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Developmental and epileptic encephalopathies (DEEs) are neurodevelopmental diseases characterized by refractory epilepsy, distinct electroencephalographic and neuroradiological features, and various degrees of developmental delay. Mutations in KCNQ2, KCNQ3, and, more rarely, KCNQ5 genes encoding voltage-gated potassium channel subunits variably contributing to excitability control of specific neuronal populations at distinct developmental stages have been associated to DEEs. In the present work, the clinical features of two DEE patients carrying de novo KCNQ5 variants affecting the same residue in the pore region of the Kv7.5 subunit (G347S/A) are described. The in vitro functional properties of channels incorporating these variants were investigated with electrophysiological and biochemical techniques to highlight pathophysiological disease mechanisms. Currents carried by Kv7.5 G347 S/A channels displayed: 1) large (10 times) increases in maximal current density, 2) the occurrence of a voltage-independent component, 3) slower deactivation kinetics, and 4) hyperpolarization shift in activation. All these functional features are consistent with a gain-of-function (GoF) pathogenetic mechanism. Similar functional changes were also observed when the same variants were introduced at the corresponding position in Kv7.2 subunits. Nonstationary noise analysis revealed that GoF effects observed for both Kv7.2 and Kv7.5 variants were mainly attributable to an increase in single-channel open probability, without changes in membrane abundance or single-channel conductance. The mutation-induced increase in channel opening probability was insensitive to manipulation of membrane levels of the critical Kv7 channel regulator PIP2. These results reveal a pathophysiological mechanism for KCNQ5-related DEEs, which might be exploited to implement personalized treatments.

Background In December 2019, a novel human-infecting coronavirus, SARS-CoV-2, had emerged. The WHO has classified the epidemic as a “public health emergency of international concern”. A dramatic situation has unfolded with thousands of deaths, occurring mainly in the aged and very ill people. Epidemiological studies suggest that immune system function is impaired in elderly individuals and these subjects often present a deficiency in fat-soluble and hydrosoluble vitamins. Methods We searched for reviews describing the characteristics of autoimmune diseases and the available therapeutic protocols for their treatment. We set them as a paradigm with the purpose to uncover common pathogenetic mechanisms between these pathological conditions and SARS-CoV-2 infection. Furthermore, we searched for studies describing the possible efficacy of vitamins A, D, E, and C in improving the immune system function. Results SARS-CoV-2 infection induces strong immune system dysfunction characterized by the development of an intense proinflammatory response in the host, and the development of a life-threatening condition defined as cytokine release syndrome (CRS). This leads to acute respiratory syndrome (ARDS), mainly in aged people. High mortality and lethality rates have been observed in elderly subjects with CoV-2-related infection. Conclusions Vitamins may shift the proinflammatory Th17-mediated immune response arising in autoimmune diseases towards a T-cell regulatory phenotype. This review discusses the possible activity of vitamins A, D, E, and C in restoring normal antiviral immune system function and the potential therapeutic role of these micronutrients as part of a therapeutic strategy against SARS-CoV-2 infection.

Seizures are the most common neurological manifestation in the newborn period, with an estimated incidence of 1.8–3.5 per 1000 live births. Prolonged or intractable seizures have a detrimental effect on cognition and brain function in experimental animals and are associated with adverse long-term neurodevelopmental sequelae and an increased risk of post-neonatal epilepsy in humans. The developing brain is particularly susceptible to the potentially severe effects of epilepsy, and epilepsy, especially when refractory to medications, often results in a developmental and epileptic encephalopathy (DEE) with developmental arrest or regression. DEEs can be primarily attributed to genetic causes. Given the critical role of potassium (K+) currents with distinct subcellular localization, biophysical properties, modulation, and pharmacological profile in regulating intrinsic electrical properties of neurons and their responsiveness to synaptic inputs, it is not too surprising that genetic research in the past two decades has identified several K+ channel genes as responsible for a large fraction of DEE. In the present article, we review the genetically determined epileptic channelopathies affecting three members of the Kv7 family, namely Kv7.2 (KCNQ2), Kv7.3 (KCNQ3), and Kv7.5 (KCNQ5); we review the phenotypic spectrum of Kv7-related epileptic channelopathies, the different genetic and pathogenetic mechanisms, and the emerging genotype-phenotype correlations which may prove crucial for prognostic predictions, disease management, parental counseling, and individually tailored therapeutic attempts.

Mitochondrial K+ permeability regulates neuronal apoptosis, energy metabolism, autophagy, and protection against ischemia–reperfusion injury. Kv7.4 channels have been recently shown to regulate K+ permeability in cardiac mitochondria and exert cardioprotective effects. Here, the possible expression and functional role of Kv7.4 channels in regulating membrane potential, radical oxygen species (ROS) production, and Ca2+ uptake in neuronal mitochondria was investigated in both clonal (F11 cells) and native brain neurons. In coupled mitochondria isolated from F11 cells, K+-dependent changes of mitochondrial membrane potential (ΔΨ) were unaffected by the selective mitoBKCa channel blocker iberiotoxin and only partially inhibited by the mitoKATP blockers glyburide or ATP. Interestingly, K+-dependent ΔΨ decrease was significantly reduced by the Kv7 blocker XE991 and enhanced by the Kv7 activator retigabine. Among Kv7s, western blot experiments showed the expression of only Kv7.4 subunits in F11 mitochondrial fractions; immunocytochemistry experiments showed a strong overlap between the Kv7.4 fluorescent signal and that of the mitochondrial marker Mitotracker. Silencing of Kv7.4 expression significantly suppressed retigabine-dependent decrease in ΔΨ in intact F11 cells. Expression of Kv7.4 subunits was also detected by western blot in isolated mitochondria from total mouse brain and by immunofluorescence in mouse primary cortical neurons. Pharmacological experiments revealed a relevant functional role for Kv7.4 channels in regulating membrane potential and Ca2+ uptake in isolated neuronal mitochondria, as well as ΔΨ and ROS production in intact cortical neurons. In conclusion, these findings provide the first experimental evidence for the expression of Kv7.4 channels and their contribution in regulating K+ permeability of neuronal mitochondria.

