RESUMO
A major driver of neuronal hyperexcitability is dysfunction of K+ channels, including voltage-gated KCNQ2/3 channels. Their hyperpolarized midpoint of activation and slow activation and deactivation kinetics produce a current that regulates membrane potential and impedes repetitive firing. Inherited mutations in KCNQ2 and KCNQ3 are linked to a wide spectrum of neurodevelopmental disorders (NDDs), ranging from benign familial neonatal seizures to severe epileptic encephalopathies and autism spectrum disorders. However, the impact of these variants on the molecular mechanisms underlying KCNQ3 channel function remains poorly understood and existing treatments have significant side effects. Here, we use voltage clamp fluorometry, molecular dynamic simulations, and electrophysiology to investigate NDD-associated variants in KCNQ3 channels. We identified two distinctive mechanisms by which loss- and gain-of function NDD-associated mutations in KCNQ3 affect channel gating: one directly affects S4 movement while the other changes S4-to-pore coupling. MD simulations and electrophysiology revealed that polyunsaturated fatty acids (PUFAs) primarily target the voltage-sensing domain in its activated conformation and form a weaker interaction with the channel's pore. Consistently, two such compounds yielded partial and complete functional restoration in R227Q- and R236C-containing channels, respectively. Our results reveal the potential of PUFAs to be developed into therapies for diverse KCNQ3-based channelopathies.
Assuntos
Canal de Potássio KCNQ3 , Simulação de Dinâmica Molecular , Mutação , Transtornos do Neurodesenvolvimento , Canal de Potássio KCNQ3/genética , Canal de Potássio KCNQ3/metabolismo , Humanos , Transtornos do Neurodesenvolvimento/genética , Transtornos do Neurodesenvolvimento/tratamento farmacológico , Ativação do Canal Iônico/efeitos dos fármacos , Animais , Células HEK293 , Potenciais da Membrana/efeitos dos fármacosRESUMO
Bone cancer pain (BCP) represents a prevalent symptom among cancer patients with bone metastases, yet its underlying mechanisms remain elusive. This study investigated the transcriptional regulation mechanism of Kv7(KCNQ)/M potassium channels in DRG neurons and its involvement in the development of BCP in rats. We show that HDAC2-mediated transcriptional repression of kcnq2/kcnq3 genes, which encode Kv7(KCNQ)/M potassium channels in dorsal root ganglion (DRG), contributes to the sensitization of DRG neurons and the pathogenesis of BCP in rats. Also, HDAC2 requires the formation of a corepressor complex with MeCP2 and Sin3A to execute transcriptional regulation of kcnq2/kcnq3 genes. Moreover, EREG is identified as an upstream signal molecule for HDAC2-mediated kcnq2/kcnq3 genes transcription repression. Activation of EREG/EGFR-ERK-Runx1 signaling, followed by the induction of HDAC2-mediated transcriptional repression of kcnq2/kcnq3 genes in DRG neurons, leads to neuronal hyperexcitability and pain hypersensitivity in tumor-bearing rats. Consequently, the activation of EREG/EGFR-ERK-Runx1 signaling, along with the subsequent transcriptional repression of kcnq2/kcnq3 genes by HDAC2 in DRG neurons, underlies the sensitization of DRG neurons and the pathogenesis of BCP in rats. These findings uncover a potentially targetable mechanism contributing to bone metastasis-associated pain in cancer patients.
