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1.
Int J Mol Sci ; 22(17)2021 Aug 25.
Artigo em Inglês | MEDLINE | ID: mdl-34502077

RESUMO

The cloning of connexins cDNA opened the way to the field of gap junction channelopathies. Thus far, at least 35 genetic diseases, resulting from mutations of 11 different connexin genes, are known to cause numerous structural and functional defects in the central and peripheral nervous system as well as in the heart, skin, eyes, teeth, ears, bone, hair, nails and lymphatic system. While all of these diseases are due to connexin mutations, minimal attention has been paid to the potential diseases of cell-cell communication caused by mutations of Cx-associated molecules. An important Cx accessory protein is calmodulin (CaM), which is the major regulator of gap junction channel gating and a molecule relevant to gap junction formation. Recently, diseases caused by CaM mutations (calmodulinopathies) have been identified, but thus far calmodulinopathy studies have not considered the potential effect of CaM mutations on gap junction function. The major goal of this review is to raise awareness on the likely role of CaM mutations in defects of gap junction mediated cell communication. Our studies have demonstrated that certain CaM mutants affect gap junction channel gating or expression, so it would not be surprising to learn that CaM mutations known to cause diseases also affect cell communication mediated by gap junction channels.


Assuntos
Calmodulina/genética , Canalopatias/genética , Conexinas/genética , Doenças Genéticas Inatas/genética , Animais , Calmodulina/metabolismo , Comunicação Celular , Canalopatias/metabolismo , Conexinas/metabolismo , Doenças Genéticas Inatas/metabolismo , Humanos , Mutação
2.
Elife ; 102021 08 04.
Artigo em Inglês | MEDLINE | ID: mdl-34346310

RESUMO

Gap junctions between neurons serve as electrical synapses, in addition to conducting metabolites and signaling molecules. During development, early-appearing gap junctions are thought to prefigure chemical synapses, which appear much later. We present evidence for this idea at a central, glutamatergic synapse and provide some mechanistic insights. Loss or reduction in the levels of the gap junction protein Gjd2b decreased the frequency of glutamatergic miniature excitatory postsynaptic currents (mEPSCs) in cerebellar Purkinje neurons (PNs) in larval zebrafish. Ultrastructural analysis in the molecular layer showed decreased synapse density. Further, mEPSCs had faster kinetics and larger amplitudes in mutant PNs, consistent with their stunted dendritic arbors. Time-lapse microscopy in wild-type and mutant PNs reveals that Gjd2b puncta promote the elongation of branches and that CaMKII may be a critical mediator of this process. These results demonstrate that Gjd2b-mediated gap junctions regulate glutamatergic synapse formation and dendritic elaboration in PNs.


Assuntos
Conexinas/genética , Proteínas do Olho/genética , Junções Comunicantes/genética , Plasticidade Neuronal/fisiologia , Células de Purkinje/fisiologia , Sinapses/fisiologia , Proteínas de Peixe-Zebra/genética , Peixe-Zebra/fisiologia , Animais , Conexinas/metabolismo , Proteínas do Olho/metabolismo , Junções Comunicantes/metabolismo , Peixe-Zebra/genética , Proteínas de Peixe-Zebra/metabolismo
3.
Int J Mol Sci ; 22(15)2021 Jul 30.
Artigo em Inglês | MEDLINE | ID: mdl-34360970

RESUMO

Anterior cruciate ligament (ACL) ruptures are usually treated with autograft implantation to prevent knee instability. Tissue engineered ACL reconstruction is becoming promising to circumvent autograft limitations. The aim was to evaluate the influence of cyclic stretch on lapine (L) ACL fibroblasts on embroidered scaffolds with respect to adhesion, DNA and sulphated glycosaminoglycan (sGAG) contents, gene expression of ligament-associated extracellular matrix genes, such as type I collagen, decorin, tenascin C, tenomodulin, gap junctional connexin 43 and the transcription factor Mohawk. Control scaffolds and those functionalized by gas phase fluorination and cross-linked collagen foam were either pre-cultured with a suspension or with spheroids of LACL cells before being subjected to cyclic stretch (4%, 0.11 Hz, 3 days). Stretch increased significantly the scaffold area colonized with cells but impaired sGAGs and decorin gene expression (functionalized scaffolds seeded with cell suspension). Stretching increased tenascin C, connexin 43 and Mohawk but decreased decorin gene expression (control scaffolds seeded with cell suspension). Pre-cultivation of functionalized scaffolds with spheroids might be the more suitable method for maintaining ligamentogenesis in 3D scaffolds compared to using a cell suspension due to a significantly higher sGAG content in response to stretching and type I collagen gene expression in functionalized scaffolds.