Significance Variants in genes encoding neuronally expressed potassium channel subunits are frequent causes of developmental and epileptic encephalopathies (DEEs). Characterization of their functional consequences is critical to confirm diagnosis, assess prognosis, and implement personalized treatments. In the present work, we describe two patients carrying variants in KCNQ5 , a gene very recently and rarely found involved in DEEs, and reveal that they both cause remarkable gain-of-function consequences on channel activity. A PIP 2 -independent increase in open probability, without effects on membrane abundance or single-channel conductance, was responsible for the observed mutation-induced functional changes, thus revealing a pathomolecular disease mechanism for DEEs.

Neuronal Kv7 channels represent important pharmacological targets for hyperexcitability disorders including epilepsy. Retigabine is the prototype Kv7 activator clinically approved for seizure treatment; however, severe side effects associated with long-term use have led to its market discontinuation. Building upon the recently described cryoEM structure of Kv7.2 complexed with retigabine and on previous structure-activity relationship studies, a small library of retigabine analogues has been designed, synthesized, and characterized for their Kv7 opening ability using both fluorescence- and electrophysiology-based assays. Among all tested compounds, 60 emerged as a potent and photochemically stable neuronal Kv7 channel activator. Compared to retigabine, compound 60 displayed a higher brain/plasma distribution ratio, a longer elimination half-life, and more potent and effective anticonvulsant effects in an acute seizure model in mice. Collectively, these data highlight compound 60 as a promising lead compound for the development of novel Kv7 activators for the treatment of hyperexcitability diseases.

Kv7 K+ channels represent attractive pharmacological targets for the treatment of different neurological disorders, including epilepsy. In this paper, 42 conformationally restricted analogues of the prototypical Kv7 activator retigabine have been synthesized and tested by electrophysiological patch-clamp experiments as Kv7 agonists. When compared to retigabine (0.93 ± 0.43 μM), the EC50s for Kv7.2 current enhancements by compound 23a (0.08 ± 0.04 μM) were lower, whereas no change in potency was observed for 24a (0.63 ± 0.07 μM). In addition, compared to retigabine, 23a and 24a showed also higher potency in activating heteromeric Kv7.2/Kv7.3 and homomeric Kv7.4 channels. Molecular modeling studies provided new insights into the chemical features required for optimal interaction at the binding site. Stability studies evidenced improved chemical stability of 23a and 24a in comparison with retigabine. Overall, the present results highlight that the N5-alkylamidoindole moiety provides a suitable pharmacophoric scaffold for the design of chemically stable, highly potent and selective Kv7 agonists.

Alzheimer's disease (AD) is a neurodegenerative disorder that represents the first cause of dementia. Although there has been significant progress in AD research, the actual mechanisms underlying this pathology remain largely unknown. There is increasing evidence that oxidative stress, metabolic alterations, and mitochondrial dysfunction are key players in the development and worsening of AD. As a result, in the past few years, remarkable attempts have been made to develop neuroprotective strategies against the impairment of mitochondrial dynamics and cell redox status. In the present study, we reveal a novel antioxidant K

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.

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.

Benign familial infantile epilepsy (BFIE) is the most genetically heterogeneous phenotype among early-onset familial infantile epilepsies. It has an autosomal dominant inheritance pattern with incomplete penetrance. Although PRRT2 is the most mutated gene detected in families with BFIE, other mutations in KCNQ2, SCN2A, and GABRA6 genes have also been described. To date, KCNQ3 mutations have been detected in only four patients with BFIE. Here, we describe the clinical pattern and course of an additional individual with BFIE associated with a novel missense heterozygous KCNQ3 c.1850G>C variant inherited by his unaffected father. The incidence of KCNQ3 mutations among BFIE patients is reported to be low in the literature, however, whether this is underestimated is unclear as not all current epilepsy gene panels include KCNQ3.

