Neonatal Epileptic Encephalopathy Research Articles

Pyridoxal 5'-Phosphate Biosynthesis by Pyridox-(am)-ine 5'-Phosphate Oxidase: Species-Specific Features.

Enzymes reliant on pyridoxal 5'-phosphate (PLP), the metabolically active form of vitamin B6, hold significant importance in both biology and medicine. They facilitate various biochemical reactions, particularly in amino acid and neurotransmitter metabolisms. Vitamin B6 is absorbed by organisms in its non-phosphorylated form and phosphorylated within cells via pyridoxal kinase (PLK) and pyridox-(am)-ine 5'-phosphate oxidase (PNPOx). The flavin mononucleotide-dependent PNPOx enzyme converts pyridoxine 5'-phosphate and pyridoxamine 5'-phosphate into PLP. PNPOx is vital for both biosynthesis and salvage pathways in organisms producing B6 vitamers. However, for those depending on vitamin B6 as a nutrient, PNPOx participates only in the salvage pathway. Transferring the PLP produced via PNPOx to client apo-enzymes is indispensable for their catalytic function, proper folding and targeting of specific organelles. PNPOx activity deficiencies due to inborn errors lead to severe neurological pathologies, particularly neonatal epileptic encephalopathy. PNPOx maintains PLP homeostasis through highly regulated mechanisms, including structural alterations throughout the catalytic cycle and allosteric PLP binding, influencing substrate transformation at the active site. Elucidation at the molecular level of the mechanisms underlying PNPOx activity deficiencies is a requirement to develop personalized approaches to treat related disorders. Finally, despite shared features, the few PNPOx enzymes molecularly and functionally studied show species-specific regulatory properties that open the possibility of targeting it in pathogenic organisms.

MLe-KCNQ2: An Artificial Intelligence Model for the Prognosis of Missense KCNQ2 Gene Variants.

Despite the increasing availability of genomic data and enhanced data analysis procedures, predicting the severity of associated diseases remains elusive in the absence of clinical descriptors. To address this challenge, we have focused on the KV7.2 voltage-gated potassium channel gene (KCNQ2), known for its link to developmental delays and various epilepsies, including self-limited benign familial neonatal epilepsy and epileptic encephalopathy. Genome-wide tools often exhibit a tendency to overestimate deleterious mutations, frequently overlooking tolerated variants, and lack the capacity to discriminate variant severity. This study introduces a novel approach by evaluating multiple machine learning (ML) protocols and descriptors. The combination of genomic information with a novel Variant Frequency Index (VFI) builds a robust foundation for constructing reliable gene-specific ML models. The ensemble model, MLe-KCNQ2, formed through logistic regression, support vector machine, random forest and gradient boosting algorithms, achieves specificity and sensitivity values surpassing 0.95 (AUC-ROC > 0.98). The ensemble MLe-KCNQ2 model also categorizes pathogenic mutations as benign or severe, with an area under the receiver operating characteristic curve (AUC-ROC) above 0.67. This study not only presents a transferable methodology for accurately classifying KCNQ2 missense variants, but also provides valuable insights for clinical counseling and aids in the determination of variant severity. The research context emphasizes the necessity of precise variant classification, especially for genes like KCNQ2, contributing to the broader understanding of gene-specific challenges in the field of genomic research. The MLe-KCNQ2 model stands as a promising tool for enhancing clinical decision making and prognosis in the realm of KCNQ2-related pathologies.

Novel Homozygous Variants of SLC13A5 Expand the Functional Heterogeneity of a Homogeneous Syndrome of Early Infantile Epileptic Encephalopathy

BackgroundEarly infantile epileptic encephalopathy 25 (EIEE25) is a distinct type of neonatal epileptic encephalopathy caused by autosomal recessive mutations in the SLC13A5 gene. SLC13A5 encodes a transmembrane sodium/citrate cotransporter required for regulating citrate entry into cells. MethodsFour families with recessively inherited epileptic encephalopathy were sequenced by clinically accredited laboratories using commercially available epilepsy gene panels. Patients were examined by a neurologist and were clinically diagnosed with infantile epileptic encephalopathy. ResultsWe present four families with global developmental delay, intellectual disability, and defective tooth development with four novel homozygous mutations in SLC13A5. The neurological examination showed spastic quadriplegia with increased deep tendon reflexes. Brain magnetic resonance imaging showed nonspecific signal abnormality of the bilateral hemispheric white matter. Despite similar clinical features, the conditions were based on different molecular mechanisms acting on SLC13A5 (abnormal splicing, large-scale deletions, and tandem-residue insertion). ConclusionsOur results extend the landscape of autosomal recessive inherited homozygous mutations in SLC13A5 that cause a distinctive syndrome of severe neonatal epileptic encephalopathy. Our observations confirm the homogeneity of epileptic encephalopathy and dental abnormalities as a distinct clinical marker for EIEE25 despite the heterogeneous functional and mutational background.

