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Introduction: The KCNQ2/KCNQ3 genes encode the voltage-gated K channel underlying the neuronal M-current, regulating neuronal excitability. Loss-of-function (LoF) variants cause neonatal epilepsy, treatable with the M-current-opener retigabine, which is no longer marketed due to side effects. Gain-of-function (GoF) variants cause developmental encephalopathy and autism that could be amenable to M-current, but such therapies are not clinically available. In this translational project, we investigated whether donepezil, a cholinergic drug used in Alzheimer's, suppresses M currents in vitro and improves cognitive symptoms in patients with GoF variants. Methods: (1) The effect of 1 µM donepezil on the amplitude of the M-current was measured in excitatory and inhibitory neurons of mouse primary cultured hippocampal cells. M-current was measured using the standard deactivation protocol (holding at 0 mV and deactivation at -60 mV) in the voltage-clamp configuration of the whole-cell patch clamp technique. The impact of donepezil was also examined on the spontaneous firing activity of hippocampal neurons in the current-clamp configuration. (2) Four children with autism, aged 2.5-8 years, with the following GoF variants were enrolled: KCNQ2 (p. Arg144Gln) and KCNQ 3 (p.Arg227Gln, p.Arg230Cys). Patients were treated off-label with donepezil 2.5-5 mg/d for 12 months and assessed with: clinical Global Impression of Change (CGI-c), Childhood Autism Rating Scale 2 (CARS-2), Adaptive Behavior Assessment System-II (ABAS-II), and Child Development Inventory (CDI). Results: (1) Application of donepezil for at least 6 min produced a significant inhibition of the M-current with an IC50 of 0.4 µM. At 1 µM, donepezil reduced by 67% the M-current density of excitatory neurons (2.4 ± 0.46 vs. 0.89 ± 0.15 pA/pF, p < 0.05*). In inhibitory neurons, application of 1 µM donepezil produced a lesser inhibition of 59% of the M-current density (1.39 ± 0.43 vs. 0.57 ± 0.21, p > 0.05). Donepezil (1 µM) potently increased by 2.6-fold the spontaneous firing frequency, which was prevented by the muscarinic receptor antagonist atropine (10 µM). (2) The CARS-2 decreased by 3.8 ± 4.9 points (p > 0.05), but in two patients with KCNQ3 variants, the improvement was over the 4.5 clinically relevant threshold. The global clinical change was also clinically significant in these patients (CGI-c = 1). The CDI increased by 65% (p < 0.05*), while the ABAS-II remained unchanged. Discussion: Donepezil should be repurposed as a novel alternative treatment for GoF variants in KCNQ2/KCNQ3 encephalopathy.

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Chronic pain is common in our population, and most of these patients are inadequately treated, making the development of safer analgesics a high priority. Knee osteoarthritis (OA) is a primary cause of chronic pain and disability worldwide, and lower extremity OA is a major contributor to loss of quality-adjusted life-years. In this study we tested the hypothesis that a novel JDNI8 replication-defective herpes simplex-1 viral vector (rdHSV) incorporating a modified carbonic anhydrase-8 transgene (CA8*) produces analgesia and treats monoiodoacetate-induced (MIA) chronic knee pain due to OA. We observed transduction of lumbar DRG sensory neurons with these viral constructs (vHCA8*) (~40% of advillin-positive cells and ~ 50% of TrkA-positive cells colocalized with V5-positive cells) using the intra-articular (IA) knee joint (KJ) route of administration. vHCA8* inhibited chronic mechanical OA knee pain induced by MIA was dose- and time-dependent. Mechanical thresholds returned to Baseline by D17 after IA KJ vHCA8* treatment, and exceeded Baseline (analgesia) through D65, whereas negative controls failed to reach Baseline responses. Weight-bearing and automated voluntary wheel running were improved by vHCA8*, but not negative controls. Kv7 voltage-gated potassium channel-specific inhibitor XE-991 reversed vHCA8*-induced analgesia. Using IHC, IA KJ of vHCA8* activated DRG Kv7 channels via dephosphorylation, but negative controls failed to impact Kv7 channels. XE-991 stimulated Kv7.2-7.5 and Kv7.3 phosphorylation using western blotting of differentiated SH-SY5Y cells, which was inhibited by vHCA8* but not by negative controls. The observed prolonged dose-dependent therapeutic effects of IA KJ administration of vHCA8* on MIA-induced chronic KJ pain due to OA is consistent with the specific activation of Kv7 channels in small DRG sensory neurons. Together, these data demonstrate for the first-time local IA KJ administration of vHCA8* produces opioid-independent analgesia in this MIA-induced OA chronic pain model, supporting further therapeutic development.

