Ion Channel Mutations in Neuronal Diseases - American Chemical

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Ion Channel Mutations in Neuronal Diseases: A Genetics Perspective Ortrud K. Steinlein* Institute of Human Genetics, University Hospital, Ludwig-Maximilians-University, Goethestr. 29, D-80336 Munich, Germany 5.1.1. Phenotype−SCN1A Genotype Correlations 5.1.2. GEFS+ and SCN1A Mutations: Contradictory Results from Functional Studies 6. Neurons and Voltage-Dependent Calcium Channels 6.1. CACNA1A Causes More than One Type of Ataxia 7. Migraine: A Multichannelopathy? 8. Startle Disease: Glycine Receptors 9. Acquired Channelopathies 9.1. Channelopathies Caused by Autoantibodies 9.2. Transcriptional Channelopathies 10. Epilepsy: Are Ion Channels the Whole Story? 11. Gene Therapy Approaches for Channelopathies: Too Early? 12. Conclusions and Future Perspectives Author Information Corresponding Author Notes Biography Acknowledgments References

CONTENTS 1. Introduction 1.1. Ion Channels: Main and Accessory Subunits Have Different Roles 1.2. Importance of Ion Channels for Brain Function 1.3. Ion Channels and Nonexcitable Brain and Spinal Cord Cells 2. Nicotinic Acetylcholine Receptor Family 2.1. Acetylcholine Receptors in Multifactorial Disorders 2.1.1. Acetylcholine Receptor Agonists: New Therapeutic Targets in Parkinson Disease? 2.1.2. Association between CHRNA7 and Sensory Gating Defects in Schizophrenia 2.1.3. Acetylcholine Receptors in Attention Deficit Hyperactivity Disorder 2.1.4. Acetylcholine Receptors, Smoking, and Cancer 2.2. Monogenic Disorders Linked to Acetylcholine Receptor Subunit Genes 2.2.1. Autosomal Dominant Nocturnal Frontal Lobe Epilepsy 3. Potassium Channel Dysfunction and Brain Excitability 3.1. Benign Familial Neonatal Convulsions: An M-current Defect 3.2. Potassium Channel Gene Variants May Act As Epilepsy Modifiers 4. GABAA Receptors: The Brain Pacifier 4.1. Rare Familial Epilepsies Caused by GABAA Mutations 4.2. Autism, Epilepsy, and GABAA Receptors 5. Voltage-Gated Sodium Channels and the GEFS+ Spectrum 5.1. Many Faces of SCN1A © 2012 American Chemical Society

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1. INTRODUCTION For every word we hear, every joke we tell, or every thought we have, an enormous number of ion channels has to become busy in our brain. The opening and closing of their gated pores controls the ion movement through the cell membrane, which otherwise would provide an impenetrable barrier for them. Many ion channels contain a central pore formed by 2−6 transmembrane domains. The latter are arranged around the water-filled pore like the staves of a barrel. These transmembrane domains might be encoded by a single large ion channel gene such as the α subunits of the sodium channel family or composed of four or five subunits that combine to build a homomeric or heteromeric ion channel. The latter types of subunits often belong to multimember subunit families and can combine in a great many variations. Such multisubunit families are therefore able to significantly increase ion channel diversity with respect to both function and location. Functional diversity within the superfamily of ion channels is also created by the individuality of their gates. There are gates that open an ion channel pore when triggered by changes in electricity (voltage-gated channels). Other gates specifically react to chemical or mechanical signals such as pH, redox state,

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dx.doi.org/10.1021/cr300044d | Chem. Rev. 2012, 112, 6334−6352

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ation, phagocytosis, secretions of multiple cytokines or chemokines, and promotion of repair. They are called into action by cell-surface molecules that act as a kind of sensor to detect changes in the microglia environment. Apart from different kinds of cell-surface receptors and adhesion molecules, these sensors also include multiple ion channels from the chloride, potassium, proton, and calcium subfamilies. In excitable cells such as neurons, these channels are mainly responsible for the generation and control of action potentials. Nonexcitable cells such as microglia are obviously using these ion channel genes to help them exercise their role as immune cells. The exact mechanisms by which ion channels are doing this are not yet known but seem to include changes of intracellular ion homeostasis that activate or inactivate different signaling pathways.7 Migration to the site of tissue injury or inflammation, increased production of cytokines, chemokines, and other bioactive molecules, and phagocytosis are the main responses by which microglia reacts to perturbations such as injury or infection. The blockade of sodium channels by phenytoin or TTX significantly attenuated these response mechanisms, while exposure to hypoxia, ATP, or LPS treatment increased the expression of certain potassium channels in postnatal rats, an observation that was accompanied by an increased expression of cytokines such as IL-1β and TNF-α.8 These results indicate that ion channels play an important role in the activation and function of microglia.

osmolarity, or mechanical stretch. Another class of ion channel gates answers to specific ligands including glutamate, acetylcholine, γ-aminobutyric acid, or ATP.1−3 Despite their functional differences, all members of the vast family of ion channels have in common that they are able to rapidly and accurately transmit information among cells and to coordinate distant functions. 1.1. Ion Channels: Main and Accessory Subunits Have Different Roles

The principal α subunits by themselves are able to build an ion channel that contains the main functional elements needed for ion conductance, pore gating, and regulation. These α subunits contribute the transmembrane domains that build the walls of the central pore, as well as the means by which an ion channel is activated or inactivated. The latter could be voltage sensors, which are stretches of positively charged amino acids that detect changes in the membrane potential and transfer these to the pore. Other main α subunits form three-dimensional structures that provide binding sites for ligands such as acetylcholine and glutamate, or chemical signals including ATP and calcium. However, ion channel subunit families often consist not only of several pore-forming main α subunits but also of one or more accessory or β subunits. Most accessory subunits are membrane-bound proteins that possess a single transmembrane domain (compared to 4−24 transmembrane domains owned by a typical main α subunit). Formerly named auxiliary, these accessory subunits are now increasingly recognized as important modulators of ion channel function. The accessory subunits interact with specific main subunits, altering their physiological properties and subcellular localization. They have been shown to modify gating and kinetics of main subunits in a cell type-specific and channel subtype-specific manner. The accessory subunits can be truly astonishingly versatile little proteins combining conducting with nonconducting functions. A good example for this are the accessory subunits of the voltage-gated sodium channels. These accessory subunits not only modulate sodium current kinetics but are also members of the immunoglobulin superfamily of cell-adhesion molecules. In the latter role they participate in cell adhesion-related activities such as cellular aggregation and the establishment of cell−cell contacts.4−6

2. NICOTINIC ACETYLCHOLINE RECEPTOR FAMILY The nicotinic acetylcholine receptors (nAChRs) can be divided into muscular and neuronal types. Muscular nAChRs exist in only two different subtypes, a fetal one that is expressed during prenatal life and is being downregulated around birth to give place to the adult subtype. Both the fetal and the adult muscular nAChRs differ with respect to only one subunit, γ and ε, respectively. The neuronal nAChRs, however, represent a superfamily of ligand-gated ion channels with an unknown number of subtypes. Up to 12 genes (CHRNA2-CHRNA10, CHRNB2-CHRNB4) code for neuronal nAChR subunits (α2−α10 (α8 has only been found in avian), β2−β4) that are expressed in neuronal and (contrary to their name) nonneuronal tissues. Heteromeric combinations of lower-number α subunits and β subunits (α2−α6, β2−β4) assemble to build bungarotoxin-insensitive receptors. The higher-number α subunits form either homomeric (α7, α8, and α9) or heteropentameric (for example, α7α8, α9α10) α-bungarotoxin-sensitive receptors. The two subclasses of neuronal nAChRs differ with respect to the number of their ligand-binding sites with five binding sites (one on each subunit interface) present on α-subunit-only receptors and two binding sites on nAChRs built from a combination of α and β subunits. The later nAChRs contribute mainly to the marked diversity of nAChRs by building either more simple subtypes such as α2β2, α2β4, α3β2, α3β4, α6β2, and α6β4 or more complex subtypes that contain three different subunits (for example, α4α6β2, α3α4β2, α3α4β4, and α2α4β2) (Figure 1). It is not known how many of the more than 2000 possible combinations of nAChR subtypes are indeed expressed in vivo. All subtypes that have been characterized in greater detail so far showed highly individual profiles with respect to expression pattern and functional and pharmacological profiles. Some subtypes are ubiquitously expressed in brain (for example, the α4β2 nAChR) while others are found only in rather restricted areas in the brain or certain non-neuronal tissues. Not only the

1.2. Importance of Ion Channels for Brain Function

There is barely an ion channel family that does not have at least several members that are expressed in the brain, often exclusively. This is not surprising because in higher organisms the central nervous system constitutes the one organ with the highest number of excitable cells. Ion channels are involved both in rare monogenic neurological disorders and in common multifactorial traits. The monogenic disorders caused by ion channel mutations can be roughly divided into three groups: paroxysmal movement disorders, epilepsies, and headache disorders. 1.3. Ion Channels and Nonexcitable Brain and Spinal Cord Cells

Within the central nervous system microglia cells act as immune cells that respond to injury or other perturbations of brain and spinal cord with an array of weapons. Microglia cells monitor the state of health of the central nervous system and show significant activation in neurodegenerative disorders including Morbus Alzheimer or Parkinson’s disease. Their arsenal includes diverse function such as migration, prolifer6335


