RESUMO
Ticks transmit a variety of pathogens, including rickettsia and viruses, when they feed on blood, afflicting humans and other animals. Bioactive components acting on inflammation, coagulation, and the immune system were reported to facilitate ticks' ability to suck blood and transmit tick-borne diseases. In this study, a novel peptide, IstTx, from an Ixodes scapularis cDNA library was analyzed. The peptide IstTx, obtained by recombinant expression and purification, selectively inhibited a potassium channel, TREK-1, in a dose-dependent manner, with an IC50 of 23.46 ± 0.22 µM. The peptide IstTx exhibited different characteristics from fluoxetine, and the possible interaction of the peptide IstTx binding to the channel was explored by molecular docking. Notably, extracellular acidification raised its inhibitory efficacy on the TREK-1 channel. Our results found that the tick-derived peptide IstTx blocked the TREK-1 channel and provided a novel tool acting on the potassium channel.
Assuntos
Peptídeos , Canais de Potássio de Domínios Poros em Tandem , Canais de Potássio de Domínios Poros em Tandem/metabolismo , Canais de Potássio de Domínios Poros em Tandem/genética , Canais de Potássio de Domínios Poros em Tandem/antagonistas & inibidores , Canais de Potássio de Domínios Poros em Tandem/química , Animais , Humanos , Peptídeos/farmacologia , Peptídeos/química , Peptídeos/metabolismo , Ixodes/metabolismo , Simulação de Acoplamento Molecular , Sequência de Aminoácidos , Células HEK293 , Bloqueadores dos Canais de Potássio/farmacologia , Bloqueadores dos Canais de Potássio/química , Carrapatos/metabolismoRESUMO
BACKGROUND: The metastasis of tumors into bone tissue typically leads to intractable pain that is both very disabling and particularly difficult to manage. We investigated here whether riluzole could have beneficial effects for the treatment of prostate cancer-induced bone pain and how it could influence the development of bone metastasis. METHODS: We used a bone pain model induced by intratibial injection of human PC3 prostate cancer cells into male SCID mice treated or not with riluzole administered in drinking water. We also used riluzole in vitro to assess its possible effect on PC3 cell viability and functionality, using patch-clamp. RESULTS: Riluzole had a significant preventive effect on both evoked and spontaneous pain involving the TREK-1 potassium channel. Riluzole did not interfere with PC3-induced bone loss or bone remodeling in vivo. It also significantly decreased PC3 cell viability in vitro. The antiproliferative effect of riluzole is correlated with a TREK-1-dependent membrane hyperpolarization in these cells. CONCLUSION: The present data suggest that riluzole could be very useful to manage evoked and spontaneous hypersensitivity in cancer-induced bone pain and has no significant adverse effect on cancer progression.
Assuntos
Analgésicos , Neoplasias Ósseas , Dor do Câncer , Proliferação de Células , Camundongos SCID , Canais de Potássio de Domínios Poros em Tandem , Riluzol , Riluzol/farmacologia , Animais , Canais de Potássio de Domínios Poros em Tandem/metabolismo , Masculino , Neoplasias Ósseas/tratamento farmacológico , Neoplasias Ósseas/metabolismo , Neoplasias Ósseas/secundário , Neoplasias Ósseas/patologia , Neoplasias Ósseas/complicações , Humanos , Dor do Câncer/tratamento farmacológico , Dor do Câncer/metabolismo , Analgésicos/farmacologia , Proliferação de Células/efeitos dos fármacos , Células PC-3 , Camundongos , Sobrevivência Celular/efeitos dos fármacos , Neoplasias da Próstata/tratamento farmacológico , Neoplasias da Próstata/patologia , Neoplasias da Próstata/metabolismo , Linhagem Celular TumoralRESUMO
Breast cancer is the most prevalent malignancy and the most significant contributor to mortality in female oncology patients. Potassium Two Pore Domain Channel Subfamily K Member 1 (KCNK1) is differentially expressed in a variety of tumors, but the mechanism of its function in breast cancer is unknown. In this study, we found for the first time that KCNK1 was significantly up-regulated in human breast cancer and was correlated with poor prognosis in breast cancer patients. KCNK1 promoted breast cancer proliferation, invasion, and metastasis in vitro and vivo. Further studies unexpectedly revealed that KCNK1 increased the glycolysis and lactate production in breast cancer cells by binding to and activating lactate dehydrogenase A (LDHA), which promoted histones lysine lactylation to induce the expression of a series of downstream genes and LDHA itself. Notably, increased expression of LDHA served as a vicious positive feedback to reduce tumor cell stiffness and adhesion, which eventually resulted in the proliferation, invasion, and metastasis of breast cancer. In conclusion, our results suggest that KCNK1 may serve as a potential breast cancer biomarker, and deeper insight into the cancer-promoting mechanism of KCNK1 may uncover a novel therapeutic target for breast cancer treatment.