Assuntos
Ligamento Cruzado Anterior/fisiologia , Esferoides Celulares/citologia , Engenharia Tecidual/métodos , Tecidos Suporte/química , Animais , Ligamento Cruzado Anterior/citologia , Adesão Celular , Proliferação de Células , Células Cultivadas , Conexinas/genética , Conexinas/metabolismo , Decorina/genética , Decorina/metabolismo , Proteínas da Matriz Extracelular/genética , Proteínas da Matriz Extracelular/metabolismo , Feminino , Fibroblastos/fisiologia , Proteínas de Homeodomínio/genética , Proteínas de Homeodomínio/metabolismo , Homeostase , Masculino , Poliésteres/química , Coelhos , Regeneração , Esferoides Celulares/metabolismo , Estresse Mecânico
4.
Immunity ; 54(8): 1715-1727.e7, 2021 08 10.
Artigo em Inglês | MEDLINE | ID: mdl-34283971

RESUMO

Allergic airway inflammation is driven by type-2 CD4+ T cell inflammatory responses. We uncover an immunoregulatory role for the nucleotide release channel, Panx1, in T cell crosstalk during airway disease. Inverse correlations between Panx1 and asthmatics and our mouse models revealed the necessity, specificity, and sufficiency of Panx1 in T cells to restrict inflammation. Global Panx1-/- mice experienced exacerbated airway inflammation, and T-cell-specific deletion phenocopied Panx1-/- mice. A transgenic designed to re-express Panx1 in T cells reversed disease severity in global Panx1-/- mice. Panx1 activation occurred in pro-inflammatory T effector (Teff) and inhibitory T regulatory (Treg) cells and mediated the extracellular-nucleotide-based Treg-Teff crosstalk required for suppression of Teff cell proliferation. Mechanistic studies identified a Salt-inducible kinase-dependent phosphorylation of Panx1 serine 205 important for channel activation. A genetically targeted mouse expressing non-phosphorylatable Panx1S205A phenocopied the exacerbated inflammation in Panx1-/- mice. These data identify Panx1-dependent Treg:Teff cell communication in restricting airway disease.


Assuntos
Asma/imunologia , Comunicação Celular/imunologia , Conexinas/metabolismo , Proteínas do Tecido Nervoso/metabolismo , Proteínas Serina-Treonina Quinases/metabolismo , Linfócitos T Reguladores/imunologia , Animais , Linhagem Celular , Proliferação de Células/fisiologia , Conexinas/genética , Modelos Animais de Doenças , Células HEK293 , Humanos , Células Jurkat , Camundongos , Camundongos Endogâmicos C57BL , Camundongos Knockout , Proteínas do Tecido Nervoso/genética , Sistema Respiratório/imunologia
5.
Invest Ophthalmol Vis Sci ; 62(9): 27, 2021 07 01.
Artigo em Inglês | MEDLINE | ID: mdl-34283211

RESUMO

Purpose: The purpose of this study was to determine whether retinal gap junctions (GJs) via connexin 36 (Cx36, mediating coupling of many retinal cell types) and horizontal cell (HC-HC) coupling, are involved in emmetropization. Methods: Guinea pigs (3 weeks old) were monocularly form deprived (FD) or raised without FD (in normal visual [NV] environment) for 2 days or 4 weeks; alternatively, they wore a -4 D lens (hyperopic defocus [HD]) or 0 D lens for 2 days or 1 week. FD and NV eyes received daily subconjunctival injections of a nonspecific GJ-uncoupling agent, 18-ß-Glycyrrhetinic Acid (18-ß-GA). The amounts of total Cx36 and of phosphorylated Cx36 (P-Cx36; activated state that increases cell-cell coupling), in the inner and outer plexiform layers (IPLs and OPLs), were evaluated by quantitative immunofluorescence (IF), and HC-HC coupling was evaluated by cut-loading with neurobiotin. Results: FD per se (excluding effect of light-attenuation) increased HC-HC coupling in OPL, whereas HD did not affect it. HD for 2 days or 1 week had no significant effect on retinal content of Cx36 or P-Cx36. FD for 4 weeks decreased the total amounts of Cx36 and P-Cx36, and the P-Cx36/Cx36 ratio, in the IPL. Subconjunctival 18-ß-GA induced myopia in NV eyes and increased the myopic shifts in FD eyes, while reducing the amounts of Cx36 and P-Cx36 in both the IPL and OPL. Conclusions: These results suggest that cell-cell coupling via GJs containing Cx36 (particularly those in the IPL) plays a role in emmetropization and form deprivation myopia (FDM) in mammals. Although both FD and 18-ß-GA induced myopia, they had opposite effects on HC-HC coupling. These findings suggest that HC-HC coupling in the OPL might not play a significant role in emmetropization and myopia development.


