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Potassium channel genes and benign familial neonatal epilepsy

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Snezana Maljevic1, Holger Lerche Department of Neurology and Epileptology, Hertie Institute for Clinical Brain Research, University of Tu¨bingen, Tu¨bingen, Germany 1 Corresponding author: Tel.: +49-7071-29-81922; Fax: +49-7071-29-4698, e-mail address: [email protected]

Abstract Several potassium channel genes have been implicated in different neurological disorders including genetic and acquired epilepsy. Among them, KCNQ2 and KCNQ3, coding for KV7.2 and KV7.3 voltage-gated potassium channels, present an example how genetic dissection of an epileptic disorder can lead not only to a better understanding of disease mechanisms but also broaden our knowledge about the physiological function of the affected proteins and enable novel approaches in the antiepileptic therapy design. In this chapter, we focus on the neuronal KV7 channels and associated genetic disorders—channelopathies, in particular benign familial neonatal seizures, epileptic encephalopathy, and peripheral nerve hyperexcitability (neuromyotonia, myokymia) caused by KCNQ2 or KCNQ3 mutations. Furthermore, strategies using KV7 channels as targets or tools for the treatment of epileptic diseases caused by neuronal hyperexcitability are being addressed.

Keywords KCNQ2, KCNQ3, M-current, retigabine, heterologous expression, dominant-negative effect, haploinsufficiency, developmental expression

1 INTRODUCTION Each of approximately 85 billion neurons in the human brain greatly relies in its function on the specific expression of relatively small proteins—ion channels—in its membrane. These proteins provide a unique milieu in which information can be generated and transmitted to control both movement of the little toe and creation of a space shuttle or The Fifth Symphony. In other words, ion channels form selective pores for different ions, which can open and close in a regulated manner and thus determine the ion flux over membrane, presenting the basis of the electrical excitability. Essentially, changes in membrane potential allow opening of voltage-gated Progress in Brain Research, Volume 213, ISSN 0079-6123, http://dx.doi.org/10.1016/B978-0-444-63326-2.00002-8 © 2014 Elsevier B.V. All rights reserved.

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ion channels, whereas binding of specific chemical messengers (neurotransmitters) evokes ion passage through ligand-gated ion channels (Lerche et al., 2005). Voltage-gated ion channels are responsible for the generation of action potentials and their conduction along the axons, as well as for establishing and revoking membrane potential at rest. When action potentials arrive on the presynaptic membrane, they induce Ca2+ influx and the release of neurotransmitters, which bind to the ligand-gated postsynaptic channels and provide the information transmission between cells. Neurons can be distinguished by the chemical messengers they release: excitatory neurons communicate via glutamate or acetylcholine, whereas neurotransmitters produced by inhibitory neurons are g-aminobutyric acid (GABA) and glycine. Ion channels are further characterized by specific temporal and spatial distribution and can inhabit different neuronal compartments. In excitatory pyramidal cells, the specific voltage-gated sodium channels, such as NaV1.2 and NaV1.6 (Liao et al., 2010), or potassium channels, such as KV7.2 and KV7.3 (Maljevic et al., 2008), are expressed in the axon initial segments (AISs), the origin site of action potentials. In contrast, the NaV1.1 sodium channel is found at the AISs of the inhibitory neurons (Ogiwara et al., 2007). Ligand-gated channels occupy postsynaptic membranes in dendrites, but some voltage-gated ion channels are also found at these sites (Vacher et al., 2008). The majority of genetic defects detected thus far in idiopathic epilepsies affect ion channels. Genetic alterations can affect channel function and thereby alter the electrical impulse, modifying neuronal excitability and driving networks of neurons into synchronous activity, which can finally lead to an epileptic seizure. Moreover, mutations within the postsynaptic receptors can affect the conduction between cells and thus present an epilepsy-causing defect. Within a healthy brain, ion channels, ingrained in membranes of excitatory and inhibitory neurons, are providing a neuronal balance. Epileptic seizures can be elicited by disruption of this balance caused by ion channel defects and treated by anticonvulsants that are mainly affecting ion channels. It is a challenge to our understanding how and why genetic alterations resulting in epileptic seizures do not cause disease phenotype interictally. Furthermore, genetic epilepsy disorders occur at certain age and can in some cases remit spontaneously, indicating that specific patterns of ion channel function or expression may be responsible for the seizure precipitation. Potassium channel genes cover a number of important physiological functions and have, therefore, been under a detailed investigation in relation to genetic epilepsies. Indeed, in the past 20 years, several potassium channels have been associated with epilepsy. Especially one potassium channel family, the KCNQ channels, drew much attention since mutations in the KCNQ genes have been linked to different human inherited diseases. Mutations in KCNQ2 and KCNQ3 genes were the first potassium channel mutations associated with an epileptic phenotype in benign familial neonatal seizures (BFNS) (Biervert et al., 1998; Charlier et al., 1998; Schroeder et al., 1998). In the meantime, the phenotype spectrum related to these channels extended, including among others severe epileptic encephalopathy

2 Potassium channels

(EE) (Weckhuysen et al., 2012). In parallel, development of the newly approved drug retigabine, which is acting as an opener of these channels, has started a new era in the development of antiepileptic drugs.

2 POTASSIUM CHANNELS Subunits of potassium (K+) channels are encoded by approximately 80 genes (KCN) in mammals, and present the most divergent of all ion channel families. They are widely expressed throughout the body having various physiological functions (Coetzee et al., 1999). The specificity of these channels for K+ over other cations is defined by a highly conserved amino acid sequence, the so-called GYG signature sequence, which enables selective transmission of K+ by replacing the six water molecules that surround these ions. The K+ channel from a Streptomyces lividans bacterium KscA was the first crystallized ion channel (Doyle et al., 1998). Subsequently determined crystal structures of mammalian channels revealed that conformational changes, which open and close the pore, take place within its inner part in response to membrane depolarization, binding of Ca2+ or other regulatory mechanisms (Long et al., 2005). Based on the number of transmembrane (TM)-spanning regions in each subunit and their physiological and pharmacological characteristics, K+ channels are grouped into 2TM, 4TM, and 6TM or 7TM families (Gutman et al., 2005). All potassium channel genes are thought to emerge by gene duplication from a single ancestor gene ( Jegla et al., 2009) having 2TM segments. This structure is characteristic for the inward-rectifier K+ channel family (KIR), including ATP-sensitive K+ channels which associate with sulfonylurea subunits to regulate cellular metabolism and G-protein-coupled KIR channels. As in the majority of K+ channel families, functional pore is formed by four subunits (Hibino et al., 2010). As a matter of fact, the 4TM K+ channel family is the only one in which the functional pore is formed by two subunits. These channels are unique because they contain two instead of one pore-forming loop. The 4TM, responsible for the leak currents in neuronal cells, are active at rest and have constitutively open channel gate (Plant et al., 2013). K+ channels, which are voltage-insensitive and activated by low concentrations of internal Ca2+, comprise the 6TM family of “small-conductance” (SK) and “intermediate-conductance” (IK) KCa channels. Ca2+ does not bind directly on these channels but is instead bound to calmodulin (CaM), which induces conformational changes resulting in pore opening (Wei et al., 2005). In the 7TM KCa1.1, so-called big-conductance (BK) channels, the N-terminus makes a seventh pass through the membrane to the extracellular side. These channels are expressed in a broad variety of cells and binding of Ca2+ is not dependent on its association with CaM (Shieh et al., 2000). The largest family of K+ channels is encoded by about 40 genes and encompasses voltage-gated (KV) channels. KV channels consist of four a-subunits, each containing 6TM regions, which form a single pore (Fig. 1) (Gutman et al., 2005). A short

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FIGURE 1 Structure and function of KV channels. Functional voltage-gated potassium channels (KV) are made of four subunits. A typical structure of a single KV channel subunit is shown on the left. Due to their different characteristics, KV channels play diverse physiological roles. Whereas several KV channels are directly implicated in the membrane repolarization during an action potential, KV7.2/KV7.3 are active in the subthreshold range and important for the regulation of resting membrane potential and prevention of repetitive firing (middle). Examples for specific somatodendritic and axonal localization of KV channels are presented on the right.

amino acid sequence, containing positively charged arginine residues, forms the fourth TM segment S4 responsible for the channel regulation by voltage and therefore named the voltage sensor. In response to changes in membrane potential, conformational changes within this region will affect the movement of the channel gate in the intracellular side of the pore-forming S5–S6 loop. Amino and C-terminal domain are located inside the cell and can vary in their length in different subunits. The subunit assembly domain is usually found at the N-terminus, except in KCNQ (KV7) and hERG (KV11) channels, where it is located in the C-terminus. These parts of the channel also contain binding domains for auxiliary subunits or other regulatory proteins (Gutman et al., 2005). Functional diversity of potassium channels is further increased by their heteromerization into dimers (KIR) or tetramers (KV) and interaction with a number of auxiliary subunits. mRNA splicing and posttranslational modifications also contribute to the K+ channel diversity (Gutman et al., 2005; Shieh et al., 2000).

