The KCNT1 gene encodes the sodium-activated potassium channel Slack (KCNT1, KNa1.1), a regulator of neuronal excitability. Gain-of-function mutations in humans cause cortical network hyperexcitability, seizures, and severe intellectual disability. Using a mouse model expressing the Slack-R455H mutation, we find that Na+-dependent K+ (KNa) and voltage-dependent sodium (NaV) currents are increased in both excitatory and inhibitory cortical neurons. These increased currents, however, enhance the firing of excitability neurons but suppress that of inhibitory neurons. We further show that the expression of NaV channel subunits, particularly that of NaV1.6, is upregulated and that the length of the axon initial segment and of axonal NaV immunostaining is increased in both neuron types. Our study on the coordinate regulation of KNa currents and the expression of NaV channels may provide an avenue for understanding and treating epilepsies and other neurological disorders.

More than twenty recurrent missense gain-of-function (GOF) mutations have been identified in the sodium-activated potassium (KNa) channel gene KCNT1 in patients with severe developmental and epileptic encephalopathies (DEEs), most of which are resistant to current therapies. Defining the neuron types most vulnerable to KCNT1 GOF will advance our understanding of disease mechanisms and provide refined targets for precision therapy efforts. Here, we assessed the effects of heterozygous expression of a Kcnt1 GOF variant (Y777H) on KNa currents and neuronal physiology among cortical glutamatergic and GABAergic neurons in mice, including those expressing vasoactive intestinal polypeptide (VIP), somatostatin (SST), and parvalbumin (PV), to identify and model the pathogenic mechanisms of autosomal dominant KCNT1 GOF variants in DEEs. Although the Kcnt1-Y777H variant had no effects on glutamatergic or VIP neuron function, it increased subthreshold KNa currents in both SST and PV neurons but with opposite effects on neuronal output; SST neurons became hypoexcitable with a higher rheobase current and lower action potential (AP) firing frequency, whereas PV neurons became hyperexcitable with a lower rheobase current and higher AP firing frequency. Further neurophysiological and computational modeling experiments showed that the differential effects of the Y777H variant on SST and PV neurons are not likely due to inherent differences in these neuron types, but to an increased persistent sodium current in PV, but not SST, neurons. The Y777H variant also increased excitatory input onto, and chemical and electrical synaptic connectivity between, SST neurons. Together, these data suggest differential pathogenic mechanisms, both direct and compensatory, contribute to disease phenotypes, and provide a salient example of how a pathogenic ion channel variant can cause opposite functional effects in closely related neuron subtypes due to interactions with other ionic conductances.

Autosomal dominant sleep-related hypermotor epilepsy (ADSHE) is a rare heritable form of epilepsy. It is characterized by hypermotor seizures occurring mainly during sleep. Seizures are typically abrupt in onset and offset and tend to increase in complexity and duration during the night. ADSHE is inherited in an autosomal dominant manner, and penetrance is estimated to be 70%. We describe two brothers with ADSHE with a previously unreported variant in CHRNA4, and the effect of medical treatment with carbamazepine. We highlight the relevance of genetic testing in patients with atypical and clustering episodes of nightmares, night terrors, or panic attacks, as these patients could be misdiagnosed, and instead be suffering from ADSHE, a potentially treatable condition.

Gain-of-function (GOF) variants in K+ channels cause severe childhood epilepsies, but there are no mechanisms to explain how increased K+ currents lead to network hyperexcitability. Here, we introduce a human Na+-activated K+ (KNa) channel variant (KCNT1-Y796H) into mice and, using a multiplatform approach, find motor cortex hyperexcitability and early-onset seizures, phenotypes strikingly similar to those of human patients. Although the variant increases KNa currents in cortical excitatory and inhibitory neurons, there is an increase in the KNa current across subthreshold voltages only in inhibitory neurons, particularly in those with non-fast-spiking properties, resulting in inhibitory-neuron-specific impairments in excitability and action potential (AP) generation. We further observe evidence of synaptic rewiring, including increases in homotypic synaptic connectivity, accompanied by network hyperexcitability and hypersynchronicity. These findings support inhibitory-neuron-specific mechanisms in mediating the epileptogenic effects of KCNT1 channel GOF, offering cell-type-specific currents and effects as promising targets for therapeutic intervention.

