**4.2 GEFS+ and ADJME**

As mentioned, GEFS+ can also result from mutation in the GABRG2 gene encoding the γ2 subunit of the GABAA receptor (Baulac et al., 2001). Mutations in the α1 subunit gene (GABRA1) have been linked to Autosomal Dominant Juvenile Myoclonic Epilepsy (ADJME) (Cossette et al., 2002), an idiopathic epilepsy that is not associated with febrile seizures. This disorder typically manifests itself between the ages of 12 and 18 with myoclonic seizures occurring early in the morning and with additional tonic-clonic and absence seizures in some patients.

#### **4.3 How mutant GABAA receptor subunits can cause seizures**

It has been shown that mutations in the γ2 subunit of the GABAA receptor cause retention of the receptor in the endoplasmatic reticulum (ER) (Harkin et al., 2002; Kang and Macdonald, 2004). Similarly, the A322D mutation in the α1 subunit causes rapid ER associated degradation of the subunit through the ubiquitin-proteasome system (Gallagher et al., 2007).

Epileptic Channelopathies and Dysfunctional

**5.2 Benign neonatal familial convulsions** 

neurotransmitter released.

Excitability - From Gene Mutations to Novel Treatments 199

Kv7 channels are primarily localized at the axon initial segment, the site where synaptic inputs are integrated and action potentials are generated (Pan et al., 2006; Rasmussen et al., 2007). Additionally, immunohistochemical studies have demonstrated a widespread presynaptic distribution of some Kv7 channel subunits (Cooper et al., 2000), where they may play a role in depolarization-induced neurotransmitter release (Martire et al., 2004; Martire et al., 2007). Activation of pre-synaptic M-current may hyperpolarize the nerve endings, thus reducing Ca2+ influx through voltage-gated Ca2+ channels and limiting the amount of

Benign Neonatal Familial Convulsions (BNFC) is a rare autosomal dominant idiopathic form of epilepsy. It is characterized by tonic-clonic seizures that typically begin around three days after birth and remit after 3-4 months. Yet ~16% of patients also experience seizures later in life (Ronen et al., 1993). BNFC is caused by mutations in the genes encoding

All characterized mutations in Kv7.2 and Kv7.3 cause a reduction in M-current, either by changing the channel kinetics (Dedek et al., 2001), by altering the trafficking of the channel to the cell membrane (Schwake et al., 2000), or by decreasing the subunit stability (Soldovieri et al., 2006). The mutations usually cause a reduction in M-current of about 25% (Schroeder et al., 1998). Considering the role of Kv7 channels in controlling neuronal excitability, it is not surprising that a reduction in M-current can cause hyperexcitability and predispose to seizures. Several transgenic strategies have been employed to examine how Kv7.2 and Kv7.3 malfunction lead to BNFC. The traditional knock-out approach resulted in mice that died within a few hours after birth due to pulmonary atelectasis (collapse of the lung) (Watanabe et al., 2000). Even though BNFC often results from Kv7.2 haploinsufficiency, heterozygous KCNQ2+/- mice did not experience neonatal seizures and appeared normal. They did, however, have increased susceptibility to chemically induced seizures (Watanabe et al., 2000). To overcome the postnatal lethality Dirk Isbrandt and colleagues developed mice conditionally expressing dominant-negative Kv7.2 subunits (Kv7.2-G279S) where expression of the transgene could be turned on after birth (Peters et al., 2005). These mice showed spontaneous seizures, exhibited behavioural hyperactivity and had morphological changes in the hippocampus. Mark Leppert and co-workers developed orthologous mouse models carrying disease-causing mutations in the *KCNQ2* (A306T) or *KCNQ3* (G311V) gene (Singh et al., 2008). Mice heterozygous or homozygous for either mutation had reduced seizure threshold, but only mice homozygous for the mutations exhibited spontaneous seizures. The epileptic phenotype was dependent on the specific mutation, the genetic background, sex, and seizure model (Otto et al., 2009). Hence, none of these transgenic strategies have fully recapitulated the human condition but nevertheless

Kv7.2 (Biervert et al., 1998; Singh et al., 1998) or Kv7.3 (Charlier et al., 1998).

provide us with important clues regarding the pathophysiology of BNFC.

