RESUMO
Microglia are resident immune cells of the brain and regulate its inflammatory state. In neurodegenerative diseases, microglia transition from a homeostatic state to a state referred to as disease-associated microglia (DAM). DAM express higher levels of proinflammatory signaling molecules, like STAT1 and TLR2, and show transitions in mitochondrial activity toward a more glycolytic response. Inhibition of Kv1.3 decreases the proinflammatory signature of DAM, though how Kv1.3 influences the response is unknown. Our goal was to identify the potential proteins interacting with Kv1.3 during transition to DAM. We utilized TurboID, a biotin ligase, fused to Kv1.3 to evaluate potential interacting proteins with Kv1.3 via mass spectrometry in BV-2 microglia following TLR4-mediated activation. Electrophysiology, Western blotting, and flow cytometry were used to evaluate Kv1.3 channel presence and TurboID biotinylation activity. We hypothesized that Kv1.3 contains domain-specific interactors that vary during a TLR4-induced inflammatory response, some of which are dependent on the PDZ-binding domain on the C terminus. We determined that the N terminus of Kv1.3 is responsible for trafficking Kv1.3 to the cell surface and mitochondria (e.g., NUDC, TIMM50). Whereas, the C terminus interacts with immune signaling proteins in a lipopolysaccharide-induced inflammatory response (e.g., STAT1, TLR2, and C3). There are 70 proteins that rely on the C-terminal PDZ-binding domain to interact with Kv1.3 (e.g., ND3, Snx3, and Sun1). Furthermore, we used Kv1.3 blockade to verify functional coupling between Kv1.3 and interferon-mediated STAT1 activation. Overall, we highlight that the Kv1.3 potassium channel functions beyond conducting the outward flux of potassium ions in an inflammatory context and that Kv1.3 modulates the activity of key immune signaling proteins, such as STAT1 and C3.
RESUMO
Kv1.3 potassium channels, expressed by proinflammatory central nervous system mononuclear phagocytes (CNS-MPs), are promising therapeutic targets for modulating neuroinflammation in Alzheimer's disease (AD). The molecular characteristics of Kv1.3-high CNS-MPs and their cellular origin from microglia or CNS-infiltrating monocytes are unclear. While Kv1.3 blockade reduces amyloid beta (Aß) burden in mouse models, the downstream immune effects on molecular profiles of CNS-MPs remain unknown. We show that functional Kv1.3 channels are selectively expressed by a subset of CD11b+CD45+ CNS-MPs acutely isolated from an Aß mouse model (5xFAD) as well as fresh postmortem human AD brain. Transcriptomic profiling of purified CD11b+Kv1.3+ CNS-MPs, CD11b+CD45int Kv1.3neg microglia, and peripheral monocytes from 5xFAD mice revealed that Kv1.3-high CNS-MPs highly express canonical microglial markers (Tmem119, P2ry12) and are distinct from peripheral Ly6chigh/Ly6clow monocytes. Unlike homeostatic microglia, Kv1.3-high CNS-MPs express relatively lower levels of homeostatic genes, higher levels of CD11c, and increased levels of glutamatergic transcripts, potentially representing phagocytic uptake of neuronal elements. Using irradiation bone marrow CD45.1/CD45.2 chimerism in 5xFAD mice, we show that Kv1.3+ CNS-MPs originate from microglia and not blood-derived monocytes. We show that Kv1.3 channels regulate membrane potential and early signaling events in microglia. Finally, in vivo blockade of Kv1.3 channels in 5xFAD mice by ShK-223 reduced Aß burden, increased CD11c+ CNS-MPs, and expression of phagocytic genes while suppressing proinflammatory genes (IL1b). Our results confirm the microglial origin and identify unique molecular features of Kv1.3-expressing CNS-MPs. In addition, we provide evidence for CNS immunomodulation by Kv1.3 blockers in AD mouse models resulting in a prophagocytic phenotype.
Assuntos
Doença de Alzheimer/metabolismo , Encéfalo/metabolismo , Canal de Potássio Kv1.3/metabolismo , Microglia/metabolismo , Células Mieloides/metabolismo , Doença de Alzheimer/genética , Peptídeos beta-Amiloides/metabolismo , Animais , Modelos Animais de Doenças , Feminino , Humanos , Canal de Potássio Kv1.3/genética , Masculino , CamundongosRESUMO
Impaired cerebellar Purkinje neuron firing resulting from reduced expression of large-conductance calcium-activated potassium (BK) channels is a consistent feature in models of inherited neurodegenerative spinocerebellar ataxia (SCA). Restoring BK channel expression improves motor function and delays cerebellar degeneration, indicating that BK channels are an attractive therapeutic target. Current BK channel activators lack specificity and potency and are therefore poor templates for future drug development. We implemented an automated patch clamp platform for high-throughput drug discovery of BK channel activators using the Nanion SyncroPatch 384PE system. We screened over 15,000 compounds for their ability to increase BK channel current amplitude under conditions of lower intracellular calcium that is present in disease. We identified several novel BK channel activators that were then retested on the SyncroPatch 384PE to generate concentration-response curves (CRCs). Compounds with favorable CRCs were subsequently tested for their ability to improve irregular cerebellar Purkinje neuron spiking, characteristic of BK channel dysfunction in SCA1 mice. We identified a novel BK channel activator, 4-chloro-N-(5-chloro-2-cyanophenyl)-3-(trifluoromethyl)benzene-1-sulfonamide (herein renamed BK-20), that exhibited a more potent half-maximal activation of BK current (pAC50 = 4.64) than NS-1619 (pAC50 = 3.7) at a free internal calcium concentration of 270 nM in a heterologous expression system and improved spiking regularity in SCA1 Purkinje neurons. BK-20 had no activity on small-conductance calcium-activated potassium (SK)1-3 channels but interestingly was a potent blocker of the T-type calcium channel, Cav3.1 (IC50 = 1.05 µM). Our work describes both a novel compound for further drug development in disorders with irregular Purkinje spiking and a unique platform for drug discovery in degenerative ataxias. SIGNIFICANCE STATEMENT: Motor impairment associated with altered Purkinje cell spiking due to dysregulation of large-conductance calcium-activated potassium (BK) channel expression and function is a shared feature of disease in many degenerative ataxias. BK channel activators represent an outstanding therapeutic agent for ataxia. We have developed a high-throughput platform to screen for BK channel activators and identified a novel compound that can serve as a template for future drug development for the treatment of these disabling disorders.
