RESUMEN
Many aspects of mammalian physiology are mechanically regulated. One set of molecules that can mediate mechanotransduction are the mechanically activated ion channels. These ionotropic force sensors are directly activated by mechanical inputs, resulting in ionic flux across the plasma membrane. While there has been much research focus on the role of mechanically activated ion channels in touch sensation and hearing, recent data have highlighted the broad expression pattern of these molecules in mammalian cells. Disruption of mechanically activated channels has been shown to impact (a) the development of mechanoresponsive structures, (b) acute mechanical sensing, and (c) mechanically driven homeostatic maintenance in multiple tissue types. The diversity of processes impacted by these molecules highlights the importance of mechanically activated ion channels in mammalian physiology.
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Canales Iónicos , Mecanotransducción Celular , Animales , Humanos , Canales Iónicos/metabolismo , Mamíferos , Mecanotransducción Celular/fisiología , Tacto/fisiologíaRESUMEN
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.
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Evolución Molecular , Filogenia , Canales de Potasio de Dominio Poro en Tándem , Pez Cebra , Animales , Pez Cebra/genética , Pez Cebra/embriología , Canales de Potasio de Dominio Poro en Tándem/genética , Canales de Potasio de Dominio Poro en Tándem/metabolismo , Regulación del Desarrollo de la Expresión Génica , Embrión no Mamífero/metabolismo , Proteínas de Pez Cebra/genética , Proteínas de Pez Cebra/metabolismo , Desarrollo Embrionario/genéticaRESUMEN
Intrauterine infection during pregnancy can enhance uterine contractions. A two-pore K+ channel TREK1 is crucial for maintaining uterine quiescence and reducing contractility, with its properties regulated by pH changes in cell microenvironment. Meanwhile, the sodium hydrogen exchanger 1 (NHE1) plays a pivotal role in modulating cellular pH homeostasis, and its activation increases smooth muscle tension. By establishing an infected mouse model of Escherichia coli (E. coli) and lipopolysaccharide (LPS), we used Western blotting, real-time quantitative polymerase chain reaction, and immunofluorescence to detect changes of TREK1 and NHE1 expression in the myometrium, and isometric recording measured the uterus contraction. The NHE1 inhibitor cariporide was used to explore the effect of NHE1 on TREK1. Finally, cell contraction assay and siRNA transfection were performed to clarify the relationship between NHE1 and TREK1 in vitro. We found that the uterine contraction was notably enhanced in infected mice with E. coli and LPS administration. Meanwhile, TREK1 expression was reduced, whereas NHE1 expression was upregulated in infected mice. Cariporide alleviated the increased uterine contraction and promoted myometrium TREK1 expression in LPS-injected mice. Furthermore, suppression of NHE1 with siRNA transfection inhibited the contractility of uterine smooth muscle cells and activated the TREK1. Altogether, our findings indicate that infection increases the uterine contraction by downregulating myometrium TREK1 in mice, and the inhibition of TREK1 is attributed to the activation of NHE1.NEW & NOTEWORTHY Present work found that infection during pregnancy will increase myometrium contraction. Infection downregulated NHE1 and followed TREK1 expression and activation decrease in myometrium, resulting in increased myometrium contraction.
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Guanidinas , Lipopolisacáridos , Miometrio , Canales de Potasio de Dominio Poro en Tándem , Intercambiador 1 de Sodio-Hidrógeno , Sulfonas , Animales , Femenino , Ratones , Embarazo , Escherichia coli , Lipopolisacáridos/toxicidad , Miometrio/metabolismo , ARN Interferente Pequeño/metabolismo , Contracción Uterina/fisiología , Canales de Potasio de Dominio Poro en Tándem/metabolismo , Intercambiador 1 de Sodio-Hidrógeno/metabolismoRESUMEN
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.
