfonylurea and glinide drugs, which bind to SUR1, close the channel through a pathway independent of ATP and are now the primary therapy for neonatal diabetes mellitus caused by mutations in the genes encoding KATP channel subunits. Insight into the molecular details of drug and nucleotide regulation of channel activity has been illuminated by cryo-electron microscopy structures that reveal the atomic-level organization of the KATP channel complex. Here we review how these structures aid our understanding of how the various mutations in the genes encoding Kir6.2 (KCNJ11) and SUR1 (ABCC8) lead to a reduction in ATP inhibition and thereby neonatal diabetes mellitus. We also provide an update on known mutations and sulfonylurea therapy in neonatal diabetes mellitus.


Assuntos
Diabetes Mellitus/congênito , Diabetes Mellitus/genética , Doenças do Recém-Nascido/genética , Canais KATP/genética , Mutação , Canais de Potássio Corretores do Fluxo de Internalização/genética , Receptores Sulfonilureia/genética , Animais , Diabetes Mellitus/tratamento farmacológico , Humanos , Recém-Nascido , Doenças do Recém-Nascido/tratamento farmacológico , Secreção de Insulina/genética , Mutação/fisiologia , Compostos de Sulfonilureia/uso terapêutico
7.
PLoS Comput Biol ; 16(4): e1007749, 2020 04.
Artigo em Inglês | MEDLINE | ID: mdl-32251469

RESUMO

The renal outer medullary potassium (ROMK) channel is essential for potassium transport in the kidney, and its dysfunction is associated with a salt-wasting disorder known as Bartter syndrome. Despite its physiological significance, we lack a mechanistic understanding of the molecular defects in ROMK underlying most Bartter syndrome-associated mutations. To this end, we employed a ROMK-dependent yeast growth assay and tested single amino acid variants selected by a series of computational tools representative of different approaches to predict each variants' pathogenicity. In one approach, we used in silico saturation mutagenesis, i.e. the scanning of all possible single amino acid substitutions at all sequence positions to estimate their impact on function, and then employed a new machine learning classifier known as Rhapsody. We also used two additional tools, EVmutation and Polyphen-2, which permitted us to make consensus predictions on the pathogenicity of single amino acid variants in ROMK. Experimental tests performed for selected mutants in different classes validated the vast majority of our predictions and provided insights into variants implicated in ROMK dysfunction. On a broader scope, our analysis suggests that consolidation of data from complementary computational approaches provides an improved and facile method to predict the severity of an amino acid substitution and may help accelerate the identification of disease-causing mutations in any protein.


Assuntos
Canais de Potássio Corretores do Fluxo de Internalização/genética , Substituição de Aminoácidos , Síndrome de Bartter/genética , Síndrome de Bartter/metabolismo , Biologia Computacional/métodos , Humanos , Rim/metabolismo , Rim/patologia , Mutação , Mutação de Sentido Incorreto/genética , Potássio/metabolismo , Canais de Potássio Corretores do Fluxo de Internalização/metabolismo , Saccharomyces cerevisiae/genética
8.
Proc Natl Acad Sci U S A ; 117(14): 7803-7813, 2020 04 07.
Artigo em Inglês | MEDLINE | ID: mdl-32213593

RESUMO

Protein-lipid interactions are a key element of the function of many integral membrane proteins. These potential interactions should be considered alongside the complexity and diversity of membrane lipid composition. Inward rectifier potassium channel (Kir) Kir2.2 has multiple interactions with plasma membrane lipids: Phosphatidylinositol (4, 5)-bisphosphate (PIP2) activates the channel; a secondary anionic lipid site has been identified, which augments the activation by PIP2; and cholesterol inhibits the channel. Molecular dynamics simulations are used to characterize in molecular detail the protein-lipid interactions of Kir2.2 in a model of the complex plasma membrane. Kir2.2 has been simulated with multiple, functionally important lipid species. From our simulations we show that PIP2 interacts most tightly at the crystallographic interaction sites, outcompeting other lipid species at this site. Phosphatidylserine (PS) interacts at the previously identified secondary anionic lipid interaction site, in a PIP2 concentration-dependent manner. There is interplay between these anionic lipids: PS interactions are diminished when PIP2 is not present in the membrane, underlining the need to consider multiple lipid species when investigating protein-lipid interactions.