2.1 HOW POTASSIUM CHANNELS REGULATE NEURONAL EXCITABILITY KV channels usually colocalize with voltage-dependent Na+(NaV) or Ca2+(CaV) channels in excitable cells and are responsible for the cell membrane repolarization or hyperpolarization. During an action potential, cell membrane is depolarizing ought to the influx of Na+ ions through the NaV channels and the repolarization phase is determined by the inactivation of NaV channels, as well as by efflux of K+ ions due to the concentration gradient across the membrane upon the opening of KV channels

2 Potassium channels

(Lehmann-Horn and Jurkat-Rott, 1999). Slower than the NaV channels, some KV channel subunits generate fast K+ currents across the membrane, which can also inactivate and are recognized as A-type potassium currents (Shieh et al., 2000). Inactivation is a state of the channel protein in which, although still in the open conformation, the channel pore is not permeable due to occlusion by an amino terminal sequence (fast, N-type or ball-and-chain sequence inactivation) or a conformational change within a pore (slow, P- or C-type inactivation). An important potassium current in neurons is the so-called M-current, a noninactivating slow current which is activated at subthreshold voltages and can be regulated by muscarinic agonists, which is where the name comes from (Brown and Adams, 1980). Physiologically, the A-currents will have a larger impact on the initial action potentials within a spike train whereas M-current will determine the response to multiple spikes, when A-current is inactivated (Bean, 2007; Brown and Adams, 1980). Typical A-type KV channels are found in KV1–KV4 subfamilies, while KV7 (KCNQ) and KV11 (hERG) produce the M-currents (Fig. 1) (Shieh et al., 2000). Within the central and peripheral nervous systems, the a subunits of KV channel family are expressed in both neurons and glial cells and besides excitability also affects Ca2+ signaling, secretion, volume regulation, proliferation, and migration. Within a single neuron, they can occupy different subdomains indicating their specialized physiological roles ( Jensen et al., 2011). For instance, KV2 and KV4 present somatodendritic channels, KV1 subunits are found on axons and nerve terminals, KV7 reside mainly at AISs and nodes of Ranvier, and KV3 are expressed in dendritic or axonal domains, depending on the neuronal cell type or a splice variant (Fig. 1) (Vacher et al., 2008). A variety of molecular mechanisms, including interactions with other neuronal proteins, determine specific distribution of KV channels in neuronal membrane subdomains, which is also dependent on and regulated by neuronal activity ( Jensen et al., 2011; Misonou and Trimmer, 2004).

2.2 POTASSIUM CHANNELS IN EPILEPSY AND RELATED DISORDERS The major physiological roles that potassium channels play in the nervous system indicate they may be involved in a number of neuronal disorders characterized by increased excitability, such as epilepsy, migraine, naturopathic pain, ataxia, and others. Diseases caused by dysfunction of ion channels are called “channelopathies.” Before we concentrate on the neonatal seizures and the associated neuronal KCNQ2/3 channelopathies, we will shortly address the involvement of other potassium channels in epilepsy and pertinent diseases.

2.2.1 Mutations in KV1.1 Cause Episodic Ataxia KCNA1 gene encodes KV1.1 channel, which is the human homolog of the Shaker potassium channel of the fruit fly Drosophila melanogaster. Mutations causing a loss of function of the Shaker channel in fruit flies are related to the leg-shaking phenotype occurring episodically or upon ether anesthesia. As mentioned before, KV1.1 channels mediate the fast-inactivating A-currents known to regulate the repolarizing

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phase of an action potential (Shieh et al., 2000). Mutations in KCNA1 have been associated with episodic ataxia type 1 (EA-1), a human equivalent of the Shaker mutation phenotype, characterized by seconds-to-minutes-long ataxia and repetitive discharges in distal musculature (myokymia) occurring interictally (Browne et al., 1994). In some cases, complex partial or tonic–clonic seizures have been reported. As a matter of fact, compared to healthy individuals EA-1 patients are about 10 times more likely to develop seizures (Rajakulendran et al., 2007). Interestingly, the KV1.1 knock-out mouse model also exhibits an epilepsy phenotype and reveals altered axonal conduction of action potentials (Smart et al., 1998). Among the loss-of-function mechanisms caused by distinctive KCNA1 mutations are altered kinetics, reduced current amplitudes, or trafficking defects of the KV1.1 channel (Rajakulendran et al., 2007). Expression of KV1.1 mutations in neurons suggested their major effect was increased neurotransmitter release (Heeroma et al., 2009).

2.2.2 KCa1.1 Mutation Linked to Paroxysmal Dyskinesia and Epilepsy

BK channels are ubiquitously expressed and open in response to both Ca2+ increase and voltage. A KCa1.1 (gene KCNMA1) mutation has been detected in a large family with generalized epilepsy and paroxysmal dyskinesia (Du et al., 2005). Paroxysmal dyskinesias present a heterogeneous group of rare neurological disorders featuring sudden, unpredictable, disabling attacks of involuntary movement (hyperkinesias), which may require life-long treatment. Functional studies revealed an increased calcium sensitivity predicting a gain of function and neuronal hyperexcitability by a presumably faster action potential repolarization (Du et al., 2005).

2.2.3 KV4.2 and Acquired Epilepsy KV4 channels control somatodendritic excitability by generating subthreshold A-type currents. In the experimental model of temporal lobe epilepsy, pilocarpine-induced status epilepticus decreased protein levels and increased posttranslational modifications of KV4.2 (encoded by KCND2) enhancing dendritic excitability and neuronal activity and thus promoting the seizure initiation and/or propagation (Bernard et al., 2004).

3 BIOLOGY OF KCNQ2 AND KCNQ3 CHANNELS In the following, we will introduce the major features of KV7 voltage-gated potassium channels and focus on the physiological role, clinical pictures, genetics, and pathophysiology of KCNQ2/3-associated channelopathies.

3.1 MEET THE KCNQS Five voltage-gated delayed rectifier K+ channels (KV7.1–5), encoded by the KCNQ gene family and often referred to as KCNQ1–5 channels, gained a VIP (very important potassium) channels status very soon after their discovery. The excitement was

3 Biology of KCNQ2 and KCNQ3 channels

not only because it was recognized that four genes from this small gene family associate with hereditary human diseases, but also that among them first two potassium channel genes related to genetic epilepsy were found. Even more, the products of these two epilepsy genes were shown to be mainly responsible for the generation of the slow potassium M-current, previously known for almost two decades as one of the important regulators of neuronal excitability. Lastly, a drug synthesized during the clinical evaluation of flupirtine was proven to be specifically binding to the pore sequence of KCNQ channels expressed in brain and has recently been introduced into the market as a first-in-class anticonvulsive exploiting opening of KV channels as a mechanism. If we have your attention now, it is time to meet the key players.

3.1.1 KCNQ1 KCNQ1 gene, coding for KV7.1 channel proteins, was the first cloned channel of this family and has been identified using a positional cloning approach on chromosome 11p15.5 in families with long QT syndrome type 1 (Wang et al., 1996). Like in all the other KV channels, KV7.1 subunits assemble into tetramers, but present the only KV7 subunit that cannot form heterotetramers with other KV7 family members. Instead, KV7.1 a-subunits coassemble with auxiliary KCNE1 b-subunits, also known as minK or IsK, to create channels that generate the slow delayed rectifier K+ current, IKs, which plays a key role in cardiac late-phase action potential repolarization (Barhanin et al., 1996; Sanguinetti et al., 1996). Besides in the heart, KV7.1/KCNE1 channels are expressed in the inner ear, thyroid gland, lung, gastrointestinal tract, the small intestine, pancreas, forebrain neuronal networks and brainstem nuclei, and in the ovaries (Goldman et al., 2009; Jespersen et al., 2005). These channels are also found in the proximal and distal tubule of the nephron (Vallon et al., 2001), which together with the Kcne1 ( / ) mice phenotype, including hypokalemia, urinary and fecal salt wasting, and volume depletion, suggests the importance of these channels for the kidney function (Arrighi et al., 2001; Vallon et al., 2001; Warth and Barhanin, 2002). Long QT syndrome (LQTS) presents a disorder of cardiac repolarization, which predisposes affected individuals to ventricular torsade de pointes tachyarrhythmias and cardiac sudden death. In fact, two syndromes characterized by LQTS have been associated with KCNQ1 loss-of-function mutations: autosomal dominant Romano– Ward and the recessive Jervell and Lange-Nielsen syndrome. In the latter, long QT is combined with congenital deafness (Wang et al., 1996). The KV7.1 mutations often cause a strong suppression of the remaining WT currents, i.e., the dominantnegative effect (Maljevic et al., 2010; Schmitt et al., 2000). Since mouse models carrying LQTS mutations develop spontaneous seizures, a possible role of KV7.1 in epileptogenesis has also been suggested (Goldman et al., 2009).