The HCN1 p.Ser399Pro variant causes epileptic encephalopathy with super-refractory status epilepticus

HCN1 is one of four genes encoding hyperpolarization-activated cyclic nucleotide-gated channels. The phenotypic spectrum associated with HCN1 variants ranges from neonatal developmental and epileptic encephalopathy to idiopathic generalized epilepsy. We report a Japanese patient with repetitive focal seizures and super-refractory status epilepticus since early infancy caused by a de novo HCN1 variant, NM_021072.4, c.1195T>C, p.(Ser399Pro). This variant might have a dominant-negative effect on channel function, leading to severe epileptic encephalopathy.

Ultrasound-induced seizures in a mouse model of KCNQ2-NEO-DEE

PurposeKCNQ2 neonatal developmental and epileptic encephalopathy (NEO-DEE) is characterized by intractable seizures accompanied by an abnormal neurodevelopment. In a mouse model of NEO-DEE carrying the p.(Thr274Met) variant of Kcnq2, spontaneous generalized seizures occur unexpectedly preventing controlled studies and highlighting the necessity for a customized setup to trigger seizures on demand. We aimed to obtain a stable and objective read-out to control the efficacy of new antiepileptic drugs or to test seizure susceptibility. We developed a protocol to trigger ultrasound-induced seizures (UIS) on demand in this model. MethodsWe tested the ability of our protocol to induce seizures at four developmental stages in the Kcnq2p.(Thr274Met/+) mouse model. We mapped the activated brain regions using c-fos protein labeling 2 h after seizure induction. ResultsWe show that the UIS have the same phenotypic expression and the same severity as spontaneous generalized seizures (SGS) in the Kcnq2-NEO-DEE mouse model. The developmental period during which mice exhibit SGS corresponds to the period during which Kcnq2p.(Thr274Met/+) mice are the most susceptible to US. C-fos labeling reveals a subset of 6 brain regions activated 2 h after the induction of the seizure. The same regions were identified in the context of seizure induction in other rodent models. ConclusionThis study provides a non-invasive and easy to use method to induce seizures in a Kcnq2-NEO-DEE mouse model and documents early neuronal activation in specific brain regions. This method can be used to test the efficacy of new antiepileptic approaches for this intractable form of genetic epilepsy.

Ohtahara Syndrome: Case Report and Literature Review

Ohtahara syndrome or Early Infantile Epileptic Encephalopathiy (EIEE) is a rare form of neonatal epileptic encephalopathy. It has particular electro-clinical, therapeutic and outcome features. Its etiologies remains obscure and are essentially secondary to a congenital or acquired structural malformation of cortical development. We report a case of a term newborn presented with seizures on week two of live. Seizures were intractable. Metabolic workup for seizures was normal. Brain MRI did not reveal any structural abnormality. Electroencephalogram showed suppression pattern suggestive of ohtahara syndrome.

Automatic and Quantitative Electroencephalographic Characterization of Drug-resistant Epilepsy in Neonatal KCNQ2 Epileptic Encephalopathy.

KCNQ2 epileptic encephalopathy is relatively common in early-onset neonatal epileptic encephalopathy and seizure severity varied widely, categorized as drug-sensitive epilepsy and drug-resistant epilepsy. However, in clinical practice, anti-seizure medicines need to be gradually adjusted based on seizure control which undoubtedly increases the economic burden of patients, so further positive anti-seizure regimens depend on whether seizure severity can be predicted in advance. In this paper, we proposed a reliable assessment to differentiate between drug-sensitive epilepsy and drug-resistant epilepsy caused by KCNQ2 pathogenic variants. Based on the electroencephalogram (EEG) and electrooculogram (EOG) signals, twenty-four classical temporal and spectral domain features were extracted and Gradient Boosting Decision Tree (GBDT) was employed to distinguish between patients with drug-sensitive epilepsy and drug-resistant epilepsy. In addition, we also systematically investigated the impact of channel combination and feature combination based on the forward stepwise selection strategy. By employing selected channels and features, the classification accuracy can reach 81.25% with a sensitivity of 57.14% and specificity of 100%. Compared with the state-of-the-art techniques, including the functional network, effective network, and common spatial patterns, the improvement of accuracy ranges from 37.5% to 56.25%, indicating the superiority of our proposed method. Overall, the proposed method may provide a promising tool to distinguish different seizure outcomes of KCNQ2 epileptic encephalopathy.