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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.

Asunto(s)

Neoplasias Óseas , Dolor en Cáncer , Receptores ErbB , Ganglios Espinales , Histona Desacetilasa 2 , Canal de Potasio KCNQ2 , Animales , Histona Desacetilasa 2/metabolismo , Histona Desacetilasa 2/genética , Canal de Potasio KCNQ2/genética , Canal de Potasio KCNQ2/metabolismo , Ganglios Espinales/metabolismo , Ganglios Espinales/patología , Neoplasias Óseas/genética , Neoplasias Óseas/metabolismo , Neoplasias Óseas/secundario , Neoplasias Óseas/patología , Ratas , Dolor en Cáncer/genética , Dolor en Cáncer/metabolismo , Dolor en Cáncer/patología , Receptores ErbB/metabolismo , Receptores ErbB/genética , Canal de Potasio KCNQ3/genética , Canal de Potasio KCNQ3/metabolismo , Transcripción Genética , Proteína 2 de Unión a Metil-CpG/genética , Proteína 2 de Unión a Metil-CpG/metabolismo , Complejo Correpresor Histona Desacetilasa y Sin3/genética , Transducción de Señal/genética , Subunidad alfa 2 del Factor de Unión al Sitio Principal/genética , Subunidad alfa 2 del Factor de Unión al Sitio Principal/metabolismo , Humanos , Femenino , Quinasas MAP Reguladas por Señal Extracelular/metabolismo , Proteínas Represoras/genética , Proteínas Represoras/metabolismo , Ratas Sprague-Dawley , Sistema de Señalización de MAP Quinasas/genética

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OBJECTIVE: To summarize the clinical features and genetic mutation characteristics of Chinese children with KCNQ2-related epilepsy. METHODS: A cohort of children with genetically caused epilepsy was evaluated at Linyi People's Hospital from January 2017 to December 2023. After next-generation sequencing and pathogenicity analysis, we summarized the medical records and genetic testing data of the children who had KCNQ2 gene mutations. RESULTS: We identified 23 KCNQ2 gene mutations. 73.9% (n = 17) of the mutation sites were located in S5-S6 segments and the C-terminal region. In addition to the common phenotypes, 2 new phenotypes were identified: infantile convulsion with paroxysmal choreoathetosis (ICCA) and febrile seizure plus (FS+). Of all the cases with abnormal video-electro-encephalography, three cases with self-limited familial infantile epilepsy (SeLNE) exhibited a small number of multifocal discharges. Of the patients who have taken a particular antiepileptic drug, the statistics on the number of patients who have responded to the drug are as follows: oxcarbazepine (8/9, 88.9%), levetiracetam (5/7, 71.4%), phenobarbital (9/16, 56.3%), and topiramate (2/5, 40.0%). However, the efficacy of phenobarbital varied widely in treating SeLNE and KCNQ2-DEE. At the final follow-up, 1 case with SeLNE had a transient developmental regression and 7 cases with KCNQ2-DEE had mild to severe developmental backwardness. SIGNIFICANCE: Although clinically rare, we report 10 new KCNQ2 mutations and two new phenotypes: ICCA and FS+. This further expands genetic and phenotypic spectrum of KCNQ2-related epilepsy. The gene mutation sites are mostly located in S5-S6 segments and the C-terminal region, and the former is usually associated with KCNQ2-DEE. Sodium channel blockers (including oxcarbazepine and topiramate) and levetiracetam should be prioritized over phenobarbital for KCNQ2-DEE. Some cases with KCNQ2-related epilepsy may have transient developmental regression during periods of frequent seizures. Early treatment and early seizure control may be beneficial for willing outcomes in children with KCNQ2-DEE. PLAIN LANGUAGE SUMMARY: This article reports 23 cases of children with KCNQ2-related epilepsy, including 10 new mutation sites and 2 new phenotypes. It further expands the genetic and phenotypic spectrum of KCNQ2-related epilepsy. In addition, the article summarizes the gene mutation characteristics and clinical manifestations of children with KCNQ2-related epilepsy, with the expectation of providing a certain theoretical basis for the diagnosis and treatment of such patients.