3.1.2 KCNQ2 and KCNQ3 Two different approaches were used to identify KCNQ2 and KCNQ3 genes: screening of a human brain cDNA library using a KCNQ1-derived sequence (Yang et al., 1998) and positional cloning in families with BFNS (Biervert et al., 1998; Charlier et al., 1998; Schroeder et al., 1998). The corresponding protein subunits KV7.2 and

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KV7.3 are found expressed throughout different brain regions and can form homoand heterotetrameric channels, which conduct slowly activating and deactivating current elicited at subthreshold membrane potentials, the so-called M-current (Wang et al., 1998). A number of mutations associated with neonatal seizures, Rolandic epilepsy (Neubauer et al., 2008), or more severe EE (Weckhuysen et al., 2012) phenotypes have been detected so far, with the majority of them affecting the KCNQ2 channels. One rare single-nucleotide polymorphism in KCNQ3 has been linked with autism spectrum disorders (Gilling et al., 2013).

3.1.3 KCNQ4 The KCNQ4 gene has been cloned from human retina cDNA library using a KCNQ3 partial cDNA probe. In parallel, a missense dominant-negative mutation was identified in this gene, which cosegregated with an inherited autosomal dominant form of nonsyndromic progressive hearing loss (DFNA2) ( Jentsch, 2000; Kubisch et al., 1999). KCNQ4 mRNA is expressed in outer hair cells of the inner ear and low expression is found in the brain (Kubisch et al., 1999), restricted to the structures of the brainstem, predominantly within the nuclei contributing to the central auditory pathway (Kharkovets et al., 2000). The cochlear nerve appeared not to be KV7.4 immunoreactive (Kharkovets et al., 2000). KV7.4 subunits can form homo- and heterotetrameric channels with KV7.3, yielding M-like currents (Kubisch et al., 1999). Detected DFNA2 mutations show a loss of function either by a haploinsufficiency mechanism or by a dominantnegative effect (Maljevic et al., 2010). It has been proposed that dominant-negative mutations preferentially cause all-frequency hearing loss with younger onset, while mutations following a haploinsufficiency mechanism are related to a late-onset hearing impairment affecting only high frequencies (Topsakal et al., 2005). Two generated mouse models, a Kcnq4 / knock-out and a mouse carrying a dominant-negative DFNA2 mutation (KCNQ4dn/+), exhibited a hearing loss over several weeks with KCNQ4dn/+ mice showing a slower progression (Kharkovets et al., 2006). The analysis revealed depolarization and degeneration of outer hair cells, indicating that a disrupted potassium efflux due to the absence of KV7.4 currents can lead to the potassium overload of cells and their progressive devolution (Kharkovets et al., 2006).

3.1.4 KCNQ5 The last cloned member of the KCNQ gene family, encoding the KV7.5 subunit, is the KCNQ5. It was cloned from a human brain cDNA library by homology screening with KCNQ3 (Schroeder et al., 2000) and use of the KCNQ5 gene sequence identified from a GenBank search (Lerche et al., 2000). KV7.5 can form heteromers with KV7.3 and its distribution is similar to KV7.2 and KV7.3: the splice variant I is found in the brain and splice variant II and III in skeletal muscles (Lerche et al., 2000; Schroeder et al., 2000). No mutations related to epilepsy or other hereditary human disorders have been identified so far (Kananura et al., 2000; Maljevic et al., 2010).

3 Biology of KCNQ2 and KCNQ3 channels

3.2 STRUCTURAL AND FUNCTIONAL HALLMARKS OF KV7.2/3 CHANNELS All KV7 channel share a typical structure of other voltage-gated potassium channels, meaning that each subunit comprises six transmembrane (TM/S1–6) regions, a voltage-sensing, arginine-rich S4 segment and a pore formed by loops between S5 and S6 harboring the GYG sequence. With no crystal structure available so far, the length and borders of the six TM segments are based on hydrophobicity prediction or use of homology modeling based on the known structure of other potassium channels (Doyle et al., 1998; Long et al., 2005). The amino (N-) and carboxy (C-) terminal domains are positioned intracellularly. As in other KV channels, four subunits interact to form a functional channel pore. In contrast to the majority of KV channels with a tetramerization (T1) domain at their N-terminus, the assembly of KV7 channel subunits occurs via a domain localized at the C-terminus (Maljevic et al., 2003; Schmitt et al., 2000; Schwake et al., 2003). Furthermore, the KV7 C-terminus is exceptionally long and contains many regulatory domains (see below). KV7.2–KV7.5 homotetramers, as well as their heteromeric combinations with KV7.3 channels, produce the M-current, a slow subthreshold potassium current which can be abrogated by the activation of muscarinic acetylcholine receptors (Brown and Adams, 1980; Wang et al., 1998). As previously described, the M-current is important for the control of the membrane potential and can impede repetitive neuronal firing. In heterologous systems, the homomeric KV7.3 currents are not greater than background potassium currents (Schroeder et al., 1998; Wang et al., 1998). On the other hand, coexpression of KV7.3 and KV7.2 in an equimolar ratio generates at least 10-fold larger currents in Xenopus oocytes than KV7.2 alone, suggesting the formation of heteromers. Differential sensitivity to TEA, a common KV channel blocker, with KV7.2 being more sensitive than KV7.3, was used to confirm the formation of heteromers. Expression of tandem KV7.3/7.2 constructs in a nonneuronal cell line revealed an intermediate TEA sensitivity, which was indistinguishable from the one obtained for the M-current recorded from the cervical superior ganglion SCG in adult rats (Hadley et al., 2003; Wang et al., 1998). Thus, the suggested stoichiometry of KV7.2 and KV7.3 subunits in the SCG is 1:1. Moreover, a particular amino acid residue, localized in the proximity of the GYG sequence in the KV7.3 pore domain, was shown as responsible for the detainment of the KV7.3 homotetramers in the endoplasmic reticulum in neurons. In contrast, when combined with KV7.2, KV7.3 subunits are able to reach the surface membrane as part of the heterotetrameric complex (Gomez-Posada et al., 2010). One possible explanation for the current augmentation of KV7.2/KV7.3 heterotetramers is that compared to their surface expression in the homomeric constellation, the number of KV7.2 subunits reaching the plasma membrane when they are part of this heteromeric channel complex with KV7.3 is significantly increased (Schwake et al., 2000). Apart from the effects on trafficking to the surface, other molecular

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mechanisms involving regulatory actions of certain parts of channel proteins or individual amino acids have been proposed (Etxeberria et al., 2004; Maljevic et al., 2003). Role of regulating proteins, such as CaM, will be discussed below.

3.2.1 What Happens at the C-terminus? KV7 channels’ particularly long C-terminus is involved in the assembly, trafficking, and gating of these channels (Haitin and Attali, 2008). The predicted secondary structure shows four helical regions (helices A–D), which are conserved in all family members (Yus-Najera et al., 2002). The proximally located helices A and B are responsible for the interaction with CaM and involved in channel trafficking and gating. Distally located helices C and D are thought to form coiled-coil assemblies (Haitin and Attali, 2008).

3.2.1.1 Assembly of KV7 Channels The study of Schmitt et al. (2000) was the first to reveal that the C-terminal part may play a role in the assembly of the KV7 subunits. They defined a short amino acid stretch in the C-terminus essential for the functional expression of these channels and could further show that one of the mutations associated with the Jervell and Lange-Nielsen syndrome affected the subunit assembly via the C-terminus. This finding prompted research on other KV7 channels. Using a chimeric approach, parts of KV7.1, which does not interact with any other KV7 subunit, were exchanged with KV7.2 and KV7.3, to demonstrate that the assembly of KV7.2 and KV7.3 channels also happens at the C-terminus via a so-called A-domain (Maljevic et al., 2003; Schwake et al., 2003). Ensuing biochemical and structural dissection of the subunit interaction domain (Schwake et al., 2006; Wehling et al., 2007), including its crystallization in the KV7.4 (Howard et al., 2007), revealed two coil-coiled stretches, corresponding to helices C and D. Whereas helix C shows high conservation among KV7 channels, the more divergent helix D is probably the one determining subunit assembly specificity.