Assuntos
Ataxia Cerebelar , Canais de Potássio Cálcio-Ativados , Ataxias Espinocerebelares , Animais , Ataxia , Cálcio/metabolismo , Ataxia Cerebelar/tratamento farmacológico , Canais de Potássio Ativados por Cálcio de Condutância Alta , Camundongos , Potássio/metabolismo , Ataxias Espinocerebelares/metabolismoRESUMO
Microglia-mediated inflammation exerts adverse effects in ischemic stroke and in neurodegenerative disorders such as Alzheimer's disease (AD). Expression of the voltage-gated potassium channel Kv1.3 is required for microglia activation. Both genetic deletion and pharmacological inhibition of Kv1.3 are effective in reducing microglia activation and the associated inflammatory responses, as well as in improving neurological outcomes in animal models of AD and ischemic stroke. Here we sought to elucidate the molecular mechanisms underlying the therapeutic effects of Kv1.3 inhibition, which remain incompletely understood. Using a combination of whole-cell voltage-clamp electrophysiology and quantitative PCR (qPCR), we first characterized a stimulus-dependent differential expression pattern for Kv1.3 and P2X4, a major ATP-gated cationic channel, both in vitro and in vivo. We then demonstrated by whole-cell current-clamp experiments that Kv1.3 channels contribute not only to setting the resting membrane potential but also play an important role in counteracting excessive membrane potential changes evoked by depolarizing current injections. Similarly, the presence of Kv1.3 channels renders microglia more resistant to depolarization produced by ATP-mediated P2X4 receptor activation. Inhibiting Kv1.3 channels with ShK-223 completely nullified the ability of Kv1.3 to normalize membrane potential changes, resulting in excessive depolarization and reduced calcium transients through P2X4 receptors. Our report thus links Kv1.3 function to P2X4 receptor-mediated signaling as one of the underlying mechanisms by which Kv1.3 blockade reduces microglia-mediated inflammation. While we could confirm previously reported differences between males and females in microglial P2X4 expression, microglial Kv1.3 expression exhibited no gender differences in vitro or in vivo. MAIN POINTS: The voltage-gated K+ channel Kv1.3 regulates microglial membrane potential. Inhibition of Kv1.3 depolarizes microglia and reduces calcium entry mediated by P2X4 receptors by dissipating the electrochemical driving force for calcium.
Assuntos
Potenciais da Membrana , Trifosfato de Adenosina , Doença de Alzheimer , Animais , Cálcio , Feminino , Inflamação , Microglia , Receptores Purinérgicos P2 , Receptores Purinérgicos P2X4RESUMO
Microglia significantly contribute to the pathophysiology of Alzheimer's disease but an effective microglia-targeted therapeutic approach is not yet available clinically. The potassium channels Kv1.3 and Kir2.1 play important roles in regulating immune cell functions and have been implicated by in vitro studies in the 'M1-like pro-inflammatory' or 'M2-like anti-inflammatory' state of microglia, respectively. We here found that amyloid-ß oligomer-induced expression of Kv1.3 and Kir2.1 in cultured primary microglia. Likewise, ex vivo microglia acutely isolated from the Alzheimer's model 5xFAD mice co-expressed Kv1.3 and Kir2.1 as well as markers traditionally associated with M1 and M2 activation suggesting that amyloid-ß oligomer induces a microglial activation state that is more complex than previously thought. Using the orally available, brain penetrant small molecule Kv1.3 blocker PAP-1 as a tool, we showed that pro-inflammatory and neurotoxic microglial responses induced by amyloid-ß oligomer required Kv1.3 activity in vitro and in hippocampal slices. Since we further observed that Kv1.3 was highly expressed in microglia of transgenic Alzheimer's mouse models and human Alzheimer's disease brains, we hypothesized that pharmacological Kv1.3 inhibition could mitigate the pathology induced by amyloid-ß aggregates. Indeed, treating APP/PS1 transgenic mice with a 5-month oral regimen of PAP-1, starting at 9 months of age, when the animals already manifest cognitive deficits and amyloid pathology, reduced neuroinflammation, decreased cerebral amyloid load, enhanced hippocampal neuronal plasticity, and improved behavioural deficits. The observed decrease in cerebral amyloid deposition was consistent with the in vitro finding that PAP-1 enhanced amyloid-ß uptake by microglia. Collectively, these results provide proof-of-concept data to advance Kv1.3 blockers to Alzheimer's disease clinical trials.