Background The therapeutic algorithm in persistently active Rheumatoid Arthritis, despite conventional synthetic DMARDs(csDMARDs), identifies TNFα blockers and other biologics as first line treatment, without clear indications of which biologic should be adopted first in persistently active patients. Objectives In the AR-BIOM trial we analyzed several biomarkers to define which one could help to identify the best responder to IL6-R blockade. Methods Sixty-nine RA persistently active despite csDMARDs treatment, were enrolled in this Interventional Phase IV, prospective, multicenter, non-randomized, no-profit study(Clinical Trial:n.2012-001760-30) and followed for 18 months after Tocilizumab(TCZ) treatment monotherapy or in combination with csDMARDs. The study was conducted in 10 outpatient clinics of “Gruppo Italiano di Studio sulle Early Arthritis” network(GISEA Study Group) in Italy. The primary end point was the clinical response to TCZ, as LDA(DAS Results During the 18 months of the study, 9/69 patients(13.0%) discontinued the study because of treatment failure, 2(2.9%) for an AE, 2 for a SAE, and 5(7.2%) for other reasons. At 12 months FU, LDA was achieved in 75.0% and DAS-R and SDAI-R in 63.3% and 41.7% respectively, of RA patients, without significant differences between mono and combination therapy. Considering baseline biomarkers predictive of LDA at 12 months FU, IL23 plasma levels ≥43.6pg/ml arose as potent predictor [OR(95%CIs):20.0(1.9-211.2)], whereas the best predictors of DAS-R were baseline BAFF plasma levels ≥563.3pg/ml [OR(95%CIs):3.9(1.1-14.3)] and IL23 plasma levels ≥43.6pg/ml [OR(95%CIs):4.1(1.1-15.2)]. In addition, Albumin levels ≥4.25g/dl at 3 months FU, arose as a biochemical parameter predicting DAS-R [OR(95%CIs):26.0(3.9-173.1)] and SDAI-R [OR(95%CIs):5.3(1.2-22.9)] at 12 months FU. Conclusion IL23 and BAFF related inflammation are targets of IL6 blockade that significantly increases Albumin levels. High plasma levels of IL23 and BAFF at baseline represent biomarkers for LDA and DAS-R achievement in RA inflammation driven by IL6. Albumin after three months of therapy represents the strongest predictor of remission at 12 months. Disclosure of Interests Gianfranco Ferraccioli Speakers bureau: BMS, Roche, Barbara Tolusso: None declared, Florenzo Iannone Consultant for: F Iannone has received consultancy fees and/or speaker honoraria from Pfizer, AbbVie, MSD, BMS, Novartis, Lilly, UCB outside this work, Speakers bureau: F Iannone has received consultancy fees and/or speaker honoraria from Pfizer, AbbVie, MSD, BMS, Novartis, Lilly, UCB outside this work, Maurizio Rossini: None declared, Piercarlo Sarzi-Puttini Speakers bureau: Novartis, Marcello Govoni Paid instructor for: Pfizer, Roche, Speakers bureau: Pfizer, Abbvie, MSD, Roche, Eli-Lilly, Celgene, Sanofi, Janssen, Rosario Foti: None declared, Andrea Doria: None declared, Francesco Paolo Cantatore Speakers bureau: PFIZER, ROCHE, Giovanni Francesco Miceli: None declared, oscar massimiliano epis: None declared, Anna Laura Fedele: None declared, Antonio Carletto: None declared, Nicola Maruotti: None declared, Elena De Stefani: None declared, Giorgio Amato: None declared, Elisa Gremese Consultant for: AbbVie, BMS, Celgene, Janssen, Lilly, MSD, Novartis, Sanofi, UCB, Roche, and Pfizer, Speakers bureau: BMS, Speakers bureau: Roche, Speakers bureau: AbbVie, BMS, Celgene, Janssen, Lilly, MSD, Novartis, Sanofi, UCB, Roche, and Pfizer, Giovanni Lapadula: None declared

Kv7.2 subunits encoded by the KCNQ2 gene provide a major contribution to the M-current (IKM), a voltage-gated K+ current crucially involved in the regulation of neuronal excitability. Heterozygous missense variants in Kv7.2 are responsible for epileptic diseases characterized by highly heterogeneous genetic transmission and clinical severity, ranging from autosomal-dominant Benign Familial Neonatal Seizures (BFNS) to sporadic cases of severe epileptic and developmental encephalopathy (DEE). Here, we describe a patient with neonatal onset DEE, carrying a previously undescribed heterozygous KCNQ2 c.418G &gt, C, p.Glu140Gln (E140Q) variant. Patch-clamp recordings in CHO cells expressing the E140Q mutation reveal dramatic loss of function (LoF) effects. Multistate structural modelling suggested that the E140Q substitution impeded an intrasubunit electrostatic interaction occurring between the E140 side chain in S2 and the arginine at position 210 in S4 (R210), this interaction is critically involved in stabilizing the activated configuration of the voltage-sensing domain (VSD) of Kv7.2. Functional results from coupled charge reversal or disulfide trapping experiments supported such a hypothesis. Finally, retigabine restored mutation-induced functional changes, reinforcing the rationale for the clinical use of Kv7 activators as personalized therapy for DEE-affected patients carrying Kv7.2 LoF mutations.

Kv7.2-Kv7.5 channels mediate the M-current (IKM), a K+-selective current regulating neuronal excitability and representing an attractive target for pharmacological therapy against hyperexcitability diseases such as pain. Kv7 channels interact functionally with transient receptor potential vanilloid 1 (TRPV1) channels activated by endogenous and/or exogenous pain-inducing substances, such as bradykinin (BK) or capsaicin (CAP), respectively, however, whether Kv7 channels of specific molecular composition provide a dominant contribution in BK- or CAP-evoked responses is yet unknown. To this aim, Kv7 transcripts expression and function were assessed in F11 immortalized sensorial neurons, a cellular model widely used to assess nociceptive molecular mechanisms. In these cells, the effects of the pan-Kv7 activator retigabine were investigated, as well as the effects of ICA-27243 and (S)-1, two Kv7 activators acting preferentially on Kv7.2/Kv7.3 and Kv7.4/Kv7.5 channels, respectively, on BK- and CAP-induced changes in intracellular Ca2+ concentrations ([Ca2+]i). The results obtained revealed the expression of transcripts of all Kv7 genes, leading to an IKM-like current. Moreover, all tested Kv7 openers inhibited BK- and CAP-induced responses by a similar extent (~60%), at least for BK-induced Ca2+ responses, the potency of retigabine (IC50~1 &micro, M) was higher than that of ICA-27243 (IC50~5 &micro, M) and (S)-1 (IC50~7 &micro, M). Altogether, these results suggest that IKM activation effectively counteracts the cellular processes triggered by TRPV1-mediated pain-inducing stimuli, and highlight a possible critical contribution of Kv7.4 subunits.