Clinical Study of 30 Novel KCNQ2 Variants/Deletions in KCNQ2-Related Disorders.

BackgroundKCNQ2-related disorder is typically characterized as neonatal onset seizure and epileptic encephalopathy. The relationship between its phenotype and genotype is still elusive. This study aims to provide clinical features, management, and prognosis of patients with novel candidate variants of the KCNQ2 gene.MethodsWe enrolled patients with novel variants in the KCNQ2 gene from the China Neonatal Genomes Project between January 2018 and January 2021. All patients underwent next-generation sequencing tests and genetic data were analyzed by an in-house pipeline. The pathogenicity of variants was classified according to the guideline of the American College of Medical Genetics. Each case was evaluated by two geneticists back to back. Patients' information was acquired from clinical records.ResultsA total of 30 unrelated patients with novel variants in the KCNQ2 gene were identified, including 19 patients with single-nucleotide variants (SNVs) and 11 patients with copy number variants (CNVs). For the 19 SNVs, 12 missense variants and 7 truncating variants were identified. Of them, 36.8% (7/19) of the KCNQ2 variants were located in C-terminal regions, 15.7% (3/19) in segment S2, and 15.7% (3/19) in segment S4. Among them, 18 of 19 patients experienced seizures in the early neonatal period. However, one patient presented neurodevelopmental delay (NDD) as initial phenotype when he was 2 months old, and he had severe NDD when he was 3 years old. This patient did not present seizure but had abnormal electrographic background activity and brain imaging. Moreover, for the 11 patients with CNVs, 20q13.3 deletions involving EEF1A2, KCNQ2, and CHRNA4 genes were detected. All of them presented neonatal-onset seizures, responded to antiepileptic drugs, and had normal neurological development.ConclusionIn this study, patients with novel KCNQ2 variants have variable phenotypes, whereas patients with 20q13.3 deletion involving EEF1A2, KCNQ2, and CHRNA4 genes tend to have normal neurological development.

Drosophila carrying epilepsy-associated variants in the vitamin B6 metabolism gene PNPO display allele- and diet-dependent phenotypes

Pyridox(am)ine 5 ' -phosphate oxidase (PNPO) catalyzes the rate-limiting step in the synthesis of pyridoxal 5 ' -phosphate (PLP), the active form of vitamin B6 required for the synthesis of neurotransmitters gamma-aminobutyric acid (GABA) and the monoamines. Pathogenic variants in PNPO have been increasingly identified in patients with neonatal epileptic encephalopathy and early-onset epilepsy. These patients often exhibit different types of seizures and variable comorbidities. Recently, the PNPO gene has also been implicated in epilepsy in adults. It is unclear how these phenotypic variations are linked to specific PNPO alleles and to what degree diet can modify their expression. Using CRISPR-Cas9, we generated four knock-in Drosophila alleles, hWT , hR116Q , hD33V , and hR95H , in which the endogenous Drosophila PNPO was replaced by wild-type human PNPO complementary DNA (cDNA) and three epilepsy-associated variants. We found that these knock-in flies exhibited a wide range of phenotypes, including developmental impairments, abnormal locomotor activities, spontaneous seizures, and shortened life span. These phenotypes are allele dependent, varying with the known biochemical severity of these mutations and our characterized molecular defects. We also showed that diet treatments further diversified the phenotypes among alleles, and PLP supplementation at larval and adult stages prevented developmental impairments and seizures in adult flies, respectively. Furthermore, we found that hR95H had a significant dominant-negative effect, rendering heterozygous flies susceptible to seizures and premature death. Together, these results provide biological bases for the various phenotypes resulting from multifunction of PNPO, specific molecular and/or genetic properties of each PNPO variant, and differential allele-diet interactions.

