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Highly selective for K+ at neutral pH, the TWIK1 channel becomes permeable to Na+ upon acidification. Using molecular dynamics simulations, we identify a network of residues involved in this unique property. Between the open and closed states previously observed by electron microscopy, molecular dynamics simulations show that the channel undergoes conformational changes between pH 7.5-6 involving residues His122, Glu235, Lys246 and Phe109. A complex network of interactions surrounding the selectivity filter at high pH transforms into a simple set of stronger interactions at low pH. In particular, His122 protonated by acidification moves away from Lys246 and engages in a salt bridge with Glu235. In addition, stacking interactions between Phe109 and His122, which stabilize the selectivity filter in its K+-selective state at high pH, disappear upon acidification. This leads to dissociation of the Phe109 aromatic side chain from this network, resulting in the Na+-permeable conformation of the channel.
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hERG, the pore-forming subunit of the rapid component of the delayed rectifier K+ current, plays a key role in ventricular repolarization. Mutations in the KCNH2 gene encoding hERG are associated with several cardiac rhythmic disorders, mainly the Long QT syndrome (LQTS) characterized by prolonged ventricular repolarization, leading to ventricular tachyarrhythmias, sometimes progressing to ventricular fibrillation and sudden death. Over the past few years, the emergence of next-generation sequencing has revealed an increasing number of genetic variants including KCNH2 variants. However, the potential pathogenicity of the majority of the variants remains unknown, thus classifying them as variants of uncertain significance or VUS. With diseases such as LQTS being associated with sudden death, identifying patients at risk by determining the variant pathogenicity, is crucial. The purpose of this review is to describe, on the basis of an exhaustive examination of the 1322 missense variants, the nature of the functional assays undertaken so far and their limitations. A detailed analysis of 38 hERG missense variants identified in Long QT French patients and studied in electrophysiology also underlies the incomplete characterization of the biophysical properties for each variant. These analyses lead to two conclusions: first, the function of many hERG variants has never been looked at and, second, the functional studies done so far are excessively heterogeneous regarding the stimulation protocols, cellular models, experimental temperatures, homozygous and/or the heterozygous condition under study, a context that may lead to conflicting conclusions. The state of the literature emphasizes how necessary and important it is to perform an exhaustive functional characterization of hERG variants and to standardize this effort for meaningful comparison among variants. The review ends with suggestions to create a unique homogeneous protocol that could be shared and adopted among scientists and that would facilitate cardiologists and geneticists in patient counseling and management.
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By endowing light control of neuronal activity, optogenetics and photopharmacology are powerful methods notably used to probe the transmission of pain signals. However, costs, animal handling and ethical issues have reduced their dissemination and routine use. Here we report LAKI (Light Activated K+ channel Inhibitor), a specific photoswitchable inhibitor of the pain-related two-pore-domain potassium TREK and TRESK channels. In the dark or ambient light, LAKI is inactive. However, alternating transdermal illumination at 365 nm and 480 nm reversibly blocks and unblocks TREK/TRESK current in nociceptors, enabling rapid control of pain and nociception in intact and freely moving mice and nematode. These results demonstrate, in vivo, the subcellular localization of TREK/TRESK at the nociceptor free nerve endings in which their acute inhibition is sufficient to induce pain, showing LAKI potential as a valuable tool for TREK/TRESK channel studies. More importantly, LAKI gives the ability to reversibly remote-control pain in a non-invasive and physiological manner in naive animals, which has utility in basic and translational pain research but also in in vivo analgesic drug screening and validation, without the need of genetic manipulations or viral infection.