Assuntos
Neoplasias Ósseas , Dor do Câncer , Receptores ErbB , Gânglios Espinais , Histona Desacetilase 2 , Canal de Potássio KCNQ2 , Animais , Histona Desacetilase 2/metabolismo , Histona Desacetilase 2/genética , Canal de Potássio KCNQ2/genética , Canal de Potássio KCNQ2/metabolismo , Gânglios Espinais/metabolismo , Gânglios Espinais/patologia , Neoplasias Ósseas/genética , Neoplasias Ósseas/metabolismo , Neoplasias Ósseas/secundário , Neoplasias Ósseas/patologia , Ratos , Dor do Câncer/genética , Dor do Câncer/metabolismo , Dor do Câncer/patologia , Receptores ErbB/metabolismo , Receptores ErbB/genética , Canal de Potássio KCNQ3/genética , Canal de Potássio KCNQ3/metabolismo , Transcrição Gênica , Proteína 2 de Ligação a Metil-CpG/genética , Proteína 2 de Ligação a Metil-CpG/metabolismo , Complexo Correpressor Histona Desacetilase e Sin3/genética , Transdução de Sinais/genética , Subunidade alfa 2 de Fator de Ligação ao Core/genética , Subunidade alfa 2 de Fator de Ligação ao Core/metabolismo , Humanos , Feminino , MAP Quinases Reguladas por Sinal Extracelular/metabolismo , Proteínas Repressoras/genética , Proteínas Repressoras/metabolismo , Ratos Sprague-Dawley , Sistema de Sinalização das MAP Quinases/genéticaRESUMO
Fragile X syndrome (FXS), the most frequent monogenic form of intellectual disability, is caused by transcriptional silencing of the FMR1 gene that could render neuronal hyperexcitability. Here we show that pyramidal cells (PCs) in the dorsal CA1 region of the hippocampus elicited a larger action potential (AP) number in response to suprathreshold stimulation in juvenile Fmr1 knockout (KO) than wild-type (WT) mice. Because Kv7/M channels modulate CA1 PC excitability in rats, we investigated if their dysfunction produces neuronal hyperexcitability in Fmr1 KO mice. Immunohistochemical and western blot analyses showed no differences in the expression of Kv7.2 and Kv7.3 channel subunits between genotypes; however, the current mediated by Kv7/M channels was reduced in Fmr1 KO mice. In both genotypes, bath application of XE991 (10 µM), a blocker of Kv7/M channels: produced an increased AP number, produced an increased input resistance, produced a decreased AP voltage threshold and shaped AP medium afterhyperpolarization by increasing mean velocities. Retigabine (10 µM), an opener of Kv7/M channels, produced opposite effects to XE991. Both XE991 and retigabine abolished differences in all these parameters found in control conditions between genotypes. Furthermore, a low concentration of retigabine (2.5 µM) normalized CA1 PC excitability of Fmr1 KO mice. Finally, ex vivo seizure-like events evoked by 4-aminopyiridine (200 µM) in the dorsal CA1 region were more frequent in Fmr1 KO mice, and were abolished by retigabine (5-10 µM). We conclude that CA1 PCs of Fmr1 KO mice exhibit hyperexcitability, caused by Kv7/M channel dysfunction, and increased epileptiform activity, which were abolished by retigabine. KEY POINTS: Dorsal pyramidal cells of the hippocampal CA1 region of Fmr1 knockout mice exhibit hyperexcitability. Kv7/M channel activity, but not expression, is reduced in pyramidal cells of the hippocampal CA1 region of Fmr1 knockout mice. Kv7/M channel dysfunction causes hyperexcitability in pyramidal cells of the hippocampal CA1 region of Fmr1 knockout mice by increasing input resistance, decreasing AP voltage threshold and shaping medium afterhyperpolarization. A Kv7/M channel opener normalizes neuronal excitability in pyramidal cells of the hippocampal CA1 region of Fmr1 knockout mice. Ex vivo seizure-like events evoked in the dorsal CA1 region were more frequent in Fmr1 KO mice, and such an epileptiform activity was abolished by a Kv7/M channel opener depending on drug concentration. Kv7/M channels may represent a therapeutic target for treating symptoms associated with hippocampal alterations in fragile X syndrome.
Assuntos
Potenciais de Ação , Região CA1 Hipocampal , Proteína do X Frágil da Deficiência Intelectual , Fenilenodiaminas , Células Piramidais , Animais , Masculino , Camundongos , Antracenos/farmacologia , Região CA1 Hipocampal/fisiopatologia , Região CA1 Hipocampal/metabolismo , Carbamatos/farmacologia , Proteína do X Frágil da Deficiência Intelectual/genética , Síndrome do Cromossomo X Frágil/fisiopatologia , Síndrome do Cromossomo X Frágil/genética , Canal de Potássio KCNQ2/genética , Canal de Potássio KCNQ2/metabolismo , Canal de Potássio KCNQ3/genética , Canal de Potássio KCNQ3/metabolismo , Camundongos Endogâmicos C57BL , Camundongos Knockout , Proteínas do Tecido Nervoso , Fenilenodiaminas/farmacologia , Células Piramidais/fisiologia , Células Piramidais/metabolismo , Células Piramidais/efeitos dos fármacosRESUMO
The growing interest in Kv7.2/7.3 agonists originates from the involvement of these channels in several brain hyperexcitability disorders. In particular, Kv7.2/7.3 mutants have been clearly associated with epileptic encephalopathies (DEEs) as well as with a spectrum of focal epilepsy disorders, often associated with developmental plateauing or regression. Nevertheless, there is a lack of available therapeutic options, considering that retigabine, the only molecule used in clinic as a broad-spectrum Kv7 agonist, has been withdrawn from the market in late 2016. This is why several efforts have been made both by both academia and industry in the search for suitable chemotypes acting as Kv7.2/7.3 agonists. In this context, in silico methods have played a major role, since the precise structures of different Kv7 homotetramers have been only recently disclosed. In the present review, the computational methods used for the design of Kv.7.2/7.3 small molecule agonists and the underlying medicinal chemistry are discussed in the context of their biological and structure-function properties.