Distinctive mechanisms of epilepsy-causing mutants discovered by measuring S4 movement in KCNQ2 channels

Neuronal KCNQ voltage-gated potassium channels mediate the muscarine-regulated M-current, which contributes to maintaining the resting membrane potential and is a key regulator in preventing hyperexcitability in neurons. Mutations within KCNQ2 alter channel function and result in devastating diseases such as benign familial neonatal seizures (BFNS) and epileptic encephalopathies, conditions that currently affect 50 million people worldwide. The impact that mutations have on channel function remain poorly defined, largely because of our limited understanding of the voltage sensing mechanisms that trigger channel gating.

Pyridoxamine 5’-Phosphate Oxidase Deficiency: A Potentially Treatable Epileptic Encephalopathy

Pyridoxal phosphate-responsive neonatal epileptic encephalopathy due to pyridoxamine-5-primephosphate oxidase deficiency is a rare cause of epileptic encephalopathy. A neonate with this condition presents early in life with refractory seizures which does not respond to conventional anti-epileptic medications, depressed level of consciousness, and severe psychomotor retardation if left untreated. Early initiation of active cofactor pyridoxal-5’-phophate can be curative. We are describing a rare neurometabolic condition; the first case in the Kingdom of Bahrain to the best of our knowledge. Keywords: Pyridoxal-5’-phosphate, pyridoxamine 5’-phosphate oxidase, pyridoxine dependent epilepsy, epileptic encephalopathy, neonatal seizure

Activation of SGK1.1 Upregulates the M-current in the Presence of Epilepsy Mutations.

In the central nervous system, the M-current plays a critical role in regulating subthreshold electrical excitability of neurons, determining their firing properties and responsiveness to synaptic input. The M-channel is mainly formed by subunits Kv7.2 and Kv7.3 that co-assemble to form a heterotetrametric channel. Mutations in Kv7.2 and Kv7.3 are associated with hyperexcitability phenotypes including benign familial neonatal epilepsy (BFNE) and neonatal epileptic encephalopathy (NEE). SGK1.1, the neuronal isoform of the serum and glucocorticoids-regulated kinase 1 (SGK1), increases M-current density in neurons, leading to reduced excitability and protection against seizures. Herein, using two-electrode voltage clamp on Xenopus laevis oocytes, we demonstrate that SGK1.1 selectively activates heteromeric Kv7 subunit combinations underlying the M-current. Importantly, activated SGK1.1 increases M-channel activity in the presence of two different epilepsy mutations found in Kv7.2, R207W and A306T. In addition, proximity ligation assays in the N2a cell line allowed us to address the effect of these mutations on Kv7-SGK1.1-Nedd4 molecular associations, a proposed pathway underlying augmentation of M-channel activity by SGK1.1

Characterization of Novel Pathogenic Variants Causing Pyridox(am)ine 5'-Phosphate Oxidase-Dependent Epilepsy.

Several variants of the enzyme pyridox(am)ine 5′-phosphate oxidase (PNPO), responsible for a rare form of vitamin B6-dependent neonatal epileptic encephalopathy known as PNPO deficiency (PNPOD), have been reported. However, only a few of them have been characterised with respect to their structural and functional properties, despite the fact that the knowledge of how variants affect the enzyme may clarify the disease mechanism and improve treatment. Here, we report the characterisation of the catalytic, allosteric and structural properties of recombinantly expressed D33V, R161C, P213S, and E50K variants, among which D33V (present in approximately 10% of affected patients) is one of the more common variants responsible for PNPOD. The D33V and E50K variants have only mildly altered catalytic properties. In particular, the E50K variant, given that it has been found on the same chromosome with other known pathogenic variants, may be considered non-pathogenic. The P213S variant has lower thermal stability and reduced capability to bind the FMN cofactor. The variant involving Arg161 (R161C) largely decreases the affinity for the pyridoxine 5′-phosphate substrate and completely abolishes the allosteric feedback inhibition exerted by the pyridoxal 5′-phosphate product.

Human COQ4 deficiency: delineating the clinical, metabolic and neuroimaging phenotypes

BackgroundHuman coenzyme Q4 (COQ4) is essential for coenzyme Q10 (CoQ10) biosynthesis. Pathogenic variants in COQ4 cause childhood-onset neurodegeneration. We aimed to delineate the clinical spectrum and the cellular consequences of...