Assuntos
Conexinas/metabolismo , Emetropia/fisiologia , Junções Comunicantes/metabolismo , Hiperopia/metabolismo , Miopia/metabolismo , Retina/metabolismo , Corpo Vítreo/metabolismo , Animais , Túnica Conjuntiva/metabolismo , Túnica Conjuntiva/patologia , Modelos Animais de Doenças , Junções Comunicantes/patologia , Cobaias , Hiperopia/patologia , Hiperopia/fisiopatologia , Miopia/patologia , Miopia/fisiopatologia , Retina/patologia , Retina/fisiopatologia , Privação Sensorial , Corpo Vítreo/patologia
6.
Int J Mol Sci ; 22(14)2021 Jul 06.
Artigo em Inglês | MEDLINE | ID: mdl-34298872

RESUMO

Connexins can assemble into either gap junctions (between two cells) or hemichannels (from one cell to the extracellular space) and mediate cell-to-cell signalling. A subset of connexins (Cx26, Cx30, Cx32) are directly sensitive to CO2 and fluctuations in the level within a physiological range affect their open probability, and thus, change cell conductance. These connexins are primarily found on astrocytes or oligodendrocytes, where increased CO2 leads to ATP release, which acts on P2X and P2Y receptors of neighbouring neurons and changes excitability. CO2-sensitive hemichannels are also found on developing cortical neurons, where they play a role in producing spontaneous neuronal activity. It is plausible that the transient opening of hemichannels allows cation influx, leading to depolarisation. Recently, we have shown that dopaminergic neurons in the substantia nigra and GABAergic neurons in the VTA also express Cx26 hemichannels. An increase in the level of CO2 results in hemichannel opening, increasing whole-cell conductance, and decreasing neuronal excitability. We found that the expression of Cx26 in the dopaminergic neurons in the substantia nigra at P7-10 is transferred to glial cells by P17-21, displaying a shift from being inhibitory (to neuronal activity) in young mice, to potentially excitatory (via ATP release). Thus, Cx26 hemichannels could have three modes of signalling (release of ATP, excitatory flickering open and shut and inhibitory shunting) depending on where they are expressed (neurons or glia) and the stage of development.


Assuntos
Dióxido de Carbono/metabolismo , Conexinas/metabolismo , Neuroglia/metabolismo , Neurônios/metabolismo , Transdução de Sinais/fisiologia , Animais , Astrócitos/metabolismo , Comunicação Celular/fisiologia , Humanos
7.
Nat Commun ; 12(1): 4482, 2021 07 23.
Artigo em Inglês | MEDLINE | ID: mdl-34301959

RESUMO

Activation of Pannexin 1 (PANX1) ion channels causes release of intercellular signaling molecules in a variety of (patho)physiological contexts. PANX1 can be activated by G protein-coupled receptors (GPCRs), including α1-adrenergic receptors (α1-ARs), but how receptor engagement leads to channel opening remains unclear. Here, we show that GPCR-mediated PANX1 activation can occur via channel deacetylation. We find that α1-AR-mediated activation of PANX1 channels requires Gαq but is independent of phospholipase C or intracellular calcium. Instead, α1-AR-mediated PANX1 activation involves RhoA, mammalian diaphanous (mDia)-related formin, and a cytosolic lysine deacetylase activated by mDia - histone deacetylase 6. HDAC6 associates with PANX1 and activates PANX1 channels, even in excised membrane patches, suggesting direct deacetylation of PANX1. Substitution of basally-acetylated intracellular lysine residues identified on PANX1 by mass spectrometry either prevents HDAC6-mediated activation (K140/409Q) or renders the channels constitutively active (K140R). These data define a non-canonical RhoA-mDia-HDAC6 signaling pathway for GαqPCR activation of PANX1 channels and uncover lysine acetylation-deacetylation as an ion channel silencing-activation mechanism.


Assuntos
Conexinas/metabolismo , Desacetilase 6 de Histona/metabolismo , Proteínas do Tecido Nervoso/metabolismo , Receptores Adrenérgicos alfa 1/metabolismo , Acetilação , Animais , Células Cultivadas , Conexinas/genética , Conexinas/fisiologia , Células HEK293 , Desacetilase 6 de Histona/genética , Humanos , Células Jurkat , Lisina/genética , Lisina/metabolismo , Potenciais da Membrana/fisiologia , Camundongos Endogâmicos C57BL , Camundongos Knockout , Proteínas do Tecido Nervoso/genética , Proteínas do Tecido Nervoso/fisiologia , Técnicas de Patch-Clamp , Receptores Adrenérgicos alfa 1/genética , Transdução de Sinais/genética , Proteína rhoA de Ligação ao GTP/genética , Proteína rhoA de Ligação ao GTP/metabolismo
8.
Nat Commun ; 12(1): 4484, 2021 07 23.
Artigo em Inglês | MEDLINE | ID: mdl-34301940

RESUMO

Reactive oxygen species (ROS) represent a by-product of metabolism and their excess is toxic for hematopoietic stem and progenitor cells (HSPCs). During embryogenesis, a small number of HSPCs are produced from the hemogenic endothelium, before they colonize a transient organ where they expand, for example the fetal liver in mammals. In this study, we use zebrafish to understand the molecular mechanisms that are important in the caudal hematopoietic tissue (equivalent to the mammalian fetal liver) to promote HSPC expansion. High levels of ROS are deleterious for HSPCs in this niche, however this is rescued by addition of antioxidants. We show that Cx41.8 is important to lower ROS levels in HSPCs. We also demonstrate a new role for ifi30, known to be involved in the immune response. In the hematopoietic niche, Ifi30 can recycle oxidized glutathione to allow HSPCs to dampen their levels of ROS, a role that could be conserved in human fetal liver.