Sleep-related hypermotor epilepsy, or nocturnal frontal lobe epilepsy, as it was formerly called, is a focal epilepsy with mostly sleep-related seizures of hypermotor, tonic or dystonic semiology. Sleep-related hypermotor epilepsy may be attributed to a monogenetic cause with autosomal dominant inheritance. Mutations are described in different genes, including the genes for three subunits of the nicotinic acetylcholine receptor. We present a family with members over four generations exhibiting sleep-related hypermotor epilepsy. Genetic testing was available for three members from three generations, and revealed two variants in the alpha-4 subunit of the nicotinic acetylcholine receptor (one of them being novel) which are likely to be disease-causing. As these mutations were identified in cis configuration (on the same allele), we do not know whether one of the variants alone or a combination of the two is responsible for the pathogenicity.

Pathogenic variants in KCNT1 represent an important cause of treatment-resistant epilepsy, for which an effective therapy has been elusive. Reports about the effectiveness of quinidine, a candidate precision therapy, have been mixed. We sought to evaluate the treatment responsiveness of patients with KCNT1-related epilepsy. We performed an observational study of 43 patients using a collaborative KCNT1 patient registry. We assessed treatment efficacy based upon clinical seizure reduction, side effects of quinidine therapy, and variant-specific responsiveness to treatment. Quinidine treatment resulted in a > 50% seizure reduction in 20% of patients, with rare patients achieving transient seizure freedom. Multiple other therapies demonstrated some success in reducing seizure frequency, including the ketogenic diet and vigabatrin, the latter particularly in patients with epileptic spasms. Patients with the best quinidine response had variants that clustered distal to the NADP domain within the RCK2 domain of the protein. Half of patients did not receive a quinidine trial. In those who did, nearly half did not achieve therapeutic blood levels. More favorable response to quinidine in patients with KCNT1 variants distal to the NADP domain within the RCK2 domain may suggest a variant-specific response.

Mutations in genes coding for subunits of the neuronal nicotinic acetylcholine receptor (nAChR) have been involved in familial sleep-related hypermotor epilepsy (also named autosomal dominant nocturnal frontal lobe epilepsy, ADNFLE). Most of these mutations reside in CHRNA4 and CHRNB2 genes, coding for the α4 and β2 nAChR subunits, respectively. Two mutations with contrasting functional effects were also identified in the CHRNA2 gene coding for the α2 subunit. Here, we report the third mutation in the CHRNA2, found in a patient showing ADNFLE. The patient was examined by scalp EEG, contrast-enhanced brain magnetic resonance imaging (MRI), and nocturnal video-polysomnographic recording. All exons and the exon-intron boundaries of CHRNA2, CHRNA4, CHRNB2, CRH, KCNT1 were amplified and Sanger sequenced. In the proband, we found a c.754T>C (p.Tyr252His) missense mutation located in the N-terminal ligand-binding domain and inherited from the mother. Functional studies were performed by transient co-expression of α2 and α2 Tyr252His , with either β2 or β4, in human embryonic kidney (HEK293) cells. Equimolar amounts of subunits expression were obtained by using F2A-based multi-cistronic constructs encoding for the genes relative to the nAChR subunits of interest and for the enhanced green fluorescent protein. The mutation reduced the maximal currents by approximately 80% in response to saturating concentrations of nicotine in homo- and heterozygous form, in both the α2β4 and α2β2 nAChR subtypes. The effect was accompanied by a strong right-shift of the concentration-response to nicotine. Similar effects were observed using ACh. Negligible effects were produced by α2Tyr252His on the current reversal potential. Moreover, binding of (±)-[3H]Epibatidine revealed an approximately 10-fold decrease of both Kd and Bmax (bound ligand in saturating conditions), in cells expressing α2Tyr252His. The reduced Bmax and whole-cell currents were not caused by a decrease in mutant receptor expression, as minor effects were produced by α2Tyr252His on the level of transcripts and the membrane expression of α2β4 nAChR. Overall, these results suggest that α2Tyr252His strongly reduced the number of channels bound to the agonist, without significantly altering the overall channel expression. We conclude that mutations in CHRNA2 are more commonly linked to ADNFLE than previously thought, and may cause a loss-of-function phenotype.