Assuntos
Doença de Alzheimer , Canal de Potássio Kv1.3/metabolismo , Microglia/metabolismo , Doença de Alzheimer/tratamento farmacológico , Doença de Alzheimer/metabolismo , Doença de Alzheimer/patologia , Peptídeos beta-Amiloides/farmacologia , Precursor de Proteína beta-Amiloide/genética , Animais , Animais Recém-Nascidos , Aprendizagem da Esquiva/efeitos dos fármacos , Células Cultivadas , Modelos Animais de Doenças , Comportamento Exploratório/efeitos dos fármacos , Ficusina/uso terapêutico , Regulação da Expressão Gênica/efeitos dos fármacos , Regulação da Expressão Gênica/genética , Canal de Potássio Kv1.3/genética , Potenciais da Membrana/efeitos dos fármacos , Potenciais da Membrana/genética , Camundongos , Camundongos Endogâmicos C57BL , Camundongos Transgênicos , Microglia/efeitos dos fármacos , Mutação/genética , Fragmentos de Peptídeos/farmacologia , Bloqueadores dos Canais de Potássio/farmacologia , Presenilina-1/genética , Canais de Potássio Shab/metabolismoRESUMO
Microglia show a rich repertoire of activation patterns regulated by a complex ensemble of surface ion channels, receptors, and transporters. We and others have investigated whether microglia vary their K+ channel expression as a means to achieve functional diversity. However, most of the prior studies were conducted using in vitro models such as BV2 cells, primary microglia, or brain slices in culture, which may not accurately reflect microglia physiology in adult individuals. Here we employed an in vivo mouse model of selective innate immune activation by intracerebroventricular injection of lipopolysaccharides (ICV-LPS) to determine the role of the voltage-gated Kv1.3 channel in LPS-induced M1-like microglial activation. Using microglia acutely isolated from adult brains, we detected Kv1.3 and Kir2.1 currents, and found that ICV-LPS increased the current density and RNA expression of Kv1.3 but did not affect those of Kir2.1. Genetic knockout of Kv1.3 abolished LPS-induced microglial activation exemplified by Iba-1 immunoreactivity and expression of pro-inflammatory mediators such as IL-1ß, TNF-α, IL-6, and iNOS. Moreover, Kv1.3 knockout mitigated the LPS-induced impairment of hippocampal long-term potentiation (hLTP), suggesting that Kv1.3 activity regulates pro-inflammatory microglial neurotoxicity. Pharmacological intervention using PAP-1, a small molecule that selectively blocks homotetrameric Kv1.3 channels, achieved anti-inflammatory and hLTP-recovery effects similar to Kv1.3 knockout. We conclude that Kv1.3 is required for microglial M1-like pro-inflammatory activation in vivo. A significant implication of our in vivo data is that Kv1.3 blockers could be therapeutic candidates for neurological diseases where microglia-mediated neurotoxicity is implicated in the pathogenesis.
Assuntos
Imunidade Inata , Inflamação/metabolismo , Canal de Potássio Kv1.3/metabolismo , Microglia/metabolismo , Animais , Encéfalo/efeitos dos fármacos , Encéfalo/metabolismo , Proteínas de Ligação ao Cálcio/metabolismo , Citocinas/metabolismo , Escherichia coli , Imunidade Inata/efeitos dos fármacos , Canal de Potássio Kv1.3/antagonistas & inibidores , Canal de Potássio Kv1.3/genética , Lipopolissacarídeos , Potenciação de Longa Duração/imunologia , Camundongos Endogâmicos C57BL , Camundongos Knockout , Proteínas dos Microfilamentos/metabolismo , Microglia/efeitos dos fármacos , Canais de Potássio Corretores do Fluxo de Internalização/metabolismo , Técnicas de Cultura de TecidosRESUMO
The rodenticide tetramethylenedisulfotetramine (TETS) is a potent convulsant (lethal dose in humans 7-10 mg) that is listed as a possible threat agent by the United States Department of Homeland Security. TETS has previously been studied in vivo for toxicity and in vitro in binding assays, with the latter demonstrating it to be a non-competitive antagonist on GABAA receptors. To determine whether TETS exhibits subtype selectivity for a particular GABAA receptor combination, we used whole-cell patch-clamp to determine the potency of TETS on the major synaptic and extrasynaptic GABAA receptors associated with convulsant activity. The active component of picrotoxin, picrotoxinin, was used as a control. While picrotoxinin did not differentiate well between 13 GABAA receptors, TETS exhibited the highest activity on α2ß3γ2 (IC50 480 nM, 95% CI 320-640 nM) and α6ß3γ2 (IC50 400 nM, 95% CI 290-510 nM). Introducing ß1 or ß2 subunits into these receptor combinations reduced or abolished TETS sensitivity, suggesting that TETS preferentially affects receptors with α2/ß3 or α6/ß3 composition. Since α2ß3γ2 receptors make up 15-20% of the GABAA receptors in the mammalian CNS, we suggest that α2ß3γ2 is probably the most important GABAA receptor for the seizure-inducing activity of TETS.