Assuntos
Canal de Potássio KCNQ2 , Canal de Potássio KCNQ3 , Humanos , Canal de Potássio KCNQ2/metabolismo , Canal de Potássio KCNQ2/genética , Canal de Potássio KCNQ2/química , Canal de Potássio KCNQ3/metabolismo , Canal de Potássio KCNQ3/genética , Canal de Potássio KCNQ3/química , Canal de Potássio KCNQ3/antagonistas & inibidores , Simulação por Computador , Relação Estrutura-Atividade , Descoberta de Drogas/métodos , AnimaisRESUMO
Voltage-gated ion channels are responsible for the electrical excitability of neurons and cardiomyocytes. Thus, they are obvious targets for pharmaceuticals aimed to modulate excitability. Compounds activating voltage-gated potassium (KV) channels are expected to reduce excitability. To search for new KV-channel activators, we performed a high-throughput screen of 10,000 compounds on a specially designed Shaker KV channel. Here, we report on a large family of channel-activating compounds with a carboxyl (COOH) group as the common motif. The most potent COOH activators are lipophilic (4 < LogP <7) and are suggested to bind at the interface between the lipid bilayer and the channel's positively charged voltage sensor. The negatively charged form of the COOH-group compounds is suggested to open the channel by electrostatically pulling the voltage sensor to an activated state. Several of the COOH-group compounds also activate the therapeutically important KV7.2/7.3 channel and can thus potentially be developed into antiseizure drugs. The COOH-group compounds identified in this study are suggested to act via the same site and mechanism of action as previously studied COOH-group compounds, such as polyunsaturated fatty acids and resin acids, but distinct from sites for several other types of potassium channel-activating compounds.
Assuntos
Ativação do Canal Iônico , Animais , Ativação do Canal Iônico/efeitos dos fármacos , Superfamília Shaker de Canais de Potássio/metabolismo , Canal de Potássio KCNQ2/metabolismo , Canal de Potássio KCNQ2/agonistas , Canais de Potássio de Abertura Dependente da Tensão da Membrana/metabolismo , Canais de Potássio de Abertura Dependente da Tensão da Membrana/efeitos dos fármacos , Canal de Potássio KCNQ3/metabolismo , Humanos , Xenopus laevisRESUMO
The human voltage-gated potassium channel KCNQ2/KCNQ3 carries the neuronal M-current, which helps to stabilize the membrane potential. KCNQ2 can be activated by analgesics and antiepileptic drugs but their activation mechanisms remain unclear. Here we report cryo-electron microscopy (cryo-EM) structures of human KCNQ2-CaM in complex with three activators, namely the antiepileptic drug cannabidiol (CBD), the lipid phosphatidylinositol 4,5-bisphosphate (PIP2), and HN37 (pynegabine), an antiepileptic drug in the clinical trial, in an either closed or open conformation. The activator-bound structures, along with electrophysiology analyses, reveal the binding modes of two CBD, one PIP2, and two HN37 molecules in each KCNQ2 subunit, and elucidate their activation mechanisms on the KCNQ2 channel. These structures may guide the development of antiepileptic drugs and analgesics that target KCNQ2.
Assuntos
Analgésicos , Anticonvulsivantes , Humanos , Anticonvulsivantes/farmacologia , Microscopia Crioeletrônica , Ligantes , Potenciais da Membrana , Canal de Potássio KCNQ2/química , Canal de Potássio KCNQ2/metabolismo , Canal de Potássio KCNQ3/metabolismoRESUMO
Voltage-sensitive potassium channels play an important role in controlling membrane potential and ionic homeostasis in the gut and have been implicated in gastrointestinal (GI) cancers. Through large-scale analysis of 897 patients with gastro-oesophageal adenocarcinomas (GOAs) coupled with in vitro models, we find KCNQ family genes are mutated in â¼30% of patients, and play therapeutically targetable roles in GOA cancer growth. KCNQ1 and KCNQ3 mediate the WNT pathway and MYC to increase proliferation through resultant effects on cadherin junctions. This also highlights novel roles of KCNQ3 in non-excitable tissues. We also discover that activity of KCNQ3 sensitises cancer cells to existing potassium channel inhibitors and that inhibition of KCNQ activity reduces proliferation of GOA cancer cells. These findings reveal a novel and exploitable role of potassium channels in the advancement of human cancer, and highlight that supplemental treatments for GOAs may exist through KCNQ inhibitors.