Assuntos
Neoplasias da Mama , Proliferação de Células , Histonas , Animais , Feminino , Humanos , Camundongos , Neoplasias da Mama/patologia , Neoplasias da Mama/genética , Neoplasias da Mama/metabolismo , Linhagem Celular Tumoral , Movimento Celular/genética , Proliferação de Células/genética , Regulação Neoplásica da Expressão Gênica , Glicólise/genética , Histonas/metabolismo , L-Lactato Desidrogenase/metabolismo , L-Lactato Desidrogenase/genética , Lactato Desidrogenase 5/metabolismo , Lactato Desidrogenase 5/genética , Camundongos Endogâmicos BALB C , Camundongos Nus , Invasividade Neoplásica , Metástase Neoplásica , Canais de Potássio de Domínios Poros em Tandem/metabolismo , Canais de Potássio de Domínios Poros em Tandem/genética , Prognóstico , Regulação para Cima/genéticaRESUMO
BACKGROUND: Research has shown a connection between vasculogenic mimicry (VM) and cancer progression. However, the functions of genes related to VM in the emergence and progression of TNBC have not been completely elucidated. METHODS: A survival risk model was constructed by screening biomarkers using DESeq2 and WGCNA based on public TNBC transcriptome data. Furthermore, gene set enrichment analysis was performed, and tumor microenvironment and drug sensitivity were analyzed. The selected biomarkers were validated via quantitative PCR detection, immunohistochemical staining, and protein detection in breast cancer cell lines. Biomarkers related to the proliferation and migration of TNBC cells were validated via in vitro experiments. RESULTS: The findings revealed that 235 target genes were connected to the complement and coagulation cascade pathways. The risk score was constructed using KCND2, NRP1, and VSTM4. The prognosis model using the risk score and pathological T stage yielded good validation results. The clinical risk of TNBC was associated with the angiogenesis signaling pathway, and the low-risk group exhibited better sensitivity to immunotherapy. Quantitative PCR and immunohistochemistry indicated that the expression levels of KCND2 in TNBC tissues were higher than those in adjacent nontumor tissues. In the TNBC cell line, the protein expression of KCND2 was increased. Knockdown of KCND2 and VSTM4 inhibited the proliferation and migration of TNBC cells in vitro. CONCLUSIONS: In this study, three VM-related biomarkers were identified, including KCND2, NRP1, and VSTM4. These findings are likely to aid in deepening our understanding of the regulatory mechanism of VM in TNBC.
Assuntos
Biomarcadores Tumorais , Neovascularização Patológica , Neoplasias de Mama Triplo Negativas , Humanos , Neoplasias de Mama Triplo Negativas/genética , Neoplasias de Mama Triplo Negativas/patologia , Neoplasias de Mama Triplo Negativas/metabolismo , Feminino , Prognóstico , Neovascularização Patológica/genética , Neovascularização Patológica/metabolismo , Biomarcadores Tumorais/genética , Biomarcadores Tumorais/metabolismo , Linhagem Celular Tumoral , Regulação Neoplásica da Expressão Gênica , Microambiente Tumoral/genética , Proliferação de Células/genética , Neuropilina-1/genética , Neuropilina-1/metabolismo , Movimento Celular/genética , Transcriptoma , Canais de Potássio de Domínios Poros em Tandem/genética , Canais de Potássio de Domínios Poros em Tandem/metabolismoRESUMO
Potassium ion (K+) is one of the most essential nutrients for the growth and development of tobacco (Nicotiana tabacum L.), however, the molecular regulation of K+ concentration in tobacco remains unclear. In this study, a two-pore K (TPK) channel gene NtTPKa was cloned from tobacco, and NtTPKa protein contains the unique K+ selection motif GYGD and its transmembrane region primarily locates in the tonoplast membrane. The expression of NtTPKa gene was significantly increased under low-potassium stress conditions. The concentrations of K+ in tobacco were significantly increased in the NtTPKa RNA interference lines and CRISPR/Cas9 knockout mutants. In addition, the transport of K+ by NtTPKa was validated using patch clamp technique, and the results showed that NtTPKa channel protein exclusively transported K+ in a concentration-dependent manner. Together, our results strongly suggested that NtTPKa is a key gene in maintaining K+ homeostasis in tobacco, and it could provide a new genetic resource for increasing the concentration of K+ in tobacco.
Assuntos
Regulação da Expressão Gênica de Plantas , Nicotiana , Proteínas de Plantas , Potássio , Nicotiana/genética , Nicotiana/metabolismo , Potássio/metabolismo , Proteínas de Plantas/genética , Proteínas de Plantas/metabolismo , Canais de Potássio de Domínios Poros em Tandem/metabolismo , Canais de Potássio de Domínios Poros em Tandem/genética , Sistemas CRISPR-Cas , Canais de Potássio/metabolismo , Canais de Potássio/genéticaRESUMO
BACKGROUND: This study aimed to explore the expression, molecular mechanism and its biological function of potassium two pore domain channel subfamily K member 1 (KCNK1) in bladder cancer (BC). METHODS: We integrated large numbers of external samples (n = 1486) to assess KCNK1 mRNA expression levels and collected in-house samples (n = 245) for immunohistochemistry (IHC) experiments to validate at the KCNK1 protein level. Single-cell RNA sequencing (scRNA-seq) analysis was performed to further assess KCNK1 expression and cellular communication. The transcriptional regulatory mechanisms of KCNK1 expression were explored by ChIP-seq, ATAC-seq and ChIA-PET data. Highly expressed co-expressed genes (HECEGs) of KCNK1 were used to explore potential signalling pathways. Furthermore, the immunoassay, clinical significance and molecular docking of KCNK1 were calculated. RESULTS: KCNK1 mRNA was significantly overexpressed in BC (SMD = 0.58, 95% CI [0.05; 1.11]), validated at the protein level (p < 0.0001). Upregulated KCNK1 mRNA exhibited highly distinguishing ability between BC and control samples (AUC = 0.82 [0.78-0.85]). Further, scRNA-seq analysis revealed that KCNK1 expression was predominantly clustered in BC epithelial cells and tended to increase with cellular differentiation. BC epithelial cells were involved in cellular communication mainly through the MK signalling pathway. Secondly, the KCNK1 transcription start site (TSS) showed promoter-enhancer interactions in three-dimensional space, while being transcriptionally regulated by GRHL2 and FOXA1. Most of the KCNK1 HECEGs were enriched in cell cycle-related signalling pathways. KCNK1 was mainly involved in cellular metabolism-related pathways and regulated cell membrane potassium channel activity. KCNK1 expression was associated with the level of infiltration of various immune cells. Immunotherapy and chemotherapy (docetaxel, paclitaxel and vinblastine) were more effective in BC patients in the high KCNK1 expression group. KCNK1 expression correlated with age, pathology grade and pathologic_M in BC patients. CONCLUSIONS: KCNK1 was significantly overexpressed in BC. A complex and sophisticated three-dimensional spatial transcriptional regulatory network existed in the KCNK1 TSS and promoted the upregulated of KCNK1 expression. The high expression of KCNK1 might be involved in the cell cycle, cellular metabolism, and tumour microenvironment through the regulation of potassium channels, and ultimately contributed to the deterioration of BC.
