Potassium Channels and Human Epileptic Phenotypes: An Updated Overview

1Epilepsy is one of the most common neurological disorders characterized by abnormal electrical activity in the central nervous system (CNS) and recurrent seizures represent a cardinal clinical manifestation. The phenotypic expression of each seizure is determined by the original point of the hyperexcitability and its degree of spread in the brain. Several brain defects due to membrane instability could cause epilepsy.

2In the last two decades, gene defects underlying different forms of epilepsy have been identified and most of these genes code for ion channels, which thus appear as important players in the etiopathogenesis of idiopathic epilepsy. Indeed, several epileptic phenotypes have been associated to dysfunctions of potassium (K+) channels. It has been recently proposed to name such epilepsies as “K+ channelepsies”. These channels play a major role in neuronal excitability and their importance is related to the level of their expression in subcellular domain, individual cell, or circuit. K+ channels are also involved in setting the inward-negative resting membrane potential. Based on their structures, biophysical characteristics, pharmacological sensitivities and physiology, these channels are classified as voltage-gated (Kv), inwardly rectifying (Kir), sodium (Na)-activated channels or Ca2+-activated channels (Table 1; ).

3Herein we report an updated discussion on the role of mutations in K+ channels (Table 2) in the pathogenesis of human epilepsy.

4The Kv channels are widely expressed both in the central and in peripheral nervous system where they are involved in several processes (e.g., the regulation of the duration of action potentials, the modulation of the neurotransmitter release, the control of the electrical properties and the firing of neurons). Kv channels generally regulate outward K+ currents that contribute to membrane repolarization and hyperpolarization, thus limiting the neuronal excitability. Moreover, they actively participate in cellular and molecular signaling pathways that regulate the life and death of neurons, such as apoptosis, channel phosphorylation, or cell proliferation. In particular, neuronal cell apoptosis is correlated to an increased expression of Kv channels at the plasma membrane, thus facilitating more K+ efflux and a loss of cytosolic K+. This drop in the intracellular K+ concentration actives pro-apoptotic enzymes, such as nuclease or caspase that can trigger downstream apoptotic signals culminating in DNA fragmentation or degradation.

5In human genome, forty different genes encoding for Kv channels were reported and subdivided into twelve sub-families (Kv1 through Kv12). Mammalian Kv channels are tetramers, composed of α-subunits that line an ion pore. Each α-subunit shows six α-helical transmembrane domains (S1–S6), a membrane-reentering P loop between S5 and S6, and cytosolic N-, and C-termini. The S5-P-S6 segments constitute the ion conduction pore, while the S1–S4 sequences are critical for the voltage-sensing and gating of the channel.

6Furthermore, α-subunits can bind to regulatory β subunits (Kvβ1, Kvβ2, and Kvβ3) as well as to other Kv channel-interacting proteins. This variability in the channel interactions results in strong modifications of the channel properties.

7The following Kv subfamilies have been associated with either epilepsy or other disorders showing seizures.

8The Kv1 subfamily plays an essential role in the initiation and shaping of action potentials. These channels are expressed at the soma, axons, synaptic terminals, and proximal dendrites. The most abundant Kv1 α-subunits are Kv1.1, Kv1.2, and Kv1.4. These subunits are differentially expressed and their composition is dependent upon the brain region, cell type and subcellular localization.

9Heterozygous mutations in the KCNA1 gene, encoding the Kv1.1 α subunit, were associated with episodic ataxia type 1 (EA1), a dominantly inherited disorder characterized by generalized ataxia attacks and spontaneous muscle quivering. Interestingly, a subset of patients with familial EA1 shows epileptic seizures, suggesting that Kv1.1 dysfunctions may play a role in the pathophysiology of epilepsy. Loss-of function mutations reported in the KCNA1 gene of EA1 patients cause reduced current amplitude thus contributing to seizures susceptibility.