Assuntos
Conexinas/genética , Células-Tronco Hematopoéticas/metabolismo , Estresse Oxidativo , Oxirredutases atuantes sobre Doadores de Grupo Enxofre/genética , Transdução de Sinais/genética , Nicho de Células-Tronco , Proteínas de Peixe-Zebra/genética , Animais , Animais Geneticamente Modificados , Conexinas/metabolismo , Embrião não Mamífero/citologia , Embrião não Mamífero/embriologia , Embrião não Mamífero/metabolismo , Regulação da Expressão Gênica no Desenvolvimento , Células-Tronco Hematopoéticas/citologia , Humanos , Microscopia Confocal , Mutação , Oxirredutases atuantes sobre Doadores de Grupo Enxofre/metabolismo , Imagem com Lapso de Tempo/métodos , Peixe-Zebra/embriologia , Peixe-Zebra/genética , Proteínas de Peixe-Zebra/metabolismo
9.
Int J Mol Sci ; 22(11)2021 Jun 04.
Artigo em Inglês | MEDLINE | ID: mdl-34199748

RESUMO

BACKGROUND: Psoriasis, a chronic inflammatory disease affecting 2-3% of the population, is characterised by epidermal hyperplasia, a sustained pro-inflammatory immune response and is primarily a T-cell driven disease. Previous work determined that Connexin26 is upregulated in psoriatic tissue. This study extends these findings. METHODS: Biopsies spanning psoriatic plaque (PP) and non-involved tissue (PN) were compared to normal controls (NN). RNA was isolated and subject to real-time PCR to determine gene expression profiles, including GJB2/CX26, GJB6/CX30 and GJA1/CX43. Protein expression was assessed by immunohistochemistry. Keratinocytes and fibroblasts were isolated and used in 3D organotypic models. The pro-inflammatory status of fibroblasts and 3D cultures was assessed via ELISA and RnD cytokine arrays in the presence or absence of the connexin channel blocker Gap27. RESULTS: Connexin26 expression is dramatically enhanced at both transcriptional and translational level in PP and PN tissue compared to NN (>100x). In contrast, CX43 gene expression is not affected, but the protein is post-translationally modified and accumulates in psoriatic tissue. Fibroblasts isolated from psoriatic patients had a higher inflammatory index than normal fibroblasts and drove normal keratinocytes to adopt a "psoriatic phenotype" in a 3D-organotypic model. Exposure of normal fibroblasts to the pro-inflammatory mediator peptidoglycan, isolated from Staphylococcus aureus enhanced cytokine release, an event protected by Gap27. CONCLUSION: dysregulation of the connexin26:43 expression profile in psoriatic tissue contributes to an imbalance of cellular events. Inhibition of connexin signalling reduces pro-inflammatory events and may hold therapeutic benefit.


Assuntos
Conexinas/genética , Regulação da Expressão Gênica , Psoríase/genética , Adulto , Idoso , Biópsia , Conexinas/metabolismo , Conexinas/farmacologia , Epiderme/patologia , Fibroblastos/efeitos dos fármacos , Fibroblastos/metabolismo , Fibroblastos/patologia , Perfilação da Expressão Gênica , Regulação da Expressão Gênica/efeitos dos fármacos , Células HaCaT , Humanos , Mediadores da Inflamação , Queratinócitos/efeitos dos fármacos , Queratinócitos/metabolismo , Queratinócitos/patologia , Pessoa de Meia-Idade , Modelos Biológicos , Oligopeptídeos/farmacologia , Peptidoglicano/isolamento & purificação , Fosforilação , Processamento de Proteína Pós-Traducional/efeitos dos fármacos , Psoríase/patologia , Staphylococcus aureus/fisiologia , Adulto Jovem
10.
Eur J Med Chem ; 223: 113650, 2021 Nov 05.
Artigo em Inglês | MEDLINE | ID: mdl-34174741

RESUMO

Panx-1 is a membrane channel protein involved in some pathologies such as ischemic stroke, cancer and neuropathic pain, thus representing a promising therapeutic target. We present here a study aimed at obtaining the first class of selective Panx-1 blockers, a new topic for pharmaceutical chemistry, since all compounds used so far for the study of this channel have different primary targets. Among various scaffolds analyzed, the indole nucleous emerged, whose elaboration yielded interesting Panx-1 blockers, such as the potent 5-sulfamoyl derivatives 14c and 15b (I% = 100 at 50 µM). In vivo tests performed in the mouse model of oxaliplatin-induced neuropathy, demonstrated that the hypersensitivity was completely reverted by treatment with 15b (1 nmol, administered intrathecally), suggesting a relationship between this effect and the channel blocking ability. Finally, we decided to perform a virtual screening study on compounds 5b, 6l and 14c using a recently resolved cryo-EM structure of hPanx-1 channel, to try to relate the potency of our new inhibitors.