Assuntos
Regulação Neoplásica da Expressão Gênica , Canais de Potássio de Domínios Poros em Tandem , Neoplasias da Bexiga Urinária , Humanos , Biomarcadores Tumorais/genética , Biomarcadores Tumorais/metabolismo , Simulação de Acoplamento Molecular , Canais de Potássio de Domínios Poros em Tandem/genética , Canais de Potássio de Domínios Poros em Tandem/metabolismo , Transdução de Sinais , Neoplasias da Bexiga Urinária/genética , Neoplasias da Bexiga Urinária/metabolismo , Neoplasias da Bexiga Urinária/patologiaRESUMO
Because of the importance of potassium efflux in inflammasome activation, we investigated the role of the two-pore potassium (K2P) channel TREK-1 in macrophage inflammasome activity. Using primary alveolar macrophages (AMs) and bone marrow-derived macrophages (BMDMs) from wild-type (wt) and TREK-1-/- mice, we measured responses to inflammasome priming [using lipopolysaccharide (LPS)] and activation (LPS + ATP). We measured IL-1ß, caspase-1, and NLRP3 via ELISA and Western blot. A membrane-permeable potassium indicator was used to measure potassium efflux during ATP exposure, and a fluorescence-based assay was used to assess changes in membrane potential. Inflammasome activation induced by LPS + ATP increased IL-1ß secretion in wt AMs, whereas activation was significantly reduced in TREK-1-/- AMs. Priming of BMDMs using LPS was not affected by either genetic deficiency or pharmacological inhibition of TREK-1 with Spadin. Cleavage of caspase-1 following LPS + ATP treatment was significantly reduced in TREK-1-/- BMDMs. The intracellular potassium concentration in LPS-primed wt BMDMs was significantly lower compared with TREK-1-/- BMDMs or wt BMDMs treated with Spadin. Conversely, activation of TREK-1 with BL1249 caused a decrease in intracellular potassium in wt BMDMs. Treatment of LPS-primed BMDMs with ATP caused a rapid reduction in intracellular potassium levels, with the largest change observed in TREK-1-/- BMDMs. Intracellular K+ changes were associated with changes in the plasma membrane potential (Em), as evidenced by a more depolarized Em in TREK-1-/- BMDMs compared with wt, and Em hyperpolarization upon TREK-1 channel opening with BL1249. These results suggest that TREK-1 is an important regulator of NLRP3 inflammasome activation in macrophages.NEW & NOTEWORTHY Because of the importance of potassium efflux in inflammasome activation, we investigated the role of the two-pore potassium (K2P) channel TREK-1 in macrophage inflammasome activity. Using primary alveolar macrophages and bone marrow-derived macrophages from wild-type and TREK-1-/- mice, we measured responses to inflammasome priming (using LPS) and activation (LPS + ATP). Our results suggest that TREK-1 is an important regulator of NLRP3 inflammasome activation in macrophages.
Assuntos
Inflamassomos , Canais de Potássio de Domínios Poros em Tandem , Tetra-Hidronaftalenos , Tetrazóis , Animais , Camundongos , Inflamassomos/metabolismo , Proteína 3 que Contém Domínio de Pirina da Família NLR/metabolismo , Potássio/metabolismo , Lipopolissacarídeos/farmacologia , Lipopolissacarídeos/metabolismo , Camundongos Knockout , Canais de Potássio de Domínios Poros em Tandem/genética , Canais de Potássio de Domínios Poros em Tandem/metabolismo , Macrófagos/metabolismo , Caspase 1/metabolismo , Trifosfato de Adenosina/farmacologia , Trifosfato de Adenosina/metabolismo , Interleucina-1beta/metabolismoRESUMO
BACKGROUND: The two-pore domain potassium (K2P) channels are a major type of potassium channels that maintain the cell membrane potential by conducting passive potassium leak currents independent of voltage change. They play prominent roles in multiple physiological processes, including neuromodulation, perception of pain, breathing and mood control, and response to volatile anesthetics. Mutations in K2P channels have been linked to many human diseases, such as neuronal and cardiovascular disorders and cancers. Significant progress has been made to understand their protein structures, physiological functions, and pharmacological modifiers. However, their expression and function during embryonic development remain largely unknown. RESULTS: We employed the zebrafish model and identified 23 k2p genes using BLAST search and gene cloning. We first analyzed vertebrate K2P channel evolution by phylogenetic and syntenic analyses. Our data revealed that the six subtypes of the K2P genes have already evolved in invertebrates long before the emergence of vertebrates. Moreover, the vertebrate K2P gene number increased, most likely due to two whole-genome duplications. Furthermore, we examined zebrafish k2p gene expression during early embryogenesis by in situ hybridization. Each subgroup's genes showed similar but distinct gene expression domains with some exceptions. Most of them were expressed in neural tissues consistent with their known function of neural excitability regulation. However, a few k2p genes were expressed temporarily in specific tissues or organs, suggesting that these K2P channels may be needed for embryonic development. CONCLUSIONS: Our phylogenetic and developmental analyses of K2P channels shed light on their evolutionary history and potential roles during embryogenesis related to their physiological functions and human channelopathies.