10In support of the hypothesis of an epileptogenic role of KCNA1 mutations, several knock-out mouse models for this gene developed an epileptic phenotype. Biochemical and biophysical studies demonstrated a colocalization of Kv1.1 and Kv1.2 subunits in several subcellular brain regions and that they could form heteromeric channels, which are reported as profoundly altered by EA1 mutations.

11Notably, a Kv1.2 knock-out mouse model displayed increased seizure susceptibility. In this regard, recently identified de novo loss or gain-of-function mutations in KCNA2 gene (Table 2), encoding the Kv1.2 channel, in patients showing mild to severe epileptic encephalopathy. A role of Kv1.2 was also suggested by another case report describing a de novo mutation, leading to the p.Arg297Gln amino acid substitution in a patient affected by ataxia and myoclonic epilepsy.

12The Kv4 channels are highly expressed in the brain and mediate the main dendritic A-currents which critically regulate action potential back-propagation and the induction of specific forms of synaptic plasticity. In particular, the Kv4.2 subunit is a key component of the A-type potassium current in the CNS (IA).

13In 2006, Singh and collaborators described a truncation mutation (p.Asn587fsX1) in the Kv4.2 channel encoded by the KCND2 gene, in a patient affected by temporal lobe epilepsy (TLE). This mutation causes a frame-shift, leading to a premature termination codon and consequently to a Kv4.2 channel haploinsufficiency. Recently, a whole exome sequencing study identified a de novo gain-of-function mutation (p.Val404Met) in KCND2. The mutation was found in monozygotic twins affected by autism and severe intractable seizures and occurred at a highly conserved residue within the C-terminus of the S6 transmembrane region of the ion pore. A functional analysis of mutated channels revealed a significantly slowed channel inactivation.

14Very recently, an involvement of Kv4.3 subunits in epilepsy was also suggested by the identification of a de novo mutation (p.Arg293_Phe295dup) in the relevant KCND3 gene causing a severe channel dysfunction in a patient with complex early onset cerebellar ataxia, intellectual disability, oral apraxia and epilepsy. This mutation results in the duplication of a RVF (Arginine–Valine–Phenylalanine) motif in the S4 segment and leads to a more positively charged voltage-sensor domain, altering the voltage-dependent gating properties of the channel. In details, the p.Arg293_Phe295dup mutation induced a strong depolarizing shift in the voltage dependence of both the activation (about +59.3 mV) and inactivation (+62 mV) of the channel.

15KCNQ (Kv7) channels are low-threshold activated voltage-gated potassium channels. Among the five known isoforms, KCNQ2–5 are expressed throughout the nervous system, whereas KCNQ1 is mostly expressed in cardiac tissue. The KCNQ2 gene is the most commonly reported as mutated in epilepsy. Its mutations cause neonatal epilepsies with wide phenotypic heterogeneity, ranging from benign familial neonatal seizures (BFNS) with normal cognition and unremarkable neuroimaging to early onset epileptic encephalopathies (EOEEs) with mental retardation, suppression-burst electroencephalography (EEG) and distinct neuroradiologic features. More than 80 different mutations in KCNQ2, consisting of missense, non-sense, truncations, splice site defects and frame-shift mutations, as well as sub-microscopic deletions or duplications, were described and most of them are found in the pore region and the large intracellular C-terminal domain. Functional studies suggested a strict phenotype/genotype correlation between disease severity and functional properties of mutant channels. KCNQ2 is a primary player that mediates neuronal muscarinic (M) currents: the opening of this channel or of heterogeneous KCNQ2/KCNQ3 complexes inhibits initiation of action potential and thus suppresses neuronal excitability.

16Mutations in KCNQ3 gene have been described in families affected with benign epilepsy with variable age of onset and good outcome or in a patient with benign childhood epilepsy with centrotemporal spikes (BECTS). However, two recent reports suggested that mutations in KCNQ3, similarly to KCNQ2, can be also found in patients with more severe phenotypes, including intellectual disability. In particular, they described KCNQ3 mutations in patients with early onset epilepsy and neurocognitive deficits (;; Table 2).