Assuntos
Hidrocarbonetos Aromáticos com Pontes/metabolismo , Receptores de GABA-A/metabolismo , Rodenticidas/metabolismo , Animais , Células COS , Linhagem Celular , Chlorocebus aethiops , Fibroblastos/efeitos dos fármacos , Humanos , Camundongos , Técnicas de Patch-Clamp , Picrotoxina/análogos & derivados , Ratos , Sesterterpenos , Especificidade por Substrato , Ácido gama-Aminobutírico/metabolismoRESUMO
The intermediate-conductance Ca2+-activated K+ channel (KCa3.1) constitutes an attractive pharmacological target for immunosuppression, fibroproliferative disorders, atherosclerosis, and stroke. However, there currently is no available crystal structure of this medically relevant channel that could be used for structure-assisted drug design. Using the Rosetta molecular modeling suite we generated a molecular model of the KCa3.1 pore and tested the model by first confirming previously mapped binding sites and visualizing the mechanism of TRAM-34 (1-[(2-chlorophenyl)diphenylmethyl]-1H-pyrazole), senicapoc (2,2-bis-(4-fluorophenyl)-2-phenylacetamide), and NS6180 (4-[[3-(trifluoromethyl)phenyl]methyl]-2H-1,4-benzothiazin-3(4H)-one) inhibition at the atomistic level. All three compounds block ion conduction directly by fully or partially occupying the site that would normally be occupied by K+ before it enters the selectivity filter. We then challenged the model to predict the receptor sites and mechanisms of action of the dihydropyridine nifedipine and an isosteric 4-phenyl-pyran. Rosetta predicted receptor sites for nifedipine in the fenestration region and for the 4-phenyl-pyran in the pore lumen, which could both be confirmed by site-directed mutagenesis and electrophysiology. While nifedipine is thus not a pore blocker and might be stabilizing the channel in a nonconducting conformation or interfere with gating, the 4-phenyl-pyran was found to be a classical pore blocker that directly inhibits ion conduction similar to the triarylmethanes TRAM-34 and senicapoc. The Rosetta KCa3.1 pore model explains the mechanism of action of several KCa3.1 blockers at the molecular level and could be used for structure-assisted drug design.
Assuntos
Modelos Moleculares , Bloqueadores dos Canais de Potássio/farmacologia , Bibliotecas de Moléculas Pequenas/química , Bibliotecas de Moléculas Pequenas/farmacologia , Sequência de Aminoácidos , Sítios de Ligação , Humanos , Canais de Potássio Ativados por Cálcio de Condutância Intermediária/química , Canais de Potássio Ativados por Cálcio de Condutância Intermediária/farmacologia , Ligantes , Simulação de Acoplamento Molecular , Nifedipino/química , Nifedipino/farmacologia , Bloqueadores dos Canais de Potássio/química , Domínios Proteicos , Alinhamento de Sequência , Homologia Estrutural de Proteína , Relação Estrutura-Atividade , Tiazinas/química , Tiazinas/farmacologiaRESUMO
Microglia are highly plastic cells that can assume different phenotypes in response to microenvironmental signals. Lipopolysaccharide (LPS) and interferon-γ (IFN-γ) promote differentiation into classically activated M1-like microglia, which produce high levels of pro-inflammatory cytokines and nitric oxide and are thought to contribute to neurological damage in ischemic stroke and Alzheimer's disease. IL-4 in contrast induces a phenotype associated with anti-inflammatory effects and tissue repair. We here investigated whether these microglia subsets vary in their K+ channel expression by differentiating neonatal mouse microglia into M(LPS) and M(IL-4) microglia and studying their K+ channel expression by whole-cell patch-clamp, quantitative PCR and immunohistochemistry. We identified three major types of K+ channels based on their biophysical and pharmacological fingerprints: a use-dependent, outwardly rectifying current sensitive to the KV 1.3 blockers PAP-1 and ShK-186, an inwardly rectifying Ba2+ -sensitive Kir 2.1 current, and a Ca2+ -activated, TRAM-34-sensitive KCa 3.1 current. Both KV 1.3 and KCa 3.1 blockers inhibited pro-inflammatory cytokine production and iNOS and COX2 expression demonstrating that KV 1.3 and KCa 3.1 play important roles in microglia activation. Following differentiation with LPS or a combination of LPS and IFN-γ microglia exhibited high KV 1.3 current densities (â¼50 pA/pF at 40 mV) and virtually no KCa 3.1 and Kir currents, while microglia differentiated with IL-4 exhibited large Kir 2.1 currents (â¼ 10 pA/pF at -120 mV). KCa 3.1 currents were generally low but moderately increased following stimulation with IFN-γ or ATP (â¼10 pS/pF). This differential K+ channel expression pattern suggests that KV 1.3 and KCa 3.1 inhibitors could be used to inhibit detrimental neuroinflammatory microglia functions. GLIA 2016;65:106-121.
Assuntos
Canais de Potássio Ativados por Cálcio de Condutância Intermediária/metabolismo , Canal de Potássio Kv1.3/metabolismo , Microglia/metabolismo , Canais de Potássio Corretores do Fluxo de Internalização/metabolismo , Animais , Células Cultivadas , Interferon gama/metabolismo , Lipopolissacarídeos/farmacologia , Ativação de Macrófagos , Potenciais da Membrana , Camundongos Endogâmicos C57BLRESUMO
There is a need for fast detection methods for the banned rodenticide tetramethylenedisulfotetramine (TETS), a highly potent blocker of the γ-aminobutyric acid (GABAA ) receptors. General synthetic approach toward two groups of analogues was developed. Screening of the resulting library of compounds by FLIPR or whole-cell voltage-clamp revealed that, despite the structural differences, some of the TETS analogues retained GABAA receptor inhibition; however, their potency was an order of magnitude lower. Antibodies raised in rabbits against some of the TETS analogues conjugated to protein recognized free TETS and will be used for the development of an immunoassay for TETS.