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unknown in the majority of patients with Parkinson disease. Twin studies and family observations strongly suggest that Parkinson disease is mostly a multifactorial disorder. A combination of environmental factors and genetic risk variants are likely to determine individual disease susceptibility. Genetic variants in nAChR genes seem to be perfect candidates for such genetic risk factors; thus, it is a little bit surprising that they have not played a visible role in reported associations yet. Nonetheless, they are sought out as important targets for prospective therapies.11,12 The protecting role of nicotine has been demonstrated by epidemiological studies that reported reduced incidences of Parkinson disease in heavy smokers. Furthermore, studies in primates showed that oral nicotine reduces the neuronal loss in the nigrostriatal dopaminergic pathway observed in chemically induced Parkinson disease. Nicotine enhances striatal dopamine release and prevents toxin-induced degeneration of dopaminergic neurons.12 However, attempts to use these results in humans have not been very successful so far. Nicotine therapies provided inconsistent results in the treatment of Parkinson patients.13,14 Reason for this could be that application by nicotine patches did not result in optimal nicotine concentrations in the brain or that nicotine needs to be applied before the neuronal loss has caused the first clinical symptoms. More promising than nicotine patches seem to be newer approaches that try to develop drugs that specifically target nAChRs containing subunits that are likely to be involved in the pathogenesis of Parkinson disease. Probably the most important of these nAChR subunits is α6. Receptors containing this subunit exhibit a very restricted localization in brain. They are present in the mesolimbic and nigrostriatal dopaminergic pathways as well as in structures linked to the visual system such as the superior colliculus, the optic tract, or the retina.15 Dopamine release in both the nucleus accumbens and striatum is regulated by both α4β2 and α6β2β3 or α6α4β2β3 nAChRs. The α4β2 receptor subtype shows a broad expression pattern in the brain, but α6-containing nAChRs with their restricted expression pattern seem to be the perfect targets for antiParkinson drugs. Drugs targeting these receptors can even be developed to discriminate between early and late stages of the disorder, because loss of the α6α4β2β3 nAChR subtype is already present in disease stages with mild to moderate nigrostriatal damage, whereas the amount of α6β2β3 subtype only decreases with more severe cell loss. Still the problem remains that the irreversible neuronal loss precedes the clinical manifestations. Therapies are therefore most effective when they are started early, but no tests exist so far that are able to identify the early subclinical stages of Parkinson disease. However, experiments in rodents demonstrated that even a 50% decline of α6-containing receptors can be compensated by increases in function.16 This could mean that subtype-selective agonists might be beneficial even if the nigrostriatal damage is already in an advanced stage. 2.1.2. Association between CHRNA7 and Sensory Gating Defects in Schizophrenia. Patients with schizophrenia have difficulties in filtering sensory stimuli, a deficit that has been hypothesized to contribute to their characteristic hallucinations and delusions.17,18 The observation that background noises, which most other people can ignore, might trigger a hallucination suggests a failure in elementary inhibitory processes. In healthy people the brain uses such mechanisms to regulate the amount of sensory stimuli that it constantly has to

Figure 1. Schematic presentation of different nAChR subunit classes. Arrows indicate which subunits combine to form heteropentameric receptors.

physiological but also the pathophysiological implications of the nAChRs broad diversity are currently poorly understood. The nAChRs mediate excitatory cholinergic neurotransmission and, perhaps more important, modulate the neurotransmission by other chemical messengers, including GABA, glutamate, dopamine, norepinephrine, and serotonin. In the brain they can be located on soma, dendrites, or nerve terminals and participate in diverse functions including reward, learning and memory, mood, sensory processing, pain, and neuroprotection. 2.1. Acetylcholine Receptors in Multifactorial Disorders

The cholinergic system has a key role in various functional and pathofunctional processes. It is therefore not surprising that cholinergic dysfunctions are found in many neurological disorders including Morbus Alzheimer, Parkinson disease, and hyperactivity disorders. Furthermore, the cholinergic system has been implicated in addictive behavior and cancer but also in cognitive decline during normal aging. Mostly these disorders and traits are caused by a combination of genetic susceptibility factors and unfavorable environmental factors rather than by single gene mutations. Some of the multifactorial disorders linked to specific acetylcholine receptor subunits are discussed here. 2.1.1. Acetylcholine Receptor Agonists: New Therapeutic Targets in Parkinson Disease? Parkinson disease is a movement disorder characterized by rigidity, tremor, and bradykinesia. Many patients develop dysphagia (86%), urinary incontinence (57%), and dementia (43%).9 The degeneration of the nigrostriatal dopaminergic pathway, accompanied by the loss of its dopamine-producing and dopamine-releasing capacity, is one of the major features in patients with this debiliating disorder. Both the dopaminergic and cholinergic systems overlap in this pathway, regulating important functions linked to motor activity. It is therefore not surprising that both transmitter systems are involved in the pathogenesis of Parkinson disease. In a small fraction of Parkinson patients, the disorder is caused by “classical” mutations in genes such as parkin, alpha-synuclein, PTEN-induced kinase 1, DJ-1, ubiquitin carboxyl-terminal esterase L1, and leucine-rich repeat kinase 2.10 However, the cause of the disorder remains 6336


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essential for the pathology of ADHD and that catecholamines such as dopamine and norepinephrine are important modulators of this disorder.25,26 There is also emerging evidence that cholinergic receptors contribute to the pathophysiology of ADHD.27 Indirect evidence comes from studies that show that ADHD patients tend to start smoking earlier and heavier, and that the nicotine abusus correlates with the severity of their symptoms. 28,29 As assumed for schizophrenic patients, smoking might present a form of selfmedication for ADHD patients, too. This hypothesis is supported by several short-time/small-sample studies that found that nicotine demonstrates positive neuropsychological effects in ADHD patients including a modest reduction of symptoms and a reduction in reaction time. On the other hand, maternal smoking in pregnancy has been shown to increase the risk for ADHD in the offspring, an effect that is independent from maternal ADHD.30,31 At a first glance these observations that nicotine can either improve or cause ADHD seem to contradict each other. However, this paradox is only apparent because nicotine can have effects on both neuronal plasticity and excitability. Smoking during pregnancy exposes the developing brain of the embryo to nicotine, which is suspected to cause permanent changes in synaptic connectivity.32,33 In adolescents and adults with ADHD, nicotine exhibits a more direct and short-lived effect by stimulating different neurotransmitter systems that are activated by nAChRs. Nicotine itself has serious gastrointestinal and cardiovascular side effects that render it unsuitable as a therapeutic option in ADHD. More promising would be neuronal nAChR agonists that mimic the nicotine effect with a minimum of side effects. The existing agonists only partially fulfill these criteria. The agonist ABT-418 was the first that was evaluated in ADHD. It acts as a full agonist against α4β2 nAChRs, at least in vitro. In a clinical trial ABT-418 significantly reduced inattentive symptoms, especially those with regard to organization and planning. It was less effective against impulsivity and hyperactivity, two of the socially less acceptable symptoms of ADHD. It also had some nicotine-like side effects, such as dizziness and nausea.34 Better adapted to the needs of patients seems to be ABT-089, the next nAChR agonist that was developed and tested clinically. A double-blind, randomized multicenter study showed that ABT-089 was generally well-tolerated in adults with ADHD. Four weeks of trial resulted in significant improvement in ADHD symptomatology and symptom severity.35,36 However, as promising as these data are, they do not prove yet that ABT-089 is indeed suited for the treatment of ADHD. Included in the study were only ADHD patients without any comorbidities. Such comorbidities are very common in a “normal” sample of ADHD patients and include psychiatric diagnoses such as bipolar disorder, major depressive disorder, anxiety disorders, and substance use disorders. There is no possibility to predict the effect nAChR agonists might have with regard to these comorbidities. Furthermore, the possibility of interactions between such drugs and nicotine seems to be very real, and this is a prospective patient sample with a high rate of smokers. These questions need to be addressed before it can be decided if agonists like ABT-089 are suited for treatment. 2.1.4. Acetylcholine Receptors, Smoking, and Cancer. Chronic nicotine exposure has long been known to upregulate receptor subtypes such as α4β2 in brain. There are also several newer lines of evidence that link acetylcholine receptors to nicotine dependence. By animal studies several nAChR