17Mutations in the KCNQ1 gene were associated with a particular form of long QT syndrome, the LQT1. Interestingly, some authors observed that epilepsy occurred in mouse lines bearing dominant human LQT1 mutations in this channel, which caused syncope and sudden death. Moreover, genetic variants in the KCNQ1 gene were reported in three cases of sudden unexpected death in epilepsy (SUDEP), a catastrophic complication of human idiopathic epilepsy with unknown causes. However, the relationship of these variants to the disease remains to be elucidated. The evidence that KCNQ1 genetic variations may confer susceptibility for recurrent seizure activity increasing the risk of sudden death is further supported by the description of a pathogenic KCNQ1 variant (p.Leu273Phe) in a family featuring LQTS and epilepsy.

18The KCNV2 gene encodes the voltage-gated K+ channel Kv8.2. This subunit is electrophysiologically silent when assembled in homotetramer. Otherwise, it significantly reduces the surface expression of the resulting channels and influences their biophysical properties when involved in the formation of functional heterotetramers with Kv2 subunits. Kv2.1 and Kv8.2 show significant regional overlap: within the hippocampus, transcripts for both KCNV2 and KCNB1, which encodes Kv2.1, are detected in excitatory neurons of the pyramidal cell layers and the dentate gyrus. Similarly, both of them are abundantly expressed in the cortex. Their regional colocalization is consistent with an effect of Kv8.2 variants on Kv2.1 channels within cells critically important for seizure generation and propagation.

19A support of the involvement of KCNV2 in seizure pathogenesis was provided by the identification of non-synonymous variants in two unrelated children showing epilepsy: p.Arg7Lys and p.Met285Arg. In particular, the p.Arg7Lys was found in a patient affected by febrile and afebrile partial seizures, whereas the p.Met285Arg was reported in a case of epileptic encephalopathy and severe refractory epilepsy. The functional characterization of these variants demonstrated that they both enhanced Kv8.2-mediated suppression of Kv2.1 currents, suggesting a role in decreasing delayed rectifier K+ current in neurons, therefore increasing cells excitability. Moreover, the p.Met285Arg caused a shift in the voltage-dependence of activation as well as slower activation kinetics, in accordance with the more severe clinical phenotype of the patient.

20The human ether-a-go-go-related gene (hERG, also known as KCNH2) encodes the pore-forming subunit of the rapid component of the delayed rectifier K+ channels, Kv11.1, which are expressed in several tissues, mostly in brain and heart. In the brain, Kv11.1 channels regulate neuronal firing and modulate the excitability of GABAergic and dopaminergic neurons. The same channel exerts a different function in the heart being involved in the regulation of membrane potentials in the ventricles.

21Mutations in the KCNH2 gene were reported to cause type 2 long QT syndrome (LQT2), a rare inherited ion channel disorder characterized by prolonged QT interval and predisposing patients to ventricular arrhythmias that can lead to syncope and sudden cardiac death (SCD). LQT2 syndrome is frequently misdiagnosed as epilepsy due to seizures that are triggered by cerebral hypoperfusion during a ventricular arrhythmia, therefore suggesting a possible link between epilepsy and cardiac arrhythmias, as described by several clinical reports. In particular, a seizure phenotype was reported in about 30% of unrelated LQTS patients carrying pathogenic variants in the KCNH2 gene, suggesting that mutations in the Kv11.1 channel associated with LQTS may also predispose to seizure activity. Moreover, a post-mortem study identified nearly 13% of LQTS pathogenic variants in the KCNH2 and SCN5A genes in epileptic samples. In particular, regarding KCNH2, two non-synonymous mutations have been identified: p.Arg176Trp and p.Arg1047Leu. Another study on three families showing a history of seizures and LQTS2 lead to the identification of three novel KCNH2 mutations: p.Tyr493Phe, Ala429Pro and Thr74ArgfsTer32 (also named p.del234-241). In vitro functional analyses of all these variants showed a loss of hERG potassium channel function with a reduction of the current, suggesting a dominant negative effect. reported a case of a man with long history of epilepsy and referred for cardiologic evaluation, showing the p.Arg534Cys mutation. In addition, other authors identified a nonsense mutation (p.Arg863X) leading to a 296-amino acid deletion while a loss-of-function mutation (p.Ile82Thr) was reported in a pedigree featuring LQTS, idiopathic epilepsy and increased risk of sudden death.