Assuntos
Hidrocarbonetos Aromáticos com Pontes/síntese química , Haptenos/química , Receptores de GABA-A/metabolismo , Bibliotecas de Moléculas Pequenas/síntese química , Animais , Hidrocarbonetos Aromáticos com Pontes/farmacologia , Avaliação Pré-Clínica de Medicamentos/métodos , Fenômenos Eletrofisiológicos/fisiologia , Humanos , Imunoensaio/métodos , Concentração Inibidora 50 , Estrutura Molecular , Neurônios , Coelhos , Bibliotecas de Moléculas Pequenas/farmacologia , Relação Estrutura-AtividadeRESUMO
The voltage-gated potassium (Kv) 1.3 channel is widely regarded as a therapeutic target for immunomodulation in autoimmune diseases. ShK-186, a selective inhibitor of Kv1.3 channels, ameliorates autoimmune diseases in rodent models, and human phase 1 trials of this agent in healthy volunteers have been completed. In this study, we identified and characterized a large family of Stichodactyla helianthus toxin (ShK)-related peptides in parasitic worms. Based on phylogenetic analysis, 2 worm peptides were selected for study: AcK1, a 51-residue peptide expressed in the anterior secretory glands of the dog-infecting hookworm Ancylostoma caninum and the human-infecting hookworm Ancylostoma ceylanicum, and BmK1, the C-terminal domain of a metalloprotease from the filarial worm Brugia malayi. These peptides in solution adopt helical structures closely resembling that of ShK. At doses in the nanomolar-micromolar range, they block native Kv1.3 in human T cells and cloned Kv1.3 stably expressed in L929 mouse fibroblasts. They preferentially suppress the proliferation of rat CCR7(-) effector memory T cells without affecting naive and central memory subsets and inhibit the delayed-type hypersensitivity (DTH) response caused by skin-homing effector memory T cells in rats. Further, they suppress IFNγ production by human T lymphocytes. ShK-related peptides in parasitic worms may contribute to the potential beneficial effects of probiotic parasitic worm therapy in human autoimmune diseases.
Assuntos
Doenças Autoimunes/prevenção & controle , Venenos de Cnidários/química , Helmintos/metabolismo , Memória Imunológica/efeitos dos fármacos , Canal de Potássio Kv1.3/antagonistas & inibidores , Fragmentos de Peptídeos/farmacologia , Bloqueadores dos Canais de Potássio/farmacologia , Linfócitos T/efeitos dos fármacos , Sequência de Aminoácidos , Animais , Proliferação de Células , Células Cultivadas , Citocinas/metabolismo , Eletrofisiologia , Feminino , Fibroblastos/efeitos dos fármacos , Fibroblastos/imunologia , Fibroblastos/metabolismo , Humanos , Hipersensibilidade Tardia/prevenção & controle , Espectroscopia de Ressonância Magnética , Masculino , Camundongos , Modelos Moleculares , Dados de Sequência Molecular , Fragmentos de Peptídeos/química , Filogenia , Conformação Proteica , Ratos , Ratos Endogâmicos Lew , Receptores CCR7/metabolismo , Homologia de Sequência de Aminoácidos , Relação Estrutura-Atividade , Linfócitos T/imunologia , Linfócitos T/metabolismoRESUMO
MMP23 is a member of the matrix metalloprotease family of zinc- and calcium-dependent endopeptidases, which are involved in a wide variety of cellular functions. Its catalytic domain displays a high degree of structural homology with those of other metalloproteases, but its atypical domain architecture suggests that it may possess unique functional properties. The N-terminal MMP23 pro-domain contains a type-II transmembrane domain that anchors the protein to the plasma membrane and lacks the cysteine-switch motif that is required to maintain other MMPs in a latent state during passage to the cell surface. Instead of the C-terminal hemopexin domain common to other MMPs, MMP23 contains a small toxin-like domain (TxD) and an immunoglobulin-like cell adhesion molecule (IgCAM) domain. The MMP23 pro-domain can trap Kv1.3 but not closely-related Kv1.2 channels in the endoplasmic reticulum, preventing their passage to the cell surface, while the TxD can bind to the channel pore and block the passage of potassium ions. The MMP23 C-terminal IgCAM domain displays some similarity to Ig-like C2-type domains found in IgCAMs of the immunoglobulin superfamily, which are known to mediate protein-protein and protein-lipid interactions. MMP23 and Kv1.3 are co-expressed in a variety of tissues and together are implicated in diseases including cancer and inflammatory disorders. Further studies are required to elucidate the mechanism of action of this unique member of the MMP family.
Assuntos
Metaloproteinases da Matriz/química , Canais de Potássio/química , Transporte Proteico , Sequência de Aminoácidos , Animais , Domínio Catalítico , Humanos , Metaloproteinases da Matriz/metabolismo , Metaloproteinases da Matriz/fisiologia , Modelos Moleculares , Dados de Sequência Molecular , Potássio/metabolismo , Canais de Potássio/metabolismo , Alinhamento de SequênciaRESUMO
Voltage-gated sodium (Na(V)) and potassium (K(V)) channels are critical components of neuronal action potential generation and propagation. Here, we report that Na(V)ß1 encoded by SCN1b, an integral subunit of Na(V) channels, coassembles with and modulates the biophysical properties of K(V)1 and K(V)7 channels, but not K(V)3 channels, in an isoform-specific manner. Distinct domains of Na(V)ß1 are involved in modulation of the different K(V) channels. Studies with channel chimeras demonstrate that Na(V)ß1-mediated changes in activation kinetics and voltage dependence of activation require interaction of Na(V)ß1 with the channel's voltage-sensing domain, whereas changes in inactivation and deactivation require interaction with the channel's pore domain. A molecular model based on docking studies shows Na(V)ß1 lying in the crevice between the voltage-sensing and pore domains of K(V) channels, making significant contacts with the S1 and S5 segments. Cross-modulation of Na(V) and K(V) channels by Na(V)ß1 may promote diversity and flexibility in the overall control of cellular excitability and signaling.