Assuntos
Adenocarcinoma , Canais de Potássio KCNQ , Humanos , Canais de Potássio KCNQ/genética , Canais de Potássio KCNQ/metabolismo , Canal de Potássio KCNQ3/genética , Canal de Potássio KCNQ3/metabolismo , Canal de Potássio KCNQ2/fisiologia , Adenocarcinoma/genéticaRESUMO
Neuronal Kv7 voltage-gated potassium channels generate the M-current and regulate neuronal excitability. Here, we report that dehydroepiandrosterone sulfate (DHEAS) is an endogenous Kv7 channel modulator that attenuates Gq-coupled receptor-induced M-current suppression. DHEAS reduced muscarinic agonist-induced Kv7-current suppression of Kv7.1, Kv7.2, Kv7.4, or Kv7.5 homomeric currents and endogenous M-currents in rat sympathetic ganglion neurons. However, DHEAS per se did not alter the voltage dependence of these Kv7 homomeric channels or the m1 receptor-induced activation of phospholipase C or protein kinase C. DHEAS-treated Kv7.2 homomeric currents became resistant to depletion of phosphatidylinositol 4,5-bisphosphate (PIP2) induced by voltage-activated phosphatase, Ci-VSP or eVSP. Our computational models predicted a novel binding site for DHEAS in the cytoplasmic domain of Kv7 subunits. A single-point mutation of the predicted key histidine into cysteine in the rat Kv7.2 subunit, rKv7.2(H558C), resulted in a loss of effects of DHEAS on muscarinic Kv7 current suppression. Furthermore, in vivo administration of DHEAS in mice of both sexes reduced late phase pain responses in the formalin paw test. However, it did not have effects on early phase responses in the formalin paw test or responses in the hot plate test. Coadministration of a selective Kv7 inhibitor, XE991, and DHEAS eliminated analgesic effects of DHEAS in late phase responses in the formalin paw test. Collectively, these results suggest that DHEAS attenuates M-current suppression by stabilizing PIP2-Kv7 subunit interaction and can mitigate inflammatory pain.SIGNIFICANCE STATEMENT M-current suppression induced by stimulation of Gq-coupled receptors is a form of Kv7 current modulation that can reversibly increase neuronal excitability. This study demonstrates that DHEAS, an endogenous steroid hormone, is a novel Kv7 channel modulator that can attenuate M-current suppression without affecting basal Kv7 channel kinetics. Administration of DHEAS in vivo alleviated inflammatory pain in rodents. These results suggest that the degree of M-current suppression can be dynamically regulated by small molecules. Therefore, this novel form of Kv7 channel regulation holds promising potential as a therapeutic target for sensitized nervous activities, such as inflammatory pain.
Assuntos
Canal de Potássio KCNQ2 , Agonistas Muscarínicos , Masculino , Feminino , Camundongos , Ratos , Animais , Sulfato de Desidroepiandrosterona , Canal de Potássio KCNQ2/metabolismo , Agonistas Muscarínicos/farmacologia , Dor/tratamento farmacológico , Formaldeído , Canal de Potássio KCNQ3/genética , Canal de Potássio KCNQ3/metabolismoRESUMO
Autism spectrum disorders (ASD) represent neurodevelopmental disorders characterized by social deficits, repetitive behaviors, and various comorbidities, including epilepsy. ANK2, which encodes a neuronal scaffolding protein, is frequently mutated in ASD, but its in vivo functions and disease-related mechanisms are largely unknown. Here, we report that mice with Ank2 knockout restricted to cortical and hippocampal excitatory neurons (Ank2-cKO mice) show ASD-related behavioral abnormalities and juvenile seizure-related death. Ank2-cKO cortical neurons show abnormally increased excitability and firing rate. These changes accompanied decreases in the total level and function of the Kv7.2/KCNQ2 and Kv7.3/KCNQ3 potassium channels and the density of these channels in the enlengthened axon initial segment. Importantly, the Kv7 agonist, retigabine, rescued neuronal excitability, juvenile seizure-related death, and hyperactivity in Ank2-cKO mice. These results suggest that Ank2 regulates neuronal excitability by regulating the length of and Kv7 density in the AIS and that Kv7 channelopathy is involved in Ank2-related brain dysfunctions.
