Received: 1 June 2022 | Revised: 31 October 2022 | Accepted: 31 October 2022 DOI: 10.1111/epi.17455 RESEARCH ARTICLE De novo KCNA6 variants with attenuated KV1.6 channel deactivation in patients with epilepsy Vincenzo Salpietro1,2 | Valentina Galassi Deforie1 | Stephanie Efthymiou1 | Emer O'Connor1 | Anna Marcé-­Grau3 | Reza Maroofian1 | Pasquale Striano4,5 | Federico Zara 4,6 | Michelle M. Morrow 7 | SYNAPS Study Group | Adi Reich | Amy Blevins | Júlia Sala-­Coromina3 | Andrea Accogli4,6 7 7 | Sara Fortuna | Marie Alesandrini | P. Y. Billie Au 8 9 10 | Nilika Shah Singhal11 | Benjamin Cogne12,13 | Bertrand Isidor12,13 | Michael G. Hanna1,14 | Alfons Macaya3 | Dimitri M. Kullmann15 | Henry Houlden1 | Roope Männikkö1 1 Department of Neuromuscular Disease, UCL Institute of Neurology, University College London, London, UK 2 Department of Biotechnological and Applied Clinical Sciences (DISCAB), University of L'Aquila, L'Aquila, Italy 3 Department of Paediatric Neurology, University Hospital Vall d'Hebron, Universitat Autònoma de Barcelona, Barcelona, Spain 4 Department of Neurosciences, Rehabilitation, Ophthalmology, Genetics, Maternal and Child Health (DiNOGMI), University of Genoa, 16124 Genoa, Italy 5 Unit of Pediatric Neurology, IRCCS, Istituto “Giannina Gaslini”, Genoa 16123, Italy 6 Medical Genetics Unit, IRCCS, Istituto “Giannina Gaslini”, Genoa 16123, Italy 7 GeneDx, Maryland, Gaithersburg, USA 8 Istituto Italiano di Tecnologia (IIT), Genoa, Italy 9 Neuropediatrics Unit, Centre Hospitalier Universitaire Nantes, Nantes, France 10 Department of Medical Genetics, Alberta Children's Hospital Research Institute, Cumming School of Medicine, University of Calgary, Alberta, Calgary, Canada 11 Departments of Neurology and Pediatrics, UCSF Benioff Children's Hospital, University of California, California, San Francisco, USA 12 Centre Hospitalier Universitaire Nantes, Service de Génétique Médicale, Nantes, France 13 Université de Nantes, CNRS, INSERM, L'Institut du Thorax, Nantes, France 14 Queen Square Centre for Neuromuscular Diseases, National Hospital for Neurology and Neurosurgery, London, UK 15 Department of Clinical and Experimental Epilepsy, UCL Institute of Neurology, University College London, London, UK Correspondence Henry Houlden and Roope Männikkö, Abstract Department of Neuromuscular Disease, Objective: Mutations in the genes encoding neuronal ion channels are a com- UCL Queen Square Institute of mon cause of Mendelian neurological diseases. We sought to identify novel de Neurology, London WC1N 3BG, UK. Email: h.houlden@ucl.ac.uk and novo sequence variants in cases with early infantile epileptic phenotypes and r.mannikko@ucl.ac.uk neurodevelopmental anomalies. Vincenzo Salpietro and Valentina Galassi Deforie are equal first authors. Henry Houlden and Roope Männikkö are equal corresponding authors. This is an open access article under the terms of the Creative Commons Attribution License, which permits use, distribution and reproduction in any medium, provided the original work is properly cited. © 2022 The Authors. Epilepsia published by Wiley Periodicals LLC on behalf of International League Against Epilepsy. Epilepsia. 2023;64:443–455.  wileyonlinelibrary.com/journal/epi   | 443 444 |    SALPIETRO et al. Funding information Wellcome Trust, Grant/Award Number: Methods: Following clinical diagnosis, we performed whole exome sequencing WT093205MA;WT104033AIA;212285/ of the index cases and their parents. Identified channel variants were expressed Z/18/Z in Xenopus oocytes and their functional properties assessed using two-­electrode voltage clamp. Results: We identified novel de novo variants in KCNA6 in four unrelated indi- viduals variably affected with neurodevelopmental disorders and seizures with onset in the first year of life. Three of the four identified mutations affect the pore-­lining S6 α-­helix of KV1.6. A prominent finding of functional characteriza- tion in Xenopus oocytes was that the channel variants showed only minor effects on channel activation but slowed channel closure and shifted the voltage depend- ence of deactivation in a hyperpolarizing direction. Channels with a mutation affecting the S6 helix display dominant effects on channel deactivation when co-­ expressed with wild-­type KV1.6 or KV1.1 subunits. Significance: This is the first report of de novo nonsynonymous variants in KCNA6 associated with neurological or any clinical features. Channel variants showed a consistent effect on channel deactivation, slowing the rate of chan- nel closure following normal activation. This specific gain-­of-­function feature is likely to underlie the neurological phenotype in our patients. Our data highlight KCNA6 as a novel channelopathy gene associated with early infantile epileptic phenotypes and neurodevelopmental anomalies. KEYWORDS KV1 Shaker channel family, neurodevelopmental disorder, voltage-­gated potassium channels, whole exome sequencing 1 | I N T RO DU CT ION Key Points Epilepsy is a neurological disorder that affects ~0.5% to 1.0% of all children up to the age of 16 years of age.1 • Trio whole exome sequencing identified de novo Different behavioral, cognitive, and psychiatric comor- variants in KCNA6 in patients with different de- bidities are associated with pediatric and adult epilepsy, grees of epilepsy and intellectual disability. and in some cases with intellectual disability (ID).2 At • KCNA6 encodes voltage-­gated potassium chan- least 