It can be questioned why the disease usually only is clinically manifested in neonates when mutations in the channels cause general hyperexcitability. One possible explanation for the age-dependent remission of seizures is related to the expression of Kv7 channels. There is evidence for a developmental upregulation of Kv7 channels (Weber et al., 2006), suggesting that a reduction in M-current of 25% might have a more prominent effect in the fetal brain

**5.3 How mutations in Kv7.2 and Kv7.3 can cause BNFC** 

This reduced cell surface expression would result in decreased inhibitory GABAA receptor current, and consequently an increase in neuronal excitability and seizure susceptibility. But why are γ2 mutations associated with febrile seizures? And why are mutations in α1 not? Variations in temperature have effects on most cellular events. For example, synaptic vesicle recycling has been shown to be temperature dependent with increased temperature speeding both endo- and exocytosis, and there is evidence that inhibitory synaptic strength can be modulated within 10 min through recruitment of more functional GABAA receptors to the postsynaptic plasma membrane (Wan et al., 1997). Studies on cultured hippocamplal neurons showed that while trafficking of wild-type α1β2γ2 receptors is slightly temperature dependent with a small decrease in surface expression after incubation at 40°C for 2h, trafficking of receptors with mutations in the γ2 subunit is highly temperature dependent (Kang et al., 2006). Increases in temperature from 37°C to 40°C impaired trafficking and/or accelerated endocytosis of the mutant receptors within 10 min, suggesting that the febrile seizures may be a result of a temperature-induced reduction in GABA-mediated inhibition. The study also showed that the A322D mutation in the α1 subunit did not cause a temperature-dependent reduction in surface expression, consistent with a resulting epilepsy syndrome not associated with febrile seizures (Kang et al., 2006).

#### **5. Mutations in potassium channel genes**

#### **5.1 Kv7 channels and the M-current**

The Kv7 family of voltage-gated potassium channels consists of five members, Kv7.1 -5 (also termed KCNQ1-5). All five members share the general structure of voltage-gated potassium channels with four subunits that assemble to form functional tetramers. Each subunit consists of six transmembrane helices, S1-S6, and has a pore forming domain, which is formed by a P-loop between the fifth and the sixth helix. The P-loop contains the GYG (glycine-tyrosine-glycine) sequence, which is highly conserved among potassium channels and confers K+ selectivity. The fourth helix forms the voltage sensor; it contains several arginine residues and is therefore strongly positive. A S4-S5 linker in one subunit couples the voltage sensor to the intracellular activation gate in S6 of the adjacent subunit (Laine et al., 2003). Although heavily debated, it is believed that when the membrane potential depolarize, the voltage sensor is pushed out leading to bending of the S6 so potassium can enter the channel pore (Long et al., 2005).

All Kv7 channels are strongly inhibited upon activation of muscarinic receptors and are hence called M-channels (Schroeder et al., 2000; Selyanko et al., 2000). The current conducted by these channels, the M-current, was first described in bullfrog sympathetic ganglia as a slowly activating, slowly deactivating, sub-threshold voltage-dependent K+ current that showed no inactivation (Brown and Adams, 1980). The Kv7 channels have slow activation and deactivation kinetics, and in line with other voltage-gated potassium channels they open upon membrane depolarization. However, the threshold for activation is low compared to most other channels, approximately -60 mV. Since the channels open at voltages that are around or below the threshold for generation of an action potential they allow potassium flow that opposes the depolarization required to generate action potentials, and hence make the neuron less excitable. If the M-channels remain open during excitation of the nerve, the spike frequency is dampened, whiles inhibition of the M-current by activation of muscarinic acetylcholine receptors enables repetitive firing (Hille, 2001).