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Dor , Canais de Potássio de Domínios Poros em Tandem , Animais , Camundongos , Avaliação Pré-Clínica de Medicamentos , Nociceptores , Nematoides , Canais de Potássio de Domínios Poros em Tandem/antagonistas & inibidoresRESUMO
Two-pore domain K+ channels (K2P channels), active as dimers, produce inhibitory currents regulated by a variety of stimuli. Among them, TWIK1-related alkalinization-activated K+ channel 1 (TALK1), TWIK1-related alkalinization-activated K+ channel 2 (TALK2), and TWIK1-related acid-sensitive K+ channel 2 (TASK2) form a subfamily of structurally related K2P channels stimulated by extracellular alkalosis. The human genes encoding these proteins are clustered at chromosomal region 6p21 and coexpressed in multiple tissues, including the pancreas. The question whether these channels form functional heteromers remained open. By analyzing single-cell transcriptomic data, we show that these channels are coexpressed in insulin-secreting pancreatic ß-cells. Using in situ proximity ligation assay and electrophysiology, we show that they form functional heterodimers both upon heterologous expression and under native conditions in human pancreatic ß-cells. We demonstrate that heteromerization of TALK2 with TALK1 or with TASK2 endows TALK2 with sensitivity to extracellular alkalosis in the physiological range. We further show that the association of TASK2 with TALK1 and TALK2 increases their unitary conductance. These results provide a new example of heteromerization in the K2P channel family expanding the range of the potential physiological and pathophysiological roles of TALK1/TALK2/TASK2 channels, not only in insulin-secreting cells but also in the many other tissues in which they are coexpressed.
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Alcalose , Células Secretoras de Insulina , Canais de Potássio de Domínios Poros em Tandem , Humanos , Canais de Potássio de Domínios Poros em Tandem/genética , Canais de Potássio de Domínios Poros em Tandem/metabolismo , Células Secretoras de Insulina/metabolismo , Concentração de Íons de Hidrogênio , Insulina/metabolismo , Potássio/metabolismoRESUMO
[This corrects the article DOI: 10.3389/fphar.2021.755826.].
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Mechanoelectrical transduction is mediated by the opening of different types of force-sensitive ion channels, including Piezo1/2 and the TREK/TRAAK K2P channels. Piezo1 curves the membrane locally into an inverted dome that reversibly flattens in response to force application. Moreover, Piezo1 forms numerous preferential interactions with various membrane lipids, including cholesterol. Whether this structural architecture influences the functionality of neighboring membrane proteins is unknown. Here, we show that Piezo1/2 increase TREK/TRAAK current amplitude, slow down activation/deactivation, and remove inactivation upon mechanical stimulation. These findings are consistent with a mechanism whereby Piezo1/2 cause a local depletion of membrane cholesterol associated with a prestress of TREK/TRAAK channels. This regulation occurs in mouse fibroblasts between endogenous Piezo1 and TREK-1/2, both channel types acting in concert to delay wound healing. In conclusion, we demonstrate a community effect between different structural and functional classes of mechanosensitive ion channels.
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Ativação do Canal Iônico , Canais Iônicos/fisiologia , Mecanotransdução Celular , Canais de Potássio de Domínios Poros em Tandem/metabolismo , Animais , Colesterol/metabolismo , Fibroblastos/citologia , Fibroblastos/metabolismo , Gengiva/citologia , Gengiva/metabolismo , Células HEK293 , Humanos , Masculino , Camundongos , Camundongos Endogâmicos C57BL , Camundongos Knockout , Canais de Potássio de Domínios Poros em Tandem/genéticaRESUMO
BACKGROUND AND AIMS: Mutations in KCNH2 cause long or short QT syndromes (LQTS or SQTS) predisposing to life-threatening arrhythmias. Over 1000 hERG variants have been described by clinicians, but most remain to be characterised. The objective is to standardise and accelerate the phenotyping process to contribute to clinician diagnosis and patient counselling. In silico evaluation was also included to characterise the structural impact of the variants. METHODS: We selected 11 variants from known LQTS patients and two variants for which diagnosis was problematic. Using the Gibson assembly strategy, we efficiently introduced mutations in hERG cDNA despite GC-rich sequences. A pH-sensitive fluorescent tag was fused to hERG for efficient evaluation of channel trafficking. An optimised 35-s patch-clamp protocol was developed to evaluate hERG channel activity in transfected cells. R software was used to speed up analyses. RESULTS: In the present work, we observed a good correlation between cell surface expression, assessed by the pH-sensitive tag, and current densities. Also, we showed that the new biophysical protocol allows a significant gain of time in recording ion channel properties and provides extensive information on WT and variant channel biophysical parameters, that can all be recapitulated in a single parameter defined herein as the repolarisation power. The impacts of the variants on channel structure were also reported where structural information was available. These three readouts (trafficking, repolarisation power and structural impact) define three pathogenicity indexes that may help clinical diagnosis. CONCLUSIONS: Fast-track characterisation of KCNH2 genetic variants shows its relevance to discriminate mutants that affect hERG channel activity from variants with undetectable effects. It also helped the diagnosis of two new variants. This information is meant to fill a patient database, as a basis for personalised medicine. The next steps will be to further accelerate the process using an automated patch-clamp system.