22Kv channel functional diversity is enhanced by coassembly with a wide array of auxiliary subunits, which cannot form functional channels alone but which can greatly impact channel function upon coassembly with α-subunits to form hetero-oligomeric complexes. Defects in these subunits may affect Kv channel function and network excitability, resulting thus in an increase of seizure susceptibility. Several subunits have been identified, including β-subunit (Kvβ), leucine-rich glioma-inactivated-1 (KvLGI1) and K+ channel-interacting protein (KvKChIP).

23Kvβ subunits are cytoplasmatic proteins critical for the correct membrane localization and normal biophysical properties of voltage-gated K+ channels. Variations in the expression of different Kvβ genes and their isoforms could significantly impact K+ channel function, especially with respect to inactivation kinetics. In the mammalian genome three genes encode Kvβ subunits: Kvβ1, Kvβ2, and Kvβ3. Interestingly, Kvβ2 knockout mouse models were characterized by cold-swim induced tremors and occasional seizures, suggesting thus a role of this subunit in the regulation of neuronal excitability. An association between the severity of seizures and the loss-of-function of the KCNAB2 gene that encodes the β2 subunit was reported. In particular, the hemizygous deletion of KCNAB2 identified in this manuscript in epileptic patients suggested that haploinsufficiency of this gene may represent a significant risk factor for epilepsy: the lack of the β subunit would reduce K+ channel-mediate membrane repolarization and increase neuronal excitability.

24The leucine-rich glioma-inactivated-1 (LGI1) is the best characterized LGI family protein, highly expressed in neurons, which encodes a secreted protein containing two domains [a leucine-rich repeat domain (LRR) and a β-propeller domain called EPTP] that mediate protein-protein interactions. LGI1 binds to the presynaptic voltage-gated potassium channel Kv1.1 and prevents Kv channel inactivation mediated by the β subunit of the channel. The LG1 gene was find to be mutated in approximately 50% of ADLTE (autosomal dominant lateral TLE) families: more than 30 disease-causing mutations in LGI1 gene have been associated so far with this focal epilepsy that is characterized by good response to antiepileptic drugs and with a juvenile onset. In particular, almost all mutations are missense, splice-site, or short indels while only a single microdeletion has been reported. Certain LGI1 mutants (typically non-secreted mutants) fail to prevent channel inactivation resulting in more rapidly closing channels, which extends presynaptic depolarization and leads to increased calcium (Ca2+) influx. Consequently, release of neurotransmitter is increased excessively and may induce focal seizures. Moreover, it was demonstrated that the loss of LGI1 gene in mice induced lethal epilepsy, suggesting its essential role as an antiepileptogenic ligand. LGI1 may serve as a major determinant of brain excitation and the LGI1 gene-targeted mouse could provide a good model for human epilepsy.

25The K+ channel-interacting proteins (KChIPs 1–4) compose a subfamily of neuronal Ca2+ sensor proteins that modulate trafficking, targeting to the plasma membrane, as well as turnover and endocytosis of Kv4 channels. Among KChIPs, KChIP2 is abundantly expressed in hippocampal pyramidal cells and represents the major target of Kv4 α subunits to form a complex essential for IA regulation in hippocampal neurons. This current has been found to be reduced in the presence of a deletion in the KChIP2 gene by Wang and collaborators. The authors thus suggested that it may increase susceptibility to seizures. Moreover, they also hypothesized a role of KChIP2 in SUDEP risk, since KChIP2 knockout mice were previously shown to be highly susceptible to induced arrhythmias. In conclusion, these data suggested that loss-of-function mutations in modulatory subunits could increase the susceptibility to seizures and cardiac arrhythmias, thereby providing a unified mechanism for a neurocardiac syndrome such as SUDEP.