Assuntos
Metabolismo dos Lipídeos/genética , Lipídeos/genética , Canais de Potássio Corretores do Fluxo de Internalização/genética , Animais , Ânions/metabolismo , Membrana Celular/genética , Membrana Celular/metabolismo , Proteínas de Membrana/genética , Proteínas de Membrana/metabolismo , Simulação de Dinâmica Molecular , Fosfatidilinositol 4,5-Difosfato/metabolismo , Potássio/metabolismo , Canais de Potássio Corretores do Fluxo de Internalização/metabolismo
9.
Cytogenet Genome Res ; 160(1): 22-28, 2020.
Artigo em Inglês | MEDLINE | ID: mdl-32018267

RESUMO

We report on a novel variant of the dicentric chromosome 17;20 (dic (17;20)(p11.2;q11.2) in a patient with de novo myelodysplastic syndrome (MDS). Based on FISH and array-CGH, the variant turns out to be an insertion of chromosome 17 (17p11.2-telomere 17) into chromosome 20 with breakpoints at 20q11.22 and 20q13.33. Based on conventional chromosome analysis and G-banding patterns, the region 17p11.2-17q25 was directly inserted between 20q11.22 and 20q13.33. The breakpoint junctions occurred within KCNJ12 (17p11.2), UQCC1 (20q11.2), and CDH4 (20q13.3), leading to 5' deletions of all the genes and positioning the 3' of UQCC1 next to KCNJ12 at 17p11.2 and CDH4 next to an unknown gene at 17q25-20q13.3. In addition, the centromere of chromosome 17 was not active, transforming the primary constriction to a flat band. Therefore, the novel insertion variant is a pseudo dicentric derivative chromosome with one functional centromere: 45,XX,der(17;20)del(20)(q11.22q13.33)ins(20;17)(q11.2;p11.2q25). A review of the literature of all dic(17;20) cases is presented. For the first time, we report an array-CGH characterization of such rare variant that revealed to be an insertion.


Assuntos
Aberrações Cromossômicas , Cromossomos Humanos Par 17/genética , Cromossomos Humanos Par 20/genética , Hibridização Genômica Comparativa , Síndromes Mielodisplásicas/genética , Linhagem da Célula , Centrômero/ultraestrutura , Bandeamento Cromossômico , Deleção Cromossômica , Feminino , Rearranjo Gênico , Humanos , Cariotipagem , Proteínas de Membrana/genética , Pessoa de Meia-Idade , Canais de Potássio Corretores do Fluxo de Internalização/genética , Translocação Genética
10.
Br Poult Sci ; 61(3): 320-327, 2020 Jun.
Artigo em Inglês | MEDLINE | ID: mdl-32008360

RESUMO

1. The potassium voltage-gated channel subfamily J member 11 gene (KCNJ11) is involved in the insulin secretion pathway. Studies have shown that mutation in this gene is associated with muscle weakness. The objective of the present study was to establish the association between KCNJ11 gene polymorphism and chicken growth performance and to analyse its expression pattern. 2. A novel 163-bp insertion/deletion (indel) polymorphism was identified in the region downstream of the KCNJ11 gene in 2330 individuals from ten populations by polymerase chain reaction (PCR). An F2 resource population was used to investigate the genetic effects of the chicken KCNJ11 gene. Association analysis showed that the indel was significantly associated with chicken growth traits and that the phenotypic value of the ins-ins (II) genotype is higher than that of the ins-del (ID) and del-del (DD) genotypes. 3. Gene expression for different genotypes showed that birds carrying the II allele had a higher expression level than the DD genotypes. Analysis of tissue and spatiotemporal expression patterns indicated that the KCNJ11 gene was highly expressed in muscle tissues, with the highest levels in muscle tissue at one week of age, and that a 10% crude protein diet reduced the expression of this gene, average daily gain and muscle fibre diameter. 4. The results suggested that this novel 163-bp indel has the potential to become a new target for marker-assisted selection.