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Arritmias Cardíacas/genética , Canal de Potássio ERG1/genética , Síndrome do QT Longo/genética , Potenciais de Ação/genética , Humanos , Regulador Transcricional ERG/genética , Virulência/efeitos dos fármacosRESUMO
Inhibitory potassium channels of the TREK1/TRAAK family are integrators of multiple stimuli, including temperature, membrane stretch, polyunsaturated fatty acids and pH. How these signals affect the gating of these channels is the subject of intense research. We have previously identified a cytoplasmic domain, pCt, which plays a major role in controlling channel activity. Here, we use pharmacology to show that the effects of pCt, arachidonic acid, and extracellular pH converge to the same gate within the channel. Using a state-dependent inhibitor, fluoxetine, as well as natural and synthetic openers, we provide further evidence that the "up" and "down" conformations identified by crystallography do not correspond to open and closed states of these channels.
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Potassium channels form the largest family of ion channels with more than 80 members involved in cell excitability and signalling. Most of them exist as homomeric channels, whereas specific conditions are required to obtain heteromeric channels. It is well established that heteromerization of voltage-gated and inward rectifier potassium channels affects their function, increasing the diversity of the native potassium currents. For potassium channels with two pore domains (K2P ), homomerization has long been considered the rule, their polymodal regulation by a wide diversity of physical and chemical stimuli being responsible for the adaptation of the leak potassium currents to cellular needs. This view has recently evolved with the accumulation of evidence of heteromerization between different K2P subunits. Several functional intragroup and intergroup heteromers have recently been identified, which contribute to the functional heterogeneity of this family. K2P heteromerization is involved in the modulation of channel expression and trafficking, promoting functional and signalling diversity. As illustrated in the Abstract Figure, heteromerization of TREK1 and TRAAK provides the cell with more possibilities of regulation. It is becoming increasingly evident that K2P heteromers contribute to important physiological functions including neuronal and cardiac excitability. Since heteromerization also affects the pharmacology of K2P channels, this understanding helps to establish K2P heteromers as new therapeutic targets for physiopathological conditions.
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Canais de Potássio de Domínios Poros em Tandem , Neurônios/metabolismo , Potássio , Canais de Potássio de Domínios Poros em Tandem/genética , Canais de Potássio de Domínios Poros em Tandem/metabolismo , Transporte Proteico , Transdução de SinaisRESUMO
BACKGROUND AND PURPOSE: Opioids are effective painkillers. However, their risk-benefit ratio is dampened by numerous adverse effects and opioid misuse has led to a public health crisis. Safer alternatives are required, but isolating the antinociceptive effect of opioids from their adverse effects is a pharmacological challenge because activation of the µ opioid receptor triggers both the antinociceptive and adverse effects of opioids. EXPERIMENTAL APPROACH: The TREK1 potassium channel is activated downstream of µ receptor and involved in the antinociceptive activity of morphine but not in its adverse effects. Bypassing the µ opioid receptor to directly activate TREK1 could therefore be a safer analgesic strategy. KEY RESULTS: We developed a selective TREK1 activator, RNE28, with antinociceptive activity in naive rodents and in models of inflammatory and neuropathic pain. This activity was lost in TREK1 knockout mice or wild-type mice treated with the TREK1 blocker spadin, showing that TREK1 is required for the antinociceptive activity of RNE28. RNE28 did not induce respiratory depression, constipation, rewarding effects, or sedation at the analgesic doses tested. CONCLUSION AND IMPLICATIONS: This proof-of-concept study shows that TREK1 activators could constitute a novel class of painkillers, inspired by the mechanism of action of opioids but devoid of their adverse effects.