Assuntos
Evolução Molecular , Filogenia , Canais de Potássio de Domínios Poros em Tandem , Peixe-Zebra , Animais , Peixe-Zebra/genética , Peixe-Zebra/embriologia , Canais de Potássio de Domínios Poros em Tandem/genética , Canais de Potássio de Domínios Poros em Tandem/metabolismo , Regulação da Expressão Gênica no Desenvolvimento , Embrião não Mamífero/metabolismo , Proteínas de Peixe-Zebra/genética , Proteínas de Peixe-Zebra/metabolismo , Desenvolvimento Embrionário/genéticaRESUMO
Purpose: The purpose of this study was to elucidate the involvement of potassium two pore domain channel subfamily K member 5 (KCNK5)-mediated potassium efflux in the pathogenesis of dry eye and to unravel the underlying molecular mechanisms. Methods: To induce experimental dry eye in adult wild-type C57BL/6 mice, scopolamine was administered via subcutaneous injection, and the mice were subjected to desiccating stress. To create an in vitro model of dry eye, desiccation stress was applied to the human corneal epithelial cell line (HCE-T). Intracellular potassium concentration was quantified using inductively coupled plasma mass spectrometry. Cellular death was assessed through lactate dehydrogenase assays. Gene expression profiling was conducted through both RNA sequencing and quantitative real-time PCR. Protein analysis was carried out through Western blotting and immunofluorescence staining. Assessment of the corneal epithelial defect area was conducted through fluorescein sodium staining. Tear secretion was quantified using the phenol red cotton thread method. Results: Potassium efflux was observed to further facilitate corneal epithelial pyroptosis. KCNK5 exhibited upregulation in both in vivo and in vitro models of dry eye. The overexpression of KCNK5 was observed to induce potassium efflux and activate the NLR family pyrin domain containing 3 (NLRP3) inflammasome-mediated pyroptosis in vitro. Silencing KCNK5 effectively mitigated pyroptosis in dry eye. Additionally, the overexpression of KCNK5 results in the downregulation of TNF superfamily member 10 (TNFSF10) and subsequent impairment of autophagy. TNFSF10 supplementation could promote autophagy and mitigate pyroptosis in dry eye. Conclusions: The upregulation of KCNK5 mediates TNFSF10 to impair autophagy and induce pyroptosis in dry eye. Consequently, targeting KCNK5 may represent a novel and promising approach to therapeutic intervention in the management of dry eye.
Assuntos
Síndromes do Olho Seco , Canais de Potássio de Domínios Poros em Tandem , Ligante Indutor de Apoptose Relacionado a TNF , Animais , Humanos , Camundongos , Autofagia , Síndromes do Olho Seco/metabolismo , Células Epiteliais , Camundongos Endogâmicos C57BL , Canais de Potássio de Domínios Poros em Tandem/metabolismo , Piroptose , Ligante Indutor de Apoptose Relacionado a TNF/metabolismoRESUMO
Influenza-A virus (IAV) infects yearly an estimated one billion people worldwide, resulting in 300,000-650,000 deaths. Preventive vaccination programs and antiviral medications represent the mainstay of therapy, but with unacceptably high morbidity and mortality rates, new targeted therapeutic approaches are urgently needed. Since inflammatory processes are commonly associated with measurable changes in the cell membrane potential (Em), we investigated whether Em hyperpolarization via TREK-1 (K2P2.1) K+ channel activation can protect against influenza-A virus (IAV)-induced pneumonia. We infected mice with IAV, which after 5 days caused 10-15% weight loss and a decrease in spontaneous activity, representing a clinically relevant infection. We then started a 3-day intratracheal treatment course with the novel TREK-1 activating compounds BL1249 or ML335. We confirmed TREK-1 activation with both compounds in untreated and IAV-infected primary human alveolar epithelial cells (HAECs) using high-throughput fluorescent imaging plate reader (FLIPR) assays. In mice, TREK-1 activation with BL1249 and ML335 counteracted IAV-induced histological lung injury and decrease in lung compliance and improved BAL fluid total protein levels, cell counts, and inflammatory IL-6, IP-10/CXCL-10, MIP-1α, and TNF-α levels. To determine whether these anti-inflammatory effects were mediated by activation of alveolar epithelial TREK-1 channels, we studied the effects of BL1249 and ML335 in IAV-infected HAEC, and found that TREK-1 activation decreased IAV-induced inflammatory IL-6, IP-10/CXCL10, and CCL-2 secretion. Dissection of TREK-1 downstream signaling pathways and construction of protein-protein interaction (PPI) networks revealed NF-κB1 and retinoic acid-inducible gene-1 (RIG-1) cascades as the most likely targets for TREK-1 protection. Therefore, TREK-1 activation may represent a novel therapeutic approach against IAV-induced lung injury.