26Inwardly rectifying K+ (Kir) channels are widely expressed in several excitable and non-excitable tissues playing a key role in the maintenance of the resting membrane potential and consequently in the regulation of cell excitability. Approximately 15 Kir clones forming either homotetramers or heterotetramers were identified and grouped in seven different families based on sequence similarity and functional properties: Kir1-Kir7. Generally, Kir channels showed the greater conductance at negative potentials in respect to the equilibrium potential for K+ (EK), while an inhibition of the outward flow of K+ ions caused by both Mg2+ and polyamines was reported at more positive values. Several Kir channels have been associated with epileptic phenotypes and, in particular, Kir2.1, Kir3, Kir4 and Kir6.

27The Kir2.1 channel is encoded by the KCNJ2 gene whose expression is reported in several brain areas as well as in astrocytes where they control astrocyte-mediated K+ buffering in combination with Kir4.1.

28Several mutations impairing the channel functionality were reported in the KCNJ2 of Andersen–Tawil syndrome (ATS) patients (;;; see Table 2 for mutation details). On the other hand, Kir2.1 gain-of-function mutations cause the type-3 variant of the short QT syndrome (SQT3s) which results in QT shortening and increased risk of sudden cardiac death. Recently, some authors detected a novel mutation (p.Lys346Thr) in the KCNJ2 in monozygotic twins displaying SQT3s and autism-epilepsy phenotype, suggesting the existence of a Kir2.1 role in neuropsychiatric disorders and epilepsy. Functional studies revealed that this mutation causes an increase of the channel’s surface expression and stability at the plasma membrane, a reduction in protein degradation and an altered protein compartmentalization.

29The G-protein-coupled Kir (GIRK) channels belong to the subfamily of Kir3 that are important regulators of electrical excitability in both cardiomyocytes and neurons. Different types of neurotransmitters, such as acetylcholine, dopamine, opioids, serotonin, somatostatin, adenosine, and GABA, activate these channels by stimulating their G-protein coupled receptors (GPCRs). This results in a final membrane hyperpolarization and inhibition of cell excitability due to the activation of an outward flux of K+ ions. Mammals express four GIRK channel subunits (GIRK1-4, also named Kir3.1-3.4), encoded by KCNJ3, KCNJ6, KCNJ9, and KCNJ5, respectively. These four subunits can form homo or heterotetramers with unique biophysical properties, regulation and distribution.

30Alterations in GIRK channel function have been associated with pathophysiology of severe brain disorders, including epilepsy. In this regard, a GIRK2 knockout mouse model resulted to be more susceptible to develop both spontaneous or induced seizures in respect to wild type mice. In particular, mice carrying a p.Gly156Ser mutation displayed an epileptic phenotype. Indeed, this mutation has been found to alter the putative ion-permeable, pore-forming domain of the channel, inducing Ca2+ overload in cells and reducing channel availability, leading thus to neurodegeneration and seizures susceptibility.

31An increased expression of GIRK channels was observed in rat brain after an electroconvulsive shock, probably altering the excitability of granule cells and the functions of neurotransmitter receptors which are coupled to these channels. Another evidence in support of a role of GIRK channels in epilepsy was provided by the demonstration that ML297, a potent and selective activator of GIRK channels, showed epileptogenic properties in mice. On the other hand, the inhibition of GIRK channel activity by drugs causes seizures. All these considerations imply that changes in Kir3 channel activity may alter the susceptibility to seizures.

32Among Kir4 channels, the Kir4.1, encoded by the KCNJ10 gene, is the only one that has been associated to epilepsy. This subunit can assemble itself in homomeric channels or it can constitute heterotetramers in combination with Kir5.1 (KCNJ16). Kir4.1 expression has been detected primarily in the thalamus, cortex, brainstem and hippocampus. Kir4.1 channels play a key role in maintaining resting membrane potential by transporting K+ from the extracellular space into glial cells in the CNS.