Assuntos
Galinhas , Mutação INDEL , Canais de Potássio Corretores do Fluxo de Internalização/genética , Animais , Galinhas/crescimento & desenvolvimento , Genótipo , Fenótipo , Polimorfismo Genético
11.
Proc Natl Acad Sci U S A ; 117(6): 2938-2947, 2020 02 11.
Artigo em Inglês | MEDLINE | ID: mdl-31980523

RESUMO

The conformational changes required for activation and K+ conduction in inward-rectifier K+ (Kir) channels are still debated. These structural changes are brought about by lipid binding. It is unclear how this process relates to fast gating or if the intracellular and extracellular regions of the protein are coupled. Here, we examine the structural details of KirBac1.1 reconstituted into both POPC and an activating lipid mixture of 3:2 POPC:POPG (wt/wt). KirBac1.1 is a prokaryotic Kir channel that shares homology with human Kir channels. We establish that KirBac1.1 is in a constitutively active state in POPC:POPG bilayers through the use of real-time fluorescence quenching assays and Förster resonance energy transfer (FRET) distance measurements. Multidimensional solid-state NMR (SSNMR) spectroscopy experiments reveal two different conformers within the transmembrane regions of the protein in this activating lipid environment, which are distinct from the conformation of the channel in POPC bilayers. The differences between these three distinct channel states highlight conformational changes associated with an open activation gate and suggest a unique allosteric pathway that ties the selectivity filter to the activation gate through interactions between both transmembrane helices, the turret, selectivity filter loop, and the pore helix. We also identify specific residues involved in this conformational exchange that are highly conserved among human Kir channels.


Assuntos
Proteínas de Bactérias/química , Proteínas de Bactérias/metabolismo , Canais de Potássio Corretores do Fluxo de Internalização/química , Canais de Potássio Corretores do Fluxo de Internalização/metabolismo , Proteínas de Bactérias/genética , Domínio Catalítico , Transferência Ressonante de Energia de Fluorescência , Cinética , Espectroscopia de Ressonância Magnética , Modelos Moleculares , Fosfatidilcolinas/química , Fosfatidilcolinas/metabolismo , Fosfatidilgliceróis/química , Fosfatidilgliceróis/metabolismo , Potássio/metabolismo , Canais de Potássio Corretores do Fluxo de Internalização/genética , Conformação Proteica , Domínios Proteicos , Estrutura Secundária de Proteína
12.
Biochem J ; 477(3): 671-689, 2020 02 14.
Artigo em Inglês | MEDLINE | ID: mdl-31957808

RESUMO

ATP-sensitive potassium (KATP) channels are widely expressed and play key roles in many tissues by coupling metabolic state to membrane excitability. The SUR subunits confer drug and enhanced nucleotide sensitivity to the pore-forming Kir6 subunit, and so information transfer between the subunits must occur. In our previous study, we identified an electrostatic interaction between Kir6 and SUR2 subunits that was key for allosteric information transfer between the regulatory and pore-forming subunit. In this study, we demonstrate a second putative interaction between Kir6.2-D323 and SUR2A-Q1336 using patch clamp electrophysiological recording, where charge swap mutation of the residues on either side of the potential interaction compromise normal channel function. The Kir6.2-D323K mutation gave rise to a constitutively active, glibenclamide and ATP-insensitive KATP complex, further confirming the importance of information transfer between the Kir6 and SUR2 subunits. Sensitivity to modulators was restored when Kir6.2-D323K was co-expressed with a reciprocal charge swap mutant, SUR-Q1336E. Importantly, equivalent interactions have been identified in both Kir6.1 and Kir6.2 suggesting this is a second important interaction between Kir6 and the proximal C terminus of SUR.


Assuntos
Transportadores de Cassetes de Ligação de ATP , Canais KATP , Canais de Potássio Corretores do Fluxo de Internalização/química , Receptores Sulfonilureia/química , Transportadores de Cassetes de Ligação de ATP/química , Transportadores de Cassetes de Ligação de ATP/metabolismo , Sítio Alostérico , Células HEK293 , Humanos , Canais KATP/química , Canais KATP/metabolismo , Modelos Estruturais , Mutação , Técnicas de Patch-Clamp , Canais de Potássio Corretores do Fluxo de Internalização/genética , Canais de Potássio Corretores do Fluxo de Internalização/metabolismo , Receptores Sulfonilureia/genética , Receptores Sulfonilureia/metabolismo
13.
Nucleic Acids Res ; 48(2): e11, 2020 01 24.
Artigo em Inglês | MEDLINE | ID: mdl-31745561