This reduced cell surface expression would result in decreased inhibitory GABAA receptor current, and consequently an increase in neuronal excitability and seizure susceptibility. But why are γ2 mutations associated with febrile seizures? And why are mutations in α1 not? Variations in temperature have effects on most cellular events. For example, synaptic vesicle recycling has been shown to be temperature dependent with increased temperature speeding both endo- and exocytosis, and there is evidence that inhibitory synaptic strength can be modulated within 10 min through recruitment of more functional GABAA receptors to the postsynaptic plasma membrane (Wan et al., 1997). Studies on cultured hippocamplal neurons showed that while trafficking of wild-type α1β2γ2 receptors is slightly temperature dependent with a small decrease in surface expression after incubation at 40°C for 2h, trafficking of receptors with mutations in the γ2 subunit is highly temperature dependent (Kang et al., 2006). Increases in temperature from 37°C to 40°C impaired trafficking and/or accelerated endocytosis of the mutant receptors within 10 min, suggesting that the febrile seizures may be a result of a temperature-induced reduction in GABA-mediated inhibition. The study also showed that the A322D mutation in the α1 subunit did not cause a temperature-dependent reduction in surface expression, consistent with a resulting epilepsy

The Kv7 family of voltage-gated potassium channels consists of five members, Kv7.1 -5 (also termed KCNQ1-5). All five members share the general structure of voltage-gated potassium channels with four subunits that assemble to form functional tetramers. Each subunit consists of six transmembrane helices, S1-S6, and has a pore forming domain, which is formed by a P-loop between the fifth and the sixth helix. The P-loop contains the GYG (glycine-tyrosine-glycine) sequence, which is highly conserved among potassium channels and confers K+ selectivity. The fourth helix forms the voltage sensor; it contains several arginine residues and is therefore strongly positive. A S4-S5 linker in one subunit couples the voltage sensor to the intracellular activation gate in S6 of the adjacent subunit (Laine et al., 2003). Although heavily debated, it is believed that when the membrane potential depolarize, the voltage sensor is pushed out leading to bending of the S6 so potassium can

All Kv7 channels are strongly inhibited upon activation of muscarinic receptors and are hence called M-channels (Schroeder et al., 2000; Selyanko et al., 2000). The current conducted by these channels, the M-current, was first described in bullfrog sympathetic ganglia as a slowly activating, slowly deactivating, sub-threshold voltage-dependent K+ current that showed no inactivation (Brown and Adams, 1980). The Kv7 channels have slow activation and deactivation kinetics, and in line with other voltage-gated potassium channels they open upon membrane depolarization. However, the threshold for activation is low compared to most other channels, approximately -60 mV. Since the channels open at voltages that are around or below the threshold for generation of an action potential they allow potassium flow that opposes the depolarization required to generate action potentials, and hence make the neuron less excitable. If the M-channels remain open during excitation of the nerve, the spike frequency is dampened, whiles inhibition of the M-current by

activation of muscarinic acetylcholine receptors enables repetitive firing (Hille, 2001).

syndrome not associated with febrile seizures (Kang et al., 2006).

**5. Mutations in potassium channel genes** 

**5.1 Kv7 channels and the M-current** 

enter the channel pore (Long et al., 2005).

Kv7 channels are primarily localized at the axon initial segment, the site where synaptic inputs are integrated and action potentials are generated (Pan et al., 2006; Rasmussen et al., 2007). Additionally, immunohistochemical studies have demonstrated a widespread presynaptic distribution of some Kv7 channel subunits (Cooper et al., 2000), where they may play a role in depolarization-induced neurotransmitter release (Martire et al., 2004; Martire et al., 2007). Activation of pre-synaptic M-current may hyperpolarize the nerve endings, thus reducing Ca2+ influx through voltage-gated Ca2+ channels and limiting the amount of neurotransmitter released.

#### **5.2 Benign neonatal familial convulsions**

Benign Neonatal Familial Convulsions (BNFC) is a rare autosomal dominant idiopathic form of epilepsy. It is characterized by tonic-clonic seizures that typically begin around three days after birth and remit after 3-4 months. Yet ~16% of patients also experience seizures later in life (Ronen et al., 1993). BNFC is caused by mutations in the genes encoding Kv7.2 (Biervert et al., 1998; Singh et al., 1998) or Kv7.3 (Charlier et al., 1998).