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Analgésicos Opioides , Neuralgia , Analgésicos , Analgésicos Opioides/efeitos adversos , Animais , Camundongos , Camundongos Knockout , Morfina , Receptores Opioides muRESUMO
Mutations in the actively expressed, maternal allele of the imprinted KCNK9 gene cause Birk-Barel intellectual disability syndrome (BBIDS). Using a BBIDS mouse model, we identify here a partial rescue of the BBIDS-like behavioral and neuronal phenotypes mediated via residual expression from the paternal Kcnk9 (Kcnk9pat) allele. We further demonstrate that the second-generation HDAC inhibitor CI-994 induces enhanced expression from the paternally silenced Kcnk9 allele and leads to a full rescue of the behavioral phenotype suggesting CI-994 as a promising molecule for BBIDS therapy. Thus, these findings suggest a potential approach to improve cognitive dysfunction in a mouse model of an imprinting disorder.
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Anormalidades Craniofaciais/genética , Anormalidades Craniofaciais/metabolismo , Histonas/metabolismo , Deficiência Intelectual/genética , Deficiência Intelectual/metabolismo , Hipotonia Muscular/genética , Hipotonia Muscular/metabolismo , Canais de Potássio/genética , Animais , Comportamento Animal , Benzamidas , Encéfalo/metabolismo , Anormalidades Craniofaciais/tratamento farmacológico , Modelos Animais de Doenças , Feminino , Técnicas de Silenciamento de Genes , Impressão Genômica , Inibidores de Histona Desacetilases/farmacologia , Humanos , Deficiência Intelectual/tratamento farmacológico , Locus Cerúleo/metabolismo , Masculino , Camundongos , Camundongos Endogâmicos C57BL , Camundongos Knockout , Hipotonia Muscular/tratamento farmacológico , Mutação , Fenótipo , Fenilenodiaminas/farmacologia , Canais de Potássio/deficiência , Canais de Potássio/metabolismo , Regulação para Cima/efeitos dos fármacosRESUMO
Mutations that modulate the activity of ion channels are essential tools to understand the biophysical determinants that control their gating. Here, we reveal the conserved role played by a single amino acid position (TM2.6) located in the second transmembrane domain of two-pore domain potassium (K2P) channels. Mutations of TM2.6 to aspartate or asparagine increase channel activity for all vertebrate K2P channels. Using two-electrode voltage-clamp and single-channel recording techniques, we find that mutation of TM2.6 promotes channel gating via the selectivity filter gate and increases single channel open probability. Furthermore, channel gating can be progressively tuned by using different amino acid substitutions. Finally, we show that the role of TM2.6 was conserved during evolution by rationally designing gain-of-function mutations in four Caenorhabditis elegans K2P channels using CRISPR/Cas9 gene editing. This study thus describes a simple and powerful strategy to systematically manipulate the activity of an entire family of potassium channels.
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Potenciais da Membrana/fisiologia , Canais de Potássio de Domínios Poros em Tandem/metabolismo , Animais , Sistemas CRISPR-Cas/genética , Sistemas CRISPR-Cas/fisiologia , Caenorhabditis elegans , Proteínas de Caenorhabditis elegans/genética , Proteínas de Caenorhabditis elegans/metabolismo , Drosophila , Evolução Molecular , Humanos , Invertebrados , Potenciais da Membrana/genética , Mutação/genética , Canais de Potássio de Domínios Poros em Tandem/genética , VertebradosRESUMO
It is often unclear why some genetic mutations to a given gene contribute to neurological disorders and others do not. For instance, two mutations have previously been found to produce a dominant negative for TRESK, a two-pore-domain K+ channel implicated in migraine: TRESK-MT, a 2-bp frameshift mutation, and TRESK-C110R. Both mutants inhibit TRESK, but only TRESK-MT increases sensory neuron excitability and is linked to migraine. Here, we identify a new mechanism, termed frameshift mutation-induced alternative translation initiation (fsATI), that may explain why only TRESK-MT is associated with migraine. fsATI leads to the production of a second protein fragment, TRESK-MT2, which co-assembles with and inhibits TREK1 and TREK2, two other two-pore-domain K+ channels, to increase trigeminal sensory neuron excitability, leading to a migraine-like phenotype in rodents. These findings identify TREK1 and TREK2 as potential molecular targets in migraine and suggest that fsATI should be considered as a distinct class of mutations.