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The increasing rates of drug misuse highlight the urgency of identifying improved therapeutics for treatment. Most drug-seeking behaviours that can be modelled in rodents utilize the repeated intravenous self-administration (SA) of drugs. Recent studies examining the mesolimbic pathway suggest that Kv7/KCNQ channels may contribute to the transition from recreational to chronic drug use. However, to date, all such studies used noncontingent, experimenter-delivered drug model systems, and the extent to which this effect generalizes to rats trained to self-administer drugs is not known. Here, we tested the ability of retigabine (ezogabine), a Kv7 channel opener, to regulate instrumental behaviour in male Sprague Dawley rats. We first validated the ability of retigabine to target experimenter-delivered cocaine in a conditioned place preference (CPP) assay and found that retigabine reduced the acquisition of place preference. Next, we trained rats for cocaine-SA under a fixed-ratio or progressive-ratio reinforcement schedule and found that retigabine pretreatment attenuated the SA of low to moderate doses of cocaine. This was not observed in parallel experiments, with rats self-administering sucrose, a natural reward. Compared with sucrose-SA, cocaine-SA was associated with reductions in the expression of the Kv7.5 subunit in the nucleus accumbens, without alterations in Kv7.2 and Kv7.3. Therefore, these studies reveal a reward-specific reduction in SA behaviour and support the notion that Kv7 is a potential therapeutic target for human psychiatric diseases with dysfunctional reward circuitry.

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Carbamatos , Cocaína , Fenilendiaminas , Ratas Sprague-Dawley , Autoadministración , Sacarosa , Animales , Fenilendiaminas/farmacología , Fenilendiaminas/administración & dosificación , Carbamatos/farmacología , Carbamatos/administración & dosificación , Cocaína/farmacología , Cocaína/administración & dosificación , Masculino , Ratas , Sacarosa/administración & dosificación , Sacarosa/farmacología , Comportamiento de Búsqueda de Drogas/efectos de los fármacos , Canales de Potasio KCNQ/efectos de los fármacos , Condicionamiento Operante/efectos de los fármacos , Inhibidores de Captación de Dopamina/farmacología , Inhibidores de Captación de Dopamina/administración & dosificación

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OBJECTIVE: Pharmacological activation of neuronal Kv7 channels by the antiepileptic drug retigabine (RTG; ezogabine) has been proven effective in treating partial epilepsy. However, RTG was withdrawn from the market due to the toxicity caused by its phenazinium dimer metabolites, leading to peripheral skin discoloration and retinal abnormalities. To address the undesirable metabolic properties of RTG and prevent the formation of phenazinium dimers, we made chemical modifications to RTG, resulting in a new RTG derivative, 1025c, N,N'-{4-[(4-fluorobenzyl) (prop-2-yn-1-yl)amino]-1,2-phenylene}bis(3,3-dimethylbutanamide). METHODS: Whole-cell recordings were used to evaluate Kv7 channel openers. Site-directed mutagenesis and molecular docking were adopted to investigate the molecular mechanism underlying 1025c and Kv7.2 interactions. Mouse seizure models of maximal electroshock (MES), subcutaneous pentylenetetrazol (scPTZ), and PTZ-induced kindling were utilized to test compound antiepileptic activity. RESULTS: The novel compound 1025c selectively activates whole-cell Kv7.2/7.3 currents in a concentration-dependent manner, with half-maximal effective concentration of .91 ± .17 µmol·L-1. The 1025c compound also causes a leftward shift in Kv7.2/7.3 current activation toward a more hyperpolarized membrane potential, with a shift of the half voltage of maximal activation (ΔV1/2) of -18.6 ± 3.0 mV. Intraperitoneal administration of 1025c demonstrates dose-dependent antiseizure activities in assays of MES, scPTZ, and PTZ-induced kindling models. Moreover, through site-directed mutagenesis combined with molecular docking, a key residue Trp236 has been identified as critical for 1025c-mediated activation of Kv7.2 channels. Photostability experiments further reveal that 1025c is more photostable than RTG and is unable to dimerize. SIGNIFICANCE: Our findings demonstrate that 1025c exhibits potent and selective activation of neuronal Kv7 channels without being metabolized to phenazinium dimers, suggesting its developmental potential as an antiseizure agent for therapy.

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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.

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Canal de Potasio KCNQ2 , Canal de Potasio KCNQ3 , Humanos , Canal de Potasio KCNQ2/metabolismo , Canal de Potasio KCNQ2/genética , Canal de Potasio KCNQ2/química , Canal de Potasio KCNQ3/metabolismo , Canal de Potasio KCNQ3/genética , Canal de Potasio KCNQ3/química , Canal de Potasio KCNQ3/antagonistas & inhibidores , Simulación por Computador , Relación Estructura-Actividad , Descubrimiento de Drogas/métodos , Animales