Assuntos
Canais de Potássio de Abertura Dependente da Tensão da Membrana/metabolismo , Subunidade beta-1 do Canal de Sódio Disparado por Voltagem/metabolismo , Animais , Células CHO , Cricetinae , Cricetulus , Ativação do Canal Iônico , Cinética , Camundongos , Modelos Moleculares , Células PC12 , Canais de Potássio de Abertura Dependente da Tensão da Membrana/química , Ligação Proteica , Estrutura Terciária de Proteína , Ratos , XenopusRESUMO
Bifenthrin, a relatively stable type I pyrethroid that causes tremors and impairs motor activity in rodents, is broadly used. We investigated whether nanomolar bifenthrin alters synchronous Ca(2+) oscillations (SCOs) necessary for activity-dependent dendritic development. Primary mouse cortical neurons were cultured 8 or 9 days in vitro (DIV), loaded with the Ca(2+) indicator Fluo-4, and imaged using a Fluorescence Imaging Plate Reader Tetra. Acute exposure to bifenthrin rapidly increased the frequency of SCOs by 2.7-fold (EC50 = 58 nM) and decreased SCO amplitude by 36%. Changes in SCO properties were independent of modifications in voltage-gated sodium channels since 100 nM bifenthrin had no effect on the whole-cell Na(+) current, nor did it influence neuronal resting membrane potential. The L-type Ca(2+) channel blocker nifedipine failed to ameliorate bifenthrin-triggered SCO activity. By contrast, the metabotropic glutamate receptor (mGluR)5 antagonist MPEP [2-methyl-6-(phenylethynyl)pyridine] normalized bifenthrin-triggered increase in SCO frequency without altering baseline SCO activity, indicating that bifenthrin amplifies mGluR5 signaling independent of Na(+) channel modification. Competitive [AP-5; (-)-2-amino-5-phosphonopentanoic acid] and noncompetitive (dizocilpine, or MK-801 [(5S,10R)-(+)-5-methyl-10,11-dihydro-5H-dibenzo[a,d]cyclohepten-5,10-imine maleate]) N-methyl-d-aspartate antagonists partially decreased both basal and bifenthrin-triggered SCO frequency increase. Bifenthrin-modified SCO rapidly enhanced the phosphorylation of cAMP response element-binding protein (CREB). Subacute (48 hours) exposure to bifenthrin commencing 2 DIV-enhanced neurite outgrowth and persistently increased SCO frequency and reduced SCO amplitude. Bifenthrin-stimulated neurite outgrowth and CREB phosphorylation were dependent on mGluR5 activity since MPEP normalized both responses. Collectively these data identify a new mechanism by which bifenthrin potently alters Ca(2+) dynamics and Ca(2+)-dependent signaling in cortical neurons that have long term impacts on activity driven neuronal plasticity.
Assuntos
Cálcio/metabolismo , Córtex Cerebral/citologia , Inseticidas/toxicidade , Neurônios/efeitos dos fármacos , Piretrinas/toxicidade , Canais de Sódio Disparados por Voltagem/metabolismo , Animais , Células Cultivadas , Proteína de Ligação ao Elemento de Resposta ao AMP Cíclico/metabolismo , Potenciais da Membrana/efeitos dos fármacos , Camundongos , Rede Nervosa/efeitos dos fármacos , Rede Nervosa/fisiologia , Neuritos/efeitos dos fármacos , Neuritos/fisiologia , Neurônios/metabolismo , Fosforilação , Receptor de Glutamato Metabotrópico 5/metabolismo , Transdução de SinaisRESUMO
Matrix metalloproteases (MMPs) are endopeptidases that regulate diverse biological processes. Synthesized as zymogens, MMPs become active after removal of their prodomains. Much is known about the metalloprotease activity of these enzymes, but noncanonical functions are poorly defined, and functions of the prodomains have been largely ignored. Here we report a novel metalloprotease-independent, channel-modulating function for the prodomain of MMP23 (MMP23-PD). Whole-cell patch clamping and confocal microscopy, coupled with deletion analysis, demonstrate that MMP23-PD suppresses the voltage-gated potassium channel KV1.3, but not the closely related KV1.2 channel, by trapping the channel intracellularly. Studies with KV1.2-1.3 chimeras suggest that MMP23-PD requires the presence of the KV1.3 region from the S5 trans-membrane segment to the C terminus to modulate KV1.3 channel function. NMR studies of MMP23-PD reveal a single, kinked trans-membrane α-helix, joined by a short linker to a juxtamembrane α-helix, which is associated with the surface of the membrane and protected from exchange with the solvent. The topological similarity of MMP23-PD to KCNE1, KCNE2, and KCNE4 proteins that trap KV1.3, KV1.4, KV3.3, and KV3.4 channels early in the secretory pathway suggests a shared mechanism of channel regulation. MMP23 and KV1.3 expression is enhanced and overlapping in colorectal cancers where the interaction of the two proteins could affect cell function.