40% of epilepsies are believed to be caused by genetic nel KV1.6 expressed in the central nervous system factors,3 of which, only 1% are Mendelian epilepsies.4 In (CNS) but not associated previously with disease. the majority of these cases the heritability is still unex- • Identified KCNA6 variants are absent in the con- plained.5,6 Roughly a third of the known genes associated trol population and predicted damaging with with Mendelian epilepsies encode ion channels.7 three of four variants affecting pore lining S6 helix. The voltage-­gated potassium channel (KV) superfamily • Functional analysis showed consistent, domi- includes more than 40 individual subunits grouped into 12 nant defects in channel deactivation, whereas distinct sub-­families (KV1-­12). KV channels conduct the key activation was little affected. repolarizing current of action potentials that regulate vari- • Our data indicate KCNA6 as a novel gene asso- ous physiological processes including neuronal and muscle ciated with epilepsy with neurodevelopmental excitability and the secretion of hormones and neurotrans- disorder. mitters.8,9 They consist of four α-­subunits, each of which is composed of six transmembrane helices (S1-­S6).8,9 Helices S1-­S4 of each subunit form a regulatory voltage sensing do- channel opening (activation), whereas hyperpolarization main (VSD), whereas helices S5-­S6 of all four subunits form closes (deactivates) the pore.9 Upon prolonged depolariza- a central potassium conducting pore. Upon depolarization, tion, some KV channels undergo inactivation typically me- the VSDs undergo conformational transitions that result in diated by channel N-­terminus occluding the pore (N-­type SALPIETRO et al.    | 445 inactivation). KVβ-­subunits can modulate α-­subunit activ- Written informed consent for genetic sequencing and in- ity and induce N-­type inactivation with their N-­terminus.10 clusion in the study was obtained by the parents or legal Pathogenic variants in genes encoding KV subunits may guardians of all individuals. This study has been approved exert one or more effects on the assembly, trafficking, or the by the ethics committee of the University College London kinetics or the voltage dependence of the opening-­closing-­ (07/Q0512/26) and the ethical committees at the other par- inactivating transitions of the channel, thereby disturbing ticipating centers in Barcelona, Calgary, San Francisco, and membrane excitability or action potentials, leading to neu- Nantes. Clinical, brain imaging, and electroencephalogram rological diseases.11,12 (EEG) data were reviewed, and the individuals diagnosed The KV1 (KCNA) subfamily comprises eight homolo- with neurodevelopmental impairment including intellec- gous subunits (KV1.1-­KV1.8) that can assemble as homo-­ or tual disability (ID), developmental delay (DD), and devel- heterotetrameric channels with diverse biophysical prop- opmental epileptic encephalopathy (EE) were recruited erties.13 KV1 channels are delayed rectifiers, as they allow in the different institutions that were participating in the a sustained potassium efflux after membrane depolariza- study (individuals 1–­4; see Supporting Information for tion that repolarizes the membrane following an action case reports). Based on the International League Against potential. So far, genetic defects in only KV1.1, KV1.2, and epilepsy (ILAE) classification, an EE was defined in the KV1.4 subunits have been identified as established causes patients as refractory seizures and cognitive slowing or re- of Mendelian neurological diseases.14,15 Heterozygous gression associated with frequent, ongoing epileptiform ac- variants in KCNA1 (KV1.1; MIM 176260) have been linked tivity.29 ID was defined based on the presence of significant to episodic ataxia type 1 (EA1, MIM 160120), some- deficits in conceptual, social, and/or practical skills associ- times associated with myokymia or (less commonly) ated with significant deficits in adaptive behavior.30 Autism seizures.16–­18 Heterozygous de novo variants in KCNA2 spectrum disorders were defined based on impaired social (KV1.2; MIM 176262) were identified as a cause of early-­ communication, repetitive behaviors, and signs of sensory onset developmental epileptic encephalopathy (DEE 32, abnormalities. The diagnoses were made by specialist pedi- MIM 616366).19 Both KCNA1-­ and KCNA2-­related gen- atric neurology teams at the respective centers. Routine otypes and phenotypes continue to expand and distinct clinical genetic and metabolic screenings performed during clinical presentations may arise by the effect of specific initial workup was negative in each case, which warranted mutations on channel expression or function.15,20–­22 further investigation on a research basis. KCNA6 (MIM: 176257) encodes the KV1.6 channel predominantly expressed in the central and peripheral nervous system.23,24 Unlike the expression of KCNA1 and 2.2 | Next-­generation sequencing and KCNA2, KCNA6 expression is low in putamen and cere- bioinformatics bellum (Figure S1), but the expression of these genes co- incides in the frontal and occipital cortex where KCNA6 Genomic DNA was extracted from the whole blood or expression is highest. Expression of KV1.6 is increased in a saliva after informed consent for DNA analysis was pro- model of epilepsy and has an important impact on network vided by the families. Trio WES (index case and his/her activity.25 However, there