RESUMO

Domain recombination is a key principle in protein evolution and protein engineering, but inserting a donor domain into every position of a target protein is not easily experimentally accessible. Most contemporary domain insertion profiling approaches rely on DNA transposons, which are constrained by sequence bias. Here, we establish Saturated Programmable Insertion Engineering (SPINE), an unbiased, comprehensive, and targeted domain insertion library generation technique using oligo library synthesis and multi-step Golden Gate cloning. Through benchmarking to MuA transposon-mediated library generation on four ion channel genes, we demonstrate that SPINE-generated libraries are enriched for in-frame insertions, have drastically reduced sequence bias as well as near-complete and highly-redundant coverage. Unlike transposon-mediated domain insertion that was severely biased and sparse for some genes, SPINE generated high-quality libraries for all genes tested. Using the Inward Rectifier K+ channel Kir2.1, we validate the practical utility of SPINE by constructing and comparing domain insertion permissibility maps. SPINE is the first technology to enable saturated domain insertion profiling. SPINE could help explore the relationship between domain insertions and protein function, and how this relationship is shaped by evolutionary forces and can be engineered for biomedical applications.


Assuntos
Elementos de DNA Transponíveis/genética , Evolução Molecular , Mutagênese Insercional/genética , Canais de Potássio Corretores do Fluxo de Internalização/genética , Biblioteca Gênica , Humanos , Oligonucleotídeos , Domínios Proteicos/genética , Engenharia de Proteínas , Recombinação Genética/genética
14.
Am J Physiol Renal Physiol ; 318(1): F216-F228, 2020 01 01.
Artigo em Inglês | MEDLINE | ID: mdl-31736353

RESUMO

K+ deficiency stimulates renal salt reuptake via the Na+-Cl- cotransporter (NCC) of the distal convoluted tubule (DCT), thereby reducing K+ losses in downstream nephron segments while increasing NaCl retention and blood pressure. NCC activation is mediated by a kinase cascade involving with no lysine (WNK) kinases upstream of Ste20-related proline-alanine-rich kinase (SPAK) and oxidative stress-responsive kinase-1 (OSR1). In K+ deficiency, WNKs and SPAK/OSR1 concentrate in spherical cytoplasmic domains in the DCT termed "WNK bodies," the significance of which is undetermined. By feeding diets of varying salt and K+ content to mice and using genetically engineered mouse lines, we aimed to clarify whether WNK bodies contribute to WNK-SPAK/OSR1-NCC signaling. Phosphorylated SPAK/OSR1 was present both at the apical membrane and in WNK bodies within 12 h of dietary K+ deprivation, and it was promptly suppressed by K+ loading. In WNK4-deficient mice, however, larger WNK bodies formed, containing unphosphorylated WNK1, SPAK, and OSR1. This suggests that WNK4 is the primary active WNK isoform in WNK bodies and catalyzes SPAK/OSR1 phosphorylation therein. We further examined mice carrying a kidney-specific deletion of the basolateral K+ channel-forming protein Kir4.1, which is required for the DCT to sense plasma K+ concentration. These mice displayed remnant mosaic expression of Kir4.1 in the DCT, and upon K+ deprivation, WNK bodies developed only in Kir4.1-expressing cells. We postulate a model of DCT function in which NCC activity is modulated by plasma K+ concentration via WNK4-SPAK/OSR1 interactions within WNK bodies.


Assuntos
Hipopotassemia/metabolismo , Rim/metabolismo , Proteínas Serina-Treonina Quinases/metabolismo , Animais , Feminino , Hipopotassemia/sangue , Túbulos Renais Distais/metabolismo , Masculino , Camundongos , Camundongos Knockout , Fosforilação , Potássio/sangue , Canais de Potássio Corretores do Fluxo de Internalização/genética , Canais de Potássio Corretores do Fluxo de Internalização/metabolismo , Proteínas Serina-Treonina Quinases/genética , Transdução de Sinais/fisiologia , Membro 3 da Família 12 de Carreador de Soluto/metabolismo
15.
Toxicol Lett ; 318: 57-64, 2020 Jan.
Artigo em Inglês | MEDLINE | ID: mdl-31585160