The development of patient-specific induced pluripotent stem cells (iPSCs) offered interesting insights in modeling the pathogenesis of Charcot-Marie-Tooth (CMT) disease and thus we decided to explore the phenotypes of iPSCs derived from a single CMT patient carrying a mutant ATP1A1 allele (p.Pro600Ala). iPSCs clones generated from CMT and control fibroblasts, were induced to differentiate into neural precursors and then into post-mitotic neurons. Control iPSCs differentiated into neuronal precursors and then into post-mitotic neurons within 6-8 days. On the contrary, the differentiation of CMT iPSCs was clearly defective. Electrophysiological properties confirmed that post-mitotic neurons were less mature compared to the normal counterpart. The impairment of in vitro differentiation of CMT iPSCs only concerned with the neuronal pathway, because they were able to differentiate into mesendodermal cells and other ectodermal derivatives. ATP1A1 was undetectable in the few neuronal cells derived from CMT iPSCs. ATP1A1 gene mutation (p.Pro600Ala), responsible for a form of axonal CMT disease, is associated in vitro with a dramatic alteration of the differentiation of patient-derived iPSCs into post-mitotic neurons. Thus, the defect in neuronal cell development might lead in vivo to a decreased number of mature neurons in ATP1A1-CMT disease.

Objective Recent reports have described single individuals with neurodevelopmental disability (NDD) harboring heterozygous KCNQ3 de novo variants (DNVs). We sought to assess whether pathogenic variants in KCNQ3 cause NDD and to elucidate the associated phenotype and molecular mechanisms. Methods Patients with NDD and KCNQ3 DNVs were identified through an international collaboration. Phenotypes were characterized by clinical assessment, review of charts, electroencephalographic (EEG) recordings, and parental interview. Functional consequences of variants were analyzed in vitro by patch-clamp recording. Results Eleven patients were assessed. They had recurrent heterozygous DNVs in KCNQ3 affecting residues R230 (R230C, R230H, R230S) and R227 (R227Q). All patients exhibited global developmental delay within the first 2 years of life. Most (8/11, 73%) were nonverbal or had a few words only. All patients had autistic features, and autism spectrum disorder (ASD) was diagnosed in 5 of 11 (45%). EEGs performed before 10 years of age revealed frequent sleep-activated multifocal epileptiform discharges in 8 of 11 (73%). For 6 of 9 (67%) recorded between 1.5 and 6 years of age, spikes became near-continuous during sleep. Interestingly, most patients (9/11, 82%) did not have seizures, and no patient had seizures in the neonatal period. Voltage-clamp recordings of the mutant KCNQ3 channels revealed gain-of-function (GoF) effects. Interpretation Specific GoF variants in KCNQ3 cause NDD, ASD, and abundant sleep-activated spikes. This new phenotype contrasts both with self-limited neonatal epilepsy due to KCNQ3 partial loss of function, and with the neonatal or infantile onset epileptic encephalopathies due to KCNQ2 GoF. ANN NEUROL 2019;86:181-192

ObjectiveEarly identification of de novo KCNQ2 variants in patients with epilepsy raises prognostic issues toward optimal management. We analyzed the clinical and genetic information from a cohort of patients with de novo KCNQ2 pathogenic variants to dissect genotype-phenotype correlations.MethodsPatients with de novo KCNQ2 pathogenic variants were identified from Italy, Denmark, and Belgium. Atomic resolution Kv7.2 structures were also generated using homology modeling to map the variants.ResultsWe included 34 patients with a mean age of 4.7 years. Median seizure onset was 2 days, mainly with focal seizures with autonomic signs. Twenty-two patients (65%) were seizure free at the mean age of 1.2 years. More than half of the patients (17/32) displayed severe/profound intellectual disability; however, 4 (13%) of them had a normal cognitive outcome.A total of 28 de novo pathogenic variants were identified, most missense (25/28), and clustered in conserved regions of the protein; 6 variants recurred, and 7 were novel. We did not identify a relationship between variant position and seizure offset or cognitive outcome in patients harboring missense variants. Besides, recurrent variants were associated with overlapping epilepsy features but also variable evolution regarding the intellectual outcome.ConclusionsWe highlight the complexity of variant interpretation to assess the impact of a class of de novo KCNQ2 mutations. Genetic modifiers could be implicated, but the study paradigms to successfully address the impact of each single mutation need to be developed.

Aims: Plasmalemmal Kv7.1 (KCNQ1) channels are critical players in cardiac excitability; however, little is known on the functional role of additional Kv7 family members (Kv7.2-5) in cardiac cells. In this work, the expression, function, cellular and subcellular localization, and potential cardioprotective role against anoxic-ischaemic cardiac injury of Kv7.4 channels have been investigated. Methods and results: Expression of Kv7.1 and Kv7.4 transcripts was found in rat heart tissue by quantitative polymerase chain reaction. Western blots detected Kv7.4 subunits in mitochondria from Kv7.4-transfected cells, H9c2 cardiomyoblasts, freshly isolated adult cardiomyocytes, and whole hearts. Immunofluorescence experiments revealed that Kv7.4 subunits co-localized with mitochondrial markers in cardiac cells, with ∼ 30-40% of cardiac mitochondria being labelled by Kv7.4 antibodies, a result also confirmed by immunogold electron microscopy experiments. In isolated cardiac (but not liver) mitochondria, retigabine (1-30 μM) and flupirtine (30 μM), two selective Kv7 activators, increased Tl+influx, depolarized the membrane potential, and inhibited calcium uptake; all these effects were antagonized by the Kv7 blocker XE991. In intact H9c2 cells, reducing Kv7.4 expression by RNA interference blunted retigabine-induced mitochondrial membrane depolarization; in these cells, retigabine decreased mitochondrial Ca2+levels and increased radical oxygen species production, both effects prevented by XE991. Finally, retigabine reduced cellular damage in H9c2 cells exposed to anoxia/re-oxygenation and largely prevented the functional and morphological changes triggered by global ischaemia/reperfusion (../R) in Langendorff-perfused rat hearts. Conclusion: Kv7.4 channels are present and functional in cardiac mitochondria; their activation exerts a significant cardioprotective role, making them potential therapeutic targets against I/R-induced cardiac injury.