#### **5.3 How mutations in Kv7.2 and Kv7.3 can cause BNFC**

All characterized mutations in Kv7.2 and Kv7.3 cause a reduction in M-current, either by changing the channel kinetics (Dedek et al., 2001), by altering the trafficking of the channel to the cell membrane (Schwake et al., 2000), or by decreasing the subunit stability (Soldovieri et al., 2006). The mutations usually cause a reduction in M-current of about 25% (Schroeder et al., 1998). Considering the role of Kv7 channels in controlling neuronal excitability, it is not surprising that a reduction in M-current can cause hyperexcitability and predispose to seizures. Several transgenic strategies have been employed to examine how Kv7.2 and Kv7.3 malfunction lead to BNFC. The traditional knock-out approach resulted in mice that died within a few hours after birth due to pulmonary atelectasis (collapse of the lung) (Watanabe et al., 2000). Even though BNFC often results from Kv7.2 haploinsufficiency, heterozygous KCNQ2+/- mice did not experience neonatal seizures and appeared normal. They did, however, have increased susceptibility to chemically induced seizures (Watanabe et al., 2000). To overcome the postnatal lethality Dirk Isbrandt and colleagues developed mice conditionally expressing dominant-negative Kv7.2 subunits (Kv7.2-G279S) where expression of the transgene could be turned on after birth (Peters et al., 2005). These mice showed spontaneous seizures, exhibited behavioural hyperactivity and had morphological changes in the hippocampus. Mark Leppert and co-workers developed orthologous mouse models carrying disease-causing mutations in the *KCNQ2* (A306T) or *KCNQ3* (G311V) gene (Singh et al., 2008). Mice heterozygous or homozygous for either mutation had reduced seizure threshold, but only mice homozygous for the mutations exhibited spontaneous seizures. The epileptic phenotype was dependent on the specific mutation, the genetic background, sex, and seizure model (Otto et al., 2009). Hence, none of these transgenic strategies have fully recapitulated the human condition but nevertheless provide us with important clues regarding the pathophysiology of BNFC.

It can be questioned why the disease usually only is clinically manifested in neonates when mutations in the channels cause general hyperexcitability. One possible explanation for the age-dependent remission of seizures is related to the expression of Kv7 channels. There is evidence for a developmental upregulation of Kv7 channels (Weber et al., 2006), suggesting that a reduction in M-current of 25% might have a more prominent effect in the fetal brain

Epileptic Channelopathies and Dysfunctional

composition (Dani and Bertrand, 2007).

al., 2002).

**6.2 Autosomal Dominant Nocturnal Frontal Lobe Epilepsy** 

most patients the seizures tend to peter out in adulthood.

**6.3 How mutations in nAChRs can cause ADNFLE** 

to play a role in the generation of these sleep spindles.

Excitability - From Gene Mutations to Novel Treatments 201

in the brain have been identified (α2-α10 and β2-β4). The most widely distributed nAChRs in the human brain are the homomeric α7 and the heteromeric α4β2. The biochemical, pharmacological and biophysical characteristic of the channels are dependent on the subunit

Autosomal Dominant Nocturnal Frontal Lobe Epilepsy (ADNFLE) is a focal epilepsy characterized by clusters of brief nocturnal motor seizures with hyperkinetic or tonic manifestations. Seizures typically occur very soon after falling asleep or during the early morning hours, initiated during stage 2 non-rapid-eye-movement (NREM) sleep. Onset usually occurs around age 10 and the seizures often persist through adult life. However, in

In 1995, a missense mutation (S248F) in the nAChR α4 subunit gene (CHRNA4) was found to underlie ADNFLE (Steinlein et al., 1995). Additional disease causing mutations in this gene (Hirose et al., 1999; Steinlein et al., 1997), and mutations in the β2 subunit gene (CHRNB2) (Phillips et al., 2001) have later been identified. Because α4 and β2 subunits combine, it is not strange that mutations in either subunit produce comparable epileptic symptoms. Almost every known mutation is found in the TM2 region of the two subunits. A common finding among the mutations is that the sensitivity to ACh is increased (Bertrand et