RESUMO

3-Bromopyruvate (3-BrPA) is a promising agent that has been widely studied in the treatment of cancer and pulmonary hypertension. Rotenone is a pesticide commonly used on farms and was shown to have anti-cancer activity and delay fibrosis progression in chronic kidney disease in a recent study. However, there are few studies showing the toxicity of rotenone and 3-BrPA in the myocardium. To support further medical exploration, it is necessary to clarify the side effects of these compounds on the heart. This study was designed to examine the cardiotoxicity of 3-BrPA and rotenone by investigating electrical and structural cardiac remodeling in rats. Forty male rats were divided into 4 groups (n = 10 in each group) and injected intraperitoneally with 3-BrPA, rotenone or a combination of 3-BrPA and rotenone. The ventricular effective refractory period (VERP), corrected QT interval (QTc), and ventricular tachycardia/ventricular fibrillation (VT/VF) inducibility were measured. The expression of Cx43, Kir2.1, Kir6.2, DHPRα1, KCNH2, caspase3, caspase9, Bax, Bcl2, and P53 was detected. Masson's trichrome, TUNEL, HE, and PAS staining and transmission electron microscopy were used to detect pathological and ultrastructural changes. Our results showed that rotenone alone and rotenone combined with 3-BrPA significantly increased the risk of ventricular arrhythmias. Rotenone combined with 3-BrPA caused myocardial apoptosis, and rotenone alone and rotenone combined with 3-BrPA caused electrical and structural cardiac remodeling in rats.


Assuntos
Antineoplásicos/toxicidade , Ventrículos do Coração/efeitos dos fármacos , Inseticidas/toxicidade , Piruvatos/toxicidade , Rotenona/toxicidade , Taquicardia Ventricular/induzido quimicamente , Fibrilação Ventricular/induzido quimicamente , Remodelação Ventricular/efeitos dos fármacos , Potenciais de Ação/efeitos dos fármacos , Animais , Apoptose/efeitos dos fármacos , Proteínas Reguladoras de Apoptose/metabolismo , Cardiotoxicidade , Conexina 43/genética , Conexina 43/metabolismo , Frequência Cardíaca/efeitos dos fármacos , Ventrículos do Coração/metabolismo , Ventrículos do Coração/fisiopatologia , Ventrículos do Coração/ultraestrutura , Masculino , Canais de Potássio Corretores do Fluxo de Internalização/genética , Canais de Potássio Corretores do Fluxo de Internalização/metabolismo , Ratos Wistar , Período Refratário Eletrofisiológico/efeitos dos fármacos , Medição de Risco , Taquicardia Ventricular/metabolismo , Taquicardia Ventricular/patologia , Taquicardia Ventricular/fisiopatologia , Fibrilação Ventricular/metabolismo , Fibrilação Ventricular/patologia , Fibrilação Ventricular/fisiopatologia
16.
Am J Physiol Renal Physiol ; 318(2): F332-F337, 2020 02 01.
Artigo em Inglês | MEDLINE | ID: mdl-31841387

RESUMO

Inwardly rectifying K+ (Kir) channels are expressed in multiple organs and cell types and play critical roles in cellular function. Most notably, Kir channels are major determinants of the resting membrane potential and K+ homeostasis. The renal outer medullary K+ channel (Kir1.1) was the first renal Kir channel identified and cloned in the kidney over two decades ago. Since then, several additional members, including classical and ATP-regulated Kir family classes, have been identified to be expressed in the kidney and to contribute to renal ion transport. Although the ATP-regulated Kir channel class remains the most well known due to severe pathological phenotypes associated with their mutations, progress is being made in defining the properties, localization, and physiological functions of other renal Kir channels, including those localized to the basolateral epithelium. This review is primarily focused on the current knowledge of the expression and localization of renal Kir channels but will also briefly describe their proposed functions in the kidney.


Assuntos
Rim/metabolismo , Canais de Potássio Corretores do Fluxo de Internalização/metabolismo , Potássio/metabolismo , Animais , Regulação da Expressão Gênica , Homeostase , Humanos , Rim/fisiopatologia , Nefropatias/genética , Nefropatias/metabolismo , Nefropatias/fisiopatologia , Potenciais da Membrana , Canais de Potássio Corretores do Fluxo de Internalização/genética
17.
Int J Mol Sci ; 20(21)2019 Oct 25.
Artigo em Inglês | MEDLINE | ID: mdl-31731540