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Hyperexcitability of neuronal networks is central to the pathogenesis of Alzheimer's disease (AD). Pharmacological activation of Kv7 channels is an effective way to reduce neuronal firing. Our results showed that that pharmacologically activating the Kv7 channel with Retigabine (RTG) can alleviate cognitive impairment in mice without affecting spontaneous activity. RTG could also ameliorate damage to the Nissl bodies in cortex and hippocampal CA and DG regions in 9-month-old APP/PS1 mice. Additionally, RTG could reduce the Aß plaque number in the hippocampus and cortex of both 6-month-old and 9-month-old mice. By recordings of electroencephalogram, we showed that a decrease in the number of abnormal discharges in the brains of the AD model mice when the Kv7 channel was opened. Moreover, Western blot analysis revealed a reduction in the expression of the p-Tau protein in both the hippocampus and cortex upon Kv7 channel opening. These findings suggest that Kv7 channel opener RTG may ameliorate cognitive impairment in AD, most likely by reducing brain excitability.

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Enfermedad de Alzheimer , Precursor de Proteína beta-Amiloide , Carbamatos , Disfunción Cognitiva , Modelos Animales de Enfermedad , Hipocampo , Ratones Transgénicos , Fenilendiaminas , Animales , Fenilendiaminas/farmacología , Carbamatos/farmacología , Enfermedad de Alzheimer/tratamiento farmacológico , Enfermedad de Alzheimer/metabolismo , Enfermedad de Alzheimer/patología , Ratones , Precursor de Proteína beta-Amiloide/genética , Precursor de Proteína beta-Amiloide/metabolismo , Hipocampo/efectos de los fármacos , Hipocampo/metabolismo , Disfunción Cognitiva/tratamiento farmacológico , Disfunción Cognitiva/metabolismo , Presenilina-1/genética , Masculino , Placa Amiloide/tratamiento farmacológico , Placa Amiloide/patología , Placa Amiloide/metabolismo , Corteza Cerebral/efectos de los fármacos , Corteza Cerebral/metabolismo , Corteza Cerebral/patología , Proteínas tau/metabolismo , Conducta Animal/efectos de los fármacos , Ratones Endogámicos C57BL

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Background & Objective: Chronic peripheral neuropathic pain (PNP) is a debilitating condition that is associated with many types of injury/diseases, including diabetes mellitus. Patients with longstanding diabetes develop diabetic PNP (DPNP), which is resilient to currently available drugs. The underlying molecular mechanisms of DPNP are still illusive, but Kv7 channels that have been implicated in the pathogenesis of various types of chronic pain are likely to be involved. Indeed, using the streptozotocin (STZ) rat model of DPNP, we have previously shown that Kv7 activation with their non-selective activator retigabine attenuated neuropathic pain behavior suggesting that these channels are implicated in DPNP pathogenesis. Here, we evaluated, in the same STZ model, whether the more potent and more selective Kv7 channel openers flupirtine and ML213 attenuate STZ-induced pain hypersensitivity. Methods: Male Sprague Dawley rats (250-300 g) were used. The STZ model involved a single injection of STZ (60 mg/kg, i.p.). Behavioral testing for mechanical and heat pain sensitivity was performed using a dynamic plantar aesthesiometer and Hargreaves analgesiometer, respectively. Results: STZ rats exhibited behavioral signs of mechanical and heat hypersensitivity as indicated by significant decreases in the mean paw withdrawal threshold (PWT) and mean paw withdrawal latency (PWL), respectively, at 35 days post-STZ treatment. Single injections of flupirtine (10 mg/kg, i.p.) and ML213 (5 mg/kg, i.p.) to STZ rats (35-days after STZ treatment) caused significant increases in the mean PWT, but not PWL, indicating attenuation of mechanical, but not heat hypersensitivity. Both flupirtine and ML213 were as effective as the positive control gabapentin (10/kg, i.p.), and their anti-allodynic effects were prevented by the Kv7 channel-specific blocker XE991 (3 mg/kg, i.p.). Conclusion: The findings suggest that Kv7 channels are involved in the mechanisms of mechanical but not heat hypersensitivity associated with DPNP, and that their activation may prove to be effective in alleviating DPNP symptoms.

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GRT-X, which targets both the mitochondrial translocator protein (TSPO) and the Kv7.2/3 (KCNQ2/3) potassium channels, has been shown to efficiently promote recovery from cervical spine injury. In the present work, we investigate the role of GRT-X and its two targets in the axonal growth of dorsal root ganglion (DRG) neurons. Neurite outgrowth was quantified in DRG explant cultures prepared from wild-type C57BL6/J and TSPO-KO mice. TSPO was pharmacologically targeted with the agonist XBD173 and the Kv7 channels with the activator ICA-27243 and the inhibitor XE991. GRT-X efficiently stimulated DRG axonal growth at 4 and 8 days after its single administration. XBD173 also promoted axonal elongation, but only after 8 days and its repeated administration. In contrast, both ICA27243 and XE991 tended to decrease axonal elongation. In dissociated DRG neuron/Schwann cell co-cultures, GRT-X upregulated the expression of genes associated with axonal growth and myelination. In the TSPO-KO DRG cultures, the stimulatory effect of GRT-X on axonal growth was completely lost. However, GRT-X and XBD173 activated neuronal and Schwann cell gene expression after TSPO knockout, indicating the presence of additional targets warranting further investigation. These findings uncover a key role of the dual mode of action of GRT-X in the axonal elongation of DRG neurons.