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Inflamasomas , Canales de Potasio de Dominio Poro en Tándem , Tetrahidronaftalenos , Tetrazoles , Animales , Ratones , Inflamasomas/metabolismo , Proteína con Dominio Pirina 3 de la Familia NLR/metabolismo , Potasio/metabolismo , Lipopolisacáridos/farmacología , Lipopolisacáridos/metabolismo , Ratones Noqueados , Canales de Potasio de Dominio Poro en Tándem/genética , Canales de Potasio de Dominio Poro en Tándem/metabolismo , Macrófagos/metabolismo , Caspasa 1/metabolismo , Adenosina Trifosfato/farmacología , Adenosina Trifosfato/metabolismo , Interleucina-1beta/metabolismoRESUMEN
Our objective is to explore the protective effect of Dexmedetomidine on brain apoptosis and its mechanism through TREK-1 pathway. Forty male Sprague-Dawley rats were allocated into four groups: Sham, Cerebral Ischemia/Reperfusion Injury (CIRI), 50 µg/kg Dex, and 100 µg/kg Dex. A rat model of middle cerebral artery occlusion (MCAO) was employed to simulate cerebral embolism. Primary cortical neurons were exposed to Dex for 48 h, with some receiving additional treatment with 100 µM yohimbine hydrochloride (YOH) or TREK-1 small interfering RNA (siRNA). Neuronal damage was assessed using hematoxylin and eosin (HE) staining. Cell viability and apoptosis were measured by Cell Counting Kit-8 (CCK8) and flow cytometry, respectively. Protein and gene expression levels of Bcl-2, Bax, and TREK-1 were determined by Western blot and real-time polymerase chain reaction (PCR). Histopathological changes revealed that Dex treatment at both 50 µg/kg and 100 µg/kg significantly mitigated neuronal damage compared to the CIRI group. YOH treatment and Trek1 siRNA significantly reduced cell viability (p < 0.05). The mRNA expression and protein levels of TREK-1 and Bax were remarkably increased, while mRNA expression and protein levels of Bcl-2 was seriously decreased after CIRI modeling. In contrast, Dex treatment at both concentrations led to decreased TREK-1 and Bax expression and increased Bcl-2 expression in primary cortical neurons. Addition of 100 µM YOH and Trek1 siRNA reversed the effects of Dex on apoptosis-related genes (p < 0.05). Dex exerts neuroprotective effects through the TREK-1 pathway in vivo and in vitro.
RESUMEN
Pancreatic ductal adenocarcinoma (PDAC) remains the most lethal cancer type. PDAC is characterized by fibrotic, hypoxic, and presumably acidic tumor microenvironment (TME). Acidic TME is an important player in tumor development, progression, aggressiveness, and chemoresistance. The dysregulation of ductal ion transporters/channels might contribute to extracellular pH (pHe) acidification and PDAC progression. Our aim was to test whether H+/K+-ATPases and pH-sensitive K+ channels contribute to these processes and could be targeted by clinically approved drugs. We used human pancreatic cancer cells adapted to various pHe conditions and grown in monolayers and spheroids. First, we created cells expressing pHoran4 at the outer plasma membrane and showed that pantoprazole, the H+/K+-ATPase inhibitor, alkalinized pHe. Second, we used FluoVolt to monitor the membrane voltage (Vm) and showed that riluzole hyperpolarized Vm, most likely by opening of pH-sensitive K+ channels such as TREK-1. Third, we show that pantoprazole and riluzole inhibited cell proliferation and viability of monolayers and spheroids of cancer cells adapted to various pHe conditions. Most importantly, combination of the two drugs had significantly larger inhibitory effects on PDAC cell survival. We propose that co-targeting H+/K+-ATPases and pH-sensitive K+ channels by re-purposing of pantoprazole and riluzole could provide novel acidosis-targeted therapies of PDAC.
RESUMEN
Two-pore domain potassium (K2P) channels stabilize the resting membrane potential of both excitable and nonexcitable cells and, as such, are important regulators of cell activity. There are many conditions where pharmacological regulation of K2P channel activity would be of therapeutic benefit, including, but not limited to, atrial fibrillation, respiratory depression, pulmonary hypertension, neuropathic pain, migraine, depression, and some forms of cancer. Up until now, few if any selective pharmacological regulators of K2P channels have been available. However, recent publications of solved structures with small-molecule activators and inhibitors bound to TREK-1, TREK-2, and TASK-1 K2P channels have given insight into the pharmacophore requirements for compound binding to these sites. Together with the increasing availability of a number of novel, active, small-molecule compounds from K2P channel screening programs, these advances have opened up the possibility of rational activator and inhibitor design to selectively target K2P channels.