Epilepsy of infancy with migrating focal seizures (EIMFS) is a rare early-onset developmental epileptic encephalopathy resistant to anti-epileptic drugs. The most common cause for EIMFS is a gain-of-function mutation in the KCNT1 potassium channel gene, and treatment with the KCNT1 blocker quinidine has been suggested as a rational approach for seizure control in EIMFS patients. However, variable results on the clinical efficacy of quinidine have been reported. In the present study, we provide a detailed description of the clinical, genetic, in vitro, and in vivo electrophysiological profile and pharmacological responses to quinidine of 2 EIMFS unrelated patients with a heterozygous de novo KCNT1 mutation: c.2849G>A (p.R950Q) in patient 1 and c.2677G>A (p.E893K) in patient 2. When expressed heterologously in CHO cells, KCNT1 channels carrying each variant showed gain-of-function effects, and were more effectively blocked by quinidine when compared to wild-type KCNT1 channels. On the basis of these in vitro results, add-on quinidine treatment was started at 3 and 16 months of age in patients 1 and 2, respectively. The results obtained reveal that quinidine significantly reduced seizure burden (by about 90%) and improved quality of life in both patients, but failed to normalize developmental milestones, which persisted as severely delayed. Based on the present experience, early quinidine intervention associated with heart monitoring and control of blood levels is among the critical factors for therapy effectiveness in EIMFS patients with KCNT1 gain-of-function mutations. Multicenter studies are needed to establish a consensus protocol for patient recruitment, quinidine treatment modalities, and outcome evaluation, to optimize clinical efficacy and reduce risks as well as variability associated to quinidine use in such severe developmental encephalopathy. ELECTRONIC SUPPLEMENTARY MATERIAL: The online version of this article (10.1007/s13311-018-0657-9) contains supplementary material, which is available to authorized users.

Epilepsy of infancy with migrating focal seizures (EIMFS) is a rare early-onset developmental epileptic encephalopathy resistant to anti-epileptic drugs. The most common cause for EIMFS is a gain-of-function mutation in the KCNT1 potassium channel gene, and treatment with the KCNT1 blocker quinidine has been suggested as a rational approach for seizure control in EIMFS patients. However, variable results on the clinical efficacy of quinidine have been reported. In the present study, we provide a detailed description of the clinical, genetic, in vitro, and in vivo electrophysiological profile and pharmacological responses to quinidine of 2 EIMFS unrelated patients with a heterozygous de novo KCNT1 mutation: c.2849G>A (p.R950Q) in patient 1 and c.2677G>A (p.E893K) in patient 2. When expressed heterologously in CHO cells, KCNT1 channels carrying each variant showed gain-of-function effects, and were more effectively blocked by quinidine when compared to wild-type KCNT1 channels. On the basis of these in vitro results, add-on quinidine treatment was started at 3 and 16 months of age in patients 1 and 2, respectively. The results obtained reveal that quinidine significantly reduced seizure burden (by about 90%) and improved quality of life in both patients, but failed to normalize developmental milestones, which persisted as severely delayed. Based on the present experience, early quinidine intervention associated with heart monitoring and control of blood levels is among the critical factors for therapy effectiveness in EIMFS patients with KCNT1 gain-of-function mutations. Multicenter studies are needed to establish a consensus protocol for patient recruitment, quinidine treatment modalities, and outcome evaluation, to optimize clinical efficacy and reduce risks as well as variability associated to quinidine use in such severe developmental encephalopathy.

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.

Over one hundred mutations in the Kv7.2 (KCNQ2) gene encoding for phosphatidylinositol 4,5-bisphosphate (PIP

Summary Variants in KCNQ2 encoding for Kv7.2 neuronal K+ channel subunits lead to a spectrum of neonatal-onset epilepsies, ranging from self-limiting forms to severe epileptic encephalopathy. Most KCNQ2 pathogenic variants cause loss-of-function, whereas few increase channel activity (gain-of-function). We herein provide evidence for a new phenotypic and functional profile in KCNQ2-related epilepsy: infantile spasms without prior neonatal seizures associated with a gain-of-function gene variant. With use of an international registry, we identified four unrelated patients with the same de novo heterozygous KCNQ2 c.593G>A, p.Arg198Gln (R198Q) variant. All were born at term and discharged home without seizures or concern of encephalopathy, but developed infantile spasms with hypsarrhythmia (or modified hypsarrhythmia) between the ages of 4 and 6 months. At last follow-up (ages 3–11 years), all patients were seizure-free and had severe developmental delay. In vitro experiments showed that Kv7.2 R198Q subunits shifted current activation gating to hyperpolarized potentials, indicative of gain-of-function; in neurons, Kv7.2 and Kv7.2 R198Q subunits similarly populated the axon initial segment, suggesting that gating changes rather than altered subcellular distribution contribute to disease molecular pathogenesis. We conclude that KCNQ2 R198Q is a model for a new subclass of KCNQ2 variants causing infantile spasms and encephalopathy, without preceding neonatal seizures. A PowerPoint slide summarizing this article is available for download in the Supporting Information section here