is no established implication of parents) was performed in all individuals. Libraries were KCNA6 in human disease phenotypes. KV1.6 channels prepared from parent and patient DNA, and exomes were contain an N-­type inactivation prevention (NIP) domain captured and sequenced by WES on Illumina sequencers. in the N-­terminus and are insensitive to N-­type inactiva- Raw data were processed and filtered with established tion induced by KV1.4 subunits or KVβ1 subunits26,27 but pipelines. Exonic and donor and acceptor splicing variants sensitive to N-­type inactivation by KVβ3 subunits.28 Herein were considered for further analysis. Priority was given to we describe four unrelated individuals with seizures and/ rare variants that were present at <1% frequency in public or variable abnormalities of neurological development, in databases, such as 1000 Genomes Project, NHLBI Exome whom exome sequencing detected heterozygous de novo Variant Server, and the Genome Aggregation Database nonsynonymous variants in KCNA6. (GnomAD) and had a genomic evolutionary rate profil- ing (GERP) score >2. Synonymous variants were not con- sidered. Variants were confirmed by Sanger sequencing. 2 | M AT E R IALS AN D MET H OD S Parents of the affected individuals were recruited for seg- regation analysis, which was carried out using Sanger se- 2.1 | Consent and diagnosis quencing. All variants were determined independently by the participating centers. The contributing research cent- Affected children were recruited for genetic analysis using ers were connected with help of web-­based tools such as whole exome sequencing (WES) at four distinct centers. GeneMatcher.31 446 |    SALPIETRO et al. 2.3 | Homology model in response to tail voltage of −30 mV. At the tail voltage, WT and mutant channels activate slowly allowing for es- The Chain B of 3LUT (structure for the full-­length KV1.2, tablishment of a stable baseline after settling of capacitive 2.9 Å) with a 73.55% BLAST identity with KV1.6 based transients. Voltage dependence and time course of deac- on the Swiss Model portal32 was used as a template to tivation were assessed using a protocol where channels build KCNA6. The model underwent steepest descent were fully activated at +30 mV for 250 ms before applying minimizations to be stopped either when the maximum test-­voltage pulses to more negative voltages in 10 mV dec- force was lower than 1000.0 kJ/mol/nm or when 50 000 rements. Voltage dependence was assessed by plotting the minimization steps were performed with 0.005 kJ/mol tail current amplitude measured following the test pulses energy step size, with Verlet cutoff scheme, short-­range against voltage. Time course of deactivation was assessed electrostatic cutoff, and Van der Waals cutoff of 1.0 nm by fitting an exponential curve to the current decay during with AMBER99SB-­ILDN force field first with fixed back- the test-­voltage step. The voltage dependence of activa- bone, and then without constraints as implemented in the tion and deactivation were also assessed using the same Gromacs package v. 2016.1. The model was processed by protocols, but the duration of the pre-­pulse and the test the Orientations of Proteins in Membranes (OPM) data- pulses was only 20 ms and the activating pre-­pulse was to base.33 Mutants were built by manually mutating relevant +60 mV. residues with Swiss-­PdbViewer34 and reiterating the same To assess the voltage dependence of activation or deac- procedure as described above. tivation, the tail current amplitude was plotted against the test voltage and the current–­voltage relationship was fit by a Boltzmann function: y = 1/(1 + exp (V1/2 − V)/VSlope). 2.4 | Molecular biology and Xenopus To assess the rate of the deactivation, the current decay laevis oocytes was fitted with single exponential function. The data were analyzed using Clampfit 10.7 (Molecular devices), Origin Rat Kcna6 clone23 was a gift from A Vassilevski (Russian (OriginLabs), and GraphPad Prism software. Data are pre- Academy of Sciences, Moscow). Mutations were intro- sented as means ± standard error of the mean (SEM). The duced using the QuikChange site directed mutagenesis parameters were compared via one-­way analysis of vari- kit (Agilent technologies) and verified by sequencing ance (ANOVA) with Tukey's multiple comparisons tests whole Kcna6. Messenger RNA (mRNA) was transcribed and Welch's ANOVA with Dunnett T3 multiple compari- in vitro using mMessage mMachine kit (ThermoFisher). son tests with ranked values for nonparametric data. X. laevis oocytes were isolated according to procedures approved by the UK Animals (Scientific Procedures) Act 1986 and injected with 2.5 ng mutant or wild-­type (WT) 3 | RESULTS Kcna6 or WT KCNA1 mRNA22 and stored in a modified Barth's solution supplemented with penicillin (50 U/ml), 3.1 | Clinical synopsis streptomycin (50 μg/mL), and amikacin (100 μg/mL). The heterozygous condition was simulated by co-­injecting WT Extended case reports and patient videos are provided in and mutant mRNAs in a 1:1 ratio (2.5 total mRNA). the Supporting Information. Genotypic and phenotypic characteristics of the four individuals are summarized in Table 1. 