Stage 2 NREM sleep is characterized by the appearance of sleep spindles and slow waves, transient physiological rhythmic oscillations that are produced by synchronized synaptic potentials in cortical neurons. It appears that the seizure often arise from a sleep spindle that transforms into epileptic discharges (Picard et al., 2007). Thalamocortical circuits are thought

Thalamic relay neurons are reciprocally connected to cortical neurons by excitatory synapses. Both thalamic and cortical neurons also excite GABAergic interneurons of the nucleus reticularis, which in turn inhibit thalamic relay neurons thus forming a feedbackloop (Kandel et al., 2000). The thalamic relay neurons have two different modes of signalling activity: a transmission mode during wakefulness and rapid eye movement (REM) sleep and a burst mode during NREM sleep. In the transmission mode, the resting membrane potential of the thalamic relay neurons is near the firing threshold and incoming excitatory synaptic potentials drive the neuron to fire in a pattern that reflects the sensory stimuli. During the burst mode the thalamic relay neurons are hyperpolarized and respond to brief depolarization with a burst of action potentials, which indicates that the thalamus is unable to relay sensory information to the cortex (Kandel et al., 2000). The thalamic relay neurons can be in different modes because they possess special Ca2+ channels named T-type channels. These channels are inactivated at the resting membrane potential but become available for activation when the cell is hyperpolarized, and can then be transiently opened by depolarization (Contreras, 2006). The interneurons of the nucleus reticularis that form synapses at the relay neurons hyperpolarize the relay neurons upon activation of GABAA receptors, thus removing the inactivation of the T-type Ca2+ channels. Incoming excitatory synaptic potentials can then trigger transient opening of the T-type Ca2+ channels and the

and that this reduction is not sufficient to cause seizures later in life when expression levels of Kv7 channels are higher.

Another possible mechanism is related to developmental changes in GABA function. During the first weeks of life, GABA, the main inhibitory neurotransmitter in the adult brain, provides the main excitatory drive to immature hippocampal neurons (Ben-Ari, 2002). Due to delayed expression of a chloride exporter there is a high intracellular concentration of chloride that leads to a negative shift in the reversal potential for chloride ions, so opening of ionotropic GABA receptors leads to an efflux of negative chloride ions and therefore depolarization. When the chloride-extruding system becomes operative, chloride is efficiently transported out of the cell, and GABA begins to exert its conventional inhibitory action (Ben-Ari, 2002). Because of this, the inhibition in neonatal circuits appears mainly to be mediated through presynaptic control of neurotransmitter release. As Kv7 channels are involved in the release of neurotransmitters (Martire et al., 2004; Martire et al., 2007), it was proposed that these channels serve as the main inhibitor in neonates, and that attenuation of M-current due to mutations in Kv7.2 and Kv7.3 causes reduced inhibition that is sufficient to cause epilepsy (Peters et al., 2005). If Kv7 channels are expressed in GABAergic neurons, the reduced M-current would possibly also cause increased GABA release which further would increase excitability at this point of development. It is also important to note that the neonatal brain is particularly prone to seizures (Holmes and Ben-Ari, 1998). As development continues and overall excitability decreases, reduced M-current apparently becomes less problematic and the seizures abate.

Yet, if this is the fact, why do ∼16% of the patients experience seizures later in life after GABA has gained its inhibitory function? There is evidence to indicate that suppression of M-current within the first postnatal week can cause developmental defects, and that the resulting morphological changes in the brain, rather than reduced M-current causes the seizures in adulthood (Peters et al., 2005). In other words, it appears to become a symptomatic epilepsy with an idiopathic aetiology.