process. In the scientific literature there are several observations reported that can be regarded as evidence that the α7 nAChR subunit plays an important role in the failure of sensory stimuli regulation in schizophrenics. As a first and most obvious evidence, the fact is often cited that at 80% the rate of smokers (and especially of heavy smokers) in schizophrenics is considerably higher than in any other population subgroup. However, newer association studies suggest that other nAChR subtypes (i.e., α4, α5, β2, β3) are more important for smoking than α7 receptors. More convincing of a role of α7 in schizophrenia are post-mortem studies that showed diminished labeling of putative inhibitory neurons by α-bungarotoxin in the hippocampus and thalamus of patients. The role of α7 nAChR subunits in schizophrenia is further supported by the observation that the experimental drug GTS21 improves auditory gating in mice. GTS-21 is a compound derived from anabaseine, an alkaloid found in marine worms, and acts as a partial agonist at α7 nAChRs. However, it is also a weak competitive agonist of α4ß2 nAChRs; thus, these two effects might overlap. The most interesting evidence is still presented by the study published by Leonard et al. in 200219 in which they describe a statistically greater prevalence of certain functional alleles of α7 promoter variants in schizophrenic subjects than in controls. Studies demonstrated that the presence of these α7 promoter polymorphism alleles is associated with the failure to inhibit the P50 auditory-evoked potential response.19−21 In these studies scalp electrodes were used to record waves with a 50-ms latency (P50) following paired auditory stimuli delivered 0.5 s apart. In healthy subjects the amplitude of the second response is decreased through inhibitory neuronal pathways, most likely to restore neuronal excitability after a nerve impulse. In more than 85% of schizophrenic patients, this restoration is significantly reduced, suggesting a defect of auditory sensory gating. Interestingly, this defect seems to be partially repaired by high doses of nicotine, which led to the hypothesis that the heavy smoking observed in many schizophrenic patients could be a form of self-medication. This would fit the observation that the symptoms of schizophrenia can recur when patients temporarily stop smoking.22 It should, however, be noted that there is no direct experimental evidence showing that the effect nicotine has on P50 is indeed mediated by the α7 subunit instead of, for example, by one of the other nAChR subunits. During evolution, CHRNA7 has been partially duplicated, and this duplication is part of the chimeric gene CHRFAM7A that has been shown to regulate CHRNA7 expression in humans. CHRFAM7A exists in two orientations within human population, inversion polymorphism and a 2-bp deletion polymorphism in exon 6 of CHRFAM7A, which has been shown to be in strong linkage disequilibrium with this inversion polymorphism. Interestingly, this 2-bp polymorphism is associated with endophenotypes of schizophrenia, suggesting a functional role of CHRFAM7A in cognitive impairment. 2.1.3. Acetylcholine Receptors in Attention Deficit Hyperactivity Disorder. The most prominent symptoms of attention deficit hyperactivity disorder (ADHD) are impulsivity and hyperactivity. Additional symptoms are cognitive deficits, particularly impairments in attention and executive function that are found in many ADHD patients. Follow-up studies have shown that both hyperactivity and impulsive symptoms decline over time, but that the cognitive deficits are persistent.23,24 Neuropsychological and neuroimaging studies indicate that abnormalities in frontal and fronto-striatal networks are 6337


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subunits (α5, α7, β4) have been implicated in the physical symptoms of withdrawal. For example, CHRNB2 knockout mice displayed loss of anxiety-related behavior and aversion, suggesting that β2-containing nAChRs are involved in the affective signs of nicotine withdrawal. Non-β2-containing nAChRs seem to be more closely associated with physical signs of nicotine withdrawal because CHRNA7 knockout mice showed a loss of nicotine withdrawal-induced hyperalgesia and a reduction in somatic signs but normal nicotine tolerance.37,38 Several genome-wide association studies presented strong evidence that certain genetic variants within the CHRNA3/A5/ B4 gene cluster on chromosome 15 affect the risk for nicotine dependence. These risks are mainly associated with two nonsynonymous nucleotide polymorphisms, rs16969968 in CHRNA5 and rs1051730 in CHRNA3.39−41 The nAChR encoding gene cluster on chromosome 15 has been implicated not only in nicotine dependence but also in lung cancer susceptibility. There is evidence that this is not always simply an indirect effect related to the carcinogenic effect of smoking but a direct association between the nAChR subunit genes CHRNA3/A5/B4 and lung cancer. In 2008 a large association study found an association of genetic variants in these genes, especially rs16969968, with lung cancer. This association was present irrespective of smoking status or smoking quantity. These results have been confirmed in two independent studies that described not only an association between CHRNA3/A5/ B4 variants and lung cancer phenotypes but also found an association with an earlier age at lung cancer onset.42,43 The primary role of nAChR genes in lung cancer is further supported by the observation that the CHRNA3 gene frequently exhibits DNA hypermethylation in lung cancer.44 The reasons behind this selective hypermethylation and the functional consequences of the subsequent silencing of CHRNA3 are not yet known. One possibility would be that the silencing of CHRNA3 affects cell migration and apoptosis. Such a role in airway epithelial cells has already been shown for another nAChR subunit, α7. This subunit is expressed by basal progenitor cells involved in the regeneration of the airway epithelium. It promotes basal cell differentiation by restricting cell growth and seems also to have been involved in the regulation of inflammation and immunity.45 The α7 subunit might therefore promote lung cancer in more than one way. Prolonged exposure to nicotine in heavy smokers changes the activation−desensitization pattern of α7 nAChRs, an effect that might promote the development of cell metaplasia. Such effects have been observed in vivo after inactivation of α7 in epithelial cells.45 Second, α7 nAChRs are involved in the regulation of inflammation and immune process molecules such as tumor necrosis factor, interferon-gamma and interleukin-6, and antibody IgG1.46,47 Suppression of the immune response by nicotine may affect the clearance of transformed cells and support the growth of neoplastic lesions.48−50 Another nAChR subunit linked to cancer, especially to breast cancer, is α9. This subunit is present in both primary tumors and surrounding nonmalignant breast tissue, but its expression is increased in cancer cells. This fits to the observation that silencing of α9 in cultivated breast cancer cells reduced their proliferation and tumorigenic potential whereas overexpression enhanced proliferation and colony formation. When tumors that expressed increased levels of α9-nAChRs were transferred to wild-type mice, these hosts showed enhanced graft volumes after exposition to nicotine. Taken together these results suggest that acetylcholine receptor subunits such as α9 play an

important role in breast carcinogenesis, a hypothesis that tallies with the observation that smoking increases the risk for breast cancer.51−54 2.2. Monogenic Disorders Linked to Acetylcholine Receptor Subunit Genes

Several different monogenic disorders have already been linked to nAChRs, including congenital myasthenia and multiple pterygia syndrome (Escobar syndrome). Both disorders are caused by mutations in the muscular nAChR subtypes Some of the neuronal nAChR subunit have also been found to cause monogenic disorders, with the most prominent example being autosomal dominant nocturnal frontal lobe epilepsy (ADNFLE), which is due to mutations in at least two different neuronal nAChR subunits. 2.2.1. Autosomal Dominant Nocturnal Frontal Lobe Epilepsy. The majority of seizure disorders either have only a weak genetic background or are due to a combination of several genetic susceptibility factors and environmental factors. This is especially true for the more common types of idiopathic epilepsies such as childhood absence epilepsy or juvenile myoclonic epilepsy (Janz syndrome). Genetic epilepsies such as ADNFLE are exceptional because they are caused by single gene mutations. Unlike most multifactorial disorders, such monogenic traits are usually rare. Monogenic epilepsies are mostly channelopathies, i.e., they are known to be often caused by mutations in genes that code for either voltage-gated or ligand-gated ion channels. So far only a few epilepsy genes have been identified that are nonion channel genes. Most of these genes are of unknown function. ADNFLE is caused by mutations in either CHRNA4 or CHRNB2, the two most ubiquitously expressed nAChR subunit genes. A third gene, CHRNA2, seems to cause an ADNFLE-like phenotype but has not been confirmed in independent studies yet.55 In ADNFLE patients the motor seizures mostly occur at the time of arousal from nonrapid eye movement (non-REM) sleep stage. The seizures are often stereotyped and brief and tend to cluster. They can vary from simple arousals from sleep to bizarre, hyperkinetic events with tonic or dystonic features. Sleepwalking is also a frequently reported symptom in ADNFLE patients. Borderline intelligence or mild deficits in executive and cognitive tasks are not uncommon in ADNFLE patients, and in some families psychiatric disorders or mental retardation are frequently associated with the epilepsy trait.56,57 Comparison of the clinical data shows that the percentage of patients reported to have associated major neurological or psychiatric features is rather low for some mutations but quite high for others. Furthermore, patients with the same mutation tend to have an identical pattern of accessory major neurological or psychiatric features.58 These patients are often from different countries or even continents; thus, these associations cannot be easily explained by a shared genetic background or common environmental factors. A causal relationship between the neurological and psychiatric features and the respective ADNFLE mutation seems to be the most likely explanation. Such a theory would fit well with the wide range of neuronal dysfunctions and psychiatric disorders that nAChRs have been implicated in, although it would not explain why only some ADNFLE mutations seem to increase the risk for these additional neurological and psychiatric symptoms whereas other mutations can be described as rather benign.58 Most of these mutations are clustering within the second or third transmembrane domain of CHRNA4 and CHRNB2; thus, 6338