Mutations in the KCNQ2 and KCNQ3 genes encoding for Kv 7.2 (KCNQ2; Q2) and Kv 7.3 (KCNQ3; Q3) voltage-dependent K(+) channel subunits, respectively, cause neonatal epilepsies with wide phenotypic heterogeneity. In addition to benign familial neonatal epilepsy (BFNE), KCNQ2 mutations have been recently found in families with one or more family members with a severe outcome, including drug-resistant seizures with psychomotor retardation, electroencephalogram (EEG) suppression-burst pattern (Ohtahara syndrome), and distinct neuroradiological features, a condition that was named "KCNQ2 encephalopathy." In the present article, we describe clinical, genetic, and functional data from 17 patients/families whose electroclinical presentation was consistent with the diagnosis of BFNE. Sixteen different heterozygous mutations were found in KCNQ2, including 10 substitutions, three insertions/deletions and three large deletions. One substitution was found in KCNQ3. Most of these mutations were novel, except for four KCNQ2 substitutions that were shown to be recurrent. Electrophysiological studies in mammalian cells revealed that homomeric or heteromeric KCNQ2 and/or KCNQ3 channels carrying mutant subunits with newly found substitutions displayed reduced current densities. In addition, we describe, for the first time, that some mutations impair channel regulation by syntaxin-1A, highlighting a novel pathogenetic mechanism for KCNQ2-related epilepsies.

K v 7.4 channel subunits are expressed in central auditory pathways and in inner ear sensory hair cells and skeletal and smooth muscle cells. Openers of K v 7.4 channels have been suggested to improve hearing loss, systemic or pulmonary arterial hypertension, urinary incontinence, gastrointestinal and neuropsychiatric diseases, and skeletal muscle disorders. Scorpion venoms are a large source of peptides active on K + channels. Therefore, we have optimized a combined purification/screening procedure to identify specific modulator(s) of K v 7.4 channels from the venom of the North African scorpion Androctonus australis ( Aa ). We report the isolation and functional characterization of AaTXK β (2–64) , a novel variant of AaTXK β (1–64) , in a high-performance liquid chromatography fraction from Aa venom (named P8), which acts as the first peptide activator of K v 7.4 channels. In particular, in both Xenopus oocytes and mammalian Chinese hamster ovary cells, AaTXK β (2–64) , but not AaTXK β (1–64) , hyperpolarized the threshold voltage of current activation and increased the maximal currents of heterologously expressed K v 7.4 channels. AaTXK β (2–64) also activated K v 7.3, K v 7.2/3, and K v 7.5/3 channels, whereas homomeric K v 1.1, K v 7.1, and K v 7.2 channels were unaffected. We anticipate that these results may prove useful in unraveling the novel biologic roles of AaTXK β (2–64) -sensitive K v 7 channels and developing novel pharmacologic tools that allow subtype-selective targeting of K v 7 channels.

Background: The Kv7 (KCNQ) subfamily of voltage-gated potassium channels consists of 5 members (Kv7.1-5) each showing characteristic tissue distribution and physiological roles. Given their functional heterogeneity, Kv7 channels represent important pharmacological targets for the development of new drugs for neuronal, cardiovascular and metabolic diseases. Objective: In the present manuscript, we focus on describing the pharmacological relevance and potential therapeutic applications of drugs acting on neuronally-expressed Kv7.2/3 channels, placing particular emphasis on the different chemotypes, and highlighting their pharmacodynamic and, whenever possible, pharmacokinetic peculiarities. Methods: The present work is based on an in-depth search of the currently available scientific literature, and on our own experience and knowledge in the field of neuronal Kv7 channel pharmacology. Space limitations impeded to describe the full pharmacological potential of Kv7 channels; thus, we have chosen to focus on neuronal channels composed of Kv7.2 and Kv7.3 subunits, and to mainly concentrate on their involvement in epilepsy. Results: An astonishing heterogeneity in the molecular scaffolds exploitable to develop Kv7.2/3 modulators is evident, with important structural/functional peculiarities of distinct compound classes. Conclusion: In the present work we have attempted to show the current status and growing potential of the Kv7 pharmacology field. We anticipate a bright future for the field, and express our hopes that the efforts herein reviewed will result in an improved treatment of hyperexcitability (or any other) diseases.

Ectrodactyly-ectodermal dysplasia-clefting (EEC) syndrome is a rare autosomal dominant disease caused by mutations in the p63 gene. To date, approximately 40 different p63 mutations have been identified, all heterozygous. No definitive treatments are available to counteract and resolve the progressive corneal degeneration due to a premature aging of limbal epithelial stem cells. Here, we describe a unique case of a young female patient, aged 18 years, with EEC and corneal dysfunction, who was, surprisingly, homozygous for a novel and de novo R311K missense mutation in the p63 gene. A detailed analysis of the degree of somatic mosaicism in leukocytes from peripheral blood and oral mucosal epithelial stem cells (OMESCs) from biopsies of buccal mucosa showed that approximately 80% were homozygous mutant cells and 20% were heterozygous. Cytogenetic and molecular analyses excluded genomic alterations, thus suggesting a de novo mutation followed by an allelic gene conversion of the wild-type allele by de novo mutant allele as a possible mechanism to explain the homozygous condition. R311K-p63 OMESCs were expanded in vitro and heterozygous holoclones selected following clonal analysis. These R311K-p63 OMESCs were able to generate well-organized and stratified epithelia in vitro, resembling the features of healthy tissues. This study supports the rationale for the development of cultured autologous oral mucosal epithelial stem cell sheets obtained by selected heterozygous R311K-p63 stem cells, as an effective and personalized therapy for reconstructing the ocular surface of this unique case of EEC syndrome, thus bypassing gene therapy approaches. Significance This case demonstrates that in a somatic mosaicism context, a novel homozygous mutation in the p63 gene can arise as a consequence of an allelic gene conversion event, subsequent to a de novo mutation. The heterozygous mutant R311K-p63 stem cells can be isolated by means of clonal analysis and given their good regenerative capacity, they may be used to successfully correct the corneal defects present in this unique case of ectrodactyly-ectodermal dysplasia-clefting syndrome.

