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Gain-of-function (GoF) variants in KCNT1 channels cause severe, drug-resistant forms of epilepsy. Quinidine is a known KCNT1 blocker, but its clinical use is limited due to severe drawbacks. To identify novel KCNT1 blockers, a homology model of human KCNT1 was built and used to screen an in-house library of compounds. Among the 20 molecules selected, five ( CPK4 , 13 , 16 , 18 , and 20 ) showed strong KCNT1-blocking ability in an in vitro fluorescence-based assay. Patch-clamp experiments confirmed a higher KCNT1-blocking potency of these compounds when compared to quinidine, and their selectivity for KCNT1 over hERG and Kv7.2 channels. Among identified molecules, CPK20 displayed the highest metabolic stability; this compound also blocked KCNT2 currents, although with a lower potency, and counteracted GoF effects prompted by 2 recurrent epilepsy-causing KCNT1 variants (G288S and A934T). The present results provide solid rational basis for future design of novel compounds to counteract KCNT 1-related neurological disorders.

Ion channels in the blood-brain barrier (BBB) play a main role in controlling the interstitial fluid composition and cerebral blood flow, and their dysfunction contributes to the disruption of the BBB occurring in many neurological diseases such as epilepsy. In this study, using morphological and functional approaches, we evaluated the expression and role in the BBB of Kv7 channels, a family of voltage-gated potassium channels including five members (Kv7.1-5) that play a major role in the regulation of cell excitability and transmembrane flux of potassium ions. Immunofluorescence experiments showed that Kv7.1, Kv7.4, and Kv7.5 were expressed in rat brain microvessels (BMVs), as well as brain primary- and clonal (BEND-3) endothelial cells (ECs). Kv7.5 localized at the cell-to-cell junction sites, whereas Kv7.4 was also found in pericytes. The Kv7 activator retigabine increased transendothelial electrical resistance (TEER) in both primary ECs and BEND-3 cells; moreover, retigabine reduced paracellular dextran flux in BEND-3 cells. These effects were prevented by the selective Kv7 blocker XE-991. Exposure to retigabine also hyperpolarized cell membrane and increased tight junctions (TJs) integrity in BEND-3 cells. BMVs from rats treated with kainic acid (KA) showed a disruption of TJs and a selective reduction of Kv7.5 expression. In BEND-3 cells, retigabine prevented the increase of cell permeability and the reduction of TJs integrity induced by KA. Overall, these findings demonstrate that Kv7 channels are expressed in the BBB, where they modulate barrier properties both in physiological and pathological conditions. NEW & NOTEWORTHY This study describes for the first time the expression and the functional role of Kv7 potassium channels in the blood-brain barrier. We show that the opening of Kv7 channels reduces endothelial cell permeability both in physiological and pathological conditions via the hyperpolarization of cell membrane and the sealing of tight junctions. Therefore, activation of endothelial Kv7 channels might be a useful strategy to treat epilepsy and other neurological disorders characterized by blood-brain barrier dysfunction.

Objective: Variants in several potassium channel genes, including KCNA1 and KCNA2, cause Developmental and Epileptic Encephalopathies (DEEs). We investigated whether variants in KCNA3, another mammalian homologue of the Drosophila shaker family and encoding for Kv1.3 subunits, can cause DEE., Methods: Genetic analysis of study individuals was performed by routine exome or genome sequencing, usually of parent-offspring trios. Phenotyping was performed via a standard clinical questionnaire. Currents from wild-type and/or mutant Kv1.3 subunits were investigated by whole-cell patch-clamp upon their heterologous expression., Results: Fourteen individuals, each carrying a de novo heterozygous missense variant in KCNA3, were identified. Most (12/14; 86%) had DEE with marked speech delay with or without motor delay, intellectual disability, epilepsy, and autism spectrum disorder. Functional analysis of Kv1.3 channels carrying each variant revealed heterogeneous functional changes, ranging from "pure" loss-of-function (LoF) effects due to faster inactivation kinetics, depolarized voltage-dependence of activation, slower activation kinetics, increased current inactivation, reduced or absent currents with or without dominant-negative effects, to "mixed" loss- and gain-of-function (GoF) effects. Compared to controls, Kv1.3 currents in lymphoblasts from 1 of the proband displayed functional changes similar to those observed upon heterologous expression of channels carrying the same variant. The antidepressant drug fluoxetine inhibited with similar potency the currents from wild-type and 1 of the Kv1.3 GoF variant., Interpretation: We describe a novel association of de novo missense variants in KCNA3 with a human DEE, and provide evidence that fluoxetine might represent a potential targeted treatment for individuals carrying variants with significant GoF effects. ANN NEUROL 2024;95:365-376., (© 2023 The Authors. Annals of Neurology published by Wiley Periodicals LLC on behalf of American Neurological Association.)