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Anestesia , Preparaciones Farmacéuticas , Canales de Potasio de Dominio Poro en Tándem , HumanosRESUMEN
K2P2.1 (TREK1), a two-pore domain potassium channel, has emerged as regulator of leukocyte transmigration into the central nervous system. In the context of skeletal muscle, immune cell infiltration constitutes the pathogenic hallmark of idiopathic inflammatory myopathies (IIMs). However, the underlying mechanisms remain to be elucidated. In this study, we investigated the role of K2P2.1 in the autoimmune response of IIMs. We detected K2P2.1 expression in primary skeletal muscle and endothelial cells of murine and human origin. We observed an increased pro-inflammatory cell response, adhesion and transmigration by pharmacological blockade or genetic deletion of K2P2.1 in vitro and in in vivo myositis mouse models. Of note, our findings were not restricted to endothelial cells as skeletal muscle cells with impaired K2P2.1 function also demonstrated a strong pro-inflammatory response. Conversely, these features were abrogated by activation of K2P2.1 and improved the disease course of a myositis mouse model. In humans, K2P2.1 expression was diminished in IIM patients compared to non-diseased controls arguing for the translatability of our findings. In summary, K2P2.1 may regulate the inflammatory response of skeletal muscle. Further research is required to understand whether K2P2.1 could serve as novel therapeutic target.
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Células Endoteliales , Miositis , Humanos , Animales , Ratones , Células Endoteliales/patología , Miositis/genética , Músculo Esquelético/patología , Leucocitos/patologíaRESUMEN
The establishment of macromolecular complexes by scaffolding proteins is key to the local production of cAMP by anchored adenylyl cyclase (AC) and the subsequent cAMP signaling necessary for cardiac functions. We identify a novel AC scaffold, the Popeye domain-containing (POPDC) protein. The POPDC family of proteins is important for cardiac pacemaking and conduction, due in part to their cAMP-dependent binding and regulation of TREK-1 potassium channels. We show that TREK-1 binds the AC9:POPDC1 complex and copurifies in a POPDC1-dependent manner with AC9 activity in heart. Although the AC9:POPDC1 interaction is cAMP-independent, TREK-1 association with AC9 and POPDC1 is reduced upon stimulation of the ß-adrenergic receptor (ßAR). AC9 activity is required for ßAR reduction of TREK-1 complex formation with AC9:POPDC1 and in reversing POPDC1 enhancement of TREK-1 currents. Finally, deletion of the gene-encoding AC9 (Adcy9) gives rise to bradycardia at rest and stress-induced heart rate variability, a milder phenotype than the loss of Popdc1 but similar to the loss of Kcnk2 (TREK-1). Thus, POPDC1 represents a novel adaptor for AC9 interactions with TREK-1 to regulate heart rate control.
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Adenilil Ciclasas , Canales de Potasio , Adenilil Ciclasas/genéticaRESUMEN
Endoscopic third ventriculostomy (ETV) is a well-established surgical technique for treating hydrocephalus. Many providers have transitioned to utilizing the specialized Neuroballoon for the stoma dilation in ETV; however, these devices are intermittently unavailable during supply chain shortages. We present the experience of employing cardiac angioplasty and neurovascular balloons as substitutes for the Neuroballoon in 3 patients. The scepter balloon (Microvention), priced at $1800 compared to the standard $300 Neuroballoon (Integra), proved effective, but its pliability presented technical challenges. The substantial cost differential compared to a Neuroballoon ($300) raises economic considerations. The Cardiac TREK balloon (Abbott) was similarly effective, while also being easier to manage endoscopically and cheaper at $158. These experiences support the viability of non-neuroendoscopic specialized balloons as alternatives for ETV dilation of the floor of tuber cinereum.
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Hidrocefalia , Tercer Ventrículo , Ventriculostomía , Humanos , Ventriculostomía/métodos , Hidrocefalia/cirugía , Tercer Ventrículo/cirugía , Masculino , Femenino , Neuroendoscopía/métodos , LactanteRESUMEN
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.