Biallelic mutations in ELFN1 gene associated with developmental and epileptic encephalopathy and joint laxity

ELFN1, a transmembrane leucine rich repeat protein, is involved in signal transduction in both neural cells and ROD ON-bipolar synaptogenesis. We present three siblings with developmental and epileptic encephalopathy and co-morbidities due to ELFN1 gene mutation; this is the first report in literature defining the human phenotype of ELFN1 gene mutation. Clinical, electrophysiological, and radiological findings along with comprehensive genetic studies of the patients and their family members are presented. Developmental and epileptic encephalopathy, autistic features, pyramidal signs, joint laxity, and dysmorphic features are the characteristic findings of this new clinical entity, involving mainly nervous system and possibly connective tissue. Whole exome sequence analysis followed by Sanger sequencing in all family members revealed disease-causing 8 bp frameshift mutation depicted as NM_001128636.2: c.42_49delGGCCGCCA; p. (Ala15Profs*241) in ELFN1. The variant, located in the signal peptide domain in the ELFN1 gene, was found to be homozygous in three patients, and heterozygous in the parents and three healthy siblings. Segregation analysis in family members together with pathogenicity assessment tools strongly supported the damaging effect of the frameshift variant on the function of the ELFN1 protein. Mutations in ELFN1 gene may be considered in patients with neonatal and infantile-onset epileptic encephalopathy before the full clinical picture is apparent.

Neonates with the KCNQ2 Y755C Variants: Not Associated with Neonatal Epileptic Encephalopathy

Background: Pediatric epilepsy caused by a KCNQ2 gene mutation usually manifests the phenotype of a neonatal seizure. KCNQ2 encephalopathy in newborns continues to be reported on. Objectives: The exact mechanism and phenotype of the KCNQ2 mutation still require investigation. Methods: One hundred twenty-one patients with childhood epilepsy without an identified cause underwent KCNQ2 sequencing. KCNQ2 mutation variants were transfected into human embryonic kidney 293 (HEK293) cells to investigate functional changes. Results: Two patients with the c.2264G>G/A (p.Y755C) variant had neonatal epileptic encephalopathy: one had electroencephalography (EEG) burst suppression and the other had multiple focal spikes. However, the mutation was not found in the 80 healthy adult claiming without ever seizures before. A functional study showed that p.Y755C currents were not different from those in the wild-type and from those in the benign (p.N780T) polymorphism in homomeric and heteromeric (wild-type KCNQ2: mutant = 1:1) transfected HEK293 cells. Electrical current differences between HEK293 cells with wildtype mutations and cells transfected with the wild-type KCNQ2, KCNQ3, and p.Y755C mutations in a 1:2:1 ratio were not significant. Their seizures remitted after they turned 1 year old. Conclusion: We suggest that patients with the KCNQ2 p.Y755C mutations are not associated with neonatal epileptic encephalopathy

Pathogenic variants in KCNQ2 cause intellectual deficiency without epilepsy: Broadening the phenotypic spectrum of a potassium channelopathy.

High-throughput sequencing (HTS) improved the molecular diagnosis in individuals with intellectual deficiency (ID) and helped to broaden the phenotype of previously known disease-causing genes. We report herein four unrelated patients with isolated ID, carriers of a likely pathogenic variant in KCNQ2, a gene usually implicated in benign familial neonatal seizures (BFNS) or early onset epileptic encephalopathy (EOEE). Patients were diagnosed by targeted HTS or exome sequencing. Pathogenicity of the variants was assessed by multiple in silico tools. Patients' ID ranged from mild to severe with predominance of speech disturbance and autistic features. Three of the four variants disrupted the same amino acid. Compiling all the pathogenic variants previously reported, we observed a strong overlap between variants causing EOEE, isolated ID, and BFNS and an important intra-familial phenotypic variability, although missense variants in the voltage-sensing domain and the pore are significantly associated to EOEE (p < 0.01, Fisher test). Thus, pathogenic variants in KCNQ2 can be associated with isolated ID. We did not highlight strong related genotype-phenotype correlations in KCNQ2-related disorders. A second genetic hit, a burden of rare variants, or other extrinsic factors may explain such a phenotypic variability. However, it is of interest to study encephalopathy genes in non-epileptic ID patients.