While loss-of-function (LoF) variants in KCNQ2 are associated with a spectrum of neonatal-onset epilepsies, gain-of-function (GoF) variants cause a more complex phenotype that precludes neonatal-onset epilepsy. In the present work, the clinical features of three patients carrying a de novo KCNQ2 Y141N (n ​= ​1) or G239S variant (n ​= ​2) respectively, are described. All three patients had a mild global developmental delay, with prominent language deficits, and strong activation of interictal epileptic activity during sleep. Epileptic seizures were not reported. The absence of neonatal seizures suggested a GoF effect and prompted functional testing of the variants. In vitro whole-cell patch-clamp electrophysiological experiments in Chinese Hamster Ovary cells transiently-transfected with the cDNAs encoding Kv7.2 subunits carrying the Y141N or G239S variants in homomeric or heteromeric configurations with Kv7.2 subunits, revealed that currents from channels incorporating mutant subunits displayed increased current densities and hyperpolarizing shifts of about 10 ​mV in activation gating; both these functional features are consistent with an in vitro GoF phenotype. The antidepressant drug amitriptyline induced a reversible and concentration-dependent inhibition of current carried by Kv7.2 Y141N and G239S mutant channels. Based on in vitro results, amitriptyline was prescribed in one patient (G239S), prompting a significant improvement in motor, verbal, social, sensory and adaptive behavior skillsduring the two-year-treatment period. Thus, our results suggest that KCNQ2 GoF variants Y141N and G239S cause a mild DD with prominent language deficits in the absence of neonatal seizures and that treatment with the Kv7 channel blocker amitriptyline might represent a potential targeted treatment for patients with KCNQ2 GoF variants., Competing Interests: Declaration of competing interest The authors declare the following financial interests/personal relationships which may be considered as potential competing interests: Allan Bayat reports financial support was provided by Novo Nordisk Foundation. Francesco Miceli reports financial support was provided by Ministry of Education and Merit. Edward C. Cooper reports financial support was provided by Jack Pribaz Foundation and Miles Family Fund. Sarah Weckhuysen reports financial support was provided by FWO, GSKE, KCNQ2-Cure, Jack Pribaz Foundation, KCNQ2e.v., European Joint Programme on Rare Disease JTC 2020 (TreatKCNQ). Maurizio Taglialatela reports financial support was provided by Ministry of Education and Merit. Maurizio Taglialatela reports financial support was provided by Italian Ministry of Health. Maurizio Taglialatela reports was provided by European Joint Programme on Rare Disease JTC 2020 (TreatKCNQ). Sarah Weckhuysen reports a relationship with UCB, Xenon Pharmaceuticals, Lundbeck, Knopp Biosciences, Angelini Pharma, Roche, Biohaven and Encoded Therapeutics that includes: consulting or advisory. Edward C. Cooper reports a relationship with Xenon Pharmaceuticals and Knopp Biosciences that includes: consulting or advisory and funding grants. Rikke Steensbjerre Moller reports a relationship with EISAI, UCB, Orion and Angelini Pharma that includes: consulting or advisory and speaking and lecture fees., (Copyright © 2023 The Author(s). Published by Elsevier Inc. All rights reserved.)

KCNQ2-encoded Kv7.2 subunits play a critical role in balancing neuronal excitability. Mutations in KCNQ2 are responsible for highly-heterogenous epileptic and neurodevelopmental phenotypes ranging from self-limited familial neonatal epilepsy (SeLFNE) to severe developmental and epileptic encephalopathy (DEE). Pathogenic KCNQ2 variants cluster at the voltage sensor domain (VSD), the pore domain, and the C-terminal tail. Although several knock-in mice harboring Kcnq2 pore variants have been developed, no mouse line carrying Kcnq2 voltage-sensor mutations has been described. KCNQ2-R207W is an epilepsy-causing mutation located in the VSD, mainly affecting voltage-dependent channel gating. To study the physiological consequence of Kcnq2 VSD dysfunction, we generated a Kcnq2-R207W mouse line and analyzed the pathological and pharmacological phenotypes of mutant mice. As a result, both homozygous (Kcnq2 RW/RW ) and heterozygous (Kcnq2 RW/+ ) mice were viable. While Kcnq2 RW/RW mice displayed a short lifespan, growth retardation, and spontaneous seizures, Kcnq2 RW/+ mice survived and developed normally, although only a fraction (9/64; 14%) of them showed behavioral- and ECoG-confirmed spontaneous seizures. Kcnq2 RW/+ mice displayed increased susceptibility to evoked seizures, which was dramatically ameliorated by treatment with the novel KCNQ opener pynegabine (HN37). Our results show that the Kcnq2-R207W mouse line, the first harboring a Kcnq2 voltage-sensor mutation, exhibits a unique epileptic phenotype with both spontaneous seizures and increased susceptibility to evoked seizures. In Kcnq2-R207W mice, the potent KCNQ opener HN37, currently in clinical phase I, shows strong anticonvulsant activity, suggesting it may represent a valuable option for the severe phenotypes of KCNQ2-related epilepsy., (Copyright © 2022 The Authors. Published by Elsevier Inc. All rights reserved.)