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epilepsy. The receptor assembled from these subunits is one of the most important regulators of action potentials in the central nervous system (CNS). It provides the molecular basis for the neuronal M current, which ensures that a neuron is not constantly active and sending signals to other neurons. The KCNQ2/KCNQ3 potassium channel controls the subthreshold excitability of neurons and their responsiveness to synaptic input. Mutations in the KCNQ2 and KCNQ3 genes cause benign familial neonatal convulsions (BFNC), a rare epilepsy disorder. Heterologous expression experiments suggest that a reduction of roughly 25% in KCNQ2/KCNQ3 currents is sufficient to increase neuronal excitability to epileptogenic levels in early infancy. Seizures in BFNC patients usually begin in the first few days of life and remit spontaneously at 3−4 months of age. About 15% of individuals with BFNC will experience one or more seizures later in life, often provoked by external factors.67 So far it is unknown why the seizures in BFNC are mostly restricted to the first weeks or months of life. Most BFNC mutations only moderately reduce the potassium current by ∼25%. One possible explanation for the self-limiting nature of the disorder could therefore be that the not-fully-developed brain is sensitive even to slight disturbances. Another explanation could be that other potassium subunits are upregulated within the first months of life, compensating for the KCNQ2/KCNQ3 loss-of-function. However, no candidate gene suitable for such a compensatory mechanism has been identified yet. A mutation (R207W) within the voltage sensor of KCNQ2 was found to cause a clinical phenotype that is characterized by neonatal convulsions, followed by myokymia. The latter symptom is characterized by spontaneous involuntary contraction of muscle fiber groups that can be observed as vermiform movement of the overlying skin. Myokymia had been previously already found to be associated with another potassium channel subunit, encoded by the Shaker-related voltage-gated K+ channel gene Kv1.1 (KCNA1). Comparable to the BFNC−myokymia combination caused by KCNQ2R207W, the KCNA1 mutations too are causing a complex clinical phenotype characterized by myokymia, episodic attacks of cerebellar ataxia, and, sometimes, partial epilepsy.68 Within the voltage sensor of KCNQ2, the mutation R207W substitutes one of the positively charged arginines that directly sense the electric field over the membrane by a bulky hydrophobic residue. Expression studies showed that this amino acid substitution shifts the voltage dependence to more positive voltages and slows depolarization-induced activation. The effect depended on the length of the depolarization period, an observation that might explain why this particular mutation causes both central and peripheral neuronal symptoms. Depolarization periods in motor neurons tend to be shorter and action potentials tend to be fewer than in central neurons. At least in heterologous expression experiments, this caused potassium channel subunits carrying the KCNQ2-R207W mutation to exert a dominant negative effect, leading to a loss of current that was more severe than that seen for other BFNC mutations. This difference in firing patterns between motoneurons and central neurons could explain why this particular KCNQ2 mutant is able to cause myokymia in addition to BFNC.69 In BFNC families the course of the disorder is not always as benign as the name suggests. A pilot study including 10 BFNC families with known KCNQ2 mutations found in 4 (40%) of

the intragenic location of the mutations apparently seems to be no reason for clinical differences. However, expression studies have shown that even mutations that are only separated by a few amino acids can significantly differ from each other with respect to the changes in biopharmacological properties they induce in receptor function. These differences are found with regard to desensitization kinetics, the extent of sensitivity toward different full and partial agonists or channel blockers, and the magnitude of the evoked current. The only common trait between these mutant receptors is an increase in acetylcholine sensitivity, and even this increase varies significantly between ADNFLE mutations.59−61 Nevertheless, mutations that are frequently associated with psychiatric symptoms or mental retardation do not necessarily display larger functional changes than mutations that cause a more “benign” course of the disorder. So far no direct correlation has been found between the impact an ADNFLE mutation has on nAChR function and the severity of symptoms observed in the patient carrying this mutation.58

3. POTASSIUM CHANNEL DYSFUNCTION AND BRAIN EXCITABILITY The potassium channels are encoded by more than 80 genes in mammalians (more than 50 in humans) and can be divided into four major classes. These include calcium-activated potassium channels that open in response to the presence of calcium ions or other signaling molecules, inwardly rectifying potassium channels, tandem pore domain potassium channels that are constitutively open or possess high basal activation, and voltage-gated potassium channels that open or close in response to changes in the transmembrane voltage. Potassium channels serve diverse functions and are expressed in many cell types, both excitable and nonexcitable. They are involved in functions as diverse as cellular signaling, neurotransmitter release, heart rate, insulin secretion, epithelial electrolyte transport, smooth muscle contraction, and cell volume regulation. The disorders caused by potassium channels are equally diverse and include diseases of the cardiac, neuronal, renal, and metabolic systems. In the central and peripheral nervous system, potassium channels are responsible for both dynamic neuronal signaling and the setting of the resting potential of cells, mechanisms that renders them perfect candidates for neurological and sensory disorders. 3.1. Benign Familial Neonatal Convulsions: An M-current Defect

With their six transmembrane domains, the subunits of the main voltage-gated potassium channels (KCNQ1-KCNQ5) are the largest within their receptor family. Most of these channel subunits have by now been linked to monogenic disorders. KCNQ1 repolarizes cardiac action potentials and is involved in transepithelial potassium secretion in the inner ear. Depending on the severity of the loss of function, mutations in KCNQ1 and its accessory subunit KCNE1 can lead to cardiac arrhythmia in the autosomal dominant Long QT-Syndrome (Romano− Ward syndrome) or, with a more severe loss of function, to the autosomal recessive Jervell and Lange-Nielsen syndrome in which cardiac arrhythmia is associated with congenital deafness.62−64 KCNQ4 is prominently expressed in sensory hair cells in the inner ear, and mutations within this gene are the cause of an autosomal-dominant hearing-loss disorder.65,66 The voltage-gated potassium channel genes KCNQ2 and KCNQ3 had been among the first genes discovered in human 6339


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genes could help to explain the interindividual variability between patients with the same type of epilepsy as well as the co-occurrence of different types of epilepsy within the same family.

the 10 families included in the study at least one affected individual with delayed psychomotor development or mental retardation. In this study the most severely affected children were born to parents who had only a short period of neonatal seizures with spontaneous remission and normal psychomotor development. The type of mutation found in these families is not different from those in BFNC families with a more benign course of the disorder; thus, the apparent anticipation is most likely due to ascertainment bias rather than any genetic mechanism. Mutations associated with an unfavorable outcome tend to be located within the functionally critical S5/S6 regions of the KCNQ2 gene.70 However, the fact that mental retardation almost never affects all carriers of a particular KCNQ2 mutation strongly argues against the position of the mutation within the gene as the sole responsible factor for unfavorable outcomes in BFNC patients. It is more likely that unknown modifier genes and environmental influences are responsible for the phenotypic variability observed in BFNC families. However, without any knowledge about the nature of these risk factors, no advice on how to avoid them can be given to BFNC families.

4. GABAA RECEPTORS: THE BRAIN PACIFIER Another ligand-gated receptor family that is structurally very similar to nAChRs and is also linked to human monogenic epilepsies is the GABAA receptor. Its five subunits assemble around a central pore that is only opened when a specific ligand binds to the recognition site. With a total of 13 subunits, the GABAA receptors constitutes an ion channel family that is even more diverse than the nAChR family. The subunits are named by Greek letters (six α (GABRA1, GABRA2, GABRA3, GABRA4, GABRA5, and GABRA6), three β (GABRB1, GABRB2, and GABRB3), three γ (GABRG1, GABRG2, and GABRG3), and one δ (GABRD), ε (GABRE), π (GABRP), and θ (GABRQ) each). GABAA receptors typically contain two α and two β subunits, and the most common CNS subtype (43% of all GABAA receptor subtypes) is composed of α1, β2, and γ2 subunits (2α1:2β2:1γ) (Figure 2).73

3.2. Potassium Channel Gene Variants May Act As Epilepsy Modifiers

Potassium channel subunits are not only a cause for rare monogenic epilepsies but may also act as susceptibility factors or disease modifiers in more common multifactorial types of epilepsies. Experiments including double transgenic mice demonstrated that variants in voltage-gated ion channel genes such as Kcnq2 can modify the phenotype of a mouse model of human epilepsies. Variants that in Kcnq2 cause M-channel dysfunction were shown to promote seizure initiation and increase seizure severity when transferred to a genetic background of abnormal excitability.71 Another one of these putative modifier genes is KCNV2, encoding the voltage-gated potassium channel subunit Kv8.2 that can form functional heterotetramers with Shab family Kv2 subunits, modulating membrane translocation and channel properties of the resulting potassium channel. These delayed rectifier potassium channels are important for limiting membrane excitability, particularly under conditions of repetitive stimulation. In mice two nonsynonymous coding variants located in the coding region of Kcnv2 have been described that cause differences in seizure susceptibility between strains carrying complementary alleles of these variants.72 Transfer of the risk alleles to a mice strain carrying the epilepsy mutation Scn2a-GAL879-881QQQ exacerbated the epilepsy phenotype and accelerated mortality. Subsequent mutation screening in patients with different types of common epilepsies identified two amino acid exchanges that were not present in human genome variation databases, nor could they be found in healthy controls.72 The patients in whom the variants were found suffered from different types of epilepsies. Both patients inherited their KCNV2 variants from a healthy parent, which renders it unlikely that these variants are the sole reason for the patients' epilepsies. However, functional studies demonstrated that both variants enhance Kv8.2mediated suppression of Kv2.1 currents. Therefore, they could be predicted to decrease delayed rectifier potassium currents in neurons, resulting in increased excitability under conditions of repetitive stimulation.72 It is therefore possible that KCNV2 variants constitute a general risk factor that either worsens an already existing epilepsy or causes it but only in combination with other unknown factors. Such susceptibility

Figure 2. Simplified representation of a GABAA receptor. The positions of ligand-binding sites are indicated by arrows.