Assuntos
Ativação do Canal Iônico/fisiologia , Canal de Potássio Kv1.3/metabolismo , Metaloendopeptidases/metabolismo , Animais , Células COS , Neoplasias Colorretais/genética , Neoplasias Colorretais/metabolismo , Regulação Neoplásica da Expressão Gênica/genética , Humanos , Canal de Potássio Kv1.2/genética , Canal de Potássio Kv1.2/metabolismo , Canal de Potássio Kv1.3/genética , Metaloendopeptidases/genética , Proteínas de Neoplasias/genética , Proteínas de Neoplasias/metabolismo , Técnicas de Patch-Clamp , Estrutura Secundária de Proteína , Estrutura Terciária de Proteína , Transporte Proteico/fisiologiaRESUMO
Increasing evidence suggests ion channels have critical functions in the differentiation and plasticity of T cells. Kv1.3, a voltage-gated K(+) channel, is a functional marker and a pharmacological target for activated effector memory T cells. Selective Kv1.3 blockers have been shown to inhibit proliferation and cytokine production by human and rat effector memory T cells. We used Kv1.3 knockout (KO) mice to investigate the mechanism by which Kv1.3 blockade affects CD4(+) T cell differentiation during an inflammatory immune-mediated disease. Kv1.3 KO animals displayed significantly lower incidence and severity of myelin oligodendrocyte glycoprotein (MOG) peptide-induced experimental autoimmune encephalomyelitis. Kv1.3 was the only K(V) channel expressed in MOG 35-55-specific CD4(+) T cell blasts, and no K(V) current was present in MOG-specific CD4(+) T cell-blasts from Kv1.3 KO mice. Fewer CD4(+) T cells migrated to the CNS in Kv1.3 KO mice following disease induction, and Ag-specific proliferation of CD4(+) T cells from these mice was impaired with a corresponding cell-cycle delay. Kv1.3 was required for optimal expression of IFN-γ and IL-17, whereas its absence led to increased IL-10 production. Dendritic cells from Kv1.3 KO mice fully activated wild-type CD4(+) T cells, indicating a T cell-intrinsic defect in Kv1.3 KO mice. The loss of Kv1.3 led to a suppressive phenotype, which may contribute to the mechanism by which deletion of Kv1.3 produces an immunotherapeutic effect. Skewing of CD4(+) T cell differentiation toward Ag-specific regulatory T cells by pharmacological blockade or genetic suppression of Kv1.3 might be beneficial for therapy of immune-mediated diseases such as multiple sclerosis.
Assuntos
Encefalomielite Autoimune Experimental/imunologia , Encefalomielite Autoimune Experimental/metabolismo , Canal de Potássio Kv1.3/metabolismo , Ativação Linfocitária/imunologia , Linfócitos T Reguladores/imunologia , Animais , Western Blotting , Linfócitos T CD4-Positivos/citologia , Linfócitos T CD4-Positivos/imunologia , Diferenciação Celular/imunologia , Eletrofisiologia , Encefalomielite Autoimune Experimental/patologia , Ensaio de Imunoadsorção Enzimática , Feminino , Citometria de Fluxo , Camundongos , Camundongos Endogâmicos C57BL , Camundongos Knockout , Camundongos Transgênicos , Fenótipo , Subpopulações de Linfócitos T/citologia , Subpopulações de Linfócitos T/imunologia , Linfócitos T Reguladores/citologiaRESUMO
BACKGROUND: This article aims to demonstrate the morphology of 261 total anomalous pulmonary venous connection (TAPVC) cases operated at Children's Hospital 1 with in-hospital mortality of 19.5% (51/261). METHODS: All the surgical protocols of TAPVC cases repaired between 2008 and June 2023 were reviewed. The descriptions of TAPVC were based on operative findings by surgeons. RESULTS: A total of 261 TAPVC patients were operated, including 124 (47.5%) supra, 83 (31.8%) intra, 41 (15.7%) infra, and 13 (5%) mixed cases. The in-hospital mortality was 19.5% (51/261). Fifteen cases are associated with other anomalies of the heart. Four subtypes of 124 supra TAPVC were found, with 42 (33.9%) obstructed cases. The standard was all pulmonary veins (PVs) forming a common vein (CV) and draining into the innominate veins, then going to the superior vena cava (SVC) (100/124, 80.6%). Eleven supra TAPVC cases were vascular vise type. Ten cases had the vertical vein running from the right of the CV and draining directly into the SVC. Of 83 intracardiac TAPVCs with 9 (10.8%) obstructed cases, the most common was all PVs draining directly into the coronary sinus (60/83, 72.3%). The second was all PVs draining directly into the right atrium (RA) via separated ostia or forming a CV before entering the RA (17/83, 20.5%). Also, there were three cases with rare variants and 100% obstruction when the diagnosis was explored. The in-hospital mortality of intracardiac type was 13.3% (11/83) 41 infra TAPVC with obstructed rate of 61% (25/41) and in-hospital mortality of 29.3% (12/41). Thirteen mixed TAPVCs were repaired, with most cases having three PVs forming a CV. CONCLUSION: This article provides valuable information about the morphology of TAPVC types in Asian patients.