3.2.1.2 Regulation of the M-current Several regulatory molecules interact with the KV7 channel via their C-terminus. Among them are CaM, A-kinase anchoring proteins (AKAPs), protein kinase-C (PKC), phosphatidylinositol 4,5-bisphosphate (PIP2), and syntaxin 1A (syx) (Haitin and Attali, 2008; Regev et al., 2009). CaM, whose binding site is created by helices A and B in the KV7.2 C-terminus, is promoting folding and trafficking of the channel to the plasma membrane (Etxeberria et al., 2008; Haitin and Attali, 2008). Syntaxin is binding to a partially overlapping region with CaM, but exerts opposite effects on the channel function (Regev et al., 2009). Furthermore, a trimeric complex formed by the AKAP79/150 protein and PKC (Hoshi et al., 2003) is also binding at the C-terminus. Activated PKC inhibits the channel by phosphorylating serine residues in helix B and may, therefore, have a significant contribution in the transmitter-mediated inhibition of KV7 channels (Delmas and Brown, 2005). PIP2 is suggested to stabilize the open state of neuronal KV7 channels (Li et al.,

3 Biology of KCNQ2 and KCNQ3 channels

2005), probably by binding to the proximal part of the C-terminus (Haitin and Attali, 2008). Interestingly, these molecules can act antagonistically or synergistically with CaM, increasing the number of ways in which the function of these channels can be regulated (Bal et al., 2010; Delmas and Brown, 2005; Etzioni et al., 2011).

3.2.1.3 Targeting and Localization of KV7.2/7.3 Channels In neurons, KV7.2 and KV7.3 subunits are found in AISs and nodes of Ranvier (Figs. 1 and 2), but studies also indicate their expression in somatic and presynaptic regions (Devaux et al., 2004; Hu et al., 2007; Maljevic et al., 2008; Martire et al., 2004; Pan et al., 2006; Vacher et al., 2008). The localization of ion channel proteins in the AIS is mediated by ankyrin G, large adaptor protein coupling membrane proteins with actin–spectrin cytoskeleton. In fact, the whole organization of the AIS, site of generation of action potentials, is guided by ankyrin G (Rasband, 2010). The dense structure of AIS provides a necessary milieu for the detainment and synergistic action of voltage-gated ion channels needed for the generation and propagation of action potentials and also presents a diffusion barrier between somatodendritic and axonal compartments of neurons. It was first shown for the NaV channels that a short conserved amino acid sequence is crucial for the interaction with ankyrin G and thereby their targeting to the AIS (Garrido et al., 2003; Lemaillet et al., 2003). In KV7.2 and KV7.3, the ankyrin G interaction domain is found at their C-terminus (Pan et al., 2006), mapping distally from the helix D. Studies on neurons from the ankyrin G knock-out mice show that lack of ankyrin G abolishes AIS targeting of both NaV and KV7.2/7.3 channels. Furthermore, deletion of the KV7.3 ankyrin G binding domain had a greater impact on the AIS targeting of the heteromeric KV7.2/7.3 complex than the disruption of this domain in KV7.2 (Rasmussen et al., 2007). Interestingly, ankyrin G binding motif only emerged in the vertebrate orthologues of Nav and KV7 genes, coinciding with the development of myelination (Pan et al., 2006).

FIGURE 2 KV7.2 expresses in the axon initial segment (AIS). AIS is a neuronal compartment with a high concentration of ion channels involved in action potential generation. Immunohistochemical staining of a mouse brain section reveals the colocalization of KV7.2 and NaV1.2 channels in this region (Maljevic and Lerche, unpublished data).

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Immunohistochemical analysis revealed that the density of ion channels throughout the AIS is not homogenous and can vary during development (Fig. 2). For instance, NaV1.2 is found expressed earlier in the development at the AIS and settles in its proximal part in adult neurons. This process is paralleled by emergence of NaV1.6, occupying the distal part of the axon (Liao et al., 2010). The physiological meaning of the subdomain-specific localization is probably that low threshold distal NaV1.6 is more important for the initiation of action potentials, whereas proximal high-threshold NaV1.2 plays a role in backpropagation to the soma (Hu et al., 2009). However, the developmental expression pattern is still unclear, although it nicely explains the reminiscence of seizures caused by mutations in NaV1.2 (see below). Detailed analysis using confocal imaging and patch clamp recordings in the AIS showed that the density of KV7.2/7.3 channels is highest in the distal two-thirds of the AIS (Battefeld et al., 2014). The same study suggested that somatodendritic KV7 channels could be robustly activated by the backpropagating action potentials, to abate afterdepolarization and repetitive firing. On the other hand, axonal KV7 channels may have a role in stabilizing the resting membrane potential, which increases the availability of NaV channels and the action potential amplitude in nodes of Ranvier (Battefeld et al., 2014).

3.3 EXPRESSION PATTERN OF NEURONAL KV7 CHANNELS KV7.2 and KV7.3 channels are expressed together in different neurons of the central and peripheral nervous system. In the brain, they are found at different sites, including hippocampus, cortex, and thalamus, in both inhibitory and excitatory neurons (Cooper et al., 2000, 2001). However, in situ hybridization also indicates that KCNQ2 and KCNQ3 mRNA is not always expressed in the same ratio (Schroeder et al., 1998). This is supported by the observation that, in some neurons, only one of the two subunits can be detected using immunohistochemistry (Cooper et al., 2000). In rodents, both channels are found at low levels in the first postnatal days, and the expression increases within the first weeks of development (Geiger et al., 2006; Maljevic et al., 2008; Weber et al., 2006).

3.4 INSIGHTS FROM THE MOUSELAND Several mouse models have been created to study the effects of either gene deletion (knock-out models) or specific single amino acid exchanges found in patients and inserted at the homologous site of the mouse gene (so-called humanized knock-in mouse models). In the Kcnq2 knock-out line, / pups die right after birth due to pulmonary atelectasis. In a hemizygous +/ constellation, animals have no spontaneous seizures, but show increased sensitivity when pentylenetetrazole is used to induce them (Watanabe et al., 2000). Removal of the Kcnq3 does not produce any specific phenotype and these mice are viable (Tzingounis and Nicoll, 2008).

3 Biology of KCNQ2 and KCNQ3 channels

To understand what happens in neurons lacking one or both alleles of KV7.2, Robbins et al. (2013) studied sympathetic neurons isolated from late Kcnq2 / or +/ embryos. Expectedly, quantitative PCR revealed lack of Kcnq2 mRNA in the / and about 30% reduction in +/ neurons, translating into the absence or reduction of the resulting M-current, respectively. Interestingly, in both genotypes, an increase in the expression of Kcnq3 and Kcnq5 mRNA was found. In neurons from the adult Kcnq2 +/ mice, M-current level was same as in the WT neurons, probably due to increased expression from the remaining allele as a compensatory mechanism. To circumvent the early loss of Kcnq2 / mice, Peters et al. (2005) designed a conditional Kcnq2 knock-out model by introducing a dominant-negative mutant under the antibiotic control so that it can be activated at different time points during development. Interestingly, induction of expression of this dominant-negative Kcnq2 mutation in the right time window provoked spontaneous seizures, accompanied with cognitive impairment and morphological changes in the hippocampus. At the time of generation of this mouse model, the severe phenotype seemed at odds with the benign clinical pictures found in patients carrying KCNQ2 mutations, but as it will turn out, corresponds well with the clinical picture of KCNQ2-related EE (Weckhuysen et al., 2012). Two knock-in models, carrying either a KCNQ2 or a KCNQ3 BFNS-causing mutation, have also been created (Singh et al., 2008). Homozygous mice revealed reduced M-currents and showed spontaneous seizures throughout life, though not limited to the early period of development, thus not faithfully reproducing the BFNS phenotype. The heterozygotes exhibited a reduced seizure threshold upon application of convulsant drugs (Singh et al., 2008). The increased seizure susceptibility also occurred in a sex-, mouse strain-, and seizure test-dependent manner (Otto et al., 2009).

3.5 FUNCTIONAL ANALYSIS OF DISEASE-RELATED MUTATIONS Ion channel defects can be examined in heterologous expression systems as well as in neuronal cell lines and animal models. The former implies expression of the affected protein in a system free from endogenous channels with the same or similar function. Commonly used are different mammalian cell lines or Xenopus laevis oocytes. The cRNA or cDNA encoding the WT and mutant channel is injected or transfected in such cells and after providing enough time for production of encoded proteins, analyzed in parallel using a combination of electrophysiological, biochemical, or immunohistological techniques. The obtained results show how the mutant channel behaves or expresses compared to the WT. Channels may act differently in such expression systems in comparison to their native environment. However, since the major question is whether a mutation significantly affects channel function, data from heterologous systems are valuable initial step in the functional analysis. Furthermore, many of the obtained results could be reproduced in animal models.

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Use of neuronal expression system has certain complications. Namely, number of available neuronal cell lines is limited, and rodent primary neuronal cultures are viable only for several weeks. More importantly, neuronal cells express a whole range of different channels, including the channel of interest, so it is a challenge to recognize particular effects of the analyzed protein. As shown above, several mouse models for KCNQ channels are available. It is, however, unrealistic to expect that genetic murine models can be generated to study all detected mutations.