Kv7.2 and Kv7.3 subunits underlie the M-current, a neuronal K+ current characterized by an absolute functional requirement for phosphatidylinositol 4,5-bisphosphate (PIP2). Kv7.2 gene mutations cause early-onset neonatal seizures with heterogeneous clinical outcomes, ranging from self-limiting benign familial neonatal seizures to severe early-onset epileptic encephalopathy (Kv7.2-EE). In this study, the biochemical and functional consequences prompted by a recurrent variant (R325G) found independently in four individuals with severe forms of neonatal-onset EE have been investigated. Upon heterologous expression, homomeric Kv7.2 R325G channels were non-functional, despite biotin-capture in Western blots revealed normal plasma membrane subunit expression. Mutant subunits exerted dominant-negative effects when incorporated into heteromeric channels with Kv7.2 and/or Kv7.3 subunits. Increasing cellular PIP2 levels by co-expression of type 1γ PI(4)P5-kinase (PIP5K) partially recovered homomeric Kv7.2 R325G channel function. Currents carried by heteromeric channels incorporating Kv7.2 R325G subunits were more readily inhibited than wild-type channels upon activation of a voltage-sensitive phosphatase (VSP), and recovered more slowly upon VSP switch-off. These results reveal for the first time that a mutation-induced decrease in current sensitivity to PIP2 is the primary molecular defect responsible for Kv7.2-EE in individuals carrying the R325G variant, further expanding the range of pathogenetic mechanisms exploitable for personalized treatment of Kv7.2-related epilepsies.

Changes in voltage-dependent gating represent a common pathogenetic mechanism for genetically inherited channelopathies, such as benign familial neonatal seizures or peripheral nerve hyperexcitability caused by mutations in neuronal K(v)7.2 channels. Mutation-induced changes in channel voltage dependence are most often inferred from macroscopic current measurements, a technique unable to provide a detailed assessment of the structural rearrangements underlying channel gating behavior; by contrast, gating currents directly measure voltage-sensor displacement during voltage-dependent gating. In this work, we describe macroscopic and gating current measurements, together with molecular modeling and molecular-dynamics simulations, from channels carrying mutations responsible for benign familial neonatal seizures and/or peripheral nerve hyperexcitability; K(v)7.4 channels, highly related to K(v)7.2 channels both functionally and structurally, were used for these experiments. The data obtained showed that mutations affecting charged residues located in the more distal portion of S(4) decrease the stability of the open state and the active voltage-sensing domain configuration but do not directly participate in voltage sensing, whereas mutations affecting a residue (R4) located more proximally in S(4) caused activation of gating-pore currents at depolarized potentials. These results reveal that distinct molecular mechanisms underlie the altered gating behavior of channels carrying disease-causing mutations at different voltage-sensing domain locations, thereby expanding our current view of the pathogenesis of neuronal hyperexcitability diseases.

The K(V)7 (KCNQ) subfamily of voltage-gated K(+) channels consists of five members (K(V)7.1-K(V)7.5) giving rise to non-inactivating, and slowly activating/deactivating currents mainly expressed in cardiac (K(V)7.1) and neuronal (K(V)7.2-K(V)7.5) tissue. In the present study, using the cut-open oocyte voltage clamp, we studied the relation of the ionic currents from homomeric neuronal K(V)7 channels (K(V)7.2-K(V)7.5) with the gating currents recorded after K(+) conductance blockade from the same channels. Increasing the recording temperature from 18 degrees C to 28 degrees C accelerated activation/deactivation kinetics of the ionic currents in all homomeric K(V)7 channels (activation Q(10)s at 0 mV were 3.8, 4.1, 8.3 and 2.8 for K(V)7.2, K(V)7.3, K(V)7.4 and K(V)7.5 channels, respectively), without large changes in currents voltage-dependence; moreover, at 28 degrees C, ionic currents carried by K(V)7.4 channels also showed a significant increase in their maximal value. Gating currents were only resolved in K(V)7.4 and K(V)7.5 channels; the size of the ON gating charges at +40 mV was 1.34 +/- 0.34 nC for K(V)7.4, and 0.79 +/- 0.20 nC for K(V)7.5. At 28 degrees C, K(V)7.4 gating currents had the following salient properties: (1) similar time integral of Q(ON) and Q(OFF), indicating no charge immobilization; (2) a left-shift in the V(1/2) of the Q(ON)/V when compared to the G/V (approximately 50 mV in the presence of 2 mM extracellular Ba(2+)); (3) a Q(ON) decay faster than ionic current activation; and (4) a rising phase in the OFF gating charge after depolarizations larger than 0 mV. These observations suggest that, in K(V)7.4 channels, VSD movement is followed by a slow and/or low bearing charge step linking to pore opening, a result which may help to clarify the molecular consequence of disease-causing mutations and drugs affecting channel gating.