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Péptidos , Canales de Potasio de Dominio Poro en Tándem , Canales de Potasio de Dominio Poro en Tándem/metabolismo , Canales de Potasio de Dominio Poro en Tándem/genética , Canales de Potasio de Dominio Poro en Tándem/antagonistas & inhibidores , Canales de Potasio de Dominio Poro en Tándem/química , Animales , Humanos , Péptidos/farmacología , Péptidos/química , Péptidos/metabolismo , Ixodes/metabolismo , Simulación del Acoplamiento Molecular , Secuencia de Aminoácidos , Células HEK293 , Bloqueadores de los Canales de Potasio/farmacología , Bloqueadores de los Canales de Potasio/química , Garrapatas/metabolismoRESUMEN
Oxytocin, a significant pleiotropic neuropeptide, regulates psychological stress adaptation and social communication, as well as peripheral actions, such as uterine contraction and milk ejection. Recently, a Japanese Kampo medicine called Kamikihito (KKT) has been reported to stimulate oxytocin neurons to induce oxytocin secretion. Two-pore-domain potassium channels (K2P) regulate the resting potential of excitable cells, and their inhibition results in accelerated depolarization that elicits neuronal and endocrine cell activation. We assessed the effects of KKT and 14 of its components on a specific K2P, the potassium channel subfamily K member 2 (TREK-1), which is predominantly expressed in oxytocin neurons in the central nervous system (CNS). KKT inhibited the activity of TREK-1 induced via the channel activator ML335. Six of the 14 components of KKT inhibited TREK-1 activity. Additionally, we identified that 22 of the 41 compounds in the six components exhibited TREK-1 inhibitory effects. In summary, several compounds included in KKT partially activated oxytocin neurons by inhibiting TREK-1. The pharmacological effects of KKT, including antistress effects, may be partially mediated through the oxytocin pathway.
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Neuronas , Oxitocina , Canales de Potasio de Dominio Poro en Tándem , Animales , Humanos , Ratones , Medicamentos Herbarios Chinos/farmacología , Medicamentos Herbarios Chinos/química , Medicina Kampo , Neuronas/metabolismo , Neuronas/efectos de los fármacos , Oxitocina/farmacología , Oxitocina/metabolismo , Canales de Potasio de Dominio Poro en Tándem/metabolismo , Canales de Potasio de Dominio Poro en Tándem/antagonistas & inhibidoresRESUMEN
Alterations in intraocular and external pressure critically involve the pathogenesis of glaucoma, traumatic retinal injury (TRI), and other retinal disorders, and retinal neurons have been reported to express multiple mechanical-sensitive channels (MSCs) in recent decades. However, the role of MSCs in visual functions and pressure-related retinal conditions has been unclear. This review will focus on the variety and functional significance of the MSCs permeable to K+, Na+, and Ca2+, primarily including the big potassium channel (BK); the two-pore domain potassium channels TRAAK and TREK; Piezo; the epithelial sodium channel (ENaC); and the transient receptor potential channels vanilloid TRPV1, TRPV2, and TRPV4 in retinal photoreceptors, bipolar cells, horizontal cells, amacrine cells, and ganglion cells. Most MSCs do not directly mediate visual signals in vertebrate retinas. On the other hand, some studies have shown that MSCs can open in physiological conditions and regulate the activities of retinal neurons. While these data reasonably predict the crossing of visual and mechanical signals, how retinal light pathways deal with endogenous and exogenous mechanical stimulation is uncertain.
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Canales Iónicos , Neuronas Retinianas , Humanos , Animales , Canales Iónicos/metabolismo , Neuronas Retinianas/metabolismo , Mecanotransducción Celular , Retina/metabolismo , Retina/citologíaRESUMEN
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.
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Lesión Pulmonar Aguda , Virus de la Influenza A , Gripe Humana , Infecciones por Orthomyxoviridae , Canales de Potasio de Dominio Poro en Tándem , Animales , Humanos , Ratones , Lesión Pulmonar Aguda/patología , Quimiocina CXCL10/metabolismo , Gripe Humana/patología , Interleucina-6/metabolismo , Pulmón/metabolismo , Infecciones por Orthomyxoviridae/patología , Canales de Potasio de Dominio Poro en Tándem/genética , Canales de Potasio de Dominio Poro en Tándem/metabolismoRESUMEN
Modulation of two-pore domain potassium (K2P) channels has emerged as a novel field of therapeutic strategies as they may regulate immune cell activation and metabolism, inflammatory signals, or barrier integrity. One of these ion channels is the TWIK-related potassium channel 1 (TREK1). In the current study, we report the identification and validation of new TREK1 activators. Firstly, we used a modified potassium ion channel assay to perform high-throughput-screening of new TREK1 activators. Dose-response studies helped to identify compounds with a high separation between effectiveness and toxicity. Inside-out patch-clamp measurements of Xenopus laevis oocytes expressing TREK1 were used for further validation of these activators regarding specificity and activity. These approaches yielded three substances, E1, B3 and A2 that robustly activate TREK1. Functionally, we demonstrated that these compounds reduce levels of adhesion molecules on primary human brain and muscle endothelial cells without affecting cell viability. Finally, we studied compound A2 via voltage-clamp recordings as this activator displayed the strongest effect on adhesion molecules. Interestingly, A2 lacked TREK1 activation in the tested neuronal cell type. Taken together, this study provides data on novel TREK1 activators that might be employed to pharmacologically modulate TREK1 activity.