Assuntos
Epilepsia , Canais de Potássio KCNQ , Animais , Camundongos , Epilepsia/metabolismo , Canais de Potássio KCNQ/genética , Canal de Potássio KCNQ2/genética , Canal de Potássio KCNQ2/metabolismo , Canal de Potássio KCNQ3/metabolismo , Neurônios/metabolismo , Convulsões/genética , Convulsões/metabolismoRESUMO
Mutations in the KCNQ2 gene encoding KV7.2 subunit that mediates neuronal M-current cause a severe form of developmental and epileptic encephalopathy (DEE). Electrophysiological evaluation of KCNQ2 mutations has been proved clinically useful in improving outcome prediction and choosing rational anti-seizure medications (ASMs). In this study we described the clinical characteristics, electrophysiological phenotypes and the in vitro response to KCNQ openers of five KCNQ2 pore mutations (V250A, N258Y, H260P, A265T and G290S) from seven patients diagnosed with KCNQ2-DEE. The KCNQ2 variants were transfected into Chinese hamster ovary (CHO) cells alone, in combination with KCNQ3 (1:1) or with wild-type KCNQ2 (KCNQ2-WT) and KCNQ3 in a ratio of 1:1:2, respectively. Their expression and electrophysiological function were assessed. When transfected alone or in combination with KCNQ3, none of these mutations affected the membrane expression of KCNQ2, but most failed to induce a potassium current except A265T, in which trace currents were observed when co-transfected with KCNQ3. When co-expressed with KCNQ2-WT and KCNQ3 (1:1:2), the currents at 0 mV of these mutations were decreased by 30%-70% compared to the KCNQ2/3 channel, which could be significantly rescued by applying KCNQ openers including the approved antiepileptic drug retigabine (RTG, 10 µM), as well as two candidates subjected to clinical trials, pynegabine (HN37, 1 µM) and XEN1101 (1 µM). These newly identified pathologic variants enrich the KCNQ2-DEE mutation hotspots in the pore-forming domain. This electrophysiological study provides a rational basis for personalized therapy with KCNQ openers in DEE patients carrying loss-of-function (LOF) mutations in KCNQ2.
Assuntos
Encefalopatias , Canal de Potássio KCNQ2 , Cricetinae , Animais , Canal de Potássio KCNQ2/genética , Canal de Potássio KCNQ2/metabolismo , Canal de Potássio KCNQ3/genética , Canal de Potássio KCNQ3/metabolismo , Células CHO , Cricetulus , Mutação , Encefalopatias/genéticaRESUMO
Zinc (Zn) is an essential trace element; it serves as a cofactor for a great number of enzymes, transcription factors, receptors, and other proteins. Zinc is also an important signaling molecule, which can be released from intracellular stores into the cytosol or extracellular space, for example, during synaptic transmission. Amongst cellular effects of zinc is activation of Kv7 (KCNQ, M-type) voltage-gated potassium channels. Here, we investigated relationships between Kv7 channel inhibition by Ca2+/calmodulin (CaM) and zinc-mediated potentiation. We show that Zn2+ ionophore, zinc pyrithione (ZnPy), can prevent or reverse Ca2+/CaM-mediated inhibition of Kv7.2. In the presence of both Ca2+ and Zn2+, the Kv7.2 channels lose most of their voltage dependence and lock in an open state. In addition, we demonstrate that mutations that interfere with CaM binding to Kv7.2 and Kv7.3 reduced channel membrane abundance and activity, but these mutants retained zinc sensitivity. Moreover, the relative efficacy of ZnPy to activate these mutants was generally greater, compared with the WT channels. Finally, we show that zinc sensitivity was retained in Kv7.2 channels assembled with mutant CaM with all four EF hands disabled, suggesting that it is unlikely to be mediated by CaM. Taken together, our findings indicate that zinc is a potent Kv7 stabilizer, which may protect these channels from physiological inhibitory effects of neurotransmitters and neuromodulators, protecting neurons from overactivity.
Assuntos
Cálcio , Calmodulina , Espaço Intracelular , Canais de Potássio KCNQ , Zinco , Sinalização do Cálcio , Calmodulina/metabolismo , Canais de Potássio KCNQ/antagonistas & inibidores , Canais de Potássio KCNQ/química , Canais de Potássio KCNQ/genética , Canais de Potássio KCNQ/metabolismo , Mutação , Ligação Proteica/genética , Zinco/farmacologia , Zinco/metabolismo , Espaço Intracelular/metabolismo , Cálcio/metabolismo , Canal de Potássio KCNQ2/antagonistas & inibidores , Canal de Potássio KCNQ2/química , Canal de Potássio KCNQ2/genética , Canal de Potássio KCNQ2/metabolismo , Canal de Potássio KCNQ3/antagonistas & inibidores , Canal de Potássio KCNQ3/química , Canal de Potássio KCNQ3/genética , Canal de Potássio KCNQ3/metabolismoRESUMO
KCNQ channels participate in the physiology of several cell types. In neurons of the central nervous system, the primary subunits are KCNQ2, 3, and 5. Activation of these channels silence the neurons, limiting action potential duration and preventing high-frequency action potential burst. Loss-of-function mutations of the KCNQ channels are associated with a wide spectrum of phenotypes characterized by hyperexcitability. Hence, pharmacological activation of these channels is an attractive strategy to treat epilepsy and other hyperexcitability conditions as are the evolution of stroke and traumatic brain injury. In this work we show that triclosan, a bactericide widely used in personal care products, activates the KCNQ3 channels but not the KCNQ2. Triclosan induces a voltage shift in the activation, increases the conductance, and slows the closing of the channel. The response is independent of PIP2. Molecular docking simulations together with site-directed mutagenesis suggest that the putative binding site is in the voltage sensor domain. Our results indicate that triclosan is a new activator for KCNQ channels.