4.1. Rare Familial Epilepsies Caused by GABAA Mutations

Similar to nAChRs the GABAA receptors are found both presynaptically and postsynaptically, with the latter being their more predominant site. GABAA signaling constitutes the major inhibitory mechanism in the brain, which renders these receptors perfect targets for epilepsy mutations. Mutations in GABAA subunits can result in a loss of inhibitory neuronal firing that normally prevents the spread of paroxysmal discharge. This would cause an unbalanced ratio of excitation/inhibition that could easily give rise to a clinically manifest seizure. Given its importance in the prevention of brain hyperexcitability, it is rather surprising that, compared to some other ion channel families, the GABAA receptor family plays only a minor role with regard to genetic epilepsies. Despite its rather small role, it nevertheless is rather interesting because some of the known patients with GABAA mutations are affected by epilepsies that are not only usually multifactorial but belong to the most common epilepsy forms in humans. These mutated receptors are therefore valuable research tools that might help to provide an insight into the pathomechanisms behind the main epilepsy syndromes. Mutations in GABAA receptor subunits have been found in a few patients with childhood absence epilepsy (CAE) and autosomal dominant juvenile myoclonic epilepsy (JME). Additional but much rarer syndromes that are associated with GABAA mutations in a few patients are generalized epilepsy 6340


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Figure 3. Expansion of the disease-causing triplet repeat in Fragile X syndrome. Both the normal range repeat and the permutation are found in healthy individuals. In the germ line the permutation has the potatial to expand into a full mutation, causing the full mental retardation phenotype in males. In females the clinical expression of the full mutation is more variable, and psychomotor retardation is only found in about half of the full mutation carriers.

GABAA subunit.80 However cultures of embryonic cells taken from γ2R43Q knockin mice showed no changes in the trafficking and localization of γ2 GABAA subunits.79 These seemingly contradictory findings strongly suggest that additional mechanisms such as post-translational modifications, compensatory expression of other subunits, or regulatory factors are important modulators of the pathofunctional effects GABAA mutations have in vivo.

with febrile seizures plus and severe myoclonic epilepsy of infancy. These epilepsies will be discussed in detail in the following paragraphs. CAE predominantely affects girls with an onset at 4−8 years of life. It is characterized by frequent staring spells that are associated with bursts of 3-Hz spike and slowwave complexes in the electroencephalogram. The few GABAA mutations that have so far been found in patients with CAE affect different subunits (α1 (GABRA1), α6 (GABRA6), and β3 (GABRB3)).74,75 In vitro expression experiments indicate that they all cause a loss of function, either through the nonsensemediated mRNA degradation or by decreasing the number of functional GABAA receptors at the plasma membrane. Such mechanisms would fit with the hypothesis that loss of inhibition is a major pathomechanism in epilepsy. Interestingly, GABAA receptors seem also to contribute to the common multifactorial forms of CAE. The promoter for the exon 1A of the GABRB3 subunit gene contains a polymorphism, −897 T/C, which is more frequently found in CAE patients than in controls. This polymorphism decreases the expression of GABRB3 by affecting the affinity for binding the neuron-specific transcriptional activator N-Oct-3.76 Taken together, these data strongly suggest that the GABRB3 polymorphism −897 T/C is not able to cause epilepsy by itself but represents a risk factor for CAE. Usually starting at teenage age, JME is characterized by myoclonic jerks upon awakening. Grand mal seizures (almost in all patients) and absence seizures (in a third of patients) are also very common. Two GABAA mutations in different subunit genes (GABRA1 and GABRD) have been described.77,78 The functional consequences of these two mutations are not fully understood yet. In heterozygous expression experiments, one of the mutations showed reduced current amplitudes but unchanged activation, desensitization, and deactivation kinetics, whereas the other mutation showed no effect in the presence of the wild-type subunit (i.e., in the heretozygous state). It is possible that these mutations only have a subtle impact on GABAA function that might not be recorded in in vivo expression experiments. Animal models such as knockin mice might be as helpful for the characterization of these mutations as they have been proven to be for other GABAA mutations. That there might be crucial differences between in vitro and in vivo models has been demonstrated by γ2R43Q knockin mice. In vivo expression experiments suggest that mutation γ2R43Q causes decreased trafficking of the subunit to the cell surface, due to its retention and possible degradation within the endoplasmic reticulum (ER).79 Cell-culture experiments using heterologous transfections found that γ2R43Q even exhibited a dominant negative effect by decreasing the expression of the β2

4.2. Autism, Epilepsy, and GABAA Receptors

Autism is a clinical feature that can be found in many different genetic and nongenetic disorders. It can be either the sole symptom, as in “pure” autism disorders such as Kanner syndrome or Asperger syndrome, or a facultative symptom. The latter is usually observed in disorders that frequently present with psychomotor developmental delay and mental retardation, which itself presents the highest known risk factor for autism. Examples of typical monogenic disorders in which autism, mental retardation, and epilepsy are frequently observed clinical signs are Fragile X syndrome, tuberous sclerosis, Angelman syndrome, and Rett syndrome. Both autism spectrum disorders and epilepsies are heterogeneous disorders that have multiple etiologies and can be caused by several different pathophysiological mechanisms. On the clinical level there is a strong association between autism spectrum disorders and epilepsy. A high percentage of patients with autism spectrum disorders have epilepsy, a combination of neurological disorders that has a much poorer prognosis for cognitive outcome than each disorder for itself.81 The frequent co-occurrence of both disorders could point to a shared pathomechanism that is either active during embryonal development and/or disturbs the functioning of neuronal networks in the brain, at least in some of the autism−epilepsy syndromes. A promising candidate for such a shared pathomechanism would be the GABAA inhibitory system. Several lines of evidence already link autism spectrum disorders and epilepsy to abnormalities in the GABAergic transmitter system. Examples are a downregulation in density and distribution of GABAergic receptor subunits in the hippocampus of autism spectrum disorder patients with seizures, a loss of inhibitory interneurons in the epileptic brain, and evidence for an alteration of GABAergic neurons and circuits in the brains of patients with autism spectrum disorders.82,83 No direct evidence of GABAergic dysfunction in the brain of autism spectrum disorder patients has been found yet; however, indirect evidence for such a connection can 6341


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suggest that GABAergic dysfunction has a key role in the pathogenesis of mental retardation disorders that are frequently associated with autism and epilepsy.

be found in some of the above-mentioned monogenic disorders with autism and epilepsy. The Fragile X syndrome, one of the most common mental retardation disorders in males, is caused by the expansion of a triplet repeat within the untranslated region of the FMR1 gene (Figure 3). The expanded repeat prevents transcription of the FMR1 gene, creating a FMR1 null-phenotype in males. The knockout of Fmr1 in mice creates an animal model that displays many features of human Fragile X syndrome patients, including behavioral abnormalities and epilepsy. The developing forebrain of Fmr1 knockout mice shows a strong decrease in the expression of GABAA receptor α1, α3, and α4 subunit mRNAs. Furthermore, the Fmr1 knockout mice displayed a temporarily increase in the GABA synthetic enzyme GAD65 and a decrease in a GABA catabolic enzyme GABA-T, suggesting an elevation in brain GABA levels at different stages of forebrain development. The only significant changes that persisted into adulthood were the downregulation of the β2 subunit and the upregulation of GAD65. The selective downregulation of GABAA receptor α1 and β2 subunits in Fmr1 knockout mice may cause a reduction in excitatory activity over the first two postnatal weeks. Such an effect would be likely to create havoc during a critical developmental period in which the maturation of functional inhibitory synapses is believed to occur. The reduction in excitatory input in early life might have longlasting deleterious effects on brain development that could contribute to the aberrant morphological and cognitive profile in patients with Fragile X syndrome.84,85 Another example is Rett syndrome, a monogenic disorder that almost exclusively affects young girls. Rett syndrome usually starts between 6 and 18 months of age and causes a rapid loss of already acquired motor and cognitive skills. About 70% of Rett patients reportedly develop epilepsy, which ranges phenotypically from generalized tonic-clonic seizures, absences, myoclonic seizures, to tonic seizures. Abnormal gait, decreased head and body size, scoliosis/kyphosis, autistic behavior, prolonged QT intervals, and breathing irregularities such as hyperventilation and apnea are also typical for patients with Rett syndrome. A particularly distinctive feature of Rett syndrome is the repetitive hand-wringing that replaces purposeful hand use. The disorder is caused by mutations in the MECP2 gene that codes for methyl-CpG binding protein 2, a transcriptional regulator involved in chromatin remodelling and splicing.86 For reasons not completely understood yet, GABAergic neurons express 50% more MECP2 than nonGABAergic neurons, suggesting a specific role for the MeCP2 protein in GABAergic function. The MECP2 protein binds all over the genome with selectivity to methylated sites, but there are some sites such as BDNF (brain-derived neurotrophic factor) and GAD1/2 (glutamic acid decarboxylases 1/2) that exhibit increased MECP2 protein binding. This enrichment might be the cause for MECP2 overexpression in GABAergic neurons but does not explain why it happens in the first place. In animal models the loss of MeCP2 protein in GABAergic neurons caused a reduction in neurotransmitter release due to a reduction of the enzyme glutamic acid decarboxylase in presynaptic terminals. The resulting loss of inhibitory neurotransmission could cause hyperexcitable network activity.87 Phenotypically this loss was associated with different neuropsychiatric phenotypes including abnormal social behavior, learning/memory defects, motor function disturbances, or stereotyped behavior.88 Taken together, the findings in autism spectrum disorders, Fragile X syndrome, and Rett syndrome