Assuntos
Desenho de Fármacos , Indóis/química , Proteínas do Tecido Nervoso/metabolismo , Animais , Sítios de Ligação , Conexinas/antagonistas & inibidores , Conexinas/metabolismo , Modelos Animais de Doenças , Humanos , Indóis/farmacologia , Indóis/uso terapêutico , Masculino , Potenciais da Membrana/efeitos dos fármacos , Camundongos , Simulação de Acoplamento Molecular , Proteínas do Tecido Nervoso/antagonistas & inibidores , Neuralgia/induzido quimicamente , Neuralgia/tratamento farmacológico , Neuralgia/patologia , Oócitos/efeitos dos fármacos , Oócitos/fisiologia , Oxaliplatina/farmacologia , Relação Estrutura-Atividade
11.
Int J Mol Sci ; 22(11)2021 May 27.
Artigo em Inglês | MEDLINE | ID: mdl-34072103

RESUMO

This review highlights current knowledge on the expression and function of connexins and pannexins, transmembrane channel proteins that play an important role in intercellular communication, in both the developing and mature lymphatic vasculature. A particular focus is given to the involvement of these proteins in functions of the healthy lymphatic system. We describe their influence on the maintenance of extracellular fluid homeostasis, immune cell trafficking to draining lymph nodes and dietary nutrient absorption by intestinal villi. Moreover, new insights into connexin mutations in primary and secondary lymphedema as well as on the implication of lymphatic connexins and pannexins in acquired cardiovascular diseases are discussed, allowing for a better understanding of the role of these proteins in pathologies linked to dysfunctions in the lymphatic system.


Assuntos
Biomarcadores , Conexinas/genética , Conexinas/metabolismo , Suscetibilidade a Doenças , Homeostase , Sistema Linfático/metabolismo , Animais , Doenças Cardiovasculares/etiologia , Doenças Cardiovasculares/metabolismo , Doenças Cardiovasculares/patologia , Expressão Gênica , Humanos , Imuno-Histoquímica , Linfedema/etiologia , Linfedema/metabolismo , Linfedema/patologia , Mutação , Organogênese/genética
12.
Int J Mol Sci ; 22(10)2021 May 14.
Artigo em Inglês | MEDLINE | ID: mdl-34068881

RESUMO

Neuroinflammation is a major component of central nervous system (CNS) injuries and neurological diseases, including Alzheimer's disease, multiple sclerosis, neuropathic pain, and brain trauma. The activation of innate immune cells at the damage site causes the release of pro-inflammatory cytokines and chemokines, which alter the functionality of nearby tissues and might mediate the recruitment of leukocytes to the injury site. If this process persists or is exacerbated, it prevents the adequate resolution of the inflammation, and ultimately enhances secondary damage. Adenosine 5' triphosphate (ATP) is among the molecules released that trigger an inflammatory response, and it serves as a chemotactic and endogenous danger signal. Extracellular ATP activates multiple purinergic receptors (P2X and P2Y) that have been shown to promote neuroinflammation in a variety of CNS diseases. Recent studies have shown that Pannexin-1 (Panx1) channels are the principal conduits of ATP release from dying cells and innate immune cells in the brain. Herein, we review the emerging evidence that directly implicates Panx-1 channels in the neuroinflammatory response in the CNS.


Assuntos
Conexinas/metabolismo , Inflamação/fisiopatologia , Proteínas do Tecido Nervoso/metabolismo , Doenças do Sistema Nervoso/fisiopatologia , Animais , Humanos , Inflamação/metabolismo , Doenças do Sistema Nervoso/metabolismo
13.
Int J Mol Sci ; 22(10)2021 May 17.
Artigo em Inglês | MEDLINE | ID: mdl-34067798

RESUMO

In humans and other vertebrates pannexin protein family was discovered by homology to invertebrate gap junction proteins. Several biological functions were attributed to three vertebrate pannexins members. Six clinically significant independent variants of the PANX1 gene lead to human infertility and oocyte development defects, and the Arg217His variant was associated with pronounced symptoms of primary ovarian failure, severe intellectual disability, sensorineural hearing loss, and kyphosis. At the same time, only mild phenotypes were observed in Panx1 knockout mice. In addition, a passenger mutation was identified in a popular line of Panx1 knockout mice, questioning even those effects. Using CRISPR/Cas9, we created a new line of Panx1 knockout mice and a new line of mice with the clinically significant Panx1 substitution (Arg217His). In both cases, we observed no significant changes in mouse size, weight, or fertility. In addition, we attempted to reproduce a previous study on sleep/wake and locomotor activity functions in Panx1 knockout mice and found that previously reported effects were probably not caused by the Panx1 knockout itself. We consider that the pathological role of Arg217His substitution in Panx1, and some Panx1 functions in general calls for a re-evaluation.