Assuntos
Epilepsia , Triclosan , Epilepsia/metabolismo , Humanos , Canais de Potássio KCNQ/metabolismo , Canal de Potássio KCNQ1 , Canal de Potássio KCNQ2/química , Canal de Potássio KCNQ2/genética , Canal de Potássio KCNQ2/metabolismo , Canal de Potássio KCNQ3/química , Canal de Potássio KCNQ3/genética , Canal de Potássio KCNQ3/metabolismo , Simulação de Acoplamento Molecular , Neurotransmissores , Triclosan/farmacologiaRESUMO
KCNQ2 and KCNQ3 channels are associated with multiple neurodevelopmental disorders and are also therapeutic targets for neurological and neuropsychiatric diseases. For more than two decades, it has been thought that most KCNQ channels in the brain are either KCNQ2/3 or KCNQ3/5 heteromers. Here, we investigated the potential heteromeric compositions of KCNQ2-containing channels. We applied split-intein protein trans-splicing to form KCNQ2/5 tandems and coexpressed these with and without KCNQ3. Unexpectedly, we found that KCNQ2/5 tandems form functional channels independent of KCNQ3 in heterologous cells. Using mass spectrometry, we went on to demonstrate that KCNQ2 associates with KCNQ5 in native channels in the brain, even in the absence of KCNQ3. Additionally, our functional heterologous expression data are consistent with the formation of KCNQ2/3/5 heteromers. Thus, the composition of KCNQ channels is more diverse than has been previously recognized, necessitating a re-examination of the genotype/phenotype relationship of KCNQ2 pathogenic variants.
Assuntos
Canais de Potássio KCNQ , Canal de Potássio KCNQ3 , Animais , Encéfalo/metabolismo , Genótipo , Canais de Potássio KCNQ/genética , Canais de Potássio KCNQ/metabolismo , Canal de Potássio KCNQ2/genética , Canal de Potássio KCNQ2/metabolismo , Canal de Potássio KCNQ3/genética , Canal de Potássio KCNQ3/metabolismo , Camundongos , Proteínas do Tecido Nervoso/metabolismo , Fenótipo , Processamento de ProteínaRESUMO
Cannabidiol (CBD), a chemical found in the Cannabis sativa plant, is a clinically effective antiepileptic drug whose mechanism of action is unknown. Using a fluorescence-based thallium flux assay, we performed a large-scale screen and found enhancement of flux through heterologously expressed human Kv7.2/7.3 channels by CBD. Patch-clamp recordings showed that CBD acts at submicromolar concentrations to shift the voltage dependence of Kv7.2/7.3 channels in the hyperpolarizing direction, producing a dramatic enhancement of current at voltages near -50 mV. CBD enhanced native M-current in mouse superior cervical ganglion starting at concentrations of 30 nM and also enhanced M-current in rat hippocampal neurons. The potent enhancement of Kv2/7.3 channels by CBD may contribute to its effectiveness as an antiepileptic drug by reducing neuronal hyperexcitability.
Assuntos
Canabidiol/farmacologia , Canal de Potássio KCNQ2/metabolismo , Canal de Potássio KCNQ3/metabolismo , Neurônios/metabolismo , Animais , Células CHO , Cricetinae , Cricetulus , Fenômenos Eletrofisiológicos/efeitos dos fármacos , Regulação da Expressão Gênica/efeitos dos fármacos , Humanos , Canal de Potássio KCNQ2/genética , Canal de Potássio KCNQ3/genética , Neurônios/efeitos dos fármacos , RatosRESUMO
Sleep quality declines with age; however, the underlying mechanisms remain elusive. We found that hyperexcitable hypocretin/orexin (Hcrt/OX) neurons drive sleep fragmentation during aging. In aged mice, Hcrt neurons exhibited more frequent neuronal activity epochs driving wake bouts, and optogenetic activation of Hcrt neurons elicited more prolonged wakefulness. Aged Hcrt neurons showed hyperexcitability with lower KCNQ2 expression and impaired M-current, mediated by KCNQ2/3 channels. Single-nucleus RNA-sequencing revealed adaptive changes to Hcrt neuron loss in the aging brain. Disruption of Kcnq2/3 genes in Hcrt neurons of young mice destabilized sleep, mimicking aging-associated sleep fragmentation, whereas the KCNQ-selective activator flupirtine hyperpolarized Hcrt neurons and rejuvenated sleep architecture in aged mice. Our findings demonstrate a mechanism underlying sleep instability during aging and a strategy to improve sleep continuity.