RESUMO

An increased flux of potassium ions into the mitochondrial matrix through the ATP-sensitive potassium channel (mitoKATP) has been shown to provide protection against ischemia-reperfusion injury. Recently, it was proposed that the mitochondrial-targeted isoform of the renal outer medullary potassium channel (ROMK) protein creates a pore-forming subunit of mitoKATP in heart mitochondria. Our research focuses on the properties of mitoKATP from heart-derived H9c2 cells. For the first time, we detected single-channel activity and describe the pharmacology of mitoKATP in the H9c2 heart-derived cells. The patch-clamping of mitoplasts from wild type (WT) and cells overexpressing ROMK2 revealed the existence of a potassium channel that exhibits the same basic properties previously attributed to mitoKATP. ROMK2 overexpression resulted in a significant increase of mitoKATP activity. The conductance of both channels in symmetric 150/150 mM KCl was around 97 ± 2 pS in WT cells and 94 ± 3 pS in cells overexpressing ROMK2. The channels were inhibited by 5-hydroxydecanoic acid (a mitoKATP inhibitor) and by Tertiapin Q (an inhibitor of both the ROMK-type channels and mitoKATP). Additionally, mitoKATP from cells overexpressing ROMK2 were inhibited by ATP/Mg2+ and activated by diazoxide. We used an assay based on proteinase K to examine the topology of the channel in the inner mitochondrial membrane and found that both termini of the protein localized to the mitochondrial matrix. We conclude that the observed activity of the channel formed by the ROMK protein corresponds to the electrophysiological and pharmacological properties of mitoKATP.


Assuntos
Trifosfato de Adenosina/metabolismo , Magnésio/metabolismo , Membranas Mitocondriais/metabolismo , Proteínas Mitocondriais/metabolismo , Miocárdio/metabolismo , Canais de Potássio Corretores do Fluxo de Internalização/metabolismo , Trifosfato de Adenosina/genética , Linhagem Celular , Humanos , Proteínas Mitocondriais/genética , Canais de Potássio Corretores do Fluxo de Internalização/genética
18.
Exp Eye Res ; 189: 107852, 2019 12.
Artigo em Inglês | MEDLINE | ID: mdl-31647904

RESUMO

Mutations in KCNJ13 are associated with two retinal disorders; Leber congenital amaurosis (LCA) and snowflake vitreoretinal degeneration (SVD). We describe a novel fibrovascular proliferation in the retina of two affected members of a KCNJ13-related LCA family with a homozygous c.458C > T, p.(Thr153Ile) missense mutation. Optical coherence tomography retinal imaging of the kcnj13 mutant zebrafish (obelixtd15 c.502T > C, p.[Phe168Leu]) revealed a late onset retinal degeneration at 12 months, with retinal thinning and associated retinovascular changes, including increased vessel calibre and vitreous deposits. Both human and zebrafish variants are missense and located within the conserved transmembrane M2 protein domain, suggesting that disruption of this region may contribute to retinovascular changes as an additional feature to the previously described LCA phenotype. Close monitoring of other patients with similar mutations may be required to minimise the ensuing retinal damage.


Assuntos
Amaurose Congênita de Leber/genética , Mutação de Sentido Incorreto , Canais de Potássio Corretores do Fluxo de Internalização/genética , Retina/metabolismo , Degeneração Retiniana/genética , Adolescente , Adulto , Idoso , Idoso de 80 Anos ou mais , Animais , Criança , DNA/genética , Análise Mutacional de DNA , Modelos Animais de Doenças , Feminino , Angiofluoresceinografia/métodos , Fundo de Olho , Humanos , Amaurose Congênita de Leber/metabolismo , Amaurose Congênita de Leber/patologia , Masculino , Pessoa de Meia-Idade , Linhagem , Fenótipo , Canais de Potássio Corretores do Fluxo de Internalização/metabolismo , Domínios Proteicos , Retina/patologia , Degeneração Retiniana/metabolismo , Degeneração Retiniana/patologia , Tomografia de Coerência Óptica/métodos , Adulto Jovem , Peixe-Zebra
19.
Hum Genomics ; 13(1): 53, 2019 10 22.
Artigo em Inglês | MEDLINE | ID: mdl-31640787