Assuntos
Conexinas/metabolismo , Proteínas do Tecido Nervoso/metabolismo , Animais , Conexinas/genética , Conexinas/fisiologia , Perda Auditiva Neurossensorial/genética , Humanos , Masculino , Camundongos , Camundongos Endogâmicos C57BL , Camundongos Transgênicos , Mutação de Sentido Incorreto/genética , Proteínas do Tecido Nervoso/genética , Proteínas do Tecido Nervoso/fisiologia , Fenótipo , Sono/genética
14.
Commun Biol ; 4(1): 702, 2021 06 08.
Artigo em Inglês | MEDLINE | ID: mdl-34103655

RESUMO

Ca2+/calmodulin-dependent protein kinase II (CaMKII) binding and phosphorylation of mammalian connexin-36 (Cx36) potentiate electrical coupling. To explain the molecular mechanism of how Cx36 modifies plasticity at gap junctions, we investigated the roles of ionotropic N-methyl-D-aspartate receptors and pannexin1 (Panx1) channels in regulating Cx36 binding to CaMKII. Pharmacological interference and site-directed mutagenesis of protein interaction sites shows that NMDA receptor activation opens Cx36 channels, causing the Cx36- CaMKII binding complex to adopt a compact conformation. Ectopic Panx1 expression in a Panx1 knock-down cell line is required to restore CaMKII mediated opening of Cx36. Furthermore, blocking of Src-family kinase activation of Panx1 is sufficient to prevent the opening of Cx36 channels. Our research demonstrates that the efficacy of Cx36 channels requires convergent calcium-dependent signaling processes in which activation of ionotropic N-methyl-D-aspartate receptor, Src-family kinase, and Pannexin1 open Cx36. Our results add to the best of our knowledge a new twist to mounting evidence for molecular communication between these core components of electrical and chemical synapses.


Assuntos
Proteína Quinase Tipo 2 Dependente de Cálcio-Calmodulina/metabolismo , Conexinas/metabolismo , Proteínas do Tecido Nervoso/metabolismo , Receptores de N-Metil-D-Aspartato/metabolismo , Transdução de Sinais , Animais , Cálcio/metabolismo , Sinalização do Cálcio , Linhagem Celular , Junções Comunicantes/metabolismo , Camundongos , Neurônios/metabolismo , Mapas de Interação de Proteínas
15.
Methods Enzymol ; 654: 271-293, 2021.
Artigo em Inglês | MEDLINE | ID: mdl-34120717

RESUMO

Connexin hemichannels are permeable to both atomic ions and small molecules. Yet, they have different selectivity for ions and signaling molecules critical for biological functions. Activity of connexin hemichannels in living cells is commonly evaluated by methods that include electrophysiology and fluorescence-based approaches. Although less common, luminescence and radioactivity-based uptake/release assays have been also successfully used to determine selectivity and permeability to different molecules. The current methods, however, have important technical and quantitative limitations that make them unsuitable for simultaneously evaluating ionic and molecular permeability using different stimuli that control channel gating (e.g., voltage or extracellular Ca2+). To address this, we have recently designed a novel methodology that combines two-electrode voltage clamp (TEVC) and dye uptake assays in translucent Xenopus oocytes. This method allows for the evaluation of molecular transport kinetics in connexin hemichannels, and its utility can also be extended to other large pore channels, such as those formed by pannexin and CALHM. In this article, we describe step by step the protocol to perform the TEVC/Dye uptake assay.


Assuntos
Conexinas , Junções Comunicantes , Transporte Biológico , Conexinas/metabolismo , Junções Comunicantes/metabolismo , Íons , Cinética
16.
J Stroke Cerebrovasc Dis ; 30(8): 105932, 2021 Aug.
Artigo em Inglês | MEDLINE | ID: mdl-34148020

RESUMO

OBJECTIVES: Bone marrow mononuclear cells (BM-MNC) show a significant therapeutic effect in combination with training even in the chronic phase of stroke. However, the mechanism of this combination therapy has not been investigated. Here, we examined its effects on brain metabolism in chronic stroke mice. MATERIALS AND METHODS: BM-MNC (1x105 cells in 100 µL of phosphate-buffered saline) were intravenously transplanted at 4 weeks (chronic stage) after the middle cerebral artery occlusion. At 3 h and 10 weeks after the administration of BM-MNC, we evaluated transcription changes of the metabolism-related genes, hypoxia inducible factor 1-α (Hif-1α), prolyl hydroxylase 3 (Phd3), pyruvate dehydrogenase kinase 1 (Pdk1), Na+/K+-ATPase (Atp1α1‒3), connexins, glucose transporters, and monocarboxylate transporters, in the brain during chronic phase of stroke using quantitative polymerase chain reaction. RESULTS: The results showed transcriptional activation of the metabolism-related genes in the contralateral cortex at 3 h after BM-MNC transplantation. Behavioral tests were performed after cell therapy, and the brain metabolism of mice with improved motor function was examined at 10 weeks after cell therapy. The therapeutic efficacy of the combination therapy with BM-MNC transplantation and training was evident in the form of transcriptional activation of ipsilateral anterior cerebral artery (ACA) cortex. CONCLUSIONS: BM-MNC transplantation combined with training for chronic stroke activated gene expression in both the ipsilateral and the contralateral side.