5. VOLTAGE-GATED SODIUM CHANNELS AND THE GEFS+ SPECTRUM Sodium channels are very large membrane proteins that are a crucial component of most excitable tissues. Their porebuilding α subunits are encoded by 10 different genes and differ by their kinetics and expression profiles. Sodium channels usually consist of a single α subunit and one or two β subunits. The latter ones are much smaller than the α subunits and are members of the immunoglobulin superfamily. They possess the Ig domain typical for this gene superfamily, a conserved sequence motif that serves diverse biological functions including growth and development, signaling, cell adhesion, and protein−carbohydrate interactions. Within sodium channels the β subunits mainly act as regulators of channel gating but also form links to the intracellular cytoskeleton and the extracellular matrix.99 5.1. Many Faces of SCN1A

With regard to human genetic disorders, the large voltage-gated sodium channel gene SCN1A certainly codes for one of the most interesting ion channel subunits in humans (Table 1). It is Table 1. Clinical Importance of Voltage-Gated Sodium Channel Subunitsa gene SCN1A

SCN2A SCN3A SCN4A SCN5A SCN8A SCN9A

disorders +

GEFS , SMEI, borderline SMEI, West syndrome, Doose, myoclonic astatic epilepsy, intractable childhood epilepsy with generalized tonic-clonic seizures (ICEGTC), Panayiotopoulos syndrome, familial hemiplegic migraine (FHM), familial autism, Rasmussens’s encephalitis, Lennox-Gastaut syndrome familial febrile seizures and epilepsy, early infantile epileptic encephalopathy hyperkalemic periodic paralysis, paramyotonia congenita, potassium-aggravated myotonia Long QT syndrome, Brugada syndrome, idiopathic ventricular fibrillation erythromelalgia, paroxysmal extreme pain disorder, channelopathy-associated insensitivity to pain, SMEI, familial febrile seizures

SCN10A SCN11A

involved in different neurological disorders, which range from relatively mild phenotypes including febrile convulsions and generalized epilepsy with febrile seizures plus (GEFS+, OMIM 604403) to severe epilepsies with an overall poor or even catastrophic prognosis. The latter includes severe myoclonic epilepsy of infancy (SMEI or Dravet syndrome, OMIM 607208), borderline SMEI (SMEB), and related epilepsies such as epilepsy with myoclonic-atonic seizures (myoclonic astatic epilepsy, Doose syndrome), infantile spasms (West syndrome), intractable childhood epilepsy with generalized tonic-clonic seizures (ICEGTC) and Panayiotopoulos syndrome, familial hemiplegic migraine type 3 (FHM, OMIM 609634), familial autism, acute encephalitis, and LennoxGastaut syndrome.89−92 With 5−10% of children affected under the age of six years, febrile convulsions are the most common seizure type in humans. For most patients a polygenic background rather than 6342


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with occipital paroxysms, early-onset benign childhood occipital epilepsy, or nocturnal childhood occipital epilepsy. The International League against Epilepsy report on classification ruled these acronyms obsolete because many of the symptoms found in Panayiotopoulos syndrome are not occipital in origin. They are predominantly autonomic seizures that consist of episodes of disturbed autonomic function with nausea, retching, and vomiting. Syncopal-like attacks that render the child unresponsive occur in about one-fifth of the seizures. Seizures are often prolonged and last more than 30 min, thus constituting autonomic status epilepticus. Compared to other SCN1A-caused epilepsies, the prognosis of Panayiotopoulos syndrome is remarkably benign. Most children have only a limited number of seizures, develop normally, and have no increased risk for epilepsy in adulthood.89 Infantile spasms or West syndrome is one of the most recognized types of epileptic encephalopathy in early infancy. It constitutes a distinct and often catastrophic form of epilepsy characterized by a unique seizure type that includes flexor, extensor, and mixed flexor−extensor spasms. Affected children usually have a distinct electroencephalogram (EEG) pattern of hypsarrhythmia, characterized by randomly distributed highamplitude waves and spikes that indicate highly disorganized brain activity. About 50% of children with West syndrome later develop Lennox-Gastaut syndrome, which is characterized by different seizure types, status epilepticus, and a poor prognosis. The majority of patients with West syndrome have an associated underlying disorder such as pre- or perinatal brain damage, tuberous sclerosis, or metabolic disorders. SCN1A mutations are a rather rare cause of West syndrome, but members of GEFS+ families seem to have a slightly increased risk to develop infantile spasms.100 5.1.1. Phenotype−SCN1A Genotype Correlations. About 90% of the SCN1A mutations are found in patients with severe myoclonic epilepsy of infancy or related disabling epilepsies, and only about 10% are found in patients with the more benign phenotype of GEFS+. However, the latter number might be a cross underestimate because patients with severe epilepsies are much more likely to be genetically tested. It is therefore realistic to assume that the higher percentage of serious epilepsies associated with SCN1A mutations is at least in part due to recruitment bias and that many GEFS+ mutations remain undiagnosed. Most of the more than 600 known SCN1A mutations are private, meaning that they have been only found in one patient or one family. This makes it nearly impossible to predict the clinical outcome in very young patients or in prenatal diagnosis. In many genetic disorders a rough distinction between benign versus serious mutations can be done by assessing their probable effect on protein function. For SCN1A it is known that truncating mutation such as out-offrame deletion or insertions or premature stop codons are almost exclusively associated with severe myoclonic epilepsy of infancy and related severe epilepsies, whereas only missense mutations are found in GEFS+. Unfortunately, it is not possible to conclude that missense mutations are predictive for the more benign GEFS+ phenotype, because approximately half of the mutations found in patients with severe myoclonic epilepsy of infancy are missense mutations, too.101102 A study including 819 individuals with SCN1A mutations demonstrated that tentative relations exist between the location of the missense mutation and the clinical phenotype.101102 Missense mutations are not randomly distributed along the SCN1A gene but cluster in certain functionally important parts.

a monogenic cause of the febrile convulsions can be deduced from empirically gained recurrence risk data. In some families febrile convulsions can persist beyond the age of six years and/ or may be later followed by with different types of afebrile seizures. This familial association of febrile convulsions and epileptic seizures has been named “Generalized epilepsy with febrile seizures plus” (GEFS+).90 Afebrile seizure types in GEFS+ individuals include generalized tonic-clonic seizures, myoclonic, absence, and atonic seizures, and in some patients also partial seizures. Furthermore, most of the clinical phenotypes summarized above that are associated with SCN1A mutations can sometimes be found in GEFS+ families. It is not unusual to observe GEFS+ families in which not two affected members share the same seizure type. Febrile convulsions are not obligate in GEFS+ patients, so that it is likely that an unknown number of families are not correctly diagnosed. The mode of inheritance underlying GEFS+ is still a matter of debate. Although in some families the trait is obviously autosomal dominant, in others it is probably better described as oligogenic or as a major gene effect. The latter two inheritance patterns would at least in part be able to explain the clinical variability observed in GEFS+ families.90 Several different ion channel genes have been implicated in GEFS+SCN1B, SCN2A, and GABRG2but most of the mutations are found within the SCN1A gene, one of the genes coding for the major, pore-forming α-subunits of the voltagegated sodium channel.78,93−95 Mutations in this gene are even more frequently identified in patients with severe myoclonic epilepsy of infancy, an epileptic encephalopathy that starts at about six years of age with prolonged febrile convulsions.89 During their second year of life, affected children develop different types of afebrile seizures including myoclonic, absence, partial, and atonic seizures that are mostly therapy-resistant. Developmental arrest and even regression become obvious and usually result in severe psychomotor retardation. A closely related epilepsy syndrome that is also caused by mutations within the SCN1A gene is borderline severe myoclonic epilepsy of infancy. This disorder shows a very similar course but without myoclonic seizures and with a less severe psychomotor impairment. Intractable childhood epilepsy with generalized tonic-clonic seizures is clinically indistinguishable from borderline severe myoclonic epilepsy of infancy and can therefore be regarded as a synonym.96 Myoclonic-astatic epilepsy was first described by Hermann Doose in 1970, and 20 years later the Commission on Classification and Terminology of the International League Against Epilepsy97 renamed it epilepsy with myoclonic-atonic seizures. However myoclonic-astatic epilepsy or Doose syndrome are still the more commonly used names for this severe epilepsy. As in the severe myoclonic epilepsy of infancy syndrome, children with myoclonic-astatic epilepsy usually develop normally until onset of seizures between 7 months and 6 years of age. Seizure types might be myoclonic, astatic, myoclonic-astatic, absence, tonic, clonic, or generalized tonicclonic, and the electroencephalogram shows generalized polyspike and wave epileptiform activity. Patients with myoclonic-astatic epilepsy were among the first to be diagnosed with SCN1A mutations. Additional ion channel genes that might cause myoclonic-astatic epilepsy are the accessory sodium channel subunit SCN1B and the γ1-subunit of the γaminobutyric acid receptor (GABRG2).98 In the past Panayiotopoulos syndrome had been given different names including early-onset benign childhood epilepsy 6343