Assuntos
Lesão Pulmonar Aguda , Vírus da Influenza A , Influenza Humana , Infecções por Orthomyxoviridae , Canais de Potássio de Domínios Poros em Tandem , Animais , Humanos , Camundongos , Lesão Pulmonar Aguda/patologia , Quimiocina CXCL10/metabolismo , Influenza Humana/patologia , Interleucina-6/metabolismo , Pulmão/metabolismo , Infecções por Orthomyxoviridae/patologia , Canais de Potássio de Domínios Poros em Tandem/genética , Canais de Potássio de Domínios Poros em Tandem/metabolismoRESUMO
Adenomyosis is linked to dysmenorrhea and infertility. The pathogenesis of adenomyosis remains unclear, and little is known of the genetic and epigenetic changes in the eutopic endometrium in adenomyosis, which may predispose patients to the invasion and migration of endometrial tissues into the myometrium. Transcriptome studies have identified genes related to various cell behaviors but no targets for therapeutic intervention. The epigenetics of the eutopic endometrium in adenomyosis have rarely been investigated. Endometrial tissue was obtained from premenopausal women with (n = 32) or without adenomyosis (n = 17) who underwent hysterectomy aged 34-57 years at a tertiary hospital. The methylome and transcriptome were assessed by using a Methylation 450 K BeadChip array and Affymetrix expression microarray. Protein expression was examined by immunohistochemistry. Differential methylation analysis revealed 53 lowly methylated genes and 176 highly methylated genes with consistent gene expression in adenomyosis, including three genes encoding potassium ion channels. High expression of KCNK9 in the eutopic and ectopic endometria in patients with adenomyosis but not in normal controls was observed. Hormone-free, antibody-based KCNK9 targeting is a potential therapeutic strategy for adenomyosis-related dysmenorrhea, menorrhagia, and infertility.
Assuntos
Adenomiose , Endometriose , Infertilidade , Canais de Potássio de Domínios Poros em Tandem , Adenomiose/genética , Adenomiose/metabolismo , Adenomiose/patologia , Dismenorreia/genética , Endometriose/patologia , Endométrio/metabolismo , Epigenômica , Feminino , Humanos , Infertilidade/metabolismo , Canais de Potássio/metabolismo , Canais de Potássio de Domínios Poros em Tandem/metabolismoRESUMO
TWIK-related acid-sensitive K+ (TASK) channels are thought to contribute to the resting membrane potential in adrenal cortical (AC) cells. However, the molecular identity of TASK channels in AC cells have not yet been elucidated. Thus, immunocytochemical and molecular biological approaches were employed to investigate the expression and intracellular distribution of TASK1 and TASK3 in mouse AC cells and H295R cells derived from human adrenocortical carcinoma. Immunocytochemical study revealed that immunoreactive materials were mainly located in the cytoplasm for TASK1 and at the cell periphery for TASK3 in mouse AC cells. A similar pattern of localization was observed when GFP-TASK1 and GFP-TASK3 were exogenously expressed in H295R cells. In addition, p11 that is known to suppress the endoplasmic reticulum exit of TASK1 was localized in the cytoplasm in mouse AC and H295R cells, but not in adrenal medullary cells. Proximity ligation assay (PLA) suggested formation of heteromeric TASK1-3 channels that were found predominantly in the cytoplasm and weakly at the cell periphery. A similar distribution was observed following exogenous expression of tandem TASK1-3 channels in H295R cells. When stimulated by angiotensin II, however, tandem TASK1-3 channels were present mainly in the cytoplasm in all H295R cells. In contrast to that in H295R cells, tandem channels were exclusively located at the cell periphery in all non-stimulated and exclusively in the cytoplasm in stimulated PC12 cells, respectively. From these results, we conclude that TASK1 proteins are present mainly in the cytoplasm and minimally at the cell periphery as a heteromeric channel with TASK3, whereas the majority of TASK3 is at the cell periphery as homomeric and heteromeric channels.
Assuntos
Células Endócrinas , Proteínas do Tecido Nervoso/metabolismo , Canais de Potássio de Domínios Poros em Tandem/metabolismo , Angiotensina II/metabolismo , Animais , Células Endócrinas/metabolismo , Humanos , Camundongos , Células PC12 , Canais de Potássio/metabolismo , RatosRESUMO
The NLRP3 (NLR family, pyrin domain containing 3) inflammasome is a multi-protein complex responsible for the activation of caspase-1 and the subsequent cleavage and activation of the potent proinflammatory cytokines IL-1ß and IL-18, and pyroptotic cell death. NLRP3 is implicated as a driver of inflammation in a range of disorders including neurodegenerative diseases, type 2 diabetes, and atherosclerosis. A commonly reported mechanism contributing to NLRP3 inflammasome activation is potassium ion (K+ ) efflux across the plasma membrane. Identification of K+ channels involved in NLRP3 activation remains incomplete. Here, we investigated the role of the K+ channel THIK-1 in NLRP3 activation. Both pharmacological inhibitors and cells from THIK-1 knockout (KO) mice were used to assess THIK-1 contribution to macrophage NLRP3 activation in vitro. Pharmacological inhibition of THIK-1 inhibited caspase-1 activation and IL-1ß release from mouse bone-marrow-derived macrophages (BMDMs), mixed glia, and microglia in response to NLRP3 agonists. Similarly, BMDMs and microglia from THIK-1 KO mice had reduced NLRP3-dependent IL-1ß release in response to P2X7 receptor activation with ATP. Overall, these data suggest that THIK-1 is a regulator of NLRP3 inflammasome activation in response to ATP and identify THIK-1 as a potential therapeutic target for inflammatory disease.