2.5 | Electrophysiology In all affected children, pregnancies, family histories and birth histories were unremarkable. Neurodevelopmental Two electrode voltage-­clamp recordings were obtained impairment had been evident since early infancy in three 24–­72 h post-­injection with GeneClamp 500B amplifier affected individuals (1–­3), who presented delay of gross and Clampex 9.2 software (Molecular devices) at room motor skills such as sitting and crawling and achieved temperature. Records were filtered at 1 kHz and sampled autonomous walk between 18 months (individuals 1 at 5 kHz. Electrode resistance was 0.1–­1 MΩ when filled and 3) and 24 months (individual 2) of age. Language with KCl 3M. Bath solution composition was (in mM): 110 impairment was also present in individuals 1–­3, who at- NaCl, 10 KCl, 1.8 CaCl2, 10 HEPES, and pH 7.4. –­P/4 pro- tained meaningful speech production late (between 2 and tocol was used to subtract the small leak currents. 3 years of age). Late social smile was noticed during the The standard protocol to assess the voltage dependence first months of life in individual 1, who showed impaired of activation was composed of depolarizing test-­voltage social communication, repetitive behaviors, and autistic pulses from holding voltage of −80 mV for 250 ms in features. Mild intellectual disability was diagnosed during 10 mV increments and measuring the current amplitude childhood in individuals 2 and 3. SALPIETRO et al.    | 447 TABLE 1 Phenotypes of individuals carrying KCNA6 de novo variants Individual 1 Individual 2 Individual 3 Individual 4 Country of origin Canada Spain France USA Gender Male Female Male Female Age at last follow-­up 7 years 22 years 6 years 2 years Variant (NM_002235.3):c.783C>G; (NM_002235.3):c.1366G>C; (NM_002235.3):c.1346C>T; (NM_002235.3):c.1339G>T; (p.Asp261Glu) (p.Val456Leu) (p.Thr449Ile) (p.Val447Phe) Inheritance De novo De novo De novo De novo Family history Migraine –­ –­ Older sibling with febrile seizures Other genetic –­ –­ –­ –­ findings Pregnancy Normal Normal Normal Normal Birth Term (cesarean section) Term (cesarean section) Term (natural delivery) Induced at 41 weeks 4 days (natural delivery) Neonatal course Admitted to neonatal care Normal Normal Normal unit due to poor oral intake Birth weight 2000 g 2900 g 2900 g 3885 g Birth OFC NA NA 35 cm 35 cm Follow–­up OFC 50th <3rd centile 50th 50th Other Short stature <3rd –­ –­ –­ Developmental Yes Yes Yes No delay Sitting (months) 12 10 NA 7 Walking (months) 18 24 17 12 Language First words at age 3 years First words aged 2 years Speech delay Appropriate. First words by 12 months; 30 words by 15 months Social Late social smile –­ –­ Appropriate communication (6 months) Intellectual –­ Mild Mild No disability Seizure types –­ Focal, tonic, tonic–­clonic Focal, absences, tonic–­ Focal (fever) clonic (fever) Onset of seizures –­ 3 months 5 months 3 months Autism spectrum Yes No No No disorder Behavior Repetitive behaviors, Normal Normal Normal selective eating habits Other –­ Joint hypermobility Learning difficulties –­ Dysmorphic features Triangular face, pointed Mild retrognathia, gingival No No chin, thin upper lip hyperplasia Neurological Motor clumsiness Tremor Normal Normal examination MRI Normal Normal Normal Normal EEG Normal Focal and diffuse epileptic Focal and diffuse epileptic Left posterior epileptic abnormalities abnormalities abnormalities Other Congenital macrocytic –­ –­ –­ anemia Abbreviation: EEG, electroencephalogram; MRI, magnetic resonance imaging; OFC, occipital frontal circumference. 448 |    SALPIETRO et al. Seizures occurred in the first months of life (between (p.Asp261Glu), c.1366G>C (p.Val456Leu), c.1346C>T the third and the fifth month of life) in three children (p.Thr449Ile), c.1339G>T (p.Val447Phe), respectively] (individuals 2–­4) and presented with different semiology (Figure 1A, Table S1). The de novo KCNA6 variant in indi- (Table 1). Individual 2 first experienced multiple focal sei- vidual 1 was diagnosed via clinical trio-­based WES through zures per day, each lasting a few seconds and character- the commercial testing lab, Blueprint Genetics. In individu- ized by brief upward gaze deviation, uni-­ or bilateral upper als 2–­4, following their respective analysis pipelines,35–­37 limb flexion, and associated with altered level of con- participating centers generated a list of candidate variants sciousness; these episodes started at the age of 3 months filtered against variants from public databases according and usually occurred upon awakening. During infancy to modes of inheritance, and then compared their results and early childhood, she also manifested drug-­resistant through international research networks. All the four vari- generalized (tonic, tonic–­clonic) seizures. Individual 3 ants were absent from the GnomAD database, and all dis- had multiple seizures per day, lasting a few seconds, and played high conservation (mean: GERP 5.14) and in silico in most cases fever-­related; these episodes started at the pathogenic predictors (mean: CADD_Phred 24.14) scores age of 5 months and were characterized by clonic move- (Table S1). In the Genome Aggregation Database (gnomAD ments of the upper extremities with altered level of con- v2.1.1; https://gnomad.broad​insti​tute.org/) KCNA6 stands sciousness. In addition, she manifested absences from as a constrained gene intolerant to missense variation (Z-­ age 9 months and tonic–­clonic seizures (triggered by score: 3.05). All identified variants affect residues that are fever) from age 22 months. Individual 4 presented at age highly conserved across different species and KV1 subunits 3 months with febrile and afebrile focal seizures with eye (Figure 