Assuntos
Células da Medula Óssea/metabolismo , Transplante de Medula Óssea , Encéfalo/metabolismo , Metabolismo Energético , Infarto da Artéria Cerebral Média/terapia , Condicionamento Físico Animal , Animais , Comportamento Animal , Encéfalo/fisiopatologia , Doença Crônica , Terapia Combinada , Conexinas/genética , Conexinas/metabolismo , Modelos Animais de Doenças , Metabolismo Energético/genética , Regulação da Expressão Gênica , Subunidade alfa do Fator 1 Induzível por Hipóxia/genética , Subunidade alfa do Fator 1 Induzível por Hipóxia/metabolismo , Infarto da Artéria Cerebral Média/genética , Infarto da Artéria Cerebral Média/metabolismo , Infarto da Artéria Cerebral Média/fisiopatologia , Masculino , Proteínas de Membrana Transportadoras/genética , Proteínas de Membrana Transportadoras/metabolismo , Camundongos Endogâmicos C57BL , Camundongos SCID , Atividade Motora , Recuperação de Função Fisiológica , ATPase Trocadora de Sódio-Potássio/genética , ATPase Trocadora de Sódio-Potássio/metabolismo , Transcrição Genética
17.
Commun Biol ; 4(1): 676, 2021 06 03.
Artigo em Inglês | MEDLINE | ID: mdl-34083742

RESUMO

Myopia is the most common developmental disorder of juvenile eyes, and it has become an increasing cause of severe visual impairment. The GJD2 locus has been consistently associated with myopia in multiple independent genome-wide association studies. However, despite the strong genetic evidence, little is known about the functional role of GJD2 in refractive error development. Here, we find that depletion of gjd2a (Cx35.5) or gjd2b (Cx35.1) orthologs in zebrafish, cause changes in the biometry and refractive status of the eye. Our immunohistological and scRNA sequencing studies show that Cx35.5 (gjd2a) is a retinal connexin and its depletion leads to hyperopia and electrophysiological changes in the retina. These findings support a role for Cx35.5 (gjd2a) in the regulation of ocular biometry. Cx35.1 (gjd2b) has previously been identified in the retina, however, we found an additional lenticular role. Lack of Cx35.1 (gjd2b) led to a nuclear cataract that triggered axial elongation. Our results provide functional evidence of a link between gjd2 and refractive error.


Assuntos
Conexinas/genética , Modelos Animais de Doenças , Proteínas do Olho/genética , Mutação , Erros de Refração/genética , Proteínas de Peixe-Zebra/genética , Peixe-Zebra/genética , Animais , Catarata/genética , Conexinas/metabolismo , Proteínas do Olho/metabolismo , Perfilação da Expressão Gênica/métodos , Humanos , Miopia/genética , RNA-Seq/métodos , Retina/metabolismo , Retina/patologia , Epitélio Pigmentado da Retina/metabolismo , Epitélio Pigmentado da Retina/patologia , Análise de Célula Única/métodos , Peixe-Zebra/metabolismo , Proteínas de Peixe-Zebra/metabolismo
18.
PLoS One ; 16(5): e0252334, 2021.
Artigo em Inglês | MEDLINE | ID: mdl-34043726

RESUMO

Crohn's disease is a chronic inflammatory bowel disease and the NLRP3 inflammasome plays an important role in Crohn's disease. Previous studies have shown that Herb-partitioned moxibustion treating (at Qihai (CV 6) and Tianshu (ST 25)) prevented the excessive activation of the NLRP3 inflammasome and repaired damaged colonic mucosa in Crohn's disease. However, the mechanism by which Herb-partitioned moxibustion (at CV 6 and ST 25) regulates NLRP3 remains unclear. In this study, we treated Crohn's disease rats with herb-partitioned moxibustion (at CV 6 and ST 25) to investigate the mechanism by which Herb-partitioned moxibustion regulates the colonic NLRP3 inflammasome by observing colon length, the colon macroscopic damage indexes, and the expression of ATP, P2X7R, Pannexin-1, NF-κBp65, NLRP3, ASC, caspase-1, IL-1ß and IL-18 in the colon in Crohn's disease. Here, this study shows that herb-partitioned moxibustion (at CV 6 and ST 25) can reduce colon macroscopic damage indexes and colon histopathological scores, alleviate colon shortening and block the abnormal activation of the NLRP3 inflammasome by inhibiting the ATP content and the expression of P2X7R, Pannexin-1 and NF-κBp65, thereby reducing the release of the downstream inflammatory cytokine IL-1ß and ultimately suppressing colonic inflammation in Crohn's disease rats. This study for the first time identifies the mechanism by which herb-partitioned moxibustion (at CV 6 and ST 25) may inhibit the abnormal activation of the NLRP3 inflammasome by inhibiting the P2X7R-Pannexin-1 signaling pathway in Crohn's disease rats.