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Potenciais de Ação/genética , Transtornos de Enxaqueca/patologia , Mutação/genética , Neurônios/fisiologia , Canais de Potássio de Domínios Poros em Tandem/genética , Potenciais de Ação/efeitos dos fármacos , Animais , Células Cultivadas , Modelos Animais de Doenças , Feminino , Expressão Gênica/genética , Células HEK293 , Humanos , Masculino , Camundongos , Camundongos Endogâmicos C57BL , Camundongos Transgênicos , Transtornos de Enxaqueca/induzido quimicamente , Transtornos de Enxaqueca/genética , Transtornos de Enxaqueca/fisiopatologia , Modelos Biológicos , Modelos Moleculares , Neurotransmissores/toxicidade , Óxido Nítrico/toxicidade , Oócitos , Canais de Potássio/genética , Canais de Potássio de Domínios Poros em Tandem/metabolismo , Ratos , Ratos Sprague-Dawley , XenopusRESUMO
External acidity induces catecholamine secretion by inhibiting TASK1-like channels in rat adrenal medullary (AM) cells. TASK channels can function as a heteromer or homomer in the TASK subfamily. In this study, we elucidate the molecular identity of TASK1-like channels in mouse AM cells using gene knockout. Genetic deletion of TASK1, but not TASK3, abolished the depolarizing inward current and catecholamine secretion in response to acidity, whereas it did not affect the resting current level. Immunocytochemistry revealed that AM cells exhibited predominantly TASK1-like and little TASK3-like immunoreactivity. A proximity ligation assay showed that TASK1/3 heteromeric channels were not formed in AM cells or PC12 cells. However, the exogenous expression of p11 in PC12 cells resulted in the heteromeric formation of TASK isoforms, which were mainly located in the cytoplasm, and p11 was not expressed in rat adrenal medullae or PC12 cells. In AM cells, genetic deletion of TASK1 resulted in enhancement of the immunoreactivity of the TALK2 channel, but not TASK3. The results indicate that TASK1 homomeric channels function as acidity sensors in AM cells, and that function is facilitated by the lack of p11 expression.-Inoue, M., Matsuoka, H., Lesage, F., Harada, K. Lack of p11 expression facilitates acidity-sensing function of TASK1 channels in mouse adrenal medullary cells.
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Canais Iônicos Sensíveis a Ácido/fisiologia , Ácidos/química , Medula Suprarrenal/fisiologia , Anexina A2/deficiência , Proteínas do Tecido Nervoso/fisiologia , Canais de Potássio de Domínios Poros em Tandem/fisiologia , Canais de Potássio/fisiologia , Proteínas S100/deficiência , Animais , Masculino , Camundongos , Camundongos Endogâmicos C57BL , Camundongos Knockout , Células PC12 , RatosRESUMO
TREK/TRAAK channels are polymodal K+ channels that convert very diverse stimuli, including bioactive lipids, mechanical stretch and temperature, into electrical signals. The nature of the structural changes that regulate their activity remains an open question. Here, we show that a cytoplasmic domain (the proximal C-ter domain, pCt) exerts antagonistic effects in TREK1 and TRAAK. In basal conditions, pCt favors activity in TREK1 whereas it impairs TRAAK activity. Using the conformation-dependent binding of fluoxetine, we show that TREK1 and TRAAK conformations at rest are different, and under the influence of pCt. Finally, we show that depleting PIP2 in live cells has a more pronounced inhibitory effect on TREK1 than on TRAAK. This differential regulation of TREK1 and TRAAK is related to a previously unrecognized PIP2-binding site (R329, R330, and R331) present within TREK1 pCt, but not in TRAAK pCt. Collectively, these new data point out pCt as a major regulatory domain of these channels and suggest that the binding of PIP2 to the pCt of TREK1 results in the stabilization of the conductive conformation in basal conditions.