Background: Prior studies have revealed remarkable phenotypic heterogeneity in KCNQ2-related disorders, correlated with effects on biophysical features of heterologously expressed channels. Here, we assessed phenotypes and functional properties associated with KCNQ2 missense variants R144W, R144Q, and R144G. We also explored in vitro blockade of channels carrying R144Q mutant subunits by amitriptyline., Methods: Patients were identified using the RIKEE database and through clinical collaborators. Phenotypes were collected by a standardized questionnaire. Functional and pharmacological properties of variant subunits were analyzed by whole-cell patch-clamp recordings., Findings: Detailed clinical information on fifteen patients (14 novel and 1 previously published) was analyzed. All patients had developmental delay with prominent language impairment. R144Q patients were more severely affected than R144W patients. Infantile to childhood onset epilepsy occurred in 40%, while 67% of sleep-EEGs showed sleep-activated epileptiform activity. Ten patients (67%) showed autistic features. Activation gating of homomeric Kv7.2 R144W/Q/G channels was left-shifted, suggesting gain-of-function effects. Amitriptyline blocked channels containing Kv7.2 and Kv7.2 R144Q subunits., Interpretation: Patients carrying KCNQ2 R144 gain-of-function variants have developmental delay with prominent language impairment, autistic features, often accompanied by infantile- to childhood-onset epilepsy and EEG sleep-activated epileptiform activity. The absence of neonatal seizures is a robust and important clinical differentiator between KCNQ2 gain-of-function and loss-of-function variants. The Kv7.2/7.3 channel blocker amitriptyline might represent a targeted treatment., Funding: Supported by FWO, GSKE, KCNQ2-Cure, Jack Pribaz Foundation, European Joint Programme on Rare Disease 2020, the Italian Ministry for University and Research, the Italian Ministry of Health, the European Commission, the University of Antwerp, NINDS, and Chalk Family Foundation., Competing Interests: Declaration of interests SW received consultancy and speaker fees from UCB, Biocodex, Xenon, Zogenix, Lundbeck, Knopp Biosciences, Encoded MT received consultancy fees from Xenon. ECC received consultancy fees from Xenon, Knopp, these activities have been reviewed and approved by Baylor College of Medicine according to its policy on disclosure of outside interests. RSM received consultancy and speaker fees from EISAI and UCB. The remaining authors have no conflicts of interest., (Copyright © 2022 The Authors. Published by Elsevier B.V. All rights reserved.)

A wide phenotypic spectrum of neurological diseases is associated with KCNA1 (Kv1.1) variants. To investigate the molecular basis of such a heterogeneous clinical presentation and identify the possible correlation with in vitro phenotypes, we compared the functional consequences of three heterozygous de novo variants (p.P403S, p.P405L, and p.P405S) in Kv1.1 pore region found in four patients with severe developmental and epileptic encephalopathy (DEE), with those of a de novo variant in the voltage sensor (p.A261T) identified in two patients with mild, carbamazepine-responsive, focal epilepsy. Patch-clamp electrophysiology was used to investigate the functional properties of mutant Kv1.1 subunits, both expressed as homomers and heteromers with wild-type Kv1.1 subunits. KCNA1 pore mutations markedly decreased (p. P405S) or fully suppressed (p. P403S, p. P405L) Kv1.1-mediated currents, exerting loss-of-function (LoF) effects. By contrast, channels carrying the p.A261T variant exhibited a hyperpolarizing shift of the activation process, consistent with a gain-of-function (GoF) effect. The present results unveil a novel correlation between in vitro phenotype (GoF vs LoF) and clinical course (mild vs severe) in KCNA1-related phenotypes. The excellent clinical response to carbamazepine observed in the patients carrying the A261T variant suggests an exquisite sensitivity of KCNA1 GoF to sodium channel inhibition that should be further explored., (© 2021 The Authors. Epilepsia published by Wiley Periodicals LLC on behalf of International League Against Epilepsy.)

Heterozygous variants in the KCNQ3 gene cause epileptic and/or developmental disorders of varying severity. Here we describe the generation of induced pluripotent stem cells (iPSCs) from a 9-year-old girl with pharmacodependent neonatal-onset epilepsy and intellectual disability who carry a homozygous single-base duplication in exon 12 of KCNQ3 (NM_004519.3: KCNQ3 c.1599dup; KCNQ3 p.PHE534ILEfs*15), and from a non-carrier brother of the proband. For iPSC generation, non-integrating episomal plasmid vectors were used to transfect fibroblasts isolated from skin biopsies. The obtained iPSC lines had a normal karyotype, showed embryonic stem cell-like morphology, expressed pluripotency markers, and possessed trilineage differentiation potential., (Copyright © 2021. Published by Elsevier B.V.)