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properties on neurons obtained from transgenic or knockin mice that express specific SCN1A mutations. A few GEFS+ mutations have already been introduced into such animal models, including the mutation SCN1A-R1648H that causes a highly variable phenotype in humans. Members of the large GEFS+ family in which this mutation had been first identified showed either childhood febrile seizures, afebrile epilepsies such as generalized tonic clonic seizures (GTCS), absence seizures and myoclonic seizures, or both febrile and afebrile seizures. The R1648H mutation, which is located in the voltage-sensing S4 segment of the fourth homologous domain of SCN1A, has been analyzed both in BAC (bacterial artifical clone) clone-transgenic mice as well as in homozygous knockin mice.105,106 The transgenic mice had a lowered seizure threshold but did not exhibit spontaneous seizures, whereas some of the knockin animals developed EEG abnormalities and spontaneous generalized seizures. The observation that not all transgenic mice developed seizures would be consistent with the inhomogeneous clinical phenotype characteristically found in human GEFS+ families. Interestingly, the knockin mice also showed lower threshold temperatures for the onset of tonic febrile seizures than their wild-type litter mates, a feature that might be related to the febrile seizures that are commonly found in GEFS+ families.105 Dissociated cortical bipolar neurons from both animal models showed similar functional changes, including usedependent inactivation and slowed recovery from inactivation.101102103 In interneurons these changes would be likely to decrease the firing and to slow down high frequency trains of action potentials, resulting in reduced GABAergic inhibition. Such an effect could weaken the neuronal networks that are in charge of controlling the activity of postsynaptic neurons, rendering sudden boosts of hyperexcitability more likely. Additional animal models displaying other SCN1A mutations are needed to determine whether impairment of inhibition is a consistent pathomechanism in GEFS+.

These are the four conserved homologous domains that function as the main ion transport sequences of this sodium channel. Each of these four domains contains six transmembrane regions (TM), including TM4 that builds the voltage sensor and TM6 as part of the inner lining of the ion channel. Both TM4 and TM6 are the sequences in which many of the SCN1A missense mutations are found that are associated with the more severe clinical phenotypes, whereas missense mutations in the transmembrane segments S1−S3 are mainly found in febrile seizures or GEFS+. One of the reasons for this clustering might be that polarity changes caused by mutations in these important transmembrane regions are more likely to be critical for protein function. Another mechanism observed in this study was an increase in composition caused by mutations that, for example, substituted a wild-type amino acid with cysteine. The latter has the ability to change the tertiary structure by forming disulfide bridges with other amino acids. Missense mutation outside TM4/TM6 sometimes can also cause severe myoclonic epilepsy of infancy and related phenotypes; the mechanism underlying this genotype− phenotype correlation seems to be a significant, mutationinduced increase in molecular volume.101102 As described previously, some preliminary relations have been found between the position of a mutation and the severity of the associated disorder. However, neither the type of the mutation nor its position within the gene showed any correlation with the type of seizure observed in the individual patient. Together with the remarkable clinical inhomogeneity often present within GEFS+ families, these observations clearly indicate that the course and the severity of the disorder are not only determined by the nature of the underlying SCN1A mutation. It rather seems that the genetic background and environmental factors play an important role with respect to the clinical outcome. These latter influences are neither ascertainable nor predictable, rendering genetic counseling in GEFS+ families difficult. It is not possible to date to safely predict if affected children will have only benign, self-limiting febrile seizures or if they are in danger to develop one of the rare disabling epilepsies associated with SCN1A mutations such as myoclonic-astatic epilepsy or infantile spasms. 5.1.2. GEFS+ and SCN1A Mutations: Contradictory Results from Functional Studies. The broad range of different disorders caused by SCN1A might suggest that clinical variability arises from differences in the way the mutations alter the function of the Nav1.1 sodium channel subunit. However, functional studies so far failed to uncover reliable genotype− phenotype correlations even for one and the same disorder. This is at least partly due to the fact that the results obtained from heterologous expression experiments seem to depend on the cellular system in which the respective studies are performed. There are GEFS+ mutations such as SCN1AR1648H that increased the persistent current when expressed in HEK cells but promoted faster recovery from inactivation in Xenopus oocytes. Both mechanisms would cause an increase in sodium channel activity in vivo; thus, the effect on channel function would be roughly the same. However, GEFS+ mutations such as R859C and D1886Y displayed the opposite effects in heterologous expression systems by shifting the voltage dependence of activation and inactivation or slowing the recovery from inactivation.104 These contradictions might be resolved by using expression models that more closely resemble the in vivo situation in the patient’s brain. This could be realized by analyzing the channels

6. NEURONS AND VOLTAGE-DEPENDENT CALCIUM CHANNELS Voltage-dependent calcium channels are large integral membrane proteins that are encoded by 10 different α subunit genes. The α subunit genes determine to which subclass (highvoltage, intermedium-voltage, or low-voltage) a calcium channel belongs. They contain the major structural elements including a calcium-selective pore, the voltage sensor, and the gating mechanisms. Alternative splicing is a well-known mechanism in calcium channel α subunit mRNA processing and is responsible for the numerous structural and functional variants that exist for each α subunit. In addition to the main calcium-conducting α subunit, several ancillary or β subunits exist (β1−β4, α2δ1−α2δ4, γ1−8). They modify the biophysical properties of α subunits and are involved in intracellular transport/processing and second-messenger-dependent modulation of voltage-gated calcium channels. 6.1. CACNA1A Causes More than One Type of Ataxia

Both episodic ataxia type-2 and spinocerebellar ataxia type-6 are known to be caused by mutations in the CACNA1A gene that encode the α1 subunit of the voltage-gated calcium channel. In episodic ataxia type-2, the patients suffer random attacks of poor muscle coordination, vertigo, and dizziness that can last from a few hours to days. The attacks, which usually start before age 20 years, are often followed by longer periods of 6344


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have the potential of being a preventive therapy without interfering with normal CACNA1A function.112

fatigue. Examination between attacks usually shows nystagmus but no other neurological signs. Migraine headaches affect more than half of patients with episodic ataxia type-2, and episodes of hemiplegia have been described in a few patients. Most patients show a favorable response to acetazolamide, a carbonic anhydrase inhibitor. Episodic ataxia type-2 is inherited as an autosomal-dominant mendelian trait with no obvious genotype/phenotype correlations. The mutations that cause episodic ataxia type-2 are distributed over the whole CACNA1A gene and are presumed to cause a total or partial loss-of-function. The mechanisms behind this effect are either a decreased channel expression predicted to result from protein misfolding and attenuated transport to the plasma membrane and/or shifts in the voltage threshold for activation.107 Transcranial magnetic stimulation (TMS) measurement of corticomotor excitability demonstrated that patients with episodic ataxia type-2 have prolonged corticomotor excitability and increased intracortical facilitation after single-pulse TMS but showed normal excitability at baseline. These test results suggest that patients with episodic ataxia type-2 are impaired in tuning down facilitatory responses to strong excitatory input. The abnormal regulation of excitability is probably caused by dysfunction of the CACNA1A channel. Single-channel experiments showed that the mutated calcium channels have a slower decay of current after repetitive stimulation than normal channels, an alteration that could explain the prolonged synaptic transmission seen in TMS recordings of patients with episodic ataxia type-2.108 In spinocerebellar ataxia type-6, patients suffer from cerebellar atrophy that causes movement symptoms such as incoordination, loss of proprioception, and involuntary nystagmus. Unlike episodic ataxia type-2, spinocerebellar ataxia type-6 is not caused by point mutations or large-scale gene rearrangements within CACNA1A but by an abnormal polyglutamine expansion in the channel’s carboxyl-terminal domain. Such unstable repeats are found in several adult or lateonset neurological disorders, with Chorea Huntington as the best known example. Usually a short version of this repeat can be found in healthy individuals who pass it to the next generation without size changes. A small number of individuals carry an intermediate form of the repeat that does not cause the disorder itself but can expand into a full mutation in the germline. In patients the size of the repeat is above a critical threshold, and this full mutation usually has fatal effects for protein function. In spinocerebellar ataxia type-6, the expanded repeat causes intraneuronal protein aggregation that most likely results in neuronal dysfunction and cell death.109,110 Compared to other triplet repeat expansion disorders, spinocerebellar ataxia type-6 presents with two unusual features. First, with 20− 33 amino acids its repeat expansion is smaller than in any other triplet repeat expansion disorder known today, and second, the expanded repeat is present only in certain splice isoforms of the CACNA1A mRNA. Alternative splicing of CACNA1A mRNA leads to the production of two distinct channel isoforms that either lack or contain the C-terminal repeat expansion.111 Both CACNA1A splice variants are expressed at roughly equivalent levels, and the relative amount of protein containing the expanded repeat is one of the critical factors that determine the severity of the ataxia. This unique disease mechanism offers interesting approaches for the development of gene therapies, such as selective targeting of the repeat expansion-carrying splice variant by RNAi molecules. Such an approach would