1B). Three of four de novo non-­synonymous variants deviation and asymmetric tonic extension of the upper (p.Val447Phe, p.Thr449Ile, p.Val456Leu) cluster within the extremities, followed by bilateral asynchronous/synchro- pore lining S6 helix, and one variant (p.Asp261Glu) is lo- nous clonic movements; these episodes, usually lasting cated in the S1–­S2 loop (Figure 1C,D). seconds to minutes and occurring from drowsiness/sleep, were characterized on EEG by ictal onset over the left posterior region (Figure S2). Several anti-­seizure drugs, 3.3 | Electrophysiology studies including valproic acid, clobazam, lamotrigine, carbamaz- epine, and ethosuximide, have been trialed in individuals Functional properties of the channel variants were stud- 2–­4 (see Supporting Information case reports); beneficial ied in X. laevis oocytes using a two-­electrode voltage effects of ethosuximide treatment were noticed in individ- clamp. The data are summarized in Table 2. The volt- ual 2, who has been seizure-­free since the age of 5 years age dependence of activation was studied using 250 ms and has had normal follow-­up EEG findings. In addition, pulses to test voltages and showed an ~−10 mV shift for individual 4 responded to carbamazepine. the variants in the S6 helix compared with WT KV1.6 The clinical features summarized are consistent with a channels (Figure 2A,B). Following the test-­voltage steps, diagnosis of variable epileptic phenotypes and/or neuro- the channel closure at the tail voltage was rapid for WT developmental anomalies in the four affected individuals. channels but clearly delayed for the S6 mutant channels Other distinctive clinical features were noted in individual (Figure 2A). To study the deactivation, we applied first 1, who showed macrocytic anemia and distinctive facial an activating pre-­pulse for 250 ms to +30 mV and then features, including pointed chin, mildly flat midface, thin studied channel closure when stepping to more nega- upper lip, and short stature (Supporting Information). tive voltages (Figure 2C–­E). Compared to WT, the rate Extensive genetic and biochemical investigations for a of closure of mutant channels was slowed (Figure 2E), range of genetic conditions, including non-­syndromic in- and for variants affecting the S6 helix voltage, steps to tellectual disabilities, genetic epilepsies, and neurometa- more negative voltages were needed to close the channel bolic disorders, were normal. In addition, brain magnetic (Figure 2D), demonstrating disrupted channel deactiva- resonance imaging (MRI) studies were performed in three tion. For WT channels, the voltage dependence of acti- individuals (individuals 2–­4) and did not disclose any vation and deactivation were parallel using 250 ms test abnormality. pulses. For the channels with mutation in S6 helix, the disrupted deactivation brought forward a hysteresis in voltage dependence of channel gating: voltage depend- 3.2 | Molecular genetic analysis and ence of deactivation is shifted tens of millivolts to hy- protein prediction perpolarized voltages compared to voltage dependence of activation following 250 ms voltage steps (Figure 2F). Four de novo non-­synonymous variants in KCNA6 were These data suggest that defective deactivation is a spe- identified in individuals 1–­4, [NM_002235.3: c.783C>G cific trait of these KV1.6 mutant channels. SALPIETRO et al.    | 449 F I G U R E 1 KCNA6 intragenic de novo variants identified in this study. (A) Family trees on individuals carrying de novo KCNA6 variants. (B) Multiple alignment showing complete conservation across species and KV1 homologues of the residues affected by the variants identified in this study (highlighted). (C) Cartoon topology of the human KV1.6 channel indicating the positions of the variants identified in this study. (+) signs indicate positively charged arginine residues in the S4 transmembrane helix. (D) Homology modeling of KV1.6 channel showing the full-­length channel in membrane plane (left panel), only the transmembrane domain in membrane plane (middle panel), and from above the membrane plane (right panel). The affected residues are shown in spheres and colored as indicated in C. To simulate the heterozygous condition of the patients, with shorter (20 ms) test and pre-­pulses, a significant shift the oocytes were co-­injected with mutant and WT mRNA in voltage dependence of deactivation was found for the S6 at a 1:1 ratio. The simulated heterozygous channels deacti- mutant channels in the simulated heterozygous condition vated to almost the same degree as the WT channel during and also for homomeric p.Asp261Glu channels when com- a 250 ms pulse used to study the homomeric channels. We pared to WT channels (Figure 3). Voltage of half-­maximal did not attempt to assess the rate of deactivation in simu- activation for the simulated heterozygous channels did lated heterozygous conditions, as the rate is likely a sum of not differ from that of WT channels for any mutation several rates of KV1.6 channel tetramers composed of vary- (Figure 3B). These data demonstrate that the S6 mutant ing fractions of WT and mutant subunits. When studied channels show a dominant effect on channel deactivation 450 |    SALPIETRO et al. TABLE 2 Electrophysiology parameters of KV1.6 channel variants Activation Deactivation Clone n V1/2 (mV) SE VSlope (mV) SE V1/2 (mV) SE VSlope (mV) SE τ (@-­80 mV) (ms) SE 250 ms pulses WT 18 −8.1 0.7 13.0 0.3 −9.9 0.7 