3.6 KCNQ2 AND KCNQ3 CHANNELOPATHIES The clinical phenotype first linked to mutations in the KCNQ2 and KCNQ3 was a rare benign form of neonatal epilepsy (BFNS). Many reports of more complex phenotypes, including peripheral nerve hyperexcitability (PNH) and myokymia or Rolandic epilepsy with centrotemporal spikes, emerged over time. However, it was only recently that a systemic analysis of a cohort of severely affected children with refractory epilepsy and mental retardation introduced an EE as a clinical phenotype related to KCNQ2 mutations. This pattern with a spectrum of phenotypes associated with mutations in a single gene has already been observed in other ion channel genes associated with epilepsy, such as SCN1A or SCN2A, in which epileptic disorders range from febrile seizures combined with heterogeneous generalized epilepsy (GEFS +) to the severe myoclonic epilepsy of infancy (SMEI or Dravet syndrome), and from benign familial neonatal–infantile seizures (BFNIS) to EE, respectively (Reid et al., 2009). As a rule, more severe phenotypes are usually linked to de novo mutations affecting these genes.

3.7 KCNQ2/3 MUTATIONS IN BFNS Three epilepsy conditions beginning within the first year of life present mainly secondarily generalized focal seizures, have transient expression and generally benign outcome, and show autosomal dominant mode of inheritance. Based on the exact onset time and the genes involved we delineate among: BFNS starting typically before the fifth day of life (Rett and Teubel, 1964), BFNIS occurring between day two and 6 months of age (Kaplan and Lacey, 1983), and benign familial infantile seizures (BFIS) emerging between 3 and 8 months of age (Vigevano et al., 1992). Interestingly, specific genes have been linked to each phenotype, with KCNQ2 and KCNQ3 causing BFNS, SCN2A driving BFNIS, and PRRT2 being responsible for BFIS. Therefore, genetic analysis can help define and differentiate among these syndromes. A recent study (Zara et al., 2013) in a group of patients with all three syndromes revealed that a certain overlap exists between BFNS and BFNIS, since early occurring seizures, even if starting later than day 5 and therefore diagnosed as BFNIS, are likely to be related to KCNQ2 mutations, although this is not always the case.

3.7.1 Clinical Features and Genetics of BFNS Starting in the first days of life, seizures in BFNS often occur in clusters and remit spontaneously after weeks to months. If at all needed, treatment is only required for a short period. The onset of seizures is partial, and they are often accompanied with

3 Biology of KCNQ2 and KCNQ3 channels

hemi-tonic or -clonic symptoms, apnoeic spells, or clinically appear as generalized. Electroencephalograms (EEGs) usually show normal interictal activity, whereas the recorded ictal EEGs reveal a focal onset, and sometimes also bilateral synchrony. About 15% of patients may have recurring seizures later in life. Inheritance is autosomal dominant and a penetrance of 85% has been estimated. Corresponding to the benign outcome, the psychomotor development is in most cases normal (Maljevic et al., 2008). Sporadically, patients with mental retardation and difficult to treat epilepsies have been described (Alfonso et al., 1997; Borgatti et al., 2004; Dedek et al., 2003; Schmitt et al., 2005; Steinlein et al., 2007). A study of a cohort of BFNS families (Soldovieri et al., 2014) reported that 5 out of the 17 families included one or two individuals with more severe clinical picture, encompassing delayed psychomotor development, intellectual disability, or other neurological features. Other affected members in these families had only benign neonatal seizures. However, recent studies of cohorts of severely affected patients introduced KCNQ2 mutations as a common cause of a specific phenotype they described as KCNQ2-related EE (see below). Moreover, one of the most common epilepsies in childhood, the so-called Rolandic or benign epilepsy of childhood with centrotemporal spikes, has also been associated with KCNQ2/3 mutations (Coppola et al., 2003; Neubauer et al., 2008). More than 50 mutations in KCNQ2 and 6 mutations in KCNQ3 have been described to cause BFNS (Fig. 3). Furthermore, deletions or duplications of KCNQ2 gene are found in a significant proportion of BFNS families (Heron et al., 2007).

3.7.2 Pathogenic Mechanisms in BFNS KV7.2 and KV7.3 mutations have been analyzed in heterologous systems, such as X. laevis oocytes and mammalian cell lines, and two mouse models carrying BFNS mutations have also been generated ( Jentsch, 2000; Maljevic et al., 2010). The common feature of all studied mutations is a loss of function in both homomeric and heteromeric channel conformations. The mechanisms underlying loss of function include haploinsufficiency, gating alterations and rarely a dominant-negative effect (Fig. 4) (Maljevic et al., 2008). Especially the cytoplasmic C-terminus of KV7.2, the pore regions (S5–S6 segments) of both KV7.2 and KV7.3 channels, and the voltage sensor S4 and the S1–S2 region of KV7.2 are affected by BFNS mutations (Fig. 3). The common functional consequence of all mutations examined so far is a reduction of the resulting K+ current (Fig. 4). Even though a complete loss of function of KV7.2 or KV7.3 is often observed ( Jentsch, 2000; Lerche et al., 1999; Maljevic et al., 2010), a coexpression of wild-type (WT) and mutant KV7.2 or KV7.3 with the WT of the other subunit in a 1:1:2 ratio, translating the expected expression ratio in patients, revealed a reduction in the current size of merely 20–25% compared with coexpression of both WTs. This means that relatively small decline in the KV7.2/KV7.3 M-current appears to be sufficient to cause epileptic seizures in neonates (Bassi et al., 2005; Jentsch, 2000; Lerche et al., 1999; Maljevic et al., 2008; Schroeder et al., 1998; Singh et al., 2003; Soldovieri et al., 2014). Even in families with larger phenotypic variability, including more severe neurological outcomes, in vitro studies

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FIGURE 3 Disease-causing mutations in KCNQ2 and KCNQ3. Schematic presentation of KV7.2 and KV7.3 structure with predicted positions for mutations causing benign familial neonatal seizures (BFNS), epileptic encephalopathy (EE), and peripheral nerve hyperexcitability (PNH) and myokymia. Modified after Orhan et al. (2014).

FIGURE 4 Effects of KV7.2 disease-causing KCNQ2 mutations. Typical currents recorded from cells expressing WT or a mutant KV7.2 channel show a dramatic decrease of the current amplitude for the mutant. Molecular mechanisms are discussed in the text. Upon coexpression with KV7.3, the recorded currents are strikingly increased for the WT and the majority of mutated channels. In experiments mimicking the presumed ratio of KV7 subunits in a patient carrying a KV7.2 mutation, expected decrease of recorded currents is 20–30% for haploinsufficiency pathomechanism, or larger when the mutant exerts the dominant-negative effect. BFNS mutations incline to the former and EE mutants to the latter mechanism. Modified after Maljevic et al. (2011) and Orhan et al. (2014).

3 Biology of KCNQ2 and KCNQ3 channels

revealed a comparably mild reduction of the maximal KV7.2/KV7.3 currents, suggesting that additional genetic and environmental factors may contribute to phenotypic variability in BFNS families (Steinlein et al., 2007). Since the C-terminal domain contains many important regions, including the tetramerization domain and binding sites for other regulatory proteins, impaired tetramerization or reduced trafficking to the surface membrane could explain how C-terminal mutations reduce KV7.2 currents. For example, reduced surface expression was found for the KV7.2 mutant truncating the C-terminus (Schwake et al., 2000). Two other C-terminal mutations were shown to disrupt the binding to CaM (Richards et al., 2004) and for one of them impaired trafficking to the surface membrane could be confirmed (Etxeberria et al., 2008). A mutation causing frameshift and prolongation of the channel protein was shown to decrease protein stability (Soldovieri et al., 2006). For some mutations affecting the C-terminus impaired regulation by syntaxin-1A, part of the presynaptic SNARE complex, known to reduce KV7.2 currents, has been demonstrated (Soldovieri et al., 2014). Interestingly, majority of reported mutations affecting the pore region in KV7.2 or KV7.3 do not show a dominant-negative effect on the WT subunits, despite presence of an intact C-terminal assembly domain (Charlier et al., 1998; Hirose et al., 2000; Schroeder et al., 1998; Singh et al., 1998, 2003). These mutants probably affect ion channel conductance and reduce K+ currents by a haploinsufficiency mechanism. However, for one trafficking-defective KV7.2 mutation located in the pore region, a dominant-negative effect was reported (Maljevic et al., 2011). Remarkably, the expression of this mutant channel in the surface membrane could be partially restored by lowering the incubation temperature or by long exposure of cells to high doses of retigabine, a neuronal KV7 channel opener. Interestingly, out of six mutations in KCNQ3 reported so far (Charlier et al., 1998; Fister et al., 2013; Hahn and Neubauer, 2009; Hirose et al., 2000; Singh et al., 2003; Soldovieri et al., 2014; Zara et al., 2013), five are found in the pore region and one in the beginning of the S6 segment. Functional analyses in heterologous systems revealed a 20–40% reduction of KV7.2/KV7.3 currents ( Jentsch, 2000; Singh et al., 2003; Soldovieri et al., 2014), whereas one mutation was shown to cause a dominant-negative effect (Sugiura et al., 2009; Uehara et al., 2008). Changes in KV7.2 channel gating have been reported for the mutations perturbing the S4 voltage sensor (Dedek et al., 2001; Miraglia del Giudice et al., 2000; Singh et al., 2003; Soldovieri et al., 2007; Wuttke et al., 2007). Mutations affecting arginine residues, thus altering positive charges within the S4 segment, cause rightward shift of the activation curve accompanied with slowed activation and faster deactivation kinetics (Miraglia del Giudice et al., 2000), together with decreased voltage sensitivity (Castaldo et al., 2002), whereas mutations of noncharged residues produce atypical gating, where rightward shift of the activation curve is accompanied with a slowing of activation kinetics upon stronger depolarizing prepulses (Soldovieri et al., 2007). Two KV7.2 mutations affecting the same positive charge (R207) and exhibiting a pronounced dominant-negative effect have been associated with PNH or BFNS and myokymia (see below).