Assuntos
Envelhecimento , Neurônios/fisiologia , Orexinas/fisiologia , Privação do Sono/fisiopatologia , Sono , Vigília , Aminopiridinas/farmacologia , Animais , Sistemas CRISPR-Cas , Eletroencefalografia , Eletromiografia , Feminino , Região Hipotalâmica Lateral/fisiopatologia , Canal de Potássio KCNQ2/genética , Canal de Potássio KCNQ2/metabolismo , Canal de Potássio KCNQ3/genética , Canal de Potássio KCNQ3/metabolismo , Masculino , Camundongos , Narcolepsia/genética , Narcolepsia/fisiopatologia , Proteínas do Tecido Nervoso/genética , Proteínas do Tecido Nervoso/metabolismo , Vias Neurais , Optogenética , Técnicas de Patch-Clamp , RNA-Seq , Qualidade do SonoRESUMO
The KCNQ family (KCNQ1-KCNQ5) of voltage-gated potassium channels plays critical roles in many physiological and pathological processes. It is known that the channel opening of all KCNQs relies on the signaling lipid molecule phosphatidylinositol 4,5-bisphosphate (PIP2). However, the molecular mechanism of PIP2 in modulating the opening of the four neuronal KCNQ channels (KCNQ2-KCNQ5), which are essential for regulating neuronal excitability, remains largely elusive. Here, we report the cryoelectron microscopy (cryo-EM) structures of human KCNQ4 determined in complex with the activator ML213 in the absence or presence of PIP2. Two PIP2 molecules are identified in the open-state structure of KCNQ4, which act as a bridge to couple the voltage-sensing domain (VSD) and pore domain (PD) of KCNQ4 leading to the channel opening. Our findings reveal the binding sites and activation mechanisms of ML213 and PIP2 for neuronal KCNQ channels, providing a framework for therapeutic intervention targeting on these important channels.
Assuntos
Fosfatidilinositol 4,5-Difosfato , Canais de Potássio de Abertura Dependente da Tensão da Membrana , Microscopia Crioeletrônica , Humanos , Canais de Potássio KCNQ/metabolismo , Canal de Potássio KCNQ2/genética , Canal de Potássio KCNQ2/metabolismo , Canal de Potássio KCNQ3/metabolismo , Ligantes , Neurônios/metabolismo , Fosfatidilinositol 4,5-Difosfato/metabolismoRESUMO
AIMS: Elevated sympathetic outflow is associated with primary hypertension. However, the mechanisms involved in heightened sympathetic outflow in hypertension are unclear. The central amygdala (CeA) regulates autonomic components of emotions through projections to the brainstem. The neuronal Kv7 channel is a non-inactivating voltage-dependent K+ channel encoded by KCNQ2/3 genes involved in stabilizing the neuronal membrane potential and regulating neuronal excitability. In this study, we investigated if altered Kv7 channel activity in the CeA contributes to heightened sympathetic outflow in hypertension. METHODS AND RESULTS: The mRNA and protein expression levels of Kv7.2/Kv7.3 in the CeA were significantly reduced in spontaneously hypertensive rats (SHRs) compared with Wistar-Kyoto (WKY) rats. Lowering blood pressure with coeliac ganglionectomy in SHRs did not alter Kv7.2 and Kv7.3 channel expression levels in the CeA. Fluospheres were injected into the rostral ventrolateral medulla (RVLM) to retrogradely label CeA neurons projecting to the RVLM (CeA-RVLM neurons). Kv7 channel currents recorded from CeA-RVLM neurons in brain slices were much smaller in SHRs than in WKY rats. Furthermore, the basal firing activity of CeA-RVLM neurons was significantly greater in SHRs than in WKY rats. Bath application of specific Kv7 channel blocker 10, 10-bis (4-pyridinylmethyl)-9(10H)-anthracnose (XE-991) increased the excitability of CeA-RVLM neurons in WKY rats, but not in SHRs. Microinjection of XE-991 into the CeA increased arterial blood pressure (ABP) and renal sympathetic nerve activity (RSNA), while microinjection of Kv7 channel opener QO-58 decreased ABP and RSNA, in anaesthetized WKY rats but not SHRs. CONCLUSIONS: Our findings suggest that diminished Kv7 channel activity in the CeA contributes to elevated sympathetic outflow in primary hypertension. This novel information provides new mechanistic insight into the pathogenesis of neurogenic hypertension.