Dyshomeostatic modulation of Ca2+-activated K+ channels in a human neuronal model of KCNQ2 encephalopathy.

Mutations in KCNQ2, which encodes a pore-forming K+ channel subunit responsible for neuronal M-current, cause neonatal epileptic encephalopathy, a complex disorder presenting with severe early-onset seizures and impaired neurodevelopment. The condition is exceptionally difficult to treat, partially because the effects of KCNQ2 mutations on the development and function of human neurons are unknown. Here, we used induced pluripotent stem cells (iPSCs) and gene editing to establish a disease model and measured the functional properties of differentiated excitatory neurons. We find that patient iPSC-derived neurons exhibit faster action potential repolarization, larger post-burst afterhyperpolarization and a functional enhancement of Ca2+-activated K+ channels. These properties, which can be recapitulated by chronic inhibition of M-current in control neurons, facilitate a burst-suppression firing pattern that is reminiscent of the interictal electroencephalography pattern in patients. Our findings suggest that dyshomeostatic mechanisms compound KCNQ2 loss-of-function leading to alterations in the neurodevelopmental trajectory of patient iPSC-derived neurons.

Neuromonitoring in Neonatal-Onset Epileptic Encephalopathies.

Considering the wide spectrum of etiologies of neonatal-onset epileptic encephalopathies (EE) and their unfavorable consequences for neurodevelopmental prognoses, neuromonitoring at-risk neonates is increasingly important. EEG is highly sensitive for early identification of electrographic seizures and abnormal background activity. Amplitude-integrated EEG (aEEG) is recommended as a useful bedside monitoring method but as a complementary tool because of methodical limitations. It is of special significance in monitoring neonates with acute symptomatic as well as structural, metabolic and genetic neonatal-onset EE, being at high risk of electrographic-only and prolonged seizures. EEG/aEEG monitoring is established as an adjunctive tool to confirm perinatal hypoxic-ischemic encephalopathy (HIE). In neonates with HIE undergoing therapeutic hypothermia, burst suppression pattern is associated with good outcomes in about 40% of the patients. The prognostic specificity of EEG/aEEG is lower compared to cMRI. As infants with HIE may develop seizures after cessation of hypothermia, recording for at least 24 h after the last seizure is recommended. Progress in the identification of genetic etiology of neonatal EE constantly increases. However, presently, no specific EEG changes indicative of a genetic variant have been characterized, except for individual variants associated with typical EEG patterns (e.g., KCNQ2, KCNT1). Long-term monitoring studies are necessary to define and classify electro-clinical patterns of neonatal-onset EE.

Voltage-Sensing Mechanism of Neuronal KCNQ Channels

The voltage-gated potassium channel KCNQ2/KCNQ3 (KCNQ2/3) mediates the M-current (IKM) in neurons. The IKM channel contributes to maintaining the resting membrane potential and impedes repetitive neuronal firing, therefore playing a crucial role in controlling neuronal excitability. Mutations within the voltage-sensor (S4) of the IKM channel have been linked to Benign Familial Neonatal Seizures (BFNS) and epileptic encephalopathies, conditions that currently affect 50 million people worldwide. However, how KCNQ2/3 channels function remains largely unknown. Here, we use two-electrode voltage clamp and inside-out patch clamp in a Xenopus oocyte expression system to assess external and internal accessibility of S4 cysteine-substituted residues to the membrane-impermeable thiol reagent (MTSET) in homomeric and heteromeric KCNQ channels. We find that in homomeric KCNQ2 (and in KCNQ3) channels, extracellular MTSET labels residues N190C (Q218C) with a similar time course in both closed and open states. By contrast, extracellular MTSET modifies residues A193 to R201 in KCNQ2 (G219-S228 in KCNQ3) at depolarized potentials, but not at hyperpolarized potentials, suggesting that these residues likely lie buried in the membrane in the closed state. We also use voltage clamp fluorometry (VCF) to determine S4 movement in KCNQ2/3 channels. In homomeric KCNQ channels, the time course and voltage dependence of fluorescence F(V) and ionic current G(V) correlate, as if the S4 and the gate motion were directly coupled. Moreover, in KCNQ2, the F(V) follows the voltage dependence of the modification rate of residue A193C. Our data also indicate that the presence of KCNQ3 subunits affects the S4 and gate of KCNQ2 in heteromeric KCNQ2/3 channels.