33Alterations of Kir4.1 channels have been linked to seizure susceptibility in both mice and humans. Conditional Kir4.1 knockout mice in astrocytes have been found to display premature lethality and severe seizures prior to death, supporting the idea of a pathophysiological relationship of the Kir4.1 impairment with epilepsy. Concerning human Kir4.1, a linkage study identified a missense variation (p.Arg271Cys) as associated with epileptic phenotypes. However, the variant did not result to have functional effects in vitro. Mutations in this gene were also reported in EAST syndrome (also named SeSAME) patients, a rare condition showing epileptic seizures among other signs (;;; see Table 2 for mutation details).

34Single nucleotide variations in Kir4.1 were detected in the DNA of TLE patients presenting with hippocampal sclerosis and antecedent febrile seizures, supporting the importance of KCNJ10 as a candidate gene for seizures susceptibility.

35Interestingly, several authors reported a strong association between epilepsy and autism spectrum disorders (ASDs) and an “autism-epilepsy phenotype” has been proposed. Indeed, a mutational screening of KCNJ10 in 52 children affected by cryptogenic epilepsy identified two heterozygous mutations (p.Arg18Gln and p.Val84Met) in three children of two unrelated families displaying seizures, ASDs, and intellectual disability. The functional consequences of these mutations appeared to be a gain-of-function mechanism. These findings suggest that an abnormal K+ homeostasis in the brain may increase the susceptibility to this “autism-epilepsy phenotype”. A common mechanism between autism and epilepsy could be the impairment of astrocytic-dependent K+ buffering, altering neuronal excitability and synaptic function.

36The adenosine triphosphate (ATP)-sensitive K+ (KATP) channels are widely distributed in various tissues where they couple cell metabolism to cell excitability. These channels are assembled by an inward rectifier K+ channel pore (Kir6.1/Kir6.2) and an ATP-binding regulatory subunit, named sulfonylurea receptor (SUR1/SUR2A/SUR2B). Neuronal KATP channels are mainly constituted by a coassembly of Kir6.2/SUR1 subunits..

37Several gain-of-function mutations were detected in the Kir6.2 (KCNJ11) or the SUR1 subunit (ABCC8). These mutations are responsible for developmental delay, epilepsy and neonatal diabetes (DEND), accounting for approximately 40% of cases and caused a decrease in the ability of ATP to block the KATP channel. This results in more fully openings of the channel at physiologically relevant concentrations of ATP, thus increasing the KATP current. Nevertheless, the pathophysiological mechanism leading to epilepsy remains to be elucidated. Probably, elevated levels of extracellular glucose and intracellular ATP attenuate KATP channels, producing a more excitable state. Moreover, mice lacking Kir6.2 are vulnerable to hypoxia, exhibiting a reduced threshold for generalized seizure. Transgenic mice, overexpressing the SUR1 gene in the forebrain, show a significant increase in the threshold for kainate-induced seizures.

38The Na+-activated K+ channels (KNa) are found in neurons throughout the brain and are responsible for delayed outward currents named IKNa. These currents regulate neuronal excitability and the rate of adaption in response to repeated stimulation at high frequencies. In many cases, IKNa is mediated by the phylogenetically related KNa channel subunits Slack and Slick. Like the Kv channels, these subunits have six hydrophobic, transmembrane segments (S1–S6) with a pore P-domain between S5 and S6 and a large cytoplasmatic C-terminal domain containing two regulators of K+ conductance (RCK) domains that are likely to be sites for Na+-binding and channel gating. The Slack subunit binds with Slick to form heterotetrameric channel complexes. Slack has been associated with different epilepsy phenotypes.

39The KCNT1 gene encodes the KNa channel subunit KCNT1, called Slack (sequence like a calcium-activated potassium channel, also known as KCa4.1 or Slo2.2). KCNT1 is highly expressed in the brain but also in the heart and the kidney at lower levels. Concerning brain, it is not widely expressed in the cortex but it is found in neurons of the frontal cortex, consistent with its known role in the pathogenesis of autosomal dominant nocturnal frontal lobe epilepsy (ADNFLE). While KCNT1 channels are thought to play important roles in modulating the firing patterns and general excitability of many types of neurons, their precise function is yet to be resolved.