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Changes in channel gating have also been linked to the two mutations affecting the S1–S2 extracellular loop (Hunter et al., 2006; Wuttke et al., 2008), both revealing a significant reduction of the relative current amplitudes limited to subthreshold voltages. In reconstitution experiments supporting the presumed in vivo constellation of mutant and WT subunits, the observed changes were even smaller than the 25% seen for other mutations, but sufficient to elicit prolonged bursts of action potentials and yield a lower threshold for infinitive firing in one compartment neuronal model cell (Wuttke et al., 2008). An interaction of the mutated residues with a positive charge within the voltage sensor S4 may be an explanation for this effect. These findings emphasized in human disease model that the subthreshold voltage range is most relevant for M channels to modulate neuronal firing.

3.7.2.1 Mechanisms of Spontaneous Seizure Remission in BFNS Although our understanding of mechanisms underlining the occurrence of neonatal seizures has significantly increased in the last years, the question of their transient expression, limited to the first days of life, remains puzzling. Could developmental changes in expression patterns of KV7.2 and KV7.3 channels account for the neonatal seizure phenotype and spontaneous remission of seizures? The available data from rodent brains suggest a significant upregulation in expression of both channels within the first three postnatal weeks (Geiger et al., 2006; Maljevic et al., 2008; Weber et al., 2006), which means that only a small number of neuronal KV7 channels are responsible for an adequate control of the subthreshold membrane potential in neonates’ brain. If the critical amount of functional channels could not be reached due to even a mild loss of function caused by mutations, the M-current may be on too low level, and this can lead to the generation of seizures. By contrast, this happens only rarely in adulthood, when M channels are abundantly available, or an upregulation of other K+ channels helps compensate for the M-channel deficit. Furthermore, the proposed excitatory action of GABA in the immature brain could aggravate this effect (Okada et al., 2003). Namely, the intracellular concentration of Cl ions in neurons is increased in the early postnatal period, and binding of GABA will elicit outward Cl currents that cause membrane depolarization, just opposite to the hyperpolarizing effect of inward Cl currents in the mature brain. Hence, with GABA acting as a depolarizing signal, the M-current might have even more important role as an inhibitor of neuronal firing and its importance would diminish parallel to the inhibitory switch of the GABAergic system. In addition, the exclusive expression of a shorter, nonfunctional splice variant of KV7.2 in fetal brain which can attenuate KV7.2/ KV7.3-mediated currents (Smith et al., 2001) has been suggested to contribute to the seizure occurrence and remission.

3.8 KCNQ2-RELATED EE Although sporadic cases of KCNQ2-related severe refractory neonatal epilepsy with developmental delay have been reported previously (Borgatti et al., 2004; Dedek et al., 2003; Steinlein et al., 2007), it was a systematic screen of a cohort of EE

3 Biology of KCNQ2 and KCNQ3 channels

patients performed by Weckhuysen et al. (2012) that revealed that KCNQ2 mutations are responsible for this severe phenotype in about 10% of patients. In their first study, seven novel KCNQ2 mutations were detected, six of them occurring de novo, and ensuing research established KCNQ2-related encephalopathy as frequent early onset (neonatal) EE phenotype (Milh et al., 2013; Numis et al., 2014; Weckhuysen et al., 2013).

3.8.1 Clinical and Genetic Features Early onset EEs include a divergent group of syndromes characterized by early occurrence of seizures correlated with impaired neurological development. Patients affected with KCNQ2-related encephalopathy present with pharmacoresistant neonatal onset seizures with strong tonic component. In contrast to BFNS patients, the interictal EEG activity is characterized by burst suppression or multifocal spikes and transient T1 and T2 hyperintensities of the basal ganglia have also been reported (Weckhuysen et al., 2012). Whereas seizures generally remit by age of 3, profound intellectual disability and motor impairment persist (Milh et al., 2013; Weckhuysen et al., 2012, 2013). The level of impairment may vary as well as the ability to learn to walk or speak by the age of 3 (Milh et al., 2013; Weckhuysen et al., 2013). So far, three cohorts of patients presenting this phenotype have been screened for mutations in the KCNQ2 gene. Following initial study by Weckhuysen and colleagues, who sequenced both KCNQ2 and KCNQ3 in the cohort of 80 patients, finding six de novo KCNQ2 mutations and a mosaic mutant in a patient with a milder phenotype, two other larger cohorts have been analyzed (Milh et al., 2013; Weckhuysen et al., 2013). Percentage of patients carrying de novo mutations in these two studies varied between 13% (11/84 patients; Weckhuysen et al., 2013) and 23% (16/71 patients; Milh et al., 2013), and the number of detected de novo KCNQ2 mutations rose to about two dozen. Interestingly, using whole exome sequencing of 12 patients with Ohtahara syndrome, presenting with similar features as the KCNQ2 encephalopathy, three de novo KCNQ2 mutations could be detected (Saitsu et al., 2012). Thorough phenotypic characterization in these studies has been accompanied with the treatment response analysis (Numis et al., 2014; Weckhuysen et al., 2013). Positive response to the KV7.2 channel opener retigabine was found in one EE patient, whereas some responded well to carbamazepine. But, seizure-free status in these children did not seem to improve the severe psychomotor delay (Numis et al., 2014).

3.8.2 Pathophysiologic Mechanisms of EE Functional analysis of seven KCNQ2 encephalopathy mutations detected in the initial report (Weckhuysen et al., 2012) revealed a loss of function of the mutant KCNQ2 allele. The study done in X. laevis oocytes unveiled that five out of seven analyzed mutants exhibited a strong dominant-negative effect on the WT subunits. This effect was found for only 4 out of more than 50 known BFNS mutations, suggesting that it may present a prevailing mechanism behind the severe EE phenotype (Orhan et al., 2014).

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The EE mutations affect functionally important parts of the channel, including the voltage-sensing S4, the pore, and the C-terminus domain (Fig. 3). Not surprisingly, both mutations in the S4 segment cause large depolarizing shifts in voltage-dependent activation, especially pronounced for the R213Q mutant. This mutation affects arginine on the same position as one BFNS mutation (R213W). Miceli et al. (2013) compared the effects of the two mutations and showed a more pronounced functional defect, mainly expressed as a dramatic decrease of voltage sensitivity, for the R213Q, which is possibly an explanation for the more severe EE phenotype. When analyzed in X. laevis oocytes, the R213Q mutation had dramatically reduced current amplitudes, although the surface expression seemed unaffected, suggesting that mutated channels may fail to open in response to depolarization. However, the most important effect for both S4 mutations seems to be the prominent dominant-negative effect on coexpressed KV7.2 and KV7.3 WT subunits in a 1:1:2 ratio, mostly pronounced in the subthreshold range of an action potential (Orhan et al., 2014). Of the three pore mutations, all showing dramatically abbreviated currents, two cannot be rescued by KV7.3 coexpression, which has not been observed for any other KV7.2 mutation so far (Orhan et al., 2014). The current reduction in 1:1:2 coexpression experiments for these mutants is > 50%, whereas a mere 20–30% reduction of the KV7.2/KV7.3 current amplitude corresponding to a haploinsufficiency mechanism is typical for the majority of BNFS-causing mutations ( Jentsch, 2000; Maljevic et al., 2008). Moreover, for the only two BFNS mutations causing a dominant-negative effect by an overall current amplitude reduction, the effect on the 1:1:2 currents was less pronounced (Maljevic et al., 2011; Singh et al., 2003), marking a close genotype– phenotype correlation. The third pore mutant yields small currents on its own and an almost 50% reduction in the 1:1:2 coexpression experiments (Orhan et al., 2014). Whereas S4 and pore mutations present with a clear dominant-negative effect, functional defects of the two C-terminal mutations are comparable to those of BFNS mutations. Additionally, one of them shifts the activation curve to more depolarized potentials, which is completely reversed by its coexpression with the WT subunits. The reduced potassium currents may be related to a disrupted trafficking to the surface membrane. However, the oocyte expression system is perhaps not best suited to analyze channel trafficking and its interactions with other molecules present in a neuronal environment. Since these mutations affect channel region comprising CaM binding site, critical for the surface expression of the KV7 channels in neurons (Alaimo et al., 2009; Etxeberria et al., 2008), as well as many sequences for interaction with other regulatory proteins, or posttranslational modifications (Delmas and Brown, 2005; Haitin and Attali, 2008; Hernandez et al., 2008), the real impact of the two C-terminal EE mutations can probably only be assessed in neurons. These functional data suggest that strikingly reduced M-current in the first days of life not only leads to seizure generation but affects the normal neuromotor development of affected children. Notably, EE mutations seem to affect the critical functional parts of the channel and mutants significantly impair the function of the WT subunits. The arising question at this point is which kind of intervention could possibly improve the outcome of the disease. One of the interesting drug candidates to