Assuntos
Mortalidade Hospitalar , Veias Pulmonares , Síndrome de Cimitarra , Humanos , Feminino , Masculino , Síndrome de Cimitarra/cirurgia , Síndrome de Cimitarra/mortalidade , Vietnã/epidemiologia , Estudos Retrospectivos , Veias Pulmonares/anormalidades , Veias Pulmonares/cirurgia , Lactente , Recém-Nascido , Pré-Escolar , Procedimentos Cirúrgicos Cardíacos/métodos , CriançaRESUMO
Introduction: Mechanical thrombectomy has improved treatment options and outcomes for acute ischemic stroke with large artery occlusion. However, as the time window of endovascular thrombectomy is extended there is an increasing need to develop immunocytoprotective therapies that can reduce inflammation in the penumbra and prevent reperfusion injury. We previously demonstrated, that by reducing neuroinflammation, KV1.3 inhibitors can improve outcomes not only in young male rodents but also in female and aged animals. To further explore the therapeutic potential of KV1.3 inhibitors for stroke therapy, we here directly compared a peptidic and a small molecule KV1.3 blocker and asked whether KV1.3 inhibition would still be beneficial when started at 72 hours after reperfusion. Methods: Transient middle cerebral artery occlusion (tMCAO, 90-min) was induced in male Wistar rats and neurological deficit assessed daily. On day-8 infarction was determined by T2-weighted MRI and inflammatory marker expression in the brain by quantitative PCR. Potential interactions with tissue plasminogen activator (tPA) were evaluated in-vitro with a chromogenic assay. Results: In a direct comparison with administration started at 2 hours after reperfusion, the small molecule PAP-1 significantly improved outcomes on day-8, while the peptide ShK-223 failed to reduce infarction and neurological deficits despite reducing inflammatory marker expression. PAP-1 still provided benefits when started 72 hours after reperfusion. PAP-1 does not reduce the proteolytic activity of tPA. Discussion: Our studies suggest that KV1.3 inhibition for immunocytoprotection after ischemic stroke has a wide therapeutic window for salvaging the inflammatory penumbra and requires brain-penetrant small molecules.
RESUMO
Epileptogenic seizures, or status epilepticus (SE), leads to excitotoxic injury in hippocampal and limbic neurons in the kainic acid (KA) animal model of temporal lobe epilepsy (TLE). Here, we have further characterized neural activity regulated methylaminoisobutryic acid (MeAIB)/glutamine transport activity in mature rat hippocampal neurons in vitro that is inhibited by riluzole (IC50 = 1 µM), an anti-convulsant benzothiazole agent. We screened a library of riluzole derivatives and identified SKA-41 followed by a second screen and synthesized several novel chlorinated aminothiazoles (SKA-377, SKA-378, SKA-379) that are also potent MeAIB transport inhibitors in vitro, and brain penetrant following systemic administration. When administered before KA, SKA-378 did not prevent seizures but still protected the hippocampus and several other limbic areas against SE-induced neurodegeneration at 3d. When SKA-377 - 379, (30 mg/kg) were administered after KA-induced SE, acute neural injury in the CA3, CA1 and CA4/hilus was also largely attenuated. Riluzole (10 mg/kg) blocks acute neural injury. Kinetic analysis of SKA-378 and riluzoles' blockade of Ca2+-regulated MeAIB transport in neurons in vitro indicates that inhibition occurs via a non-competitive, indirect mechanism. Sodium channel NaV1.6 antagonism blocks neural activity regulated MeAIB/Gln transport in vitro (IC50 = 60 nM) and SKA-378 is the most potent inhibitor of NaV1.6 (IC50 = 28 µM) compared to NaV1.2 (IC50 = 118 µM) in heterologous cells. However, pharmacokinetic analysis suggests that sodium channel blockade may not be the predominant mechanism of neuroprotection here. Riluzole and our novel aminothiazoles are agents that attenuate acute neural hippocampal injury following KA-induced SE and may help to understand mechanisms involved in the progression of epileptic disease.
Assuntos
Epilepsia do Lobo Temporal , Estado Epiléptico , Ratos , Animais , Epilepsia do Lobo Temporal/induzido quimicamente , Epilepsia do Lobo Temporal/tratamento farmacológico , Riluzol/farmacologia , Cinética , Convulsões/induzido quimicamente , Convulsões/tratamento farmacológico , Convulsões/prevenção & controle , Hipocampo , Ácido Caínico/toxicidade , Modelos Animais de DoençasRESUMO
Senicapoc, a small molecule inhibitor of the calcium-activated potassium channel KCa3.1, was safe and well-tolerated in clinical trials for sickle cell anemia. We previously reported proof-of-concept data suggesting that both pharmacological inhibition and genetic deletion of KCa3.1 reduces infarction and improves neurologic recovery in rodents by attenuating neuroinflammation. Here we evaluated the potential of repurposing senicapoc for ischemic stroke. In cultured microglia, senicapoc inhibited KCa3.1 currents with an IC50 of 7 nM, reduced Ca2+ signaling induced by the purinergic agonist ATP, suppressed expression of pro-inflammatory cytokines and enzymes (iNOS and COX-2), and prevented induction of the inflammasome component NLRP3. When transient middle cerebral artery occlusion (tMCAO, 60 min) was induced in male C57BL/6 J mice, twice daily administration of senicapoc at 10 and 40 mg/kg starting 12 h after reperfusion dose-dependently reduced infarct area determined by T2-weighted magnetic resonance imaging (MRI) and improved neurological deficit on day 8. Ultra-high-performance liquid chromatography/mass spectrometry analysis of total and free brain concentrations demonstrated sufficient KCa3.1 target engagement. Senicapoc treatment significantly reduced microglia/macrophage and T cell infiltration and activation and attenuated neuronal death. A different treatment paradigm with senicapoc started at 3 h and MRI on day 3 and day 8 revealed that senicapoc reduces secondary infarct growth and suppresses expression of inflammation markers, including T cell cytokines in the brain. Lastly, we demonstrated that senicapoc does not impair the proteolytic activity of tissue plasminogen activator (tPA) in vitro. We suggest that senicapoc could be repurposed as an adjunctive immunocytoprotective agent for combination with reperfusion therapy for ischemic stroke.