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Axones , Ganglios Espinales , Receptores de GABA , Animales , Ganglios Espinales/metabolismo , Ganglios Espinales/citología , Ratones , Axones/metabolismo , Receptores de GABA/metabolismo , Receptores de GABA/genética , Canal de Potasio KCNQ2/metabolismo , Canal de Potasio KCNQ2/genética , Ratones Noqueados , Ratones Endogámicos C57BL , Células Cultivadas , Células de Schwann/metabolismo , Células de Schwann/efectos de los fármacos , Células de Schwann/citología , Técnicas de Cocultivo , Neuronas/metabolismo , Neuronas/efectos de los fármacos

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Ion channels are membrane proteins that may also have intracellular and extracellular domains that interact with other ligands. In many cases, these interaction sites are highly mobile and may undergo changes in the configuration upon binding with regulatory signaling molecules. Isothermal titration calorimetry (ITC) is a powerful technique to quantify protein-ligand interactions of purified samples in solution. This chapter describes a fragment-based analysis method using ITC to quantify the interactions between a domain of the voltage-gated Kv7 channel and the calcium-regulated protein calmodulin. This example can be used to quantify the interactions between specific domains of other ion channels and their regulatory signaling proteins.

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Calmodulina , Calorimetría , Unión Proteica , Calorimetría/métodos , Calmodulina/metabolismo , Calmodulina/química , Ligandos , Canales Iónicos/metabolismo , Canales Iónicos/química , Humanos , Sitios de Unión

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Chronic pain is common and inadequately treated, making the development of safe and effective analgesics a high priority. Our previous data indicate that carbonic anhydrase-8 (CA8) expression in dorsal root ganglia (DRG) mediates analgesia via inhibition of neuronal ER inositol trisphosphate receptor-1 (ITPR1) via subsequent decrease in ER calcium release and reduction of cytoplasmic free calcium, essential to the regulation of neuronal excitability. This study tested the hypothesis that novel JDNI8 replication-defective herpes simplex-1 viral vectors (rdHSV) carrying a CA8 transgene (vHCA8) reduce primary afferent neuronal excitability. Whole-cell current clamp recordings in small DRG neurons showed that vHCA8 transduction caused prolongation of their afterhyperpolarization (AHP), an essential regulator of neuronal excitability. This AHP prolongation was completely reversed by the specific Kv7 channel inhibitor XE-991. Voltage clamp recordings indicate an effect via Kv7 channels in vHCA8-infected small DRG neurons. These data demonstrate for the first time that vHCA8 produces Kv7 channel activation, which decreases neuronal excitability in nociceptors. This suppression of excitability may translate in vivo as non-opioid dependent behavioral- or clinical analgesia, if proven behaviorally and clinically.