De novo variants in KCNQ2 encoding for Kv7.2 voltage-dependent neuronal potassium (K + ) channel subunits are associated with developmental epileptic encephalopathy (DEE). We herein describe the clinical and electroencephalographic (EEG) features of a child with early-onset DEE caused by the novel KCNQ2 p.G310S variant. In vitro experiments demonstrated that the mutation induces loss-of-function effects on the currents produced by channels incorporating mutant subunits; these effects were counteracted by the selective Kv7 opener retigabine and by gabapentin, a recently described Kv7 activator. Given these data, the patient started treatment with gabapentin, showing a rapid and sustained clinical and EEG improvement over the following months. Overall, these results suggest that gabapentin can be regarded as a precision therapy for DEEs due to KCNQ2 loss-of-function mutations., (Copyright © 2020 The Authors. Published by Elsevier Ltd.. All rights reserved.)

Variants in KCNT1 are associated with a wide spectrum of epileptic phenotypes, including epilepsy of infancy with migrating focal seizures (EIMFS), non-EIMFS developmental and epileptic encephalopathies, autosomal dominant or sporadic sleep-related hypermotor epilepsy, and focal epilepsy. Here, we describe a girl affected by drug-resistant focal seizures, developmental delay and behavior disorders, caused by a novel, de novo heterozygous missense KCNT1 variant (c.2809A > G, p.S937G). Functional characterization in transiently transfected Chinese Hamster Ovary (CHO) cells revealed a strong gain-of-function effect determined by the KCNT1 p.S937G variant compared to wild-type, consisting in an increased maximal current density and a hyperpolarizing shift in current activation threshold. Exposure to the antidepressant drug fluoxetine inhibited currents expressed by both wild-type and mutant KCNT1 channels. Treatment of the proband with fluoxetine led to a prolonged electroclinical amelioration, with disappearance of seizures and better EEG background organization, together with an improvement in behavior and mood. Altogether, these results suggest that, based on the proband's genetic and functional characteristics, the antidepressant drug fluoxetine may be repurposed for the treatment of focal epilepsy caused by gain-of-function variants in KCNT1. Further studies are needed to verify whether this approach could be also applied to other phenotypes of the KCNT1-related epilepsies spectrum. [ABSTRACT FROM AUTHOR]

Understanding the molecular mechanisms underlying voltage-dependent gating in voltage-gated ion channels (VGICs) has been a major effort over the last decades. In recent years, changes in the gating process have emerged as common denominators for several genetically determined channelopathies affecting heart rhythm (arrhythmias), neuronal excitability (epilepsy, pain), or skeletal muscle contraction (periodic paralysis). Moreover, gating changes appear as the main molecular mechanism by which several natural toxins from a variety of species affect ion channel function. In this work, we describe the pathophysiological and pharmacological relevance of the gating process in voltage-gated K(+) channels encoded by the K(v)7 gene family. After reviewing the current knowledge on the molecular mechanisms and on the structural models of voltage-dependent gating in VGICs, we describe the physiological relevance of these channels, with particular emphasis on those formed by K(v)7.2-K(v)7.5 subunits having a well-established role in controlling neuronal excitability in humans. In fact, genetically determined alterations in K(v)7.2 and K(v)7.3 genes are responsible for benign familial neonatal convulsions, a rare seizure disorder affecting newborns, and the pharmacological activation of K(v)7.2/3 channels can exert antiepileptic activity in humans. Both mutation-triggered channel dysfunction and drug-induced channel activation can occur by impeding or facilitating, respectively, channel sensitivity to membrane voltage and can affect overlapping molecular sites within the voltage-sensing domain of these channels. Thus, understanding the molecular steps involved in voltage-sensing in K(v)7 channels will allow to better define the pathogenesis of rare human epilepsy, and to design innovative pharmacological strategies for the treatment of epilepsies and, possibly, other human diseases characterized by neuronal hyperexcitability.

Variable phenotypes, including developmental encephalopathy with (DEE) or without seizures and myoclonic epilepsy and ataxia due to potassium channel mutation, are caused by pathogenetic variants in KCNC1, encoding for Kv3.1 channel subunits. In vitro, channels carrying most KCNC1 pathogenic variants display loss‐of‐function features. Here, we describe a child affected by DEE with fever‐triggered seizures, caused by a novel de novo heterozygous missense KCNC1 variant (c.1273G>A; V425M). Patch‐clamp recordings in transiently transfected CHO cells revealed that, compared to wild‐type, Kv3.1 V425M currents (1) were larger, with membrane potentials between −40 and +40 mV; (2) displayed a hyperpolarizing shift in activation gating; (3) failed to inactivate; and (4) had slower activation and deactivation kinetics, consistent with a mixed functional pattern with prevalent gain‐of‐function effects. Exposure to the antidepressant drug fluoxetine inhibited currents expressed by both wild‐type and mutant Kv3.1 channels. Treatment of the proband with fluoxetine led to a rapid and prolonged clinical amelioration, with the disappearance of seizures and an improvement in balance, gross motor skills, and oculomotor coordination. These results suggest that drug repurposing based on the specific genetic defect may provide an effective personalized treatment for KCNC1‐related DEEs. [ABSTRACT FROM AUTHOR]