Assuntos
Conexinas/metabolismo , Doença de Crohn/terapia , Moxibustão/métodos , Proteína 3 que Contém Domínio de Pirina da Família NLR/metabolismo , Proteínas do Tecido Nervoso/metabolismo , Receptores Purinérgicos P2X7/metabolismo , Animais , Ratos , Ratos Sprague-Dawley
19.
Cancer Sci ; 112(9): 3507-3519, 2021 Sep.
Artigo em Inglês | MEDLINE | ID: mdl-34050696

RESUMO

Necroptosis is an alternative form of programmed cell death that generally occurs under apoptosis-deficient conditions. Our previous work showed that connexin32 (Cx32) promotes the malignant progress of hepatocellular carcinoma (HCC) by enhancing the ability of resisting apoptosis in vivo and in vitro. Whether triggering necroptosis is a promising strategy to eliminate the apoptosis-resistant HCC cells with high Cx32 expression remains unknown. In this study, we found that Cx32 expression was positively correlated with the expression of necroptosis protein biomarkers in human HCC specimens, cell lines, and a xenograft model. Treatment with shikonin, a well-used necroptosis inducer, markedly caused necroptosis in HCC cells. Interestingly, overexpressed Cx32 exacerbated shikonin-induced necroptosis, but downregulation of Cx32 alleviated necroptosis in vitro and in vivo. Mechanistically, Cx32 was found to bind to Src and promote Src-mediated caspase 8 phosphorylation and inactivation, which ultimately reduced the activated caspase 8-mediated proteolysis of receptor-interacting serine-threonine protein kinase 1/3, the key molecule for necroptosis activation. In conclusion, we showed that Cx32 contributed to the activation of necroptosis in HCC cells through binding to Src and then mediating the inactivation of caspase 8. The present study suggested that necroptosis inducers could be more favorable than apoptosis inducers to eliminate HCC cells with high expression of Cx32.


Assuntos
Carcinoma Hepatocelular/metabolismo , Caspase 8/metabolismo , Conexinas/metabolismo , Neoplasias Hepáticas/metabolismo , Necroptose/genética , Coativador 1 de Receptor Nuclear/metabolismo , Transdução de Sinais/genética , Animais , Apoptose/efeitos dos fármacos , Apoptose/genética , Carcinoma Hepatocelular/genética , Carcinoma Hepatocelular/patologia , Linhagem Celular Tumoral , Proliferação de Células/efeitos dos fármacos , Proliferação de Células/genética , Conexinas/genética , Técnicas de Silenciamento de Genes , Humanos , Neoplasias Hepáticas/genética , Neoplasias Hepáticas/patologia , Masculino , Camundongos , Camundongos Endogâmicos BALB C , Camundongos Nus , Naftoquinonas/administração & dosagem , Necroptose/efeitos dos fármacos , Coativador 1 de Receptor Nuclear/genética , Fosforilação/efeitos dos fármacos , Fosforilação/genética , Transdução de Sinais/efeitos dos fármacos , Transfecção , Carga Tumoral/efeitos dos fármacos , Carga Tumoral/genética
20.
Int Heart J ; 62(3): 616-626, 2021.
Artigo em Inglês | MEDLINE | ID: mdl-34054002

RESUMO

Atrial fibrillation (AF) is a relatively common complication of hypertension. Chronic hypertension induces cardiac HDAC6 catalytic activity. However, whether HDAC6 activation contributes to hypertension-induced AF is still uncertain. We examined whether chronic cardiac HDAC6 activation-induced atrial remodeling, leading to AF induction.The HDAC6 constitutively active transgenic (TG) (HDAC6 active TG) mouse overexpressing the active HDAC6 protein, specifically in cardiomyocytes, was created to examine the effects of chronic HDAC6 activation on atrial electrical and structural remodeling and AF induction in HDAC6 active TG and non-transgenic (NTG) mice. Left atrial burst pacing (S1S1 = 30 msec) for 15-30 sec significantly increased the frequency of sustained AF in HDAC6 active-TG mice compared with NTG mice. Left steady-state atrial pacing (S1S1 = 80 msec) decreased the atrial conduction velocity in isolated HDAC6 active TG compared with NTG mouse atria. The atrial size was similar between HDAC6 active TG and NTG mice. In contrast, atrial interstitial fibrosis increased in HDAC6 active TG compared with that of NTG mouse atria. While protein expression levels of both CX40 and CX43 were similar between HDAC6 active TG and NTG mouse atria, a heterogeneous distribution of CX40 and CX43 occurred in HDAC6 active-TG mouse atria but not in NTG mouse atria. Gene expression of interleukin 6 increased in HDAC6 active TG compared with NTG mouse atria.Chronic cardiac HDAC6 activation induced atrial electrical and structural remodeling, and sustained AF. Hypertension-induced cardiac HDAC6 catalytic activity may play important roles in the development of AF.


Assuntos
Fibrilação Atrial/fisiopatologia , Conexinas/metabolismo , Átrios do Coração/fisiopatologia , Desacetilase 6 de Histona/farmacologia , Interleucina-6/metabolismo , Animais , Fibrilação Atrial/etiologia , Fibrilação Atrial/metabolismo , Remodelamento Atrial , Estimulação Cardíaca Artificial/métodos , Estudos de Casos e Controles , Feminino , Fibrose , Átrios do Coração/patologia , Desacetilase 6 de Histona/metabolismo , Hipertensão/complicações , Hipertensão/metabolismo , Hipertensão/fisiopatologia , Interleucina-6/genética , Camundongos , Camundongos Endogâmicos C57BL , Camundongos Transgênicos , Modelos Animais , Miócitos Cardíacos/metabolismo
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