Assuntos
Diabetes Mellitus Tipo 2 , Inflamassomos , Canais de Potássio de Domínios Poros em Tandem/metabolismo , Trifosfato de Adenosina/metabolismo , Animais , Caspase 1/metabolismo , Inflamassomos/metabolismo , Interleucina-1beta/metabolismo , Camundongos , Camundongos Endogâmicos C57BL , Camundongos Knockout , Proteína 3 que Contém Domínio de Pirina da Família NLR/genética , Proteína 3 que Contém Domínio de Pirina da Família NLR/metabolismo , Potássio/metabolismo , Canais de PotássioRESUMO
BACKGROUND: Our previous data revealed that reduction of TASK-1 expression, as a consequence of exposure to 17ß-estradiol, could participate in neuroprotective effects in N2A cells. However, it is unclear which estrogen receptor underlies these effects of 17ß-estradiol. METHODS AND RESULTS: In this study, the knockdown experiments are carried out to clarify the estrogen receptor responsible for effects of estrogen on TASK-1 channels. Subsequently, data from QPCR measurements reveal that estrogen receptor ß (ERß), but not estrogen receptor α, serves as a binding target for 17ß-estradiol after a 48-h treatment. CONCLUSIONS: The current result suggests the implication of the ERß-dependent manner in the pro-proliferative action of estrogen via TASK-1 channels.
Assuntos
Regulação para Baixo/efeitos dos fármacos , Estradiol/farmacologia , Receptor beta de Estrogênio/metabolismo , Estrogênios/farmacologia , Proteínas do Tecido Nervoso/metabolismo , Crista Neural/metabolismo , Fármacos Neuroprotetores/farmacologia , Canais de Potássio de Domínios Poros em Tandem/metabolismo , Transdução de Sinais/efeitos dos fármacos , Animais , Linhagem Celular , Proliferação de Células/efeitos dos fármacos , Proliferação de Células/genética , Regulação para Baixo/genética , Receptor alfa de Estrogênio/genética , Receptor alfa de Estrogênio/metabolismo , Receptor beta de Estrogênio/genética , Técnicas de Silenciamento de Genes/métodos , Camundongos , Proteínas do Tecido Nervoso/genética , Reação em Cadeia da Polimerase/métodos , Canais de Potássio de Domínios Poros em Tandem/genética , Interferência de RNA , RNA Mensageiro/metabolismo , Transdução de Sinais/genética , TransfecçãoRESUMO
Long noncoding RNAs (lncRNAs) are regarded as crucial regulators in tumor progression. Potassium two pore domain channel subfamily K member 15 and WISP2 antisense RNA 1 (KCNK15-AS1) has been confirmed to inhibit the migration and invasion of pancreatic cancer (PC) cells. However, its downstream mechanism and effect on other cellular functions in PC remain unknown. This study probed the function and potential mechanism of KCNK15-AS1 in PC cell growth. RT-qPCR and western blot were employed to measure gene expression in PC cells. ISH was applied to analyze KCNK15-AS1 expression in PC tissues. Functional assays were utilized to evaluate PC cell proliferation, apoptosis, migration and EMT. Mechanical experiments were adopted to detect gene interaction in PC cells. The obtained data indicated that KCNK15-AS1 was down-regulated in PC cells and tissues. Overexpressing KCNK15-AS1 hindered cell proliferation, migration and EMT while facilitated cell apoptosis in PC. Mechanically, alkylation repair homolog protein 5 (ALKBH5) was verified to induce m6A demethylation of KCNK15-AS1 to mediate KCNK15-AS1 up-regulation. KCNK15-AS1 combined with KCNK15 5'UTR to inhibit KCNK15 translation. Moreover, KCNK15-AS1 recruited MDM2 proto-oncogene (MDM2) to promote RE1 silencing transcription factor (REST) ubiquitination, thus transcriptionally upregulating phosphatase and tensin homolog (PTEN) to inactivate AKT pathway. In conclusion, our study first confirmed that KCNK15-AS1 hinders PC cell growth by regulating KCNK15 and PTEN, suggesting KCNK15-AS1 as a potential biomarker of PC.