40Mutations in KCNT1 gene have been found in different epilepsy syndromes: ADNFLE, epilepsy of infancy with migrating focal seizures (EIMFS, previously known as malignant migrating partial seizures in infancy, MMPSI or also more recently as malignant migrating focal seizures of infancy, MMFSI) and other types of EOEEs,, including Ohtahara syndrome (OS). The involvement of KCNT1 in these distinct disorders suggests that KCNT1 mutations may cause a spectrum of focal epilepsies. Patients displaying KCNT1 mutations have a very high occurrence of severe mental and intellectual disability.

41Four missense mutations (p.Arg398Gln, p.Tyr796His, p.Met896Ile, and p.Arg928Cys) in KCNT1 gene were reported to be associated with ADNFLE cases showing comorbidities of intellectual disability and psychiatric features. This is in contrast to ADNFLE patients without mutations in KCNT1 gene, where intelligence and other neurologic functions are largely unimpaired. Mutations are clustered around the RCK and cytoplasmatic NAD+ binding domain, the site that regulates the channel sensitivity to Na+ intracellular concentrations. A complete penetrance is reported in ADNFLE families showing KCNT1 mutations with the exception of a non-penetrant case.

42Interestingly, reported that a KCNT1 mutation (p.Arg398Gln) can lead to either ADNFLE or EIMFS within the same family, indicating that genotype-phenotype correlations are not straightforward). Similarly, a more recent study showed that the p.Gly288Ser mutation could cause both phenotypes, probably due to genetic modifiers or environmental factors. Nevertheless, this association was unexpected since in vitro studies demonstrated that mutations associated with MMFSI caused a significantly larger increase in current amplitude than those associated with ADNFLE.

43Concerning EIMFS, in addition to the above mentioned p.Gly288Ser and p.Arg398Gln, several additional mutations have been identified, including p.Val271Phe, p.Arg428Gln, p.Arg474Gln, p.Met516Val, p.Lys629Asn, p.Ile760Met, p.Pro924Leu and p.Ala934Thr. These are clustered not only around the RCK and NAD+ binding domain of the protein, but also within its S5 transmembrane segment, indicating that the alteration of other regions of KCNT1 could also be pathogenic

44Finally, two KCNT1 mutations were associated with other forms of EOEEs, strengthening once again the existence of a wide phenotypic spectrum of KCNT1 mutations. In particular, the p.Phe932Ile was detected in a patient affected by EOEEs whereas the p.Ala966Thr was found in one showing OS. Both of them are clustered around the RCK and NAD+ binding domains of the protein.

45The effect of nine different mutations in KCNT1 gene that give rise to these distinct forms of epilepsy was examined and it was demonstrated that they all result in channels displaying a strong gain-of-function phenotype: all of them produced many-fold increases in current amplitude as compared with the wild-type channel. This could greatly increase the cooperativity in channel gating that is detected in clusters of multiple channels.

46Ca2+-activated K+ channels are highly conserved complexes thought to play a critical role in neuronal firing properties and circuit excitability in the human brain. Three groups of Ca2+-activated K+ channels can be distinguished: large conductance (BKCa), intermediate conductance (IKCa), and small conductance (SKCa) channels. The opening of these channels is in response to an increase in Ca2+ concentration and a depolarization of the membrane potential, which in turn causes a secondary hyperpolarization reestablishing the membrane potential as well as Ca2+ levels. Otherwise it can produce an afterhyperpolarization to potentials more negative than the resting membrane potential. To date, only the association between KCa1.1 channel and epilepsy has been demonstrated.

47KCNMA1 gene encoded the α-subunit of the large conductance KCa1.1 channels. They show the typical tetrameric structure of K+ channels, with four α-subunits each displaying seven transmembrane segments, with a unique S0 segment, and the charged S4 segment conferring the voltage-dependence. Ca2+ sensitivity comes instead from the bulky C-terminal tail that includes a negatively charged, high-affinity Ca2+ binding region and the double negative charged RCK-domain. These channels could associate with four different types of β subunits (β1-β4, each encoded by a specific gene KCNMB1-4) which modulated channel function uniquely.