Retigabine is a first-in-class potassium channel opener approved for patients with epilepsy. Unfortunately, several side effects have limited its use in clinical practice, overshadowing its beneficial effects. Multiple studies have shown that retigabine acts by enhancing the activity of members of the voltage-gated KCNQ (Kv7) potassium channel family, particularly the neuronal KCNQ channels KCNQ2-KCNQ5. However, it is currently unknown whether retigabine's action in neurons is mediated by all KCNQ neuronal channels or by only a subset. This knowledge is necessary to elucidate retigabine's mechanism of action in the central nervous system and its adverse effects and to design more effective and selective retigabine analogs. In this study, we show that the action of retigabine in excitatory neurons strongly depends on the presence of KCNQ3 channels. Deletion of Kcnq3 severely limited the ability of retigabine to reduce neuronal excitability in mouse CA1 and subiculum excitatory neurons. In addition, we report that in the absence of KCNQ3 channels, retigabine can enhance CA1 pyramidal neuron activity, leading to a greater number of action potentials and reduced spike frequency adaptation; this finding further supports a key role of KCNQ3 channels in mediating the action of retigabine. Our work provides new insight into the action of retigabine in forebrain neurons, clarifying retigabine's action in the nervous system. NEW & NOTEWORTHY Retigabine has risen to prominence as a first-in-class potassium channel opener approved by the Food and Drug Administration, with potential for treating multiple neurological disorders. Here, we demonstrate that KCNQ3 channels are the primary target of retigabine in excitatory neurons, as deleting these channels greatly diminishes the effect of retigabine in pyramidal neurons. Our data provide the first indication that retigabine controls neuronal firing properties primarily through KCNQ3 channels. [ABSTRACT FROM AUTHOR]

Pathogenic variants in KCNQ2 and KCNQ3 , paralogous genes encoding Kv7.2 and Kv7.3 voltage-gated K+ channel subunits, are responsible for early−onset developmental/epileptic disorders characterized by heterogeneous clinical phenotypes ranging from benign familial neonatal epilepsy (BFNE) to early−onset developmental and epileptic encephalopathy (DEE). KCNQ2 variants account for the majority of pedigrees with BFNE and KCNQ3 variants are responsible for a much smaller subgroup, but the reasons for this imbalance remain unclear. Analysis of additional pedigrees is needed to further clarify the nature of this genetic heterogeneity and to improve prediction of pathogenicity for novel variants. We identified a BFNE family with two siblings and a parent affected. Exome sequencing on samples from both parents and siblings revealed a novel KCNQ3 variant (c.719T>G; p.M240R), segregating in the three affected individuals. The M240 residue is conserved among human Kv7.2-5 and lies between the two arginines (R5 and R6) closest to the intracellular side of the voltage-sensing S4 transmembrane segment. Whole cell patch-clamp recordings in Chinese hamster ovary (CHO) cells revealed that homomeric Kv7.3 M240R channels were not functional, whereas heteromeric channels incorporating Kv7.3 M240R mutant subunits with Kv7.2 and Kv7.3 displayed a depolarizing shift of about 10 mV in activation gating. Molecular modeling results suggested that the M240R substitution preferentially stabilized the resting state and possibly destabilized the activated state of the Kv7.3 subunits, a result consistent with functional data. Exposure to β-hydroxybutyrate (BHB), a ketone body generated during the ketogenic diet (KD), reversed channel dysfunction induced by the M240R variant. In conclusion, we describe the first missense loss-of-function (LoF) pathogenic variant within the S4 segment of Kv7.3 identified in patients with BFNE. Studied under conditions mimicking heterozygosity, the M240R variant mainly affects the voltage sensitivity, in contrast to previously analyzed BFNE Kv7.3 variants that reduce current density. Our pharmacological results provide a rationale for the use of KD in patients carrying LoF variants in Kv7.2 or Kv7.3 subunits. [ABSTRACT FROM AUTHOR]