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Canales de Potasio de Dominio Poro en Tándem , Humanos , Canales de Potasio de Dominio Poro en Tándem/metabolismo , Células Endoteliales/metabolismo , Enfermedades Neuroinflamatorias , Encéfalo/metabolismo , Moléculas de Adhesión Celular/metabolismoRESUMEN
The Popeye domain-containing protein 3 (POPDC3), a transmembrane protein with a unique cyclic adenosine monophosphate (cAMP) binding site, is widely expressed in mammalian tissues, with the highest levels of expression in skeletal muscle. POPDC3 plays a key role in many physiological and pathological processes and is considered a candidate biomarker and potential therapeutic target of cancer. In addition, POPDC3 gene variants have been associated with limb-girdle muscular dystrophy (LGMD) type 26. However, there are only a few studies on the biological role of POPDC3, interacting proteins, potential downstream targets, and regulated signaling pathways. Therefore, this review focuses on the structure of POPDC3 protein, interacting molecules, and the role and mechanism in cancer, and in cardiac and skeletal muscle, and to review the current research progress of POPDC3 and propose possible future study directions.
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Músculo Estriado , Distrofia Muscular de Cinturas , Neoplasias , Animales , Humanos , Moléculas de Adhesión Celular/genética , Homeostasis , Mamíferos/metabolismo , Proteínas Musculares/genética , Músculo Esquelético/metabolismo , Músculo Estriado/metabolismoRESUMEN
Major depressive disorder (MDD) refers to a widespread psychiatric disorder. Astrocytes play a pivotal role in regulating inflammation which is a well-acknowledged key component in depression pathogenesis. However, the effects of the neuroinflammation-inducing A1-like astrocytes on MDD are still unknown. TWIK-related K+ channel 1 (TREK-1) has been demonstrated to regulate the action of antidepressants. Nevertheless, its mechanisms and effects on A1-like astrocyte stimulation in MDD are not clear. Therefore, we conducted in vivo and in vitro experiments using TREK-1 specific inhibitor spadin. In vivo, rats were subjected to a 6-week chronic unpredictable mild stress (CUMS) followed by spadin treatment. Behavioral tests were employed to surveil depressive-like behaviors. Hippocampal proteomic analysis was carried out with the purpose of identifying differentially expressed proteins after CUMS and spadin treatments. In vitro, astrocyte-conditioned medium and spadin were used to treat rat astrocyte cell line. The activated microglia, inflammatory factors, A1 astrocyte markers, and activated nuclear factor kappa B (NF-κB) pathway were later analyzed using immunofluorescence, western blot, and RT-qPCR. Our findings indicated that blockage of TREK-1 reduced CUMS-induced depressive-like behavior in rats, inhibited the microglial stimulation, reduced inflammatory factor levels, and suppressed the activation of A1-like reactive astrocytes in the hippocampus. We also verified that the suppression of A1-like astrocytes by spadin necessitated the NF-κB pathway. According to the findings, blocking TREK-1 inhibited the activation of A1-like reactive astrocytes via the NF-κB signaling pathway in MDD. Our study preliminarily identifies a novel antidepressant mechanism of TREK-1 action and provides a therapeutic path for MDD.