Assuntos
Homólogo AlkB 5 da RNA Desmetilase/metabolismo , Glicoproteínas de Membrana/metabolismo , Proteínas do Tecido Nervoso/metabolismo , PTEN Fosfo-Hidrolase/metabolismo , Neoplasias Pancreáticas/genética , Canais de Potássio de Domínios Poros em Tandem/metabolismo , Proteínas Proto-Oncogênicas c-akt/metabolismo , Animais , Linhagem Celular Tumoral , Proliferação de Células , Progressão da Doença , Humanos , Camundongos , Neoplasias Pancreáticas/patologia , Transdução de Sinais , Transfecção , Regulação para Cima , Neoplasias PancreáticasRESUMO
Decompressive craniectomy is an effective strategy to reduce intracranial hypertension after traumatic brain injury (TBI), but it is related to many postoperative complications, such as delayed intracranial hematoma and diffuse brain swelling. Our previous studies have demonstrated that controlled decompression (CDC) surgery attenuates brain injury and reduces the rate of complications after TBI. Here, we investigated the potential molecular mechanisms of CDC in experimental models. The in vitro experiments were performed in a traumatic neuronal injury (TNI) model following compression treatment in primary cultured cortical neurons. We found that compression aggravates TNI-induced neuronal injury, which was significantly attenuated by CDC for 2 h or 3 h. The results of immunocytochemistry showed that CDC reduced neuronal necroptosis and activation of RIP3 induced by TNI and compression, with no effect on RIP1 activity. These protective effects were associated with decreased levels of inflammatory cytokines and preserved intracellular Ca2+ homeostasis. In addition, the expression of the two-pore domain K+ channel TREK-1 and its activity was increased by compression and prolonged by CDC. Treatment with the TREK-1 blockers, spadin or SID1900, could partially prevent the effects of CDC on intracellular Ca2+ metabolism, necroptosis, and neuronal injury following TNI and compression. Using a traumatic intracranial hypertension model in rats, we found that CDC for 20 min or 30 min was effective in alleviating brain edema and locomotor impairment in vivo. CDC significantly inhibited neuronal necroptosis and neuroinflammation and increased TREK-1 activation, and the CDC-induced protection in vivo was attenuated by spadin and SID1900. In summary, CDC is effective in alleviating compressive neuronal injury both in vitro and in vivo, which is associated with the TREK-1-mediated attenuation of intracellular Ca2+ overload, neuronal necroptosis, and neuroinflammation.
Assuntos
Edema Encefálico/terapia , Lesões Encefálicas Traumáticas/complicações , Hemorragia Cerebral/terapia , Descompressão/métodos , Necroptose , Doenças Neuroinflamatórias/terapia , Canais de Potássio de Domínios Poros em Tandem/metabolismo , Animais , Edema Encefálico/etiologia , Edema Encefálico/metabolismo , Edema Encefálico/patologia , Córtex Cerebral/metabolismo , Córtex Cerebral/patologia , Hemorragia Cerebral/etiologia , Hemorragia Cerebral/metabolismo , Hemorragia Cerebral/patologia , Doenças Neuroinflamatórias/etiologia , Doenças Neuroinflamatórias/metabolismo , Doenças Neuroinflamatórias/patologia , Neurônios/metabolismo , Neurônios/patologia , Ratos , Ratos Sprague-DawleyRESUMO
Thermoregulation is crucial for human survival at various ambient temperatures. Transient receptor potential (TRP) and TWIK-related K+ (TREK) channels expressed in sensory neurons play a role in peripheral thermosensitivity for temperature detection. In addition, these channels have various physiological roles in the skeletal, nervous, immune, vascular, digestive, and urinary systems. In women, the female hormones estradiol (E2) and progesterone (P4), which fluctuate during the menstrual cycle, affect various physiological functions, such as thermoregulation in hot and cold environments. The present review describes the effect of female hormones on TRP and TREK channels and related physiological functions. The P4 decreased thermosensitivity via TRPV1. E2 facilitates temporomandibular joint disease (TRPV1), breast cancer (TRPM8), and calcium absorption in the digestive system (TRPV5 and TRPV6), inhibits the facilitation of vasoconstriction (TRPM3), nerve inflammation (TRPM4), sweetness sensitivity (TRPM5), and menstrual disorders (TRPC1), and prevents insulin resistance (TRPC5) via each channel. P4 inhibits vasoconstriction (TRPM3), sweetness sensitivity (TRPM5), ciliary motility in the lungs (TRPV4), menstrual disorder (TRPC1), and immunity (TRPC3), and facilitates breast cancer (TRPV6) via each channel as indicated. The effects of female hormones on TREK channels and physiological functions are still under investigation. In summary, female hormones influence physiological functions via some TRP channels; however, the literature is not comprehensive and future studies are needed, especially those related to thermoregulation in women.
Assuntos
Estradiol/metabolismo , Ciclo Menstrual , Canais de Potássio de Domínios Poros em Tandem/metabolismo , Progesterona/metabolismo , Sensação Térmica , Canais de Potencial de Receptor Transitório/metabolismo , Feminino , HumanosRESUMO
TREK-1 channels are expressed in small nociceptive dorsal root ganglion (DRG) neurons where they participate in acute inflammatory and neuropathic pain. However, the role of TREK-1 in persistent pain is not well understood. The aim of this study was to investigate the local peripheral and spinal participation of TREK-1 in formalin-induced acute and long-lasting nociceptive hypersensitivity. Local peripheral or intrathecal pre-treatment with spadin, selective blocker of TREK-1, increased acute flinching behavior and secondary mechanical allodynia and hyperalgesia behavior observed 6 days after formalin injection. Local peripheral or intrathecal pre-treatment with BL-1249, selective opener of TREK-1, decreased long-lasting secondary mechanical allodynia and hyperalgesia induced by formalin. Pre-treatment with BL-1249 prevented the pro-nociceptive effect of spadin on acute nociception and long-lasting mechanical allodynia and hyperalgesia in rats. Pre-treatment with two recombinant channels that produce a high TREK-1 current, S300A and S333A (non-phosphorylated state of TREK-1), reduced formalin-induced acute pain and long-lasting mechanical allodynia and hyperalgesia. Besides, post-treatment with S300A, S333A or BL-1249 reversed long-lasting mechanical allodynia and hyperalgesia induced by formalin. Formalin increased TREK-1 expression at 1 and 6 days in DRG and dorsal spinal cord in rats, whereas that it increased c-fos expression at the DRG. Intrathecal repeated transfection of rats with S300A and S333A or injection with BL-1249 reduced formalin-induced enhanced c-fos expression. Data suggest that TREK-1 activity at peripheral and spinal sites reduces neuronal excitability in the process of acute and long-lasting nociception induced by formalin in rats.