3 Biology of KCNQ2 and KCNQ3 channels

test is retigabine, a novel antiepileptic drug targeting neuronal KV7 channels (see below), which could increase the currents of the expressed WT KV7.2 allele as well as currents carried by KV7.5 and KV7.3 subunits and possibly affect some of the homo- or heteromeric channels harboring mutant KV7.2 subunits. When tested in heterologous systems, retigabine showed effect on the majority of mutant channels, expressed alone or with KV7.2 and KV7.3, by specifically increasing their currents at the subthreshold level (Miceli et al., 2013; Orhan et al., 2014). One of the mutations localized close to the known binding site of retigabine failed to respond to its application (Orhan et al., 2014). Taken together, majority of so far studied EE mutations shows a larger functional defect compared to BFNS mutations, which may account for the more severe epileptic seizures as well as for the neurodevelopmental changes seen in patients carrying the examined mutations. This confirms the observations from the conditional KV7.2 knock-out mouse model created by introducing a dominant-negative KV7.2 mutation not found in human epilepsy (Peters et al., 2005). It will be a great challenge in the next years to find ways to revert the deleterious effects of such mutations on the M-current during pre- or neonatal development.

3.9 KCNQ2 MUTATIONS AND PNH KV7.2 channels are found expressed in both central and peripheral nervous system. It is, therefore, not surprising that the PNH presents another neurological phenotype associated with the mutations in the KCNQ2 gene. Interestingly, the two so far detected mutations affect the same amino acid within the S4 segment.

3.9.1 Clinical Picture and Genetics PNH (myokymia, neuromyotonia) presents clinically with a spontaneous and continuous muscle overactivity, which includes undulating movements of distal skeletal muscle (myokymia), fasciculations, cramps, and other symptoms caused by hyperexcitability of peripheral motor neurons (Hart et al., 2002). The most common form is autoimmune-mediated PNH characterized by generation of antibodies directed against voltage-gated KV channels (Hart et al., 2002). Mutations in two different KV channel genes, KCNA1 and KCNQ2, encoding for KV1.1 and KV7.2, respectively, have been associated with PNH. A number of mutations in KCNA1 gene have been found in patients with EA-1, which implicates neuromyotonia (Baloh, 2012; Browne et al., 1994). One KCNQ2 mutation has been linked to myokymia and BFNS (Dedek et al., 2001) and a sporadic case presenting only PNH associated with a KCNQ2 mutation has also been described (Wuttke et al., 2007).

3.9.2 Mechanisms Underlying PNH The only two mutations in KV7.2 associated with a hyperexcitability of peripheral motor neurons (PNH) affect the same arginine at position 207, within the S4 voltage sensor. Functional expression of these mutations in X. laevis oocytes revealed large depolarizing shifts of the conductance–voltage relationships coupled with

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pronounced slowing of the time course of activation compared to WT channels. Interestingly, for R207W, which apart from PNH (myokymia) also causes neonatal seizures in all but one affected individual, these effects were more pronounced (Dedek et al., 2001). Coexpression of mutants with WT KV7.2 channels showed that relative current amplitudes assessed 200 ms after the onset of depolarizing voltage steps are reduced by >70%. This dramatic dominant-negative effect on KV7.2 WT currents is what distinguishes PNH-causing mutations from the majority of mutations causing BFNS alone. Interestingly, these mutations show only small reductions of relative current amplitudes at 40 mV when expressed with KV7.2 and KV7.3 in the 1:1:2 ratio (Dedek et al., 2001; Wuttke et al., 2007), which seems to be the hallmark of the S4 mutations causing EE. Hence, the functional effects of mutations affecting positive charges in S4 segment of KV7.2 channels reveal a strong genotype–phenotype correlation. Interestingly, a nerve excitability studies performed on adults with a history of BFNS, carrying KCNQ2 mutations, detected distinctive increase in peripheral nerve excitability (Tomlinson et al., 2013). This subclinical dysfunction of KV7.2 in peripheral axons suggests that, compared to the central neurons in the neonatal period, peripheral nerves might be less prone to hyperexcitability.

4 ANTIEPILEPTIC THERAPIES TARGETING KV7 CHANNELS As shown thus far, the M-current has a clear role in controlling neuronal excitability, which is indicated by the fact that even a moderate reduction of this current can lead to the generation of seizures in neonates. It is, therefore, quite plausible that augmentation of the M-current, for instance via activation of neuronal KV7 channels, can lead to a stabilization of the resting and subthreshold membrane potential by shifting them toward the K+ equilibrium potential and thereby reducing membrane excitability. Therefore, M channels are an attractive pharmacological target to treat any disease going along with neuronal hyperexcitability, such as epilepsy, PNH, neuropathic pain, migraine, and stroke. After learning about the physiological features and genetic pathomechanisms involving KV7 channels, we shall devote some space to the therapeutical approaches involving these channels, starting with the introduction of a KV7 channel opener in antiepileptic therapy and its consequences for the development of new drugs. Other, less advanced but sufficiently promising approaches, such as gene therapy and stem cell use, will also be addressed.

4.1 THE NOVEL ANTICONVULSANT COMPOUND RETIGABINE IS A KV7 CHANNEL OPENER Initially derived from flupirtine, a substance used for treatment of acute and chronic pain, retigabine (RTG), known as ezogabine in the United States and Canada, has recently been approved as adjunctive therapy for adults with partial-onset seizures

4 Antiepileptic therapies targeting KV7 channels

(Brodie et al., 2010; French et al., 2011; Orhan et al., 2012; Porter et al., 2007a). Its major effect is the selective enhancement of the activity of neuronal KV7.2–5 channels (Rundfeldt and Netzer, 2000; Tatulian et al., 2001), which is a novel mechanism of action, not exploited by any other clinically used anticonvulsant. Furthermore, RTG does not activate cardiac KV7.1 channels, which reduces the risk for cardiac side effects (Tatulian et al., 2001). Besides affecting the KV7 channels, RTG facilitates GABAergic inhibition acting directly on GABA(A) receptors in the postsynaptic membrane (Otto et al., 2002) and when applied in higher concentrations causes an unspecific inhibitory effect on sodium-, calcium-, and kainate-induced currents (Rundfeldt and Netzer, 2000). The effectiveness of RTG has been demonstrated in many seizure models, including acute seizures, genetic epilepsy, induced epilepsy, and pharmacoresistant epilepsy models (overview in Large et al., 2012; Orhan et al., 2012; Wuttke and Lerche, 2006). One phase II and two phase III randomized, double-blind, placebocontrolled studies including 1240 patients revealed significant reductions in total partial-seizure frequency as well as increased responder rate compared to placebo (Brodie et al., 2010; French et al., 2011; Porter et al., 2007b), which led to the approval of RTG as adjunctive therapy for adults with focal seizures by the European Medicines Agency and the U.S. Food and Drug Administration (Brodie et al., 2010; French et al., 2011; Orhan et al., 2012; Porter et al., 2007b).