48KCa1.1 channels play a role in promoting high neuronal frequency firing which is consistent with their predominant expression in axon and presynaptic terminals of neurons located in brain regions (e.g., hippocampus and cortex) frequently involved in epilepsy. The involvement of these channels in epilepsy was suggested not only by their localization but also by studies on animal models. In this regard, it has been demonstrated in mice highly susceptible to convulsions that the inhibition of KCa1.1 channels is sufficient to block cortical bursting activity. Moreover, the loss of β4 subunits in KCaβ4 knockout mice promoted the excitatory synaptic transmission, resulting in temporal cortex seizures. Finally, demonstrated a deficit of KCNMA1 expression in the dentate gyrus in animal models, hypothesizing therefore its critical role in the pathogenesis of mesial temporal lobe epilepsy (mTLE).

49An association between KCa1.1 channels and epilepsy has also been observed in humans. A missense mutation in KCNMA1 (p.Asp434Gly) was detected in a large family with generalized epilepsy and paroxysmal dyskinesia. Functional studies revealed an increased Ca2+ sensitivity predicting a gain-of-function and neuronal hyperexcitability by a presumably faster action potential repolarization. Additional studies suggested that depending on the distribution of the various β subunits in the brain, this mutation can differently modulate KCa1.1 channels contributing to the pathophysiology of epilepsy and dyskinesia. As far as genes different from KCNMA1, a polymorphism in KCNMB4, named rs398702, was also associated with mTLE in an Irish cohort population but the study failed to be replicated, while a truncation mutation in KCNMB3 (p.Val256TyrfsTer4) affecting synaptic inhibition and thereby increasing neuronal excitability and seizure susceptibility, was associated with idiopathic generalized epilepsy.

50Epilepsy is one of the most common chronic and heterogeneous neurological disorders, affecting 1–2% of the population, characterized by recurrent unprovoked seizures due to abnormal synchronized electrical discharges within the CNS. Since ion channels mediate the axonal conduction of action potentials and transduction through synaptic transmission, increasing evidence suggests that any mutation-induced channel malfunction directly alter brain excitability and can induce epileptic seizures. Therefore, the discovery of genetic defects and, in particular, the electrophysiological characterization of mutant ion channels in hereditary forms of epilepsy may elucidate pathophysiological concepts of hyperexcitability in the CNS. This knowledge could enable new therapeutic strategies by antagonizing the epilepsy-causing mechanisms using the defective proteins as pharmacological targets. Given these considerations, we present an overview of mutations in K+ channels and their related accessory subunits underlying different human epileptic phenotypes. Several families of K+ channels have been involved in the pathogenesis of epilepsy or other syndromes showing seizures as a clinical sign. For each channel family, the effect of reported mutations is different: loss-of-function as well as gain-of-function could be observed. The common effect of all mutations is to determine membrane hyperexcitability, thus increasing the susceptibility to seizures. Our review highlights the pleiotropic effects of some mutations in K+ channels and the lack of a direct genotype–phenotype correlation. Interestingly, K+ channels dysfunctions seem to be mainly observed in epileptic patients with neurological comorbidities, such as ASDs, intellectual disabilities or psychiatric features, in which they are associated with more clinical severity. This observation could suggest to perform a mutation screening of K+ channels in patients showing intellectual disabilities.

51In conclusion, the discovery of K+ channels encoding genes that influence susceptibility and disease progression will provide insight into the molecular events of epileptogenesis, improve molecular diagnostic utility, and identify novel therapeutic targets for treatment of human epilepsy.

52All authors listed, have made substantial, direct and intellectual contribution to the work, and approved it for publication.

53The authors declare that the research was conducted in the absence of any commercial or financial relationships that could be construed as a potential conflict of interest.