Assuntos
Pressão Arterial , Núcleo Central da Amígdala/metabolismo , Hipertensão/metabolismo , Canal de Potássio KCNQ2/metabolismo , Canal de Potássio KCNQ3/metabolismo , Bulbo/metabolismo , Potássio/metabolismo , Sistema Nervoso Simpático/fisiopatologia , Animais , Núcleo Central da Amígdala/fisiopatologia , Modelos Animais de Doenças , Hipertensão/genética , Hipertensão/fisiopatologia , Canal de Potássio KCNQ2/genética , Canal de Potássio KCNQ3/genética , Proteínas Luminescentes/genética , Proteínas Luminescentes/metabolismo , Masculino , Bulbo/fisiopatologia , Potenciais da Membrana , Camundongos Endogâmicos C57BL , Camundongos Transgênicos , Técnicas de Rastreamento Neuroanatômico , Neurônios/metabolismo , Ratos Endogâmicos SHR , Ratos Endogâmicos WKY , Transdução de Sinais , Proteína Vesicular 2 de Transporte de Glutamato/genética , Proteína Vermelha FluorescenteRESUMO
Sentrin/small ubiquitin-like modifier (SUMO)-specific protease 2 (SENP2)-deficient mice develop spontaneous seizures in early life because of a marked reduction in M currents, which regulate neuronal membrane excitability. We have previously shown that hyper-SUMOylation of the Kv7.2 and Kv7.3 channels is critically involved in the regulation of the M currents conducted by these potassium voltage-gated channels. Here, we show that hyper-SUMOylation of the Kv7.2 and Kv7.3 proteins reduced binding to the lipid secondary messenger PIP2. CaM1 has been shown to be tethered to the Kv7 subunits via hydrophobic motifs in its C termini and implicated in the channel assembly. Mutation of the SUMOylation sites on Kv7.2 and Kv7.3 specifically resulted in decreased binding to CaM1 and enhanced CaM1-mediated assembly of Kv7.2 and Kv7.3, whereas hyper-SUMOylation of Kv7.2 and Kv7.3 inhibited channel assembly. SENP2-deficient mice exhibited increased acetylcholine levels in the brain and the heart tissue because of increases in the vagal tone induced by recurrent seizures. The SENP2-deficient mice develop seizures followed by a period of sinus pauses or atrioventricular conduction blocks. Chronic administration of the parasympathetic blocker atropine or unilateral vagotomy significantly prolonged the life of the SENP2-deficient mice. Furthermore, we showed that retigabine, an M-current opener, reduced the transcription of SUMO-activating enzyme SAE1 and inhibited SUMOylation of the Kv7.2 and Kv7.3 channels, and also prolonged the life of SENP2-deficient mice. Taken together, the previously demonstrated roles of PIP2, CaM1, and retigabine on the regulation of Kv7.2 and Kv7.3 channel function can be explained by their roles in regulating SUMOylation of this critical potassium channel.
Assuntos
Cisteína Endopeptidases/metabolismo , Canal de Potássio KCNQ2/metabolismo , Canal de Potássio KCNQ3/metabolismo , Sistemas do Segundo Mensageiro , Sumoilação , Motivos de Aminoácidos , Animais , Encéfalo/metabolismo , Cisteína Endopeptidases/genética , Canal de Potássio KCNQ2/genética , Canal de Potássio KCNQ3/genética , Camundongos , Camundongos Mutantes , Miocárdio/metabolismo , Convulsões/genética , Convulsões/metabolismo , Enzimas Ativadoras de Ubiquitina/genética , Enzimas Ativadoras de Ubiquitina/metabolismoRESUMO
BACKGROUND/AIMS: The Amyloid Precursor Protein (APP) is involved in the regulation of multiple cellular functions via protein-protein interactions and has been most studied with respect to Alzheimer's disease (AD). Abnormal processing of the single transmembrane-spanning C99 fragment of APP contributes to the formation of amyloid plaques, which are causally related to AD. Pathological C99 accumulation is thought to associate with early cognitive defects in AD. Here, unexpectedly, sequence analysis revealed that C99 exhibits 24% sequence identity with the KCNE1 voltage-gated potassium (Kv) channel ß subunit, comparable to the identity between KCNE1 and KCNE2-5 (21-30%). This suggested the possibility of C99 regulating Kv channels. METHODS: We quantified the effects of C99 on Kv channel function, using electrophysiological analysis of subunits expressed in Xenopus laevis oocytes, biochemical and immunofluorescence techniques. RESULTS: C99 isoform-selectively inhibited (by 30-80%) activity of a range of Kv channels. Among the KCNQ (Kv7) family, C99 isoform-selectively inhibited, shifted the voltage dependence and/or slowed activation of KCNQ2, KCNQ3, KCNQ2/3 and KCNQ5, with no effects on KCNQ1, KCNQ1-KCNE1 or KCNQ4. C99/APP co-localized with KCNQ2 and KCNQ3 in adult rat sciatic nerve nodes of Ranvier. Both C99 and full-length APP co-immunoprecipitated with KCNQ2 in vitro, yet unlike C99, APP only weakly affected KCNQ2/3 activity. Finally, C99 altered the effects on KCNQ2/3 function of inhibitors tetraethylammounium and XE991, but not openers retigabine and ICA27243. CONCLUSION: Our findings raise the possibility of C99 accumulation early in AD altering cellular excitability by modulating Kv channel activity.