Objective: Heterozygous variants in KCNQ2 or, more rarely, KCNQ3 genes are responsible for early‐onset developmental/epileptic disorders characterized by heterogeneous clinical presentation and course, genetic transmission, and prognosis. While familial forms mostly include benign epilepsies with seizures starting in the neonatal or early‐infantile period, de novo variants in KCNQ2 or KCNQ3 have been described in sporadic cases of early‐onset encephalopathy (EOEE) with pharmacoresistant seizures, various age‐related pathological EEG patterns, and moderate/severe developmental impairment. All pathogenic variants in KCNQ2 or KCNQ3 occur in heterozygosity. The aim of this work was to report the clinical, molecular, and functional properties of a new KCNQ3 variant found in homozygous configuration in a 9‐year‐old girl with pharmacodependent neonatal‐onset epilepsy and non‐syndromic intellectual disability. Methods: Exome sequencing was used for genetic investigation. KCNQ3 transcript and subunit expression in fibroblasts was analyzed with quantitative real‐time PCR and Western blotting or immunofluorescence, respectively. Whole‐cell patch‐clamp electrophysiology was used for functional characterization of mutant subunits. Results: A novel single‐base duplication in exon 12 of KCNQ3 (NM_004519.3:c.1599dup) was found in homozygous configuration in the proband born to consanguineous healthy parents; this frameshift variant introduced a premature termination codon (PTC), thus deleting a large part of the C‐terminal region. Mutant KCNQ3 transcript and protein abundance was markedly reduced in primary fibroblasts from the proband, consistent with nonsense‐mediated mRNA decay. The variant fully abolished the ability of KCNQ3 subunits to assemble into functional homomeric or heteromeric channels with KCNQ2 subunits. Significance: The present results indicate that a homozygous KCNQ3 loss‐of‐function variant is responsible for a severe phenotype characterized by neonatal‐onset pharmacodependent seizures, with developmental delay and intellectual disability. They also reveal difference in genetic and pathogenetic mechanisms between KCNQ2‐ and KCNQ3‐related epilepsies, a crucial observation for patients affected with EOEE and/or developmental disabilities. [ABSTRACT FROM AUTHOR]

Over one hundred mutations in the Kv7.2 (KCNQ2) gene encoding for phosphatidylinositol 4,5-bisphosphate (PIP2)-sensitive voltage-gated K+ channel subunits have been identified in early-onset epilepsies with wide phenotypic variability. By contrast, only few mutations in the closely related Kv7.3 (KCNQ3) gene have been reported, mostly associated with typical benign familial neonatal seizures (BFNS). We herein describe a patient affected by early onset epileptic encephalopathy (EOEE) carrying two Kv7.3 missense mutations (p.Val359Leu/V359L and p.Asp542Asn/D542N) in compound heterozygosis, each inherited from an asymptomatic parent. Patch-clamp recordings from transiently transfected CHO cells showed that, when incorporated in physiologically relevant Kv7.2 + Kv7.3 heteromeric channels, expression of Kv7.3 V359L or Kv7.3 D542N subunits failed to affect current density, whereas a significant decrease was instead observed when these mutant subunits were both simultaneously present. Modeling and functional experiments revealed that each variant decreased PIP2-dependent current regulation, with additive effects when the two were co-expressed. Moreover, expression of Kv7.2 subunits carrying the D535N variant previously described in three sporadic EOEE cases prompted functional changes more dramatic when compared to those of the corresponding D542N variant in Kv7.3, but similar to those observed when both Kv7.3 V359L and Kv7.3 D542N subunits were expressed together. Finally, the Kv7 activator retigabine restored channel dysfunction induced by each Kv7.2 or Kv7.3 variant(s). These results provide a plausible molecular explanation for the apparent recessive inheritance of the phenotype in the family investigated, and a rational basis for personalized therapy with Kv7 channel activators in EOEE patients carrying loss-of-function mutations in Kv7.2 or Kv7.3. [ABSTRACT FROM AUTHOR]

Objective To analyze whether KCNQ2 R201C and R201H variants, which show atypical gain-of-function electrophysiologic properties in vitro , have a distinct clinical presentation and outcome. Methods Ten children with heterozygous, de novo KCNQ2 R201C or R201H variants were identified worldwide, using an institutional review board ( IRB)-approved KCNQ2 patient registry and database. We reviewed medical records and, where possible, interviewed parents and treating physicians using a structured, detailed phenotype inventory focusing on the neonatal presentation and subsequent course. Results Nine patients had encephalopathy from birth and presented with prominent startle-like myoclonus, which could be triggered by sound or touch. In seven patients, electroencephalography ( EEG) was performed in the neonatal period and showed a burst-suppression pattern. However, myoclonus did not have an EEG correlate. In many patients the paroxysmal movements were misdiagnosed as seizures. Seven patients developed epileptic spasms in infancy. In all patients, EEG showed a slow background and multifocal epileptiform discharges later in life. Other prominent features included respiratory dysfunction (perinatal respiratory failure and/or chronic hypoventilation), hypomyelination, reduced brain volume, and profound developmental delay. One patient had a later onset, and sequencing indicated that a low abundance (~20%) R201C variant had arisen by postzygotic mosaicism. Significance Heterozygous KCNQ2 R201C and R201H gain-of-function variants present with profound neonatal encephalopathy in the absence of neonatal seizures. Neonates present with nonepileptic myoclonus that is often misdiagnosed and treated as seizures. Prognosis is poor. This clinical presentation is distinct from the phenotype associated with loss-of-function variants, supporting the value of in vitro functional screening. These findings suggest that gain-of-function and loss-of-function variants need different targeted therapeutic approaches. [ABSTRACT FROM AUTHOR]