The Na+/K+/Cl- cotransporter-1 (NKCC1) and the K+/Cl- cotransporter-2 (KCC2) set the transmembrane Cl- gradient in the brain, and are implicated in epileptogenesis. We studied the postnatal distribution of NKCC1 and KCC2 in wild-type (WT) mice, and in a mouse model of sleep-related epilepsy, carrying the mutant β2-V287L subunit of the nicotinic acetylcholine receptor (nAChR). In WT neocortex, immunohistochemistry showed a wide distribution of NKCC1 in neurons and astrocytes. At birth, KCC2 was localized in neuronal somata, whereas at subsequent stages it was mainly found in the somatodendritic compartment. The cotransporters\' expression was quantified by densitometry in the transgenic strain. KCC2 expression increased during the first postnatal weeks, while the NKCC1 amount remained stable, after birth. In mice expressing β2-V287L, the KCC2 amount in layer V of prefrontal cortex (PFC) was lower than in the control littermates at postnatal day 8 (P8), with no concomitant change in NKCC1. Consistently, the GABAergic excitatory to inhibitory switch was delayed in PFC layer V of mice carrying β2-V287L. At P60, the amount of KCC2 was instead higher in mice bearing the transgene. Irrespective of genotype, NKCC1 and KCC2 were abundantly expressed in the neuropil of most thalamic nuclei since birth. However, KCC2 expression decreased by P60 in the reticular nucleus, and more so in mice expressing β2-V287L. Therefore, a complex regulatory interplay occurs between heteromeric nAChRs and KCC2 in postnatal forebrain. The pathogenetic effect of β2-V287L may depend on altered KCC2 amounts in PFC during synaptogenesis, as well as in mature thalamocortical circuits.

Mutations in the sodium-gated potassium channel subunit gene KCNT1 have recently emerged as a cause of several different epileptic disorders. This review describes the mutational and phenotypic spectrum associated with the gene and discusses the comorbidities found in patients, which include intellectual disability and psychiatric features. The gene may also be linked with cardiac disorders. KCNT1 missense mutations have been found in 39% of patients with the epileptic encephalopathy malignant migrating focal seizures of infancy (MMFSI), making it the most significant MMFSI disease-causing gene identified to date. Mutations in KCNT1 have also been described in eight unrelated cases of sporadic and familial autosomal-dominant nocturnal frontal lobe epilepsy (ADNFLE). These patients have a high frequency of associated intellectual disability and psychiatric features. Two mutations in KCNT1 have been associated with both ADNFLE and MMFSI, suggesting that the genotype-phenotype relationship for KCNT1 mutations is not straightforward. Mutations have also been described in several patients with infantile epileptic encephalopathies other than MMFSI. Notably, all mutations in KCNT1 described to date are missense mutations, and electrophysiological studies have shown that they result in increased potassium current. Together, these genetic and electrophysiological studies raise the possibility of delivering precision medicine by treating patients with KCNT1 mutations using drugs that alter the action of potassium channels to specifically target the biological effects of their disease-causing mutation. Such trials are now in progress. Better understanding of the mechanisms underlying KCNT1-related disease will produce further improvements in treatment of the associated severe seizure disorders.

Autosomal dominant nocturnal frontal lobe epilepsy (ADNFLE) is a focal epilepsy with attacks typically arising in the frontal lobe during non-rapid eye movement (NREM) sleep. It is characterized by clusters of complex and stereotyped hypermotor seizures, frequently accompanied by sudden arousals. Cognitive and psychiatric symptoms may be also observed. Approximately 12% of the ADNFLE families carry mutations on genes coding for subunits of the heteromeric neuronal nicotinic receptors (nAChRs). This is consistent with the widespread expression of these receptors, particularly the α4β2(*) subtype, in the neocortex and thalamus. However, understanding how mutant nAChRs lead to partial frontal epilepsy is far from being straightforward because of the complexity of the cholinergic regulation in both developing and mature brains. The relation with the sleep-waking cycle must be also explained. We discuss some possible pathogenetic mechanisms in the light of recent advances about the nAChR role in prefrontal regions as well as the studies carried out in murine models of ADNFLE. Functional evidence points to alterations in prefrontal GABA release, and the synaptic unbalance probably arises during the cortical circuit maturation. Although most of the available functional evidence concerns mutations on nAChR subunit genes, other genes have been recently implicated in the disease, such as KCNT1 (coding for a Na(+)-dependent K(+) channel), DEPD5 (Disheveled, Egl-10 and Pleckstrin Domain-containing protein 5), and CRH (Corticotropin-Releasing Hormone). Overall, the uncertainties about both the etiology and the pathogenesis of ADNFLE point to the current gaps in our knowledge the regulation of neuronal networks in the cerebral cortex.