#### **6. Mutations in nAChR subunit genes**

#### **6.1 Nicotinic acetylcholine receptors**

Nicotinic acetylcholine receptors (nAChRs) consist of five subunits that assemble to form functional pentamers. Each subunit consists of a long extracellular N-terminal domain, four transmembrane helices (TM1-TM4), and a short extracellular C-terminal end. The second transmembrane segment (TM2) from each subunit lines the channel pore. The amino acids that compose the TM2 are arranged in such a way that three rings of negatively charged amino acids are oriented toward the central pore of the channel. These provide a selectivity filter that ensures that only cations can pass through the pore. Brief exposure to high concentrations of Ach causes opening of the water-filled pore and permits an influx of Na+ and Ca2+ and an efflux of K+ (Waxham, 2003). After a few milliseconds, the receptor closes to a nonconducting state. Prolonged exposure to agonist causes desensitization of the channel, which stabilizes the receptor in an unresponsive, closed state (Dani and Bertrand, 2007).

The neuronal nAChRs can be either homomeric, consisting of five α subunits, or heteromeric with two α subunits and three β subunits. So far, 12 nAChR subunits expressed

and that this reduction is not sufficient to cause seizures later in life when expression levels

Another possible mechanism is related to developmental changes in GABA function. During the first weeks of life, GABA, the main inhibitory neurotransmitter in the adult brain, provides the main excitatory drive to immature hippocampal neurons (Ben-Ari, 2002). Due to delayed expression of a chloride exporter there is a high intracellular concentration of chloride that leads to a negative shift in the reversal potential for chloride ions, so opening of ionotropic GABA receptors leads to an efflux of negative chloride ions and therefore depolarization. When the chloride-extruding system becomes operative, chloride is efficiently transported out of the cell, and GABA begins to exert its conventional inhibitory action (Ben-Ari, 2002). Because of this, the inhibition in neonatal circuits appears mainly to be mediated through presynaptic control of neurotransmitter release. As Kv7 channels are involved in the release of neurotransmitters (Martire et al., 2004; Martire et al., 2007), it was proposed that these channels serve as the main inhibitor in neonates, and that attenuation of M-current due to mutations in Kv7.2 and Kv7.3 causes reduced inhibition that is sufficient to cause epilepsy (Peters et al., 2005). If Kv7 channels are expressed in GABAergic neurons, the reduced M-current would possibly also cause increased GABA release which further would increase excitability at this point of development. It is also important to note that the neonatal brain is particularly prone to seizures (Holmes and Ben-Ari, 1998). As development continues and overall excitability decreases, reduced M-current apparently becomes less

Yet, if this is the fact, why do ∼16% of the patients experience seizures later in life after GABA has gained its inhibitory function? There is evidence to indicate that suppression of M-current within the first postnatal week can cause developmental defects, and that the resulting morphological changes in the brain, rather than reduced M-current causes the seizures in adulthood (Peters et al., 2005). In other words, it appears to become a

Nicotinic acetylcholine receptors (nAChRs) consist of five subunits that assemble to form functional pentamers. Each subunit consists of a long extracellular N-terminal domain, four transmembrane helices (TM1-TM4), and a short extracellular C-terminal end. The second transmembrane segment (TM2) from each subunit lines the channel pore. The amino acids that compose the TM2 are arranged in such a way that three rings of negatively charged amino acids are oriented toward the central pore of the channel. These provide a selectivity filter that ensures that only cations can pass through the pore. Brief exposure to high concentrations of Ach causes opening of the water-filled pore and permits an influx of Na+ and Ca2+ and an efflux of K+ (Waxham, 2003). After a few milliseconds, the receptor closes to a nonconducting state. Prolonged exposure to agonist causes desensitization of the channel, which stabilizes the receptor in an unresponsive,

The neuronal nAChRs can be either homomeric, consisting of five α subunits, or heteromeric with two α subunits and three β subunits. So far, 12 nAChR subunits expressed

of Kv7 channels are higher.

problematic and the seizures abate.

symptomatic epilepsy with an idiopathic aetiology.

**6. Mutations in nAChR subunit genes** 

**6.1 Nicotinic acetylcholine receptors** 

closed state (Dani and Bertrand, 2007).

in the brain have been identified (α2-α10 and β2-β4). The most widely distributed nAChRs in the human brain are the homomeric α7 and the heteromeric α4β2. The biochemical, pharmacological and biophysical characteristic of the channels are dependent on the subunit composition (Dani and Bertrand, 2007).