RESUMO

BACKGROUND: Dysfunction in inwardly rectifying potassium channel Kir4.1 has been implicated in SeSAME syndrome, an autosomal-recessive (AR), rare, multi-systemic disorder. However, not all neurological, intellectual disability, and comorbid phenotypes in SeSAME syndrome can be mechanistically linked solely to Kir4.1 dysfunction. METHODS: We therefore performed whole-exome sequencing and identified additional genetic risk-elements that might exert causative effects either alone or in concert with Kir4.1 in a family diagnosed with SeSAME syndrome. RESULTS: Two variant prioritization pipelines based on AR inheritance and runs of homozygosity (ROH), identified two novel homozygous variants in KCNJ10 and PI4KB and five rare homozygous variants in PVRL4, RORC, FLG2, FCRL1, NIT1 and one common homozygous variant in HSPA6 segregating in all four patients. The novel mutation in KCNJ10 resides in the cytoplasmic domain of Kir4.1, a seat of phosphatidylinositol bisphosphate (PIP2) binding. The mutation altered the subcellular localization and stability of Kir4.1 in patient-specific lymphoblastoid cells (LCLs) compared to parental controls. Barium-sensitive endogenous K+ currents in patient-specific LCLs using whole-cell patch-clamp electrophysiology revealed membrane depolarization and defects in inward K+ ion conductance across the membrane, thereby suggesting a loss-of-function effect of KCNJ10 variant. CONCLUSION: Altogether, our findings implicate the role of new genes in SeSAME syndrome without electrolyte imbalance and thereby speculate the regulation of Kir4.1 channel activity by PIP2 and integrin-mediated adhesion signaling mechanisms.


Assuntos
Perda Auditiva Neurossensorial/genética , Deficiência Intelectual/genética , Fosfotransferases (Aceptor do Grupo Álcool)/genética , Canais de Potássio Corretores do Fluxo de Internalização/genética , Convulsões/genética , Adolescente , Adulto , Criança , Feminino , Perda Auditiva Neurossensorial/patologia , Homozigoto , Humanos , Deficiência Intelectual/patologia , Masculino , Mutação/genética , Fenótipo , Convulsões/patologia , Sequenciamento Completo do Exoma , Adulto Jovem
20.
Exp Suppl ; 111: 385-416, 2019.
Artigo em Inglês | MEDLINE | ID: mdl-31588541

RESUMO

In addition to the common types of diabetes mellitus, two major monogenic diabetes forms exist. Maturity-onset diabetes of the young (MODY) represents a heterogenous group of monogenic, autosomal dominant diseases. MODY accounts for 1-2% of all diabetes cases, and it is not just underdiagnosed but often misdiagnosed to type 1 or type 2 diabetes. More than a dozen MODY genes have been identified to date, and their molecular classification is of great importance in the correct treatment decision and in the judgment of the prognosis. The most prevalent subtypes are HNF1A, GCK, and HNF4A. Genetic testing for MODY has changed recently due to the technological advancements, as contrary to the sequential testing performed in the past, nowadays all MODY genes can be tested simultaneously by next-generation sequencing. The other major group of monogenic diabetes is neonatal diabetes mellitus which can be transient or permanent, and often the diabetes is a part of a syndrome. It is a severe monogenic disease appearing in the first 6 months of life. The hyperglycemia usually requires insulin. There are two forms, permanent neonatal diabetes mellitus (PNDM) and transient neonatal diabetes mellitus (TNDM). In TNDM, the diabetes usually reverts within several months but might relapse later in life. The incidence of NDM is 1:100,000-1:400,000 live births, and PNDM accounts for half of the cases. Most commonly, neonatal diabetes is caused by mutations in KCNJ11 and ABCC8 genes encoding the ATP-dependent potassium channel of the ß cell. Neonatal diabetes has experienced a quick and successful transition into the clinical practice since the discovery of the molecular background. In case of both genetic diabetes groups, recent guidelines recommend genetic testing.


Assuntos
Diabetes Mellitus Tipo 2/genética , Diabetes Mellitus/genética , Doenças do Recém-Nascido/genética , Testes Genéticos , Quinases do Centro Germinativo , Fator 1-alfa Nuclear de Hepatócito/genética , Fator 4 Nuclear de Hepatócito/genética , Humanos , Recém-Nascido , Mutação , Canais de Potássio Corretores do Fluxo de Internalização/genética , Proteínas Serina-Treonina Quinases/genética , Receptores Sulfonilureia/genética
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