13.6 0.2 9.4 0.4 D261E 16 −9.7 0.8 12.6 0.5 −14.0 1.3 15.3 0.5 17.6*** 1.2 V447F 14 −19.8*** 1.5 12.3 0.4 −34.4*** 2.2 13.0 0.5 31.3*** 3.2 T449I 17 −17.8*** 0.8 11.4 0.3 −53.6*** 1.9 14.9 0.8 74.6*** 4.6 V456L 25 −20.2*** 0.7 11.1** 0.3 −101.0*** 5.1 16.0 1.0 231.6*** 16.8 D261E+WT 16 −6.7 1.0 12.8 0.4 −8.2 1.0 13.9 0.5 V447F+WT 14 −12.6* 0.9 12.5 0.3 −16.2*** 0.8 15.7*** 0.3 T449I+WT 4 −10.6 0.9 10.7 1.2 −17.7* 1.4 14.9 1.7 V456L+WT 10 −14.1 1.7 10.9* 0.6 −19.5** 2.0 16.5 1.1 20 ms pulses WT 24 −1.1 0.7 16.3 0.4 −16.1 0.9 24.8 0.5 D261E 17 −0.7 1.0 15.1 0.6 −32.1*** 3.3 26.9 0.8 V447F 12 −7.2 2.2 13.0*** 0.3 −65.3*** 3.9 21.8*** 0.3 T449I 16 −9.4*** 1.2 12.1*** 0.2 −112.5*** 3.2 17.3*** 0.2 V456L 14 −8.2*** 0.9 12.6*** 0.4 −149.8*** 10.0 18.5 2.0 D261E+WT 16 0.4 1.2 16.4 0.3 −13.3 1.8 24.5 0.4 V447F+WT 16 −5.1 1.0 14.1** 0.3 −39.4*** 1.7 26.7 0.4 T449I+WT 15 −3.9 1.1 13.0*** 0.5 −52.2*** 4.6 24.1 0.7 V456L+WT 9 −8.8 3.2 11.5*** 0.4 −52.6** 7.7 25.7 0.6 KV1.1 18 −17.9*** 0.c9 16.3* 0.4 −23.0*** 0.9 18.3 0.3 KV1.1+WT 17 −6.9 0.8 14.8 0.2 −17.9 0.6 19.1 0.3 KV1.1+D261E 18 −13.9*** 1.0 15.7* 0.2 −22.0** 0.8 18.3 0.2 KV1.1+V447F 13 −17.5*** 0.9 14.7 0.3 −53.7*** 1.9 22.8* 0.4 KV1.1+T449I 19 −16.7*** 0.9 15.2 0.2 −49.3*** 2.3 22.2*** 0.7 KV1.1+V456L 12 −20.6*** 0.9 14.8 0.3 −54.7*** 2.5 26.2*** 0.6 Note: Wild-­type KV1.6 channel is indicated with WT, co-­expression with KV1.6 wild-­type channel is indicated with “+WT”, co-­expression with wild-­type KV1.1 channel with “KV1.1+”. *p < .05, **p < .01, ***p < .001 when compared to WT data for both 250 ms and 20 ms pulses (excluding KV1.1 co-­expression experiments) and compared to KV1.1 + WT for experiments involving Kv1.1 channel. Data are presented only for cells where both activation and deactivation protocols were studied. and implicate defective deactivation as the main patho- KCNA6 and KCNA1 mRNA. We found that all the mutant genic mechanism of the simulated heterozygous channels. channels showed a small (~−10 mV) but significant left Using 20 ms steps, the voltage dependence of activation and shift in the voltage dependence of activation compared to deactivation differed slightly for WT channels, suggesting when WT KV1.6 was expressed together with KV1.1 (20 ms that 20 ms is too short for the channel to reach steady-­state pulses) (Figure 4). The voltage dependence of deactivation at some test voltages. However, the shift between the volt- was left-­shifted more than 30 mV when the KV1.1 channel age dependence of activation and deactivation was more was co-­expressed with S6 mutant KV1.6 channels compared pronounced for the simulated heterozygous channels with to KV1.1 channels co-­expressed with WT KV1.6 (Figure 4). mutations in the S6 helix (Figure 3). These data show that the KV1.6 mutant channels can exert The subunit composition of native KV1 channels con- dominant gain-­of-­function effects on other KV1 channels. taining KV1.6 subunits is not known, but KV1.6 can form Defective deactivation of the mutant subunit is pre- functional heteromeric channels with other KV1 sub- dicted to increase the K+ currents upon repetitive stimu- units.13,27 We studied whether the defective deactivation lation compared to WT channels. Accordingly, although features of the mutant Kv1.6 channels are dominant on for WT channels the current responses to two 5 ms another KV1 isoform by co-­injecting oocytes with both pulses at 40 Hz were roughly overlapping, for simulated SALPIETRO et al.    | 451 F I G U R E 2 Activation and deactivation properties of wild-­type and mutant KV1.6 channels. Data for wild-­type channels are shown in black, for D261E channels in blue, V447F channels in green, T449I in orange, and V456L in red. Data are shown as mean ± SEM. Number of cells is shown in Table 2. (A) Representative traces depicting activation of wild-­type and mutant channels. Voltage protocol consists of 250 ms test voltage steps in 10 mV increments from holding voltage of −80 mV and a tail voltage step to −30 mV. The scale bars are 100 ms (X) and 2 μA (Y). (B) Voltage dependence of activation was assessed using data from recordings by plotting the current at the beginning of the tail voltage step against the test voltage. (C) Representative traces depicting deactivation of wild-­type and mutant channels. Voltage protocol consists of 250 ms depolarizing pre-­pulse to +30 mV from holding voltage of −80 mV, 250 ms test voltage steps in 10 mV decrements, and a tail voltage step to −30 mV. Scale bars are as in a. (D) Voltage dependence of deactivation was assessed using data from recordings by plotting the current at the beginning of the tail voltage step against the test voltage. (E) Time constant of deactivation is plotted against the test voltage. (F) Voltage dependence of activation (open symbols, data from B) and deactivation (closed symbols, data from D) are plotted for each mutant channel. Note that although for wild-­type and D261E the curves overlap, for other mutant channels the voltage dependence of activation and deactivation is clearly distinct. Solid and dashed lines in B, D, and F show fit of Boltzmann equation to mean data; the dashed lines in F show the fit for activation data and solid lines for deactivation data. Data from individual cells were normalized to the peak amplitude derived from fitting Boltzmann equation to data. heterozygous S6 mutant channels the current amplitude has been described in the context of specific mutations