4.1.1 Mapping the RTG Binding Site The main mechanism by which RTG enhances the activity of KV7.2 and KV7.3 channels is a hyperpolarizing shift of the activation curve, i.e., increase in the relative number of channels that are opened at more negative potentials (Fig. 5) (Schenzer et al., 2005; Wuttke et al., 2005). Using recombinant channel proteins, comprising parts of the RTG-insensitive KV7.1 and RTG-sensitive KV7.2 or KV7.3 channels, the probable binding site of RTG in KV7.2 and KV7.3 channels has been identified (Schenzer et al., 2005; Wuttke et al., 2005). Chimeric channels with exchanged parts of the pore region, or single amino acids revealed that the effect of RTG on KV7.2 channels could be completely abolished in two ways: (i) by an exchange of a tryptophan localized in the cytoplasmic end of S5 at position 236 (W236) against leucine found at the corresponding position in KV7.1 or (ii) by an exchange of the entire S6 TM segment against the corresponding one of KV7.1. Moreover, exchange of the tryptophan, which is conserved in all KV7 channels except KV7.1, in KV7.3–5 channels, showed a similar effect (Schenzer et al., 2005), emphasizing that a lipophilic interaction between the fluorophenyl ring of RTG and the aromatic tryptophan presents the basis for RTG sensitivity (Lange et al., 2009; Schenzer et al., 2005; Wuttke et al., 2005). Furthermore, a glycine in the S6 segment (G301), which is critically involved in opening of cation channels and therefore referred to as the “gating hinge” ( Jiang et al., 2002), was also shown as critical for the RTG effect (Wuttke et al., 2005) (Fig. 5). This was further supported by a structural computer model based on the crystal structures of KscA and MthK potassium channels. Using a structural model of KV7.3, built on the KV1.2 crystal structure, several other residues probably

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FIGURE 5 KV7.2/KV7.3 activator retigabine. Retigabine (RTG) activates the KV7.2/KV7.3 currents, stabilizing thereby the resting membrane potential by a shift to the more negative range. This presents a potent antiepileptic mechanism. At the molecular level, RTG molecule is binding to the KV7 channel pore and stabilizing its open state. Modified after Wuttke et al. (2005).

contributing to the formation of hydrophobic binding pocket for the RTG could be identified (Lange et al., 2009). We may, therefore, conclude that the effectiveness of RTG critically depends on its interaction with the tryptophan in S5 and the flexibility of S6 helix introduced by the “gating hinge” glycine, both enabling stabilization of the channel in its open conformation (Fig. 5) and thus explaining the RTG-induced hyperpolarizing shift of the activation curve. Other amino acids within the pore region provide hydrophobic environment for the drug binding.

4.1.2 Other KV7 Openers Except for retigabine, which presents the only KV7 channel opener antiepileptic drug available, another small molecule KV7 channel opener currently in the early clinical development is ICA-105665. It has demonstrated activity in different animal models of epilepsy and has further undergone phase I/II studies including healthy volunteers and patients with simple or complex partial-onset seizures. So far, no dose-limiting

4 Antiepileptic therapies targeting KV7 channels

adverse events were observed, so that this compound currently undergoes investigation in photosensitive epilepsy (Wulff et al., 2009). Another molecule developed to activate KV7 channels is ICA-27243, which is belonging to the class of substituted benzamides. This molecule shows the highest selectivity for KV7.3/KV7.3 heterotetramers (Wickenden et al., 2008) and anticonvulsant properties in preclinical models of epilepsy (Roeloffs et al., 2008). Meclofenamate and diclofenac are two anti-inflammatory drugs with an activating effect on KV7.2/7.3 and no effect on the cardiac KV7.1 channels (Peretz et al., 2005). These drugs cause a hyperpolarizing shift of the KV7.2/7.3 activation curve and slow the deactivation kinetics, which can increase the K+ efflux and stabilize the resting membrane potential. It has further been demonstrated (Peretz et al., 2010) that a channel opener NH29, which affects the voltage-sensing domain of the KV7.2 channels, can cause depression of evoked spike discharges in neurons and reduce glutamate and GABA release, indicating that apart from the pore region, the S4 voltage sensor may also present a target for the drug design to treat diseases related to hyperexcitability. Therefore, neuronal KV7 channels present attractive and promising targets for development of new therapies. So far, both novel and existing compounds revealed anticonvulsant properties via M-channel activation and further high-throughput screens of novel small compounds are underway.

4.2 NOVEL THERAPIES INVOLVING KV CHANNELS Approximately 25% of epilepsy patients have difficult to treat epilepsies, which do not respond to various applied antiepileptic drugs or their combinations (Kwan et al., 2011). As we have shown in this chapter, KV7 channelopathies as well include an increasing number of patients with severe impairments and insufficient responsiveness to available therapies. In all such cases, approaches other than drug administration and optimization, including epilepsy surgery or deep brain stimulation, may present a promising perspective. We will address here two emerging strategies, including gene therapy based on the use of KV channels as possible tools for the treatment of hyperexcitability, and stem cell approach focused on development of patient specific models potentially serving as a basis for the precision medicine.

4.2.1 KV Channel Gene Therapy One of the interesting new advances in the treatment of hyperexcitability in brain is based on the use of viral delivery systems to introduce proteins that can reduce the activity of neurons and networks in vivo. Among such proteins are, for example, light-sensitive cation and anion channels—opsins—which can be used to increase or suppress activity of neurons and thus present optogenetic tools for the treatment of hyperexcitability in epilepsy (Bentley et al., 2013). Several studies in animal models have shown that gene delivery of chloride transporters halorhodopsins suppresses neuronal firing and seizures upon activation of halorhodopsin by light (Bentley et al., 2013; Paz et al., 2013; Wykes et al., 2012).

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Even more, using the same rat model of chronic refractory focal epilepsy, generated by injection of tetanus toxin in the motor cortex, Wykes et al. (2012) showed that overexpression of the voltage-gated potassium channel KV1.1 can either completely hinder development of epilepsy or cause reduction in the seizure frequency and their remission after a few weeks. The KV1.1 was delivered via lentiviral transduction under the CMV promoter, and the analysis revealed its preferential expression the excitatory pyramidal neurons, which expectedly showed attenuated activity when the channel was expressed alone. Interestingly, coinjection of KV1.1 with the tetanus toxin completely prevented the development of epileptiform events even though only a fraction of affected neurons expressed the channel. Finally, injection of KV1.1 one week upon establishment of epileptiform activity progressively reduced the seizure frequency in the following weeks (Wykes et al., 2012). Although limited to animal models and still at the very early stages, gene therapy methods may open a new era in the treatment of drug-resistant epilepsy. Possibly, neuronal KV7 channels may arise as useful tools for such approaches in the near future.

4.2.2 Human Cellular Models of Epilepsy Development of human-induced pluripotent stem cells (hiPSCs) as a model for different human diseases presents a great opportunity to not only examine the effects of genetic alterations related to epilepsy and their role in epileptogenesis, but to do so in a patient-derived material. hiPSCs are genetically reprogrammed adult cells which exhibit a pluripotent stem cell like state of human embryonic stem cells (Faiz and Nagy, 2013; Naegele et al., 2012). They retain a potential to evolve to any differentiated cell, including neurons, as well as to retain their pluripotency state. The differentiation into neurons via neuronal stem cell stage can give rise to all three principle cell types found in the adult mammalian brain—neurons, astrocytes, and oligodendrocytes, and protocols for more regulated differentiation of specific neuronal cell types are constantly emerging (Faiz and Nagy, 2013). Differentiated neuronal cultures from hiPSC obtained from epilepsy patients with specific genetic defect may present useful tools for (i) functional assessment of pathophysiological mechanisms underlying a specific type of epilepsy in the affected individual, (ii) drug screening, and even (iii) transplantation and brain engraftment of genetically corrected autologous cells (Hayashi et al., 2013) that may treat or prevent epilepsy. So far, several hiPSC-based epilepsy models have been established for the Dravet syndrome (Higurashi et al., 2013; Jiao et al., 2013; Liu et al., 2013), a severe EE syndrome in infancy caused by loss-of-function mutations of the SCN1A gene, encoding voltage-gated sodium channels NaV1.1. The analysis of hiPSC-derived neurons partially confirmed findings from the animal models revealing reduced activity of inhibitory neurons. Many efforts from different research groups are focusing on further implantation of iPSCs, and it will be exciting to follow these developments in the years to come.

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5 CONCLUSIONS Benign familial neonatal convulsions (BFNS) were among the first epilepsy disorders linked to mutations in specific genes, the KCNQ2 and KCNQ3. A few dozens of KCNQ2 mutations were linked to the neonatal seizures with a benign outcome, but in the meantime the phenotype spectrum increased to include severe EE in a number of cases. Functional analysis, as well as available mouse models, supports haploinsufficiency as a main pathomechanism in BFNS, whereas a strong dominant-negative effect has been suggested as a common feature of EE-causing mutations. Extensive studies of rare neurological KCNQ disorders within the last two decades generated significant knowledge about the affected KV7 potassium channels on one side, and the pathophysiological mechanisms underlying hyperexcitability in affected individuals on the other. Moreover, a novel antiepileptic drug retigabine has been introduced in the therapy of refractory epilepsies and significant efforts are made to detect novel compounds exploiting augmentation of the KV7 currents to treat epilepsy. Novel diagnostic methods and therapeutic approaches should further enable better treatment of patients carrying KCNQ2 mutations producing more severe outcomes.

5.1 FIVE THINGS WE LEARNED FROM KCNQ CHANNELS INVOLVED IN EPILEPSY 1. Benign is not always benign 2. The neuronal M-current plays an essential role in the neonatal period 3. De novo KCNQ2 mutations are likely to have deleterious effects on the outcome in patients 4. Rare and benign epilepsy syndromes are good models to study disease mechanisms and develop novel therapies 5. Drugs increasing activity of neuronal KV7 channels can be used to treat epilepsy

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Potassium channel genes and benign familial neonatal epilepsy.

Several potassium channel genes have been implicated in different neurological disorders including genetic and acquired epilepsy. Among them, KCNQ2 an...
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