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We previously proposed a role for the two-pore domain potassium (K2P) channel TREK-1 in hyperoxia (HO)-induced lung injury. To determine whether redundancy among the three TREK isoforms (TREK-1, TREK-2, and TRAAK) could protect from HO-induced injury, we now examined the effect of deletion of all three TREK isoforms in a clinically relevant scenario of prolonged HO exposure and mechanical ventilation (MV). We exposed WT and TREK-1/TREK-2/TRAAK-deficient [triple knockout (KO)] mice to either room air, 72-h HO, MV [high and low tidal volume (TV)], or a combination of HO + MV and measured quasistatic lung compliance, bronchoalveolar lavage (BAL) protein concentration, histologic lung injury scores (LIS), cellular apoptosis, and cytokine levels. We determined surfactant gene and protein expression and attempted to prevent HO-induced lung injury by prophylactically administering an exogenous surfactant (Curosurf). HO treatment increased lung injury in triple KO but not WT mice, including an elevated LIS, BAL protein concentration, and markers of apoptosis, decreased lung compliance, and a more proinflammatory cytokine phenotype. MV alone had no effect on lung injury markers. Exposure to HO + MV (low TV) further decreased lung compliance in triple KO but not WT mice, and HO + MV (high TV) was lethal for triple KO mice. In triple KO mice, the HO-induced lung injury was associated with decreased surfactant protein (SP) A and SPC but not SPB and SPD expression. However, these changes could not be explained by alterations in the transcription factors nuclear factor-1 (NF-1), NKX2.1/thyroid transcription factor-1 (TTF-1) or c-jun, or lamellar body levels. Prophylactic Curosurf administration did not improve lung injury scores or compliance in triple KO mice.
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Hiperóxia/metabolismo , Lesão Pulmonar/metabolismo , Canais de Potássio de Domínios Poros em Tandem/deficiência , Canais de Potássio/deficiência , Proteínas Associadas a Surfactantes Pulmonares/biossíntese , Animais , Regulação da Expressão Gênica/efeitos dos fármacos , Regulação da Expressão Gênica/genética , Hiperóxia/genética , Hiperóxia/patologia , Lipopolissacarídeos/toxicidade , Lesão Pulmonar/genética , Lesão Pulmonar/patologia , Camundongos , Camundongos Knockout , Proteínas Associadas a Surfactantes Pulmonares/genética , Fatores de Transcrição/genética , Fatores de Transcrição/metabolismoRESUMO
Despite intensive research, the exact function of TASK potassium channels in central and peripheral chemoreception is still under debate. In this study, we investigated the respiration of unrestrained TASK-3 (TASK-3-/-) and TASK-1/TASK-3 double knockout (TASK-1/3-/-) adult male mice in vivo using a plethysmographic device. Ventilation parameters of TASK-3-/- mice were normal under control condition (21% O2) and upon hypoxia and hypercapnia they displayed the physiological increase of ventilation. TASK-1/3-/- mice showed increased ventilation under control conditions. This increase of ventilation was caused by increased tidal volumes (VT), a phenomenon similarly observed in TASK-1-/- mice. Under acute hypoxia, TASK-1/3-/- mice displayed the physiological increase of the minute volume. Interestingly, this increase was not related to an increase of the respiratory frequency (fR), as observed in wild-type mice, but was caused by a strong increase of VT. This particular respiratory phenotype is reminiscent of the respiratory phenotype of carotid body-denervated rodents in the compensated state. Acute hypercapnia (5% CO2) stimulated ventilation in TASK-1/3-/- and wild-type mice to a similar extent; however, at higher CO2 concentrations (>5% CO2) the stimulation of ventilation was more pronounced in TASK-1/3-/- mice. At hyperoxia (100% O2), TASK-1-/-, TASK-3-/- and wild-type mice showed the physiological small decrease of ventilation. In sharp contrast, TASK-1/3-/- mice exhibited an abnormal increase of ventilation under hyperoxia. In summary, these measurements showed a grossly normal respiration of TASK-3-/- mice and a respiratory phenotype of TASK-1/3-/- mice that was characterized by a markedly enhanced tidal volume, similar to the one observed in TASK-1-/- mice. The abnormal hyperoxia response, exclusively found in TASK-1/3-/- double mutant mice, indicates that both TASK-1 and TASK-3 are essential for the hyperoxia-induced hypoventilation. The peculiar respiratory phenotype of TASK-1/3 knockout mice is reminiscent of the respiration of animals with long-term carotid body dysfunction. Taken together, TASK-1 and TASK-3 appear to serve specific and distinct roles in the complex processes underlying chemoreception and respiratory control.