at holding voltage and in response to the second pulse was leading to gain-­of-­function effects of different KV chan- clearly augmented compared to the first pulse (Figure S3). nels, including KV1.2, KV4.2, and KV7.3.19,40–­42 The p.Thr449Ile and p.Val456Leu variants, identified in children affected with epilepsy and developmental 4 | DI S C USSION anomalies (including delayed motor milestones, mild ID, and speech difficulties) exhibited the most pro- We identified four KCNA6 variants in patients with varying nounced KV1.6 gain-­of-­function features among the degrees of neurodevelopmental disorder and seizures. The variants tested in this study. The p.Val447Phe variant, variability of phenotypes is consistent with that reported identified in a 2-­year-­old girl affected with epilepsy but in other KV-­related genetic channelopathies featuring without evidence of neurodevelopmental impairment, early-­onset epilepsy variably associated with DD, ID, au- showed clear gain-­of-­function features but smaller than tism spectrum disorder, and speech difficulties .15,21,22,38–­40 p.Thr449Ile and p.Val456Leu variants. Lack of neurode- Broad neurological impairment involving multiple devel- velopmental features may correlate with milder defect opmental domains (motor, cognition, language, behavior) on channel function, although the interpretation of the 452 |    SALPIETRO et al. F I G U R E 3 Voltage dependence of activation and deactivation of simulated heterozygous mutant KV1.6 channels. Data for wild-­type channels are shown in black, for D261E channels in blue, V447F channels in green, for T449I in orange, and for V456L in red. Data are shown as mean ± SEM. Number of cells is shown in Table 2. Voltage protocols are as in Figure 2A,C, except the duration test pulse and pre-­ pulse is 20 ms and the pre-­pulse voltage is +60 mV. (A) Representative traces of oocytes injected with wild-­type (top, black), V456L (middle, red), mRNA alone or both mRNAs together (pink, bottom). Left panel shows current activation in response to test pulses ranging from −40 to 0 mV from holding voltage of −80 mV. Right panel shows current deactivation in response to test pulses ranging from −20 to −110 mV from pre-­pulse voltage of +60 mV. (B) Voltage dependence of activation assessed as in Figure 2A, but the test pulses were only 20 ms in duration. Data are shown for each variant in homomeric (solid symbols and lines) and simulated heterozygous (open symbols, dashed lines) condition. Data for wild-­type channels are shown in black in each graph. (C) Voltage dependence of deactivation assessed as in Figure 2C, but the pre-­ and test pulses were only 20 ms in duration. Symbols and lines are as in B. significance of p.Val447Phe on the clinical phenotype Our functional data implicate defective deactivation may be complicated by the early age of the affected child (gain-­of-­function) as a pathogenic mechanism for KCNA6-­ (individual 4), and long-­term follow-­up will be needed to related neurological disorders. The defect is dominant, even assess developmental (motor, cognitive, and language) on another KV1 isoform (Figure 4), and is likely to cause outcomes. Individual 1, found to harbor a de novo non-­ specific changes in the firing properties of the neurons car- synonymous variant in the S1–­S2 loop (p.Asp261Glu) rying the mutant KV1.6 channels. Because the relative con- did not exhibit seizures and presented neurodevelop- tribution of KV1.6 on total KV1 current is not described for mental impairment with delayed motor milestones, any specific neuron, it is difficult to model the effects on fir- speech difficulties, and autistic features. The effects of ing properties. However, as the mutations minimally affect the p.Asp261Glu variant on channel function were mod- channel activation, firing threshold and the shape of the est compared to the S6 helix mutant channels, although first action potential following rest are predicted to be little the mutant channel displayed some gain-­of-­function disturbed. Compromised deactivation will lead to increased features qualitatively similar to the other mutant chan- K+ currents following an initial action potential that likely nels (Figures 2E and 3C). These defects were no longer suppresses the firing of consequent action potentials and/ detected when the mutant was co-­expressed with KV1.6 or affects their shape (Figure S3). Other molecular features WT subunits. It is unlikely that p.Asp261Glu underlies not assessed in this study, such as N-­type inactivation in- the complex clinical phenotype of patient 1 (Table 1, duced by KVβ3 subunits,28 may be altered by the mutations. Supporting Information) but it may contribute to the In addition, normal inactivation may limit the impact of neurodevelopmental features. None of the affected in- defective deactivation on cell excitability. dividuals carrying KCNA6 de novo variants exhibited KV1.6 channels are broadly expressed in the brain ataxia or cerebellar involvement, which are frequent fea- and implicated in a range of neurons and networks tures in the neurological and neuroradiological pheno- (Figure S3).23–­25 Consequently, it is difficult to predict types related to genetic defects in KV1.116–­18,22 and KV1.2 how altered firing of action potentials in neurons express- channels.19,40 This is consistent with lower cerebellar ex- ing KV1.6 channel leads to over-­excitability of the brain pression of KCNA6 when compared to both KCNA1 and and which network mechanisms underlie the epilepti- KCNA2 expression (Figure S1). form activity. Gain-­of-­function KV channel variants are SALPIETRO et al.    | 453 mutations may alter the firing patterns of these interneu- rons and consequently the network activity. At a molecular level, channel activation is little affected by the mutations, suggesting that the mutations have a minor effect on channel resting state. Following activation, the rate of closure of the mutant channels is slower than for WT channels and voltage dependence of deactivation is shifted to more hyperpolarized voltages compared to the voltage dependence of activation. This hysteretic behavior is compatible with the notion that following channel acti- vation the open state of the channel is stabilized. All three variants affecting the S6 helix (p.Val447Phe, p.Thr449Ile, p.Val456Leu) increase the hydrophobic surface area and the volume of affected residues, potentially stabilizing the open state through increased hydrophobic interactions or steric hindrance in the chains packing. The charge-­ conserving p.Asp261Glu variant affects the S1-­S2 loop of the voltage sensor of KV1.6 channels, and likely slows the deactivation of the channel by stabilizing the voltage sen- sor in the activated state. Gain-­of-­function mutations of ion channels are ther- apeutically amenable to drugs that block or reduce the current through the overactive channel.43 KV channel blockers may thus be of therapeutic interest for individu- als affected with KCNA6-­related epilepsy with associated gain-­of-­function mechanisms. However, to date and to our knowledge, no KV1.6-­ specific small molecule inhibitors of KV1.6 have been reported. F I G U R E 4 Voltage dependence of activation and deactivation 5 | CONC LUSIONS of KV1.6 variants co-­expressed with KV1.1 subunits. Data for wild-­ type KV1.1 channels are shown in black, for KV1.1 co-­expressed with Our study shows that the de novo KCNA6 variants have a wild-­type KV1.6 in gray, with KV1.6-­D261E in blue, with KV1.6-­ specific and similar effect on KV1.6-­channel deactivation. V447F channels in green, with KV1.6-­T449I in orange, and with This gain-­of-­function effect of the variants identified in KV1.6-­V456L in red. Data are shown as mean ± SEM. Number of the S6 helix effect is dominant, even on another KV1 iso- cells is shown in Table 2. (A) Shows voltage dependence of activation form. These data yield solid support for the notion that the (voltage protocols are as in Figure 3B) and (B) shows voltage KV1.6 variants we discovered are pathogenic and causative dependence of deactivation (voltage protocols are as in Figure 3C). for a spectrum of infantile-­onset epileptic phenotypes and Solid lines show fit of Boltzmann equation to mean data. neurodevelopmental anomalies. Even though our cohort is small, it establishes KCNA6 as a gene associated with predicted to dampen the excitability of the neuron. This is neurodevelopmental disorders and epilepsy. The small predicted to lead to increased electrical activity associated number of discovered cases with GoF variants may be a with epilepsy if the GoF variant is expressed in inhibitory consequence of the specific, unusual effect of the variants interneurons. In this model, dampening of the electrical on compromising channel deactivation. Low tolerance of activity of the inhibitory interneuron results in overactiv- KCNA6 for missense variation suggests that further vari- ity of the network controlled by the interneuron. KCNA6 ants, including loss-­of-­function variants are likely to be mRNA and KV1.6 expression were increased in the piri- discovered associated with neurological disorders. form cortex parvalbumin interneurons following induc- tion of epilepsy,25 demonstrating KV1.6 expression in AUTHOR CONTRIBUTIONS inhibitory interneurons. Increased expression was associ- Vincenzo Salpietro led the collection of genetic and clini- ated with reduced diversity of interneuron firing patterns, cal data. Valentina Galassi Deforie led the collection of suggesting that changes in KV1.6 currents introduced by electrophysiological data. Stephanie Efthymiou, Emer 454 |    SALPIETRO et al. O'Connor, and Reza Maroofian coordinated the collection Pasquale Striano https://orcid.org/0000-0002-6065-1476 and assembly of clinical and genetic data from all partici- Michelle M. Morrow 0000-0001-9475-0934 pating centers. P. Y. Billie Au collected the clinical and Andrea Accogli https://orcid.org/0000-0001-8724-6721 genetic data of individual 1. Anna Marcé-­Grau, Júlia Sala-­ P. Y. Billie Au https://orcid.org/0000-0002-9533-7217 Coromina, and Alfons Macaya collected the clinical and Dimitri M. Kullmann https://orcid. genetic data of individual 2. Marie Alesandrini, Benjamin org/0000-0001-6696-3545 Cogne, and Bertrand Isidor collected the clinical and ge- Roope Männikkö https://orcid.org/0000-0002-8727-9184 netic data of individual 3. Michelle M. Morrow, Adi Reich, Amy Blevins, and Nilika Shah Singhal collected the clini- REFERENCES cal and genetic data of individual 4. Sara Fortuna created 1. Reilly C, Atkinson P, Das KB, Chin RF, Aylett SE, Burch V, models of KV1.6 variants. Pasquale Striano, Federico Zara, et al. Features of developmental coordination disorder in active and Andrea Accogli contributed to the analysis and revi- childhood epilepsy: a population-­based study. 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