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Introduction: Epileptiform activity is the most striking result of hyperexcitation of a group of neurons that can occur in different brain regions and then spread to other sites. Later it was shown that these rhythms have a cellular correlate in vitro called paroxysmal depolarization shift (PDS). In 13-15 DIV neuron-glial cell culture, inhibition of the GABA(A) receptors induces bursts of action potential in the form of clasters PDS and oscillations of intracellular Ca2+ concentration ([Ca2+]i). We demonstrate that GABAergic neurons expressing calcium-permeable AMPA receptors (CP-AMPARs) as well as Kv7-type potassium channels regulate hippocampal glutamatergic neurons' excitability during epileptiform activity in culture. Methods: A combination of whole-cell patch-clamp in current clamp mode and calcium imaging microscopy was used to simultaneously register membrane potential and [Ca2+]i level. To identify GABAergic cell cultures were fixed and stained with antibodies against glutamate decarboxylase GAD 65/67 and neuron-specific enolase (NSE) after vital [Ca2+]i imaging. Results and discussion: It was shown that CP-AMPARs are involved in the regulation of the PDS clusters and [Ca2+]i pulses accompanied them. Activation of CP-AMPARs of GABAergic neurons is thought to cause the release of GABA, which activates the GABA(B) receptors of other GABAergic interneurons. It is assumed that activation of these GABA(B) receptors leads to the release of beta-gamma subunits of Gi protein, which activate potassium channels, resulting in hyperpolarization and inhibition of these interneurons. The latter causes disinhibition of glutamatergic neurons, the targets of these interneurons. In turn, the CP-AMPAR antagonist, NASPM, has the opposite effect. Measurement of membrane potential in GABAergic neurons by the patch-clamp method in whole-cell configuration demonstrated that NASPM suppresses hyperpolarization in clusters and individual PDSs. It is believed that Kv7-type potassium channels are involved in the control of hyperpolarization during epileptiform activity. The blocker of Kv7 channels, XE 991, mimicked the effect of the CP-AMPARs antagonist on PDS clusters. Both drugs increased the duration of the PDS cluster. In turn, the Kv7 activator, retigabine, decreased the duration of the PDS cluster and Ca2+ pulse. In addition, retigabine led to deep posthyperpolarization at the end of the PDS cluster. The Kv7 channel is believed to be involved in the formation of PDS, as the channel blocker reduced the rate of hyperpolarization in the PDS almost three times. Thus, GABAergic neurons expressing CP-AMPARs, regulate the membrane potential of innervated glutamatergic neurons by modulating the activity of postsynaptic potassium channels of other GABAergic neurons.

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The KCNQ family is comprised of five genes and the expression products form voltage-gated potassium channels (Kv7.1-7.5) that have a major impact upon cellular physiology in many cell types. Each functional Kv7 channel forms as a tetramer that often associates with proteins encoded by the KCNE gene family (KCNE1-5) and is critically reliant upon binding of phosphatidylinositol bisphosphate (PIP2) and calmodulin. Other modulators like A-kinase anchoring proteins, ubiquitin ligases and Ca-calmodulin kinase II alter Kv7 channel function and trafficking in an isoform specific manner. It has now been identified that for Kv7.4, G protein ßγ subunits (Gßγ) can be added to the list of key regulators and is paramount for channel activity. This article provides an overview of this nascent field of research, highlighting themes and directions for future study.

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BACKGROUND AND PURPOSE: Neuropathic pain, a debilitating condition with unmet medical needs, can be characterised as hyperexcitability of nociceptive neurons caused by dysfunction of ion channels. Voltage-gated potassium channels type 7 (Kv7), responsible for maintaining neuronal resting membrane potential and thus excitability, reside under tight control of G protein-coupled receptors (GPCRs). Calcium-sensing receptor (CaSR) is a GPCR that regulates the activity of numerous ion channels, but whether CaSR can control Kv7 channel function has been unexplored until now. EXPERIMENTAL APPROACH: Experiments were conducted in recombinant cell models, mouse dorsal root ganglia (DRG) neurons and human induced pluripotent stem cell (hiPSC)-derived nociceptive-like neurons using patch-clamp electrophysiology and molecular biology techniques. KEY RESULTS: Our results demonstrate that CaSR is expressed in recombinant cell models, hiPSC-derived nociceptive-like neurons and mouse DRG neurons, and its activation induced depolarisation via Kv7.2/7.3 channel inhibition. The CaSR-Kv7.2/7.3 channel crosslink was mediated via the Gi/o protein-adenylate cyclase-cyclicAMP-protein kinase A signalling cascade. Suppression of CaSR function demonstrated a potential to rescue hiPSC-derived nociceptive-like neurons from algogenic cocktail-induced hyperexcitability. CONCLUSION AND IMPLICATIONS: This study demonstrates that the CaSR-Kv7.2/7.3 channel crosslink, via a Gi/o protein signalling pathway, effectively regulates neuronal excitability, providing a feasible pharmacological target for neuronal hyperexcitability management in neuropathic pain.

Asunto(s)

Ganglios Espinales , Células Madre Pluripotentes Inducidas , Receptores Sensibles al Calcio , Transducción de Señal , Humanos , Receptores Sensibles al Calcio/metabolismo , Células Madre Pluripotentes Inducidas/metabolismo , Animales , Ratones , Ganglios Espinales/metabolismo , Ganglios Espinales/citología , Subunidades alfa de la Proteína de Unión al GTP Gi-Go/metabolismo , Nociceptores/metabolismo , Células Cultivadas , Células HEK293