The Ciona intestinalis voltage sensor–containing phosphatase (Ci-VSP) shares high homology with the phosphatidylinositol phosphatase enzyme known as PTEN (phosphatase and tensin homologue deleted on chromosome 10). We have taken advantage of the similarity between these proteins to inquire about the coupling between the voltage sensing and the phosphatase domains in Ci-VSP. Recently, it was shown that four basic residues (R11, K13, R14, and R15) in PTEN are critical for its binding onto the membrane, required for its catalytic activity. Ci-VSP has three of the basic residues of PTEN. Here, we show that when R253 and R254 (which are the homologues of R14 and R15 in PTEN) are mutated to alanines in Ci-VSP, phosphatase activity is disrupted, as revealed by a lack of effect on the ionic currents of KCNQ2/3, where current decrease is a measure of phosphatase activity. The enzymatic activity was not rescued by the introduction of lysines, indicating that the binding is an arginine-specific interaction between the phosphatase binding domain and the membrane, presumably through the phosphate groups of the phospholipids. We also found that the kinetics and steady-state voltage dependence of the S4 segment movement are affected when the arginines are not present, indicating that the interaction of R253 and R254 with the membrane, required for the catalytic action of the phosphatase, restricts the movement of the voltage sensor.

The K(v)7.2 subunits are the main molecular determinants of the M-current, a widespread K(+) current regulating neuronal excitability. Mutations in the K(v)7.2 gene cause benign familial neonatal seizures, an autosomally inherited human epilepsy. The benign familial neonatal seizure-causing mutations include those at arginine residues at positions 207 and 214 in the S(4) segment of K(v)7.2. In this study, each of the six S(4) arginines was individually replaced with neutral glutamines, and the functional properties of mutant channels were studied by whole-cell and single-channel voltage-clamp measurements. The results obtained suggest that each S(4) arginine residue plays a relevant role in the voltage-dependent gating of K(v)7.2 channels. In particular, a decreased positive charge at the N-terminal end of S(4) stabilized the activated state of the voltage-sensor, whereas positive-charge neutralization at the C-terminal end of S(4) favored the resting conformation. Strikingly, neutralization of a single arginine at position 201 was sufficient to cause a significant loss of voltage dependence in channel activation. Moreover, by comparing the functional properties of glutamine versus tryptophan substitution, we found steric bulk to play a relevant role at position 207, but not at position 214, in which the main functional effect of this disease-causing mutation seems to be a consequence of the loss of the positive charge.

Voltage-gated ion channels (VGICs) are membrane proteins that switch from a closed to open state in response to changes in membrane potential, thus enabling ion fluxes across the cell membranes. The mechanism that regulate the structural rearrangements occurring in VGICs in response to changes in membrane potential still remains one of the most challenging topic of modern biophysics. Na(+), Ca(2+) and K(+) voltage-gated channels are structurally formed by the assembly of four similar domains, each comprising six transmembrane segments. Each domain can be divided into two main regions: the Pore Module (PM) and the Voltage-Sensing Module (VSM). The PM (helices S5 and S6 and intervening linker) is responsible for gate opening and ion selectivity; by contrast, the VSM, comprising the first four transmembrane helices (S1-S4), undergoes the first conformational changes in response to membrane voltage variations. In particular, the S4 segment of each domain, which contains several positively charged residues interspersed with hydrophobic amino acids, is located within the membrane electric field and plays an essential role in voltage sensing. In neurons, specific gating properties of each channel subtype underlie a variety of biological events, ranging from the generation and propagation of electrical impulses, to the secretion of neurotransmitters and to the regulation of gene expression. Given the important functional role played by the VSM in neuronal VGICs, it is not surprising that various VSM mutations affecting the gating process of these channels are responsible for human diseases, and that compounds acting on the VSM have emerged as important investigational tools with great therapeutic potential. In the present review we will briefly describe the most recent discoveries concerning how the VSM exerts its function, how genetically inherited diseases caused by mutations occurring in the VSM affects gating in VGICs, and how several classes of drugs and toxins selectively target the VSM.

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.

Mutations inKv7.2(KCNQ2) andKv7.3(KCNQ3) genes, encoding for voltage-gated K+channel subunits underlying the neuronal M-current, have been associated with a wide spectrum of early-onset epileptic disorders ranging from benign familial neonatal seizures to severe epileptic encephalopathies. The aim of the present work has been to investigate the molecular mechanisms of channel dysfunction caused by voltage-sensing domain mutations in Kv7.2 (R144Q, R201C, and R201H) or Kv7.3 (R230C) recently found in patients with epileptic encephalopathies and/or intellectual disability. Electrophysiological studies in mammalian cells transfected with human Kv7.2 and/or Kv7.3 cDNAs revealed that each of these four mutations stabilized the activated state of the channel, thereby producing gain-of-function effects, which are opposite to the loss-of-function effects produced by previously found mutations. Multistate structural modeling revealed that the R201 residue in Kv7.2, corresponding to R230 in Kv7.3, stabilized the resting and nearby voltage-sensing domain states by forming an intricate network of electrostatic interactions with neighboring negatively charged residues, a result also confirmed by disulfide trapping experiments. Using a realistic model of a feedforward inhibitory microcircuit in the hippocampal CA1 region, an increased excitability of pyramidal neurons was found upon incorporation of the experimentally defined parameters for mutant M-current, suggesting that changes in network interactions rather than in intrinsic cell properties may be responsible for the neuronal hyperexcitability by these gain-of-function mutations. Together, the present results suggest that gain-of-function mutations in Kv7.2/3 currents may cause human epilepsy with a severe clinical course, thus revealing a previously unexplored level of complexity in disease pathogenetic mechanisms.

KCNQ2 and KCNQ3 K+ channel subunits underlie the muscarinic-regulated K+ current (I(KM)), 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 (2043deltaT) 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 2043deltaT 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 microm) 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 2043deltaT 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.