Assuntos
Desinfetantes/farmacologia , Formaldeído/farmacologia , Gânglios Espinais , Hiperalgesia , Dor Nociceptiva , Canais de Potássio de Domínios Poros em Tandem/metabolismo , Medula Espinal , Animais , Modelos Animais de Doenças , Feminino , Gânglios Espinais/efeitos dos fármacos , Gânglios Espinais/metabolismo , Hiperalgesia/induzido quimicamente , Hiperalgesia/tratamento farmacológico , Hiperalgesia/metabolismo , Masculino , Dor Nociceptiva/induzido quimicamente , Dor Nociceptiva/tratamento farmacológico , Dor Nociceptiva/metabolismo , Peptídeos/farmacologia , Canais de Potássio de Domínios Poros em Tandem/antagonistas & inibidores , Canais de Potássio de Domínios Poros em Tandem/efeitos dos fármacos , Ratos , Ratos Wistar , Medula Espinal/efeitos dos fármacos , Medula Espinal/metabolismo , Tetra-Hidronaftalenos/farmacologia , Tetrazóis/farmacologiaRESUMO
Inflammation plays a major role in the pathogenesis of pulmonary hypertension (PH). We sought to investigate the effects of a cell-based immunomodulation in a dysimmune model of PH. PH was induced in athymic nude rats using semaxinib (Su group, n = 6). Tolerogenic macrophages (toM) were generated from monocyte isolation and then injected either the day before semaxinib injection (Prevention group, n = 6) or 3 weeks after (Reversion group, n = 6). Six athymic nude rats were used as controls. In vivo trafficking of toM was investigated with bioluminescence imaging showing that toM were mainly located into the lungs until 48 h after injection. Right ventricular (RV) end-systolic pressure and RV systolic function were assessed at 4 weeks using echocardiography. Morphometric analysis and RNA sequencing of the lungs were realized at 4 weeks. Rats treated with toM (Prevention and Reversion groups) had a significantly lower RV end-systolic pressure at 4 weeks (respectively, 25 ± 8 and 30 ± 6 mmHg vs. 67 ± 9 mmHg, P < 0.001), while RV systolic dysfunction was observed in Su and Reversion groups. Mean medial wall thickness of small arterioles was lower in Prevention and Reversion groups compared with the Su group (respectively, 10.9% ± 0.8% and 16.4% ± 1.3% vs. 28.2% ± 2.1%, P < 0.001). Similarly, cardiomyocyte area was decreased in rats treated with toM (150 ± 18 and 160 ± 86 µm2 vs. 279 ± 50 µm2, P < 0.001). A trend toward upregulation of genes involved in pulmonary arterial hypertension pathobiology was found in Su rats, while KCNK3 was significantly downregulated (fold-change = 9.8, P < 0.001). Injection of toM was associated with a less severe phenotype of PH in rats exposed to angioproliferative stress. Preserved expression of KCNK3 may explain the protective effect of toM.
Assuntos
Modelos Animais de Doenças , Hipertensão Pulmonar/terapia , Imunomodulação/imunologia , Imunoterapia/métodos , Macrófagos/imunologia , Animais , Perfilação da Expressão Gênica/métodos , Humanos , Hipertensão Pulmonar/imunologia , Hipertensão Pulmonar/fisiopatologia , Tolerância Imunológica/imunologia , Indóis/farmacologia , Pulmão/efeitos dos fármacos , Pulmão/metabolismo , Pulmão/fisiopatologia , Masculino , Proteínas do Tecido Nervoso/genética , Proteínas do Tecido Nervoso/metabolismo , Canais de Potássio de Domínios Poros em Tandem/genética , Canais de Potássio de Domínios Poros em Tandem/metabolismo , Inibidores de Proteínas Quinases/farmacologia , Pirróis/farmacologia , Ratos Nus , Roedores , Volume Sistólico/efeitos dos fármacos , Volume Sistólico/imunologia , Volume Sistólico/fisiologia , Tomografia Computadorizada de Emissão de Fóton ÚnicoRESUMO
The family of two-pore domain potassium (K2P) channels is critically involved in central cellular functions such as ion homeostasis, cell development, and excitability. K2P channels are widely expressed in different human cell types and organs. It is therefore not surprising that aberrant expression and function of K2P channels are related to a spectrum of human diseases, including cancer, autoimmune, CNS, cardiovascular, and urinary tract disorders. Despite homologies in structure, expression, and stimulus, the functional diversity of K2P channels leads to heterogeneous influences on human diseases. The role of individual K2P channels in different disorders depends on expression patterns and modulation in cellular functions. However, an imbalance of potassium homeostasis and action potentials contributes to most disease pathologies. In this review, we provide an overview of current knowledge on the role of K2P channels in human diseases. We look at altered channel expression and function, the potential underlying molecular mechanisms, and prospective research directions in the field of K2P channels.