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BACKGROUND AND PURPOSE: The analgesic action of paracetamol involves KV7 channels, and its metabolite N-acetyl-p-benzo quinone imine (NAPQI), a cysteine modifying reagent, was shown to increase currents through such channels in nociceptors. Modification of cysteine residues by N-ethylmaleimide, H2O2, or nitric oxide has been found to modulate currents through KV7 channels. The study aims to identify whether, and if so which, cysteine residues in neuronal KV7 channels might be responsible for the effects of NAPQI. EXPERIMENTAL APPROACH: To address this question, we used a combination of perforated patch-clamp recordings, site-directed mutagenesis, and mass spectrometry applied to recombinant KV7.1 to KV7.5 channels. KEY RESULTS: Currents through the cardiac subtype KV7.1 were reduced by NAPQI. Currents through all other subtypes were increased, either by an isolated shift of the channel voltage dependence to more negative values (KV7.3) or by such a shift combined with increased maximal current levels (KV7.2, KV7.4, KV7.5). A stretch of three cysteine residues in the S2-S3 linker region of KV7.2 was necessary and sufficient to mediate these effects. CONCLUSION AND IMPLICATION: The paracetamol metabolite N-acetyl-p-benzo quinone imine (NAPQI) modifies cysteine residues of KV7 subunits and reinforces channel gating in homomeric and heteromeric KV7.2 to KV7.5, but not in KV7.1 channels. In KV7.2, a triple cysteine motif located within the S2-S3 linker region mediates this reinforcement that can be expected to reduce the excitability of nociceptors and to mediate antinociceptive actions of paracetamol.

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Acetaminofén , Benzoquinonas , Cisteína , Iminas , Cisteína/metabolismo , Acetaminofén/farmacología , Benzoquinonas/farmacología , Benzoquinonas/metabolismo , Animales , Iminas/farmacología , Iminas/química , Iminas/metabolismo , Neuronas/efectos de los fármacos , Neuronas/metabolismo , Canales de Potasio KCNQ/metabolismo , Canales de Potasio KCNQ/genética , Humanos , Secuencias de Aminoácidos , Analgésicos no Narcóticos/farmacología , Células HEK293 , Ratas

RESUMEN

Cognitive dysfunction is a core symptom common in psychiatric disorders including depression that is primarily managed by antidepressants lacking efficacy in improving cognition. In this study, we report a novel dual serotonin transporter and voltage-gated potassium Kv7/KCNQ/M-channel inhibitor D01 (a 2-methyl-3-aryloxy-3-heteroarylpropylamines derivative) that exhibits both anti-depression effects and improvements in cognition. D01 inhibits serotonin transporters (Ki = 30.1 ± 6.9 nmol/L) and M channels (IC50 = 10.1 ± 2.4 µmol/L). D01 also reduces the immobility duration in the mouse FST and TST assays in a dose-dependent manner without a stimulatory effect on locomotion. Intragastric administrations of D01 (20 and 40 mg/kg) can significantly shorten the immobility time in a mouse model of chronic restraint stress (CRS)-induced depression-like behavior. Additionally, D01 dose-dependently improves the cognitive deficit induced by CRS in Morris water maze test and increases the exploration time with novel objects in normal or scopolamine-induced cognitive deficits in mice, but not fluoxetine. Furthermore, D01 reverses the long-term potentiation (LTP) inhibition induced by scopolamine. Taken together, our findings demonstrate that D01, a dual-target serotonin reuptake and M channel inhibitor, is highly effective in the treatment-resistant depression and cognitive deficits, thus holding potential for development as therapy of depression with cognitive deficits.

RESUMEN

Abnormal cellular and circuit excitability is believed to drive many core phenotypes in fragile X syndrome (FXS). The dentate gyrus is a brain area performing critical computations essential for learning and memory. However, little is known about dentate circuit defects and their mechanisms in FXS. Understanding dentate circuit dysfunction in FXS has been complicated by the presence of two types of excitatory neurons, the granule cells and mossy cells. Here we report that loss of FMRP markedly decreased excitability of dentate mossy cells, a change opposite to all other known excitability defects in excitatory neurons in FXS. This mossy cell hypo-excitability is caused by increased Kv7 function in Fmr1 knockout (KO) mice. By reducing the excitatory drive onto local hilar interneurons, hypo-excitability of mossy cells results in increased excitation/inhibition ratio in granule cells and thus paradoxically leads to excessive dentate output. Circuit-wide inhibition of Kv7 channels in Fmr1 KO mice increases inhibitory drive onto granule cells and normalizes the dentate output in response to physiologically relevant theta-gamma coupling stimulation. Our study suggests that circuit-based interventions may provide a promising strategy in this disorder to bypass irreconcilable excitability defects in different cell types and restore their pathophysiological consequences at the circuit level.