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Hiperóxia/metabolismo , Proteínas do Tecido Nervoso/deficiência , Canais de Potássio de Domínios Poros em Tandem/deficiência , Canais de Potássio/deficiência , Respiração , Animais , Dióxido de Carbono/metabolismo , Feminino , Hipercapnia/metabolismo , Hipóxia/metabolismo , Masculino , Camundongos Endogâmicos C57BL , Camundongos Knockout , Proteínas do Tecido Nervoso/genética , Fenótipo , Pletismografia Total , Canais de Potássio/genética , Canais de Potássio de Domínios Poros em Tandem/genética , Volume de Ventilação Pulmonar/fisiologiaRESUMO
Recombinant TWIK2 channels produce weak basal background K+ currents. Current amplitudes depend on the animal species the channels have been isolated from and on the heterologous system used for their re-expression. Here we show that this variability is due to a unique cellular trafficking. We identified three different sequence signals responsible for the preferential expression of TWIK2 in the Lamp1-positive lysosomal compartment. Sequential inactivation of tyrosine-based (Y308ASIP) and di-leucine-like (E266LILL and D282EDDQVDIL) trafficking motifs progressively abolishes the targeting of TWIK2 to lysosomes, and promotes its functional relocation at the plasma membrane. In addition, TWIK2 contains two N-glycosylation sites (N79AS and N85AS) on its luminal side, and glycosylation is necessary for expression in lysosomes. As shown by electrophysiology and electron microscopy, TWIK2 produces functional background K+ currents in the endolysosomes, and its expression affects the number and mean size of the lysosomes. These results show that TWIK2 is expressed in lysosomes, further expanding the registry of ion channels expressed in these organelles.
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Lisossomos/metabolismo , Canais de Potássio de Domínios Poros em Tandem/metabolismo , Domínios e Motivos de Interação entre Proteínas , Multimerização Proteica , Proteínas Recombinantes , Sequência de Aminoácidos , Animais , Cães , Expressão Gênica , Células HEK293 , Humanos , Células Madin Darby de Rim Canino , Camundongos , Canais de Potássio de Domínios Poros em Tandem/química , Canais de Potássio de Domínios Poros em Tandem/genética , Transporte Proteico , RatosRESUMO
The TWIK-related K+ channel, TREK-1, has recently emerged as an attractive therapeutic target for the development of a novel class of analgesic drugs, suggesting that activation of TREK-1 could result in pain inhibition. Here, we report the synthesis of a series of substituted acrylic acids (1-54) based on our previous work with caffeate esters. The analogues were evaluated for their ability to modulate TREK-1 channel by electrophysiology and for their in vivo antinociceptive activity (acetic acid-induced writhing and hot plate assays), leading to the identification of a series of novel molecules able to activate TREK-1 and displaying potent antinociceptive activity in vivo. Furyl analogue 36 is the most promising of the series.