Mutations in the KCNQ2 gene encoding for voltage-gated potassium channel subunits have been found in patients affected with early onset epilepsies with wide phenotypic heterogeneity, ranging from benign familial neonatal seizures (BFNS) to epileptic encephalopathy with cognitive impairment, drug resistance, and characteristic electroencephalography (EEG) and neuroradiologic features. By contrast, only few KCNQ3 mutations have been rarely described, mostly in patients with typical BFNS. We report clinical, genetic, and functional data from a family in which early onset epilepsy and neurocognitive deficits segregated with a novel mutation in KCNQ3 (c.989G>T; p.R330L). Electrophysiological studies in mammalian cells revealed that incorporation of KCNQ3 R330L mutant subunits impaired channel function, suggesting a pathogenetic role for such mutation. The degree of functional impairment of channels incorporating KCNQ3 R330L subunits was larger than that of channels carrying another KCNQ3 mutation affecting the same codon but leading to a different amino acid substitution (p.R330C), previously identified in two families with typical BFNS. These data suggest that mutations in KCNQ3, similarly to KCNQ2, can be found in patients with more severe phenotypes including intellectual disability, and that the degree of the functional impairment caused by mutations at position 330 in KCNQ3 may contribute to clinical disease severity. [ABSTRACT FROM AUTHOR]

Heteromeric assembly of KCNQ2 and KCNQ3 subunits underlie the M-current (IKM), a slowly activating and noninactivating neuronal K+ current. Mutations in KCNQ2 and KCNQ3 genes cause benign familial neonatal convulsions (BFNCs), a rare autosomal-dominant epilepsy of the newborn. In the present study, we describe the identification of a novel KCNQ2 heterozygous mutation (c587t) in a BFNC-affected family, leading to an alanine to valine substitution at amino acid position 196 located at the N-terminal end of the voltage-sensing S4 domain. The consequences on KCNQ2 subunit function prompted by the A196V substitution, as well as by the A196V/L197P mutation previously described in another BFNC-affected family, were investigated by macroscopic and single-channel current measurements in CHO cells transiently transfected with wild-type and mutant subunits. When compared with KCNQ2 channels, homomeric KCNQ2 A196V or A196V/L197P channels showed a 20 mV rightward shift in their activation voltage dependence, with no concomitant change in maximal open probability or single-channel conductance. Furthermore, current activation kinetics of KCNQ2 A196V channels displayed an unusual dependence on the conditioning prepulse voltage, being markedly slower when preceded by prepulses to more depolarized potentials. Heteromeric channels formed by KCNQ2 A196V and KCNQ3 subunits displayed gating changes similar to those of KCNQ2 A196V homomeric channels. Collectively, these results reveal a novel role for noncharged residues in the N-terminal end of S4 in controlling gating of IKM and suggest that gating changes caused by mutations at these residues may decrease IKM function, thus causing neuronal hyperexcitability, ultimately leading to neonatal convulsions. [ABSTRACT FROM AUTHOR]

KCNQ subunits encode for the M current (I[subKM]), a neuron-specific voltage-dependent K[sup+] current with a well established role in the control of neuronal excitability. In this study, by means of a combined biochemical, pharmacological, and electrophysiological approach, the role of presynaptic I[subKM] in the release of previously taken up tritiated norepineprine (NE), GABA, and D-aspartate (D-ASP) from hippocampal nerve terminals (synaptosomes) has been evaluated. Retigabine (RT) (0.01-30 µM), a specific activator of I[subKM], inhibited [³H]NE, [³H]D-ASP, and [³H]GABA release evoked by 9 mM extracellular K[sup+] ([K[sup+][sube]). RT-induced inhibition of [³H]NE release was prevented by synaptosomal entrapment of polyclonal antibodies directed against KCNQ2 subunits, an effect that was abolished by antibody preabsorption with the KCNQ2 immunizing peptide; antibodies against KCNQ3 subunits were ineffective. Flupirtine (FP), a structural analog of RT, also inhibited 9 mM [K[sup+][sube] RT-induced [³H]NE release, although its maximal inhibition was lower than that of RT. Electrophysiological studies in KCNQ2-transfected Chinese hamster ovary cells revealed that RT and FP (10 µM) caused a -19 and -9 mV hyperpolarizing shift, respectively, in the voltage dependence of activation of KCNQ2 K[sup+] channels. In the same cells, the cognition enhancer 10,10-bis (4-pyridinylmethyl)-9(10H)-anthracenone (XE-991) (10 µM) blocked KCNQ2 channels and prevented their activation by RT (1-10 µM). Finally, both XE-991 (10-100 µM) and tetraethylammonium ions (100 µM) abolished the inhibitory effect of RT (1 µM) on [³H]NE release. These findings provide novel evidence for a major regulatory role of KCNQ2 K[sup+] channel subunits in neurotransmitter release from rat hippocampal nerve endings. [ABSTRACT FROM AUTHOR]

Examines the effect of first-generation antihistamines on patients at risks of developing cardiac arrhythmias and epileptic manifestations. Cardiac toxicity potentials of antihistamines; Cardiovascular adverse effects of first-generation antihistamines; Structure and activity requirements for diphenhydramine analogues.