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Trastorno Depresivo Mayor , Canales de Potasio de Dominio Poro en Tándem , Ratas , Animales , Trastorno Depresivo Mayor/tratamiento farmacológico , FN-kappa B/metabolismo , Astrocitos/metabolismo , Potasio/metabolismo , Proteómica , Canales de Potasio de Dominio Poro en Tándem/metabolismo , Transducción de Señal , Antidepresivos/farmacología , Antidepresivos/uso terapéutico , Depresión/tratamiento farmacológico , Depresión/etiología , Estrés Psicológico/metabolismo , Hipocampo/metabolismoRESUMEN
Vagal afferents convey signals of mechanical stimulation in the gut to the brain, which is essential for the regulation of food intake. However, ion channels sensing mechanical stimuli are not fully understood. This study aimed to examine the ionic currents activated by mechanical stimulation and a possible neuro-modulatory role of nitric oxide on vagal afferents. Nodose neuronal currents and potentials, and intestinal afferent firing by mechanical stimulation were measured by whole-cell patch clamp, and in vitro afferent recording, respectively. Osmotically activated cation and two-pore domain K+ currents were identified in nodose neurons. The membrane potential displayed a biphasic change under hypotonic stimulation. Cation channel-mediated depolarization was followed by a hyperpolarization mediated by K+ channels. The latter was inhibited by l-methionine (TREK1 channel inhibitor) and l-NNA (nitric oxide synthase inhibitor). Correspondingly, mechanical stimulation activated opposing cation and TREK1 currents. NOS inhibition decreased TREK1 currents and potentiated jejunal afferent nerve firing induced by mechanical stimuli. This study suggested a novel activation mechanism of ion channels underlying adaptation under mechanical distension in vagal afferent neurons. The guts' ability to perceive mechanical stimuli is vital in determining how it responds to food intake. The mechanosensation through ion channels could initiate and control gut function.
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Óxido Nítrico , Ganglio Nudoso , Ganglio Nudoso/fisiología , Nervio Vago , Neuronas Aferentes/fisiología , NeuronasRESUMEN
Echinochrome A (Ech A), a naphthoquinoid pigment from sea urchins, is known to have anti-inflammatory and analgesic effects that have been suggested to be mediated by antioxidant activity and intracellular signaling modulation. In addition to these mechanisms, the ion channels in keratinocytes, immune cells, and nociceptive neurons may be the target for the pharmacological effects. Here, using the patch clamp technique, we investigated the effects of Ech A on the Ca2+-permeable TRPV3, TRPV1 and Orai1 channels and the two-pore domain K+ (K2P) channels (TREK/TRAAK, TASK-1, and TRESK) overexpressed in HEK 293 cells. Ech A inhibited both the TRPV3 and Orai1 currents, with IC50 levels of 2.1 and 2.4 µM, respectively. The capsaicin-activated TRPV1 current was slightly augmented by Ech A. Ech A alone did not change the amplitude of the TREK-2 current (ITREK2), but pretreatments with Ech A markedly facilitated ITREK2 activation by 2-APB, arachidonic acid (AA), and acidic extracellular pH (pHe). Similar facilitation effects of Ech A on TREK-1 and TRAAK were observed when they were stimulated with 2-APB and AA, respectively. On the contrary, Ech A did not affect the TRESK and TASK-1 currents. Interestingly, the ITREK2 maximally activated by the combined application of 2-APB and Ech A was not inhibited by norfluoxetine but was still completely inhibited by ruthenium red. The selective loss of sensitivity to norfluoxetine suggested an altered molecular conformation of TREK-2 by Ech A. We conclude that the Ech A-induced inhibition of the Ca2+-permeable cation channels and the facilitation of the TREK/TRAAK K2P channels may underlie the analgesic and anti-inflammatory effects of Ech A.
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Naftoquinonas , Humanos , Células HEK293 , Fenómenos Fisiológicos de la PielRESUMEN
Oligomeric ion channels are abundant in nature. However, the recombinant expression in cell culture-based systems remains tedious and challenging due to negative side effects, limiting the understanding of their role in health and disease. Accordingly, in this work, we demonstrate the cell-free synthesis (CFS) as an alternative platform to study the assembly of two-pore domain potassium channels (K2P) within endogenous endoplasmic reticulum-derived microsomes. Exploiting the open nature of CFS, we investigate the cotranslational translocation of TREK-2 into the microsomes and suggest a cotranslational assembly with typical single-channel behavior in planar lipid-bilayer electrophysiology. The heteromeric assembly of K2P channels is a contentious matter, accordingly we prove the successful assembly of TREK-2 with TWIK-1 using a biomolecular fluorescence complementation assay, Western blot analysis and autoradiography. The results demonstrate that TREK-2 homodimer assembly is the initial step, followed by heterodimer formation with the nascent TWIK-1, providing evidence of the intergroup heterodimerization of TREK-2 and TWIK-1 in eukaryotic CFS. Since K2P channels are involved in various pathophysiological conditions, including pain and nociception, CFS paves the way for in-depth functional studies and related pharmacological interventions. This study highlights the versatility of the eukaryotic CFS platform for investigating ion channel assembly in a native-like environment.