Asunto(s)

Síndrome del Cromosoma X Frágil , Trastornos Mentales , Ratones , Animales , Neuronas/fisiología , Interneuronas/metabolismo , Modelos Animales de Enfermedad , Giro Dentado/fisiología , Proteína de la Discapacidad Intelectual del Síndrome del Cromosoma X Frágil/genética , Proteína de la Discapacidad Intelectual del Síndrome del Cromosoma X Frágil/metabolismo

RESUMEN

The medial nucleus of the trapezoid body (MNTB) has been intensively investigated as a primary source of inhibition in brainstem auditory circuitry. MNTB-derived inhibition plays a critical role in the computation of sound location, as temporal features of sounds are precisely conveyed through the calyx of Held/MNTB synapse. In adult gerbils, cholinergic signaling influences sound-evoked responses of MNTB neurons via nicotinic acetylcholine receptors (nAChRs; Zhang et al., 2021) establishing a modulatory role for cholinergic input to this nucleus. However, the cellular mechanisms through which acetylcholine (ACh) mediates this modulation in the MNTB remain obscure. To investigate these mechanisms, we used whole-cell current and voltage-clamp recordings to examine cholinergic physiology in MNTB neurons from Mongolian gerbils (Meriones unguiculatus) of both sexes. Membrane excitability was assessed in brain slices, in pre-hearing (postnatal days 9-13) and post-hearing onset (P18-20) MNTB neurons during bath application of agonists and antagonists of nicotinic (nAChRs) and muscarinic receptors (mAChRs). Muscarinic activation induced a potent increase in excitability most prominently prior to hearing onset with nAChR modulation emerging at later time points. Pharmacological manipulations further demonstrated that the voltage-gated K+ channel KCNQ (Kv7) is the downstream effector of mAChR activation that impacts excitability early in development. Cholinergic modulation of Kv7 reduces outward K+ conductance and depolarizes resting membrane potential. Immunolabeling revealed expression of Kv7 channels as well as mAChRs containing M1 and M3 subunits. Together, our results suggest that mAChR modulation is prominent but transient in the developing MNTB and that cholinergic modulation functions to shape auditory circuit development.

Asunto(s)

Receptores Nicotínicos , Cuerpo Trapezoide , Animales , Femenino , Masculino , Cuerpo Trapezoide/fisiología , Gerbillinae , Transmisión Sináptica/fisiología , Neuronas/fisiología , Receptores Nicotínicos/metabolismo , Colinérgicos , Vías Auditivas/fisiología

RESUMEN

BACKGROUND: Variants in the KCNQ1 gene, encoding the α-subunit of the slow component of delayed rectifier K+ channel Kv7.1, cause long QT syndrome (LQTS) type 1. The location of variants may be one of the factors in determining prognosis. However, detailed genotype-phenotype relationships associated with C-terminus variants remain unelucidated. OBJECTIVE: We investigated the clinical characteristics and variant-specific arrhythmic risks in patients with LQTS carrying Kv7.1 C-terminus variants. METHODS: The study comprises 202 consecutive patients with LQTS (98 probands and 104 family members) who carry a rare heterozygous variant in the Kv7.1 C-terminus. Their clinical characteristics and arrhythmic events were investigated. RESULTS: We identified 36 unique C-terminus variants (25 missense and 11 non-missense). The p.R366W variant was identified in 8 families, and p.T587M was identified in 21 families in large numbers from northwestern Japan. As for the location of the variant, we found that the variants in highly conserved regions and nonhelical domains were associated with longer QTc intervals compared with the variants in other regions. Both p.R366W and p.T587M variants are located in the highly conserved and functionally pivotal regions close to helices A and D, which are associated with calmodulin binding and channel assembly (tetramerization), respectively. The probands carrying p.T587M and p.R366W variants had worse arrhythmia outcomes compared with those with other C-terminus variants. The haplotype analysis of p.T587M families was suggestive of a founder effect. CONCLUSION: The arrhythmic risk of C-terminus variants in Kv7.1 in LQTS is not homogeneous, and locations of variants can be a determining factor for prognosis.

Asunto(s)

Canal de Potasio KCNQ1 , Síndrome de QT Prolongado , Síndrome de Romano-Ward , Adolescente , Adulto , Niño , Femenino , Humanos , Masculino , Persona de Mediana Edad , Adulto Joven , ADN/genética , Análisis Mutacional de ADN , Electrocardiografía , Predisposición Genética a la Enfermedad , Japón/epidemiología , Canal de Potasio KCNQ1/genética , Síndrome de QT Prolongado/genética , Síndrome de QT Prolongado/fisiopatología , Linaje , Fenotipo , Pronóstico , Síndrome de Romano-Ward/genética , Síndrome de Romano-Ward/fisiopatología