Mutations in the KCNQ2 gene, encoding for voltage-gated Kv7.2 K + channel subunits, are responsible for early-onset epileptic diseases with widely-diverging phenotypic presentation, ranging from Benign Familial Neonatal Seizures (BFNS) to epileptic encephalopathy. In the present study, Kv7.2 BFNS-causing mutations (W344R, L351F, L351V, Y362C, and R553Q) have been investigated for their ability to interfere with calmodulin (CaM) binding and CaM-induced channel regulation. To this aim, semi-quantitative (Far-Western blotting) and quantitative (Surface Plasmon Resonance and dansylated CaM fluorescence) biochemical assays have been performed to investigate the interaction of CaM with wild-type or mutant Kv7.2 C-terminal fragments encompassing the CaM-binding domain; in parallel, mutation-induced changes in CaM-dependent Kv7.2 or Kv7.2/Kv7.3 current regulation were investigated by patch-clamp recordings in Chinese Hamster Ovary (CHO) cells co-expressing Kv7.2 or Kv7.2/Kv7.3 channels and CaM or CaM 1234 (a CaM isoform unable to bind Ca 2+ ). The results obtained suggest that each BFNS-causing mutation prompts specific biochemical and/or functional consequences; these range from slight alterations in CaM affinity which did not translate into functional changes (L351V), to a significant reduction in the affinity and functional modulation by CaM (L351F, Y362C or R553Q), to a complete functional loss without significant alteration in CaM affinity (W344R). CaM overexpression increased Kv7.2 and Kv7.2/Kv7.3 current levels, and partially (R553Q) or fully (L351F) restored normal channel function, providing a rationale pathogenetic mechanism for mutation-induced channel dysfunction in BFNS, and highlighting the potentiation of CaM-dependent Kv7.2 modulation as a potential therapeutic approach for Kv7.2-related epilepsies. [ABSTRACT FROM AUTHOR]

Abstract: Benign Familial Neonatal Seizures (BFNS) is a rare, autosomal-dominant epilepsy of the newborn caused by mutations in Kv7.2 (KCNQ2) or Kv7.3 (KCNQ3) genes encoding for neuronal potassium (K+) channel subunits. In this study, we describe a sporadic case of BFNS; the affected child carried heterozygous missense mutations in both Kv7.2 (D212G) and Kv7.3 (P574S) alleles. Electrophysiological experiments revealed that the Kv7.2 D212G substitution, neutralizing a unique negatively-charged residue in the voltage sensor of Kv7.2 subunits, altered channel gating, leading to a marked destabilization of the open state, a result consistent with structural analysis of the Kv7.2 subunit, suggesting a possible pathogenetic role for BFNS of this Kv7.2 mutation. By contrast, no significant functional changes appeared to be prompted by the Kv7.3 P574S substitution. Computational modelling experiments in CA1 pyramidal cells revealed that the gating changes introduced by the Kv7.2 D212G increased cell firing frequency, thereby triggering the neuronal hyperexcitability which underlies the observed neonatal epileptic condition. [Copyright &y& Elsevier]

KCNQ2 and KCNQ3 K+ channel subunits underlie the muscarinic-regulated K+ current (IKM), a widespread regulator of neuronal excitability. Mutations in KCNQ2- or KCNQ3-encoding genes cause benign familiar neonatal convulsions (BFNCs), a rare autosomal-dominant idiopathic epilepsy of the newborn. In the present study, we have investigated, by means of electrophysiological, biochemical, and immunocytochemical techniques in transiently transfected cells, the consequences prompted by a BFNC-causing 1-bp deletion (2043ΔT) in the KCNQ2 gene; this frameshift mutation caused the substitution of the last 163 amino acids of the KCNQ2 C terminus and the extension of the subunit by additional 56 residues. The 2043ΔT mutation abolished voltage-gated K+ currents produced upon homomeric expression of KCNQ2 subunits, dramatically reduced the steady-state cellular levels of KCNQ2 subunits, and prevented their delivery to the plasma membrane. Metabolic labeling experiments revealed that mutant KCNQ2 subunits underwent faster degradation; 10-h treatment with the proteasomal inhibitor MG132 (20 μM) at least partially reversed such enhanced degradation. Co-expression with KCNQ3 subunits reduced the degradation rate of mutant KCNQ2 subunits and led to their expression on the plasma membrane. Finally, co-expression of KCNQ2 2043ΔT together with KCNQ3 subunits generated functional voltage-gated K+ currents having pharmacological and biophysical properties of heteromeric channels. Collectively, the present results suggest that mutation-induced reduced stability of KCNQ2 subunits may cause epilepsy in neonates. [ABSTRACT FROM AUTHOR]