7. MIGRAINE: A MULTICHANNELOPATHY? Migraine is an episodic neurological disorder characterized by severe headaches. It predominantly affects woman (∼18% female/∼6% male). In about one-third of the patients, the headache attacks are preceded by focal transient neurological symptoms. Such an aura mostly lasts for 20−60 min and is often characterized by sensory symptoms such as disturbances of vision or paresthesias. The aura is thought to be caused by cortical spreading depression, an intensive depolarization of neuronal and glial cells that spreads like a wave slowly progressing over the cortex, followed by a period of cell inactivity. Twin and family studies have demonstrated that genetic factors play an important role in the pathogenesis of migraine. Nevertheless, most migraines have an oligogenic or multigenic background whereas the monogenetic forms of epilepsy are rare. The latter are regarded as model systems that have the potential to help researchers to elucidate the molecular mechanisms that cause this common disorder. Studies regarding these rare types of migraine have not only provided new insights but have already overthrown previous theories about the basic pathomechanisms in migraine etiology. Initially migraine was considered a cerebrovascular condition that was mainly due to the dilation of pain-sensitive cerebral vessels. This hypothesis seemed to be supported by the observation that vasodilators can cause headaches and that a comorbidity of hypertension and migraine is well-known. Today the idea that just dilation or constriction can explain migraine pain seems to be rather simplistic. The rare monogenic migraine syndromes have shown that ion channels are major players with regard to migraine etiology. Most of our current knowledge originates from studies of families with familial hemiplegic migraine, an autosomal dominant type of migraine in which the attacks are often accompanied by prolonged hemiparesis. Familial hemiplegic migraine is caused by mutations in at least three different ion channel genes: CACNA1A, encoding the α1 subunit of a neuronal calcium channel; ATP1A2, encoding the α2 subunit of Na+/K+ ATPase pumps; and the gene for the α1 subunit of the voltage-gated sodium channels, SCNA1. Two additional genes that are indirectly involved with ion channels, SLC1A3 and SLC4A4, encoding the glial glutamate transporter EAAT and the Na+−HCO3− cotransporter NBCe1 have been described recently in a single family and a sporadic patient with hemiplegic migraine, respectively. The results of these studies strongly suggest that not only the rare monogenic forms of migraine are channelopathies but that this pathophysiological concept might also apply to common, polygenic forms of migraine. It is tempting to speculate that common forms of migraine might be due to perturbation of ion balance, resulting in altered excitability of certain areas of the brain. Preliminary results from large, multicenter wholegenome association studies that included more than 2000 migraine patients seem to support this hypothesis. The first of these studies pointed toward a region on chromosome 8q22.1 that contains two interesting genes, MTDH (metadherin) and PGCP (plasma glutamate carboxypeptidase). These genes are involved in the regulation of extracellular glutamate, the most abundant excitatory neurotransmitter in the brain. Interestingly, elevated levels of glutamic acid are found in platelets, plasma, and cerebrospinal fluid of migraine patients, particularly in 6345


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nervous system; however, it only seems to play a minor role with respect to human disease. Mutations in this receptor have been linked to a rare neurological disorder named startle disease, hyperekplexia, or stiff baby syndrome.116 Startle disease is caused by mutations in different genes that either code for glycine receptor subunits (GLRA1 and GLRB) or proteins such as gephyrin, GlyT2 transporter, or collybistin that are functionally associated with this class of ion channels (Table 2).117−119 Patients with startle

those with aura. The role of glutamate in migraine pathophysiology is further supported by the observation that ingestion of glutamate-rich food provokes, in predisposed subjects, migraine attacks. A second whole-genome association study also pointed to a gene involved in the glutamate pathway. LRP1 (low-density lipoprotein receptor-related protein 1) is an endocytic receptor highly expressed by neurons that can be found in association with NMDA (N-Methyl-D-aspartate) receptors in dendritic synapses. It coprecipitates with NMDA receptors from neuronal cell lysates and can be blocked by NMDA, suggesting that LRP1 is involved in transmitterdependent postsynaptic responses.113 Evidence that not only the glutamatergic neurotransmitter system but also potassium channels might be directly involved in the pathogenesis of migraine with aura came from a combined candidate gene/linkage approach. This family-based approach identified the KCNK18 gene, a member of the twopore potassium channel family. A two-bp deletion that causes a frame shift and subsequent premature termination of TRESK, the protein encoded by KCNK18, was found to cosegregate with migraine in a large family. Functional studies demonstrated that the mutant potassium channel subunit suppresses wild-type channel function through a dominant-negative effect. No other mutations have been found so far in this gene, a fact that does not prevent researchers from regarding it as a promising candidate target for migraine therapy. The KCNK18 gene is expressed in areas of the central and peripheral neuronal system that are crucial for the development of migraine, including the trigeminal ganglion, the cortex, and the dorsal root ganglion. Furthermore, it has no close paralogues, a fact that provides the advantage that drugs can be developed that specifically target this channel subunit. Another advantage would be that for targeting KCNK18 at locations such as the trigeminal ganglion, drugs would not need to possess the ability to penetrate the blood−brain barrier. Both the brainstem and the peripheral neurons have long been suspected to be major players in migraine pathogenesis. This hypothesis is supported by different observations. Positron emission tomography has shown that areas such as the brainstem are activated during acute migraine attacks and cortical spreading depression causes activation of the trigeminovascular system along with dilatation of the middle meningeal artery. Targeting the KCNK18 gene in these regions could be a promising new approach in migraine therapy, assuming that the potassium channel encoded by this gene is indeed involved in the pathogenesis of common forms of migraine.114,115 However, it should be noted that many of the polymorphisms found to be linked to common forms of migraine in whole-genome association studies are located in genes that have no known functional connection to ion channels. It is therefore likely that additional migraine pathomechanisms exist that still wait to be discovered.

Table 2. Genes Involved in Glycinergic Transmission and Their Role in Startle Disease gene

chromosomal localization

protein

startle disease

glycine receptors α1 α2 α3] α4 β

GLRA1 GLRA2 GLRA3 GLRA4 GLRB

5q33.1 Xp22.2 4q34.1 Xq22.2 4q32.1

GlyR GlyR GlyR GlyR GlyR

+

GPHN ARHGEF9

14q23.3 Xq11.1

Gephyrin Collybistin

+ +

SLC6A9 SLC6A5 SLC32A1

1p34.1 11p15.1 20q11.23

GlyT1 GlyT2 VIAAT

+

+

receptor clustering proteins

glycine transporters

disease display a pronounced startle response to unexpected tactile or acoustic stimuli. The symptoms are most prominent during the first year of life. The startle reflex causes closure of the eyes and extension of the extremities, followed by a period of generalized stiffness. Additional symptoms might include episodic neonatal apnea, excessive sleep movement, and myoclonic epilepsy. EEG recordings can be helpful to distinguish startle disease from reflex epilepsy, a probably not-so-uncommon misclassification. In infancy patients often show an unsteady gait, which might be misinterpreted as a sign of cerebellar disorder. Instead, the noticeable gait is caused by the patient’s fear of falling, which is a frequent occurrence due to muscle hypertonicity. The episodes of acute hypertonia and apnea can be interrupted by the Vigevano maneuver in which the head and limbs are flexed toward the body trunk (forced kneechest position). The course of the disease is highly variable. Some patients are severely affected during the first year of life but improve considerably over the years whereas in other patients the full symptoms of startle disease arise later in life.120,121 Missense, nonsense, and frameshift mutations within the GLRA1 gene are the main cause of startle disease. The mutations either disrupt channel function or reduce surface expression of the glycine receptor. The effects the mutations exhibit can be either autosomal-dominant or autosomalrecessive, depending on their location within the gene. Dominant mutations are preferentially located within the pore-lining transmembrane segment and adjacent regions whereas recessive mutations result from mutations within TM1 and, less often, null alleles. The mutations are likely to disrupt or modify the inhibition of the startle response mediated by glycine receptors within the spinal cord. The

8. STARTLE DISEASE: GLYCINE RECEPTORS So far five subunits (four α (GLRA1, GLRA2, GLRA3, and GLRA4) and one β subunit (GLRB)) have been identified that form heteropentameric glycine receptors. Glycine receptors share common structural and functional characteristics with other members of the Cys-loop ion channel superfamily that includes the nicotinic acetylcholine receptor, the 5-hydroxytryptamine receptor (5HT3R), and the GABAA receptor. The ionotropic glycine receptor is one of the most widely distributed inhibitory receptors in the central and peripheral 6346


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bodies against the NMDA receptor NR2B in a subset of patients.130 Still unknown is why these autoantibodies attack only one hemisphere of the patient’s brain.131 As in other autoimmune disorders, treatment with steroids, immunoglobulin, or plasmapheresis can be useful during the acute phase. In the chronic phase hemispherectomy is often very effective in reducing seizures but not advisible if the left hemisphere that controls language is affected. Myasthenia gravis is characterized by fluctuating weakness of voluntary muscles and fatigability. It is caused by antibodies against nAChRs and associated proteins located at the neuromuscular junction. The patient’s condition characteristically worsens during periods of activity and improves after rest. In newborns of myasthenic mothers, antibodies crossing the placenta barrier can cause the rare fetal acetylcholine receptor inactivation syndrome. The affected children express typical myasthenic features during the first 4−5 weeks of their life. In some of these children, the inactivation of fetal AChRs by maternal antibodies and the subsequent impaired fetal movements result in permanent muscle defects.

most commonly affected amino acid is R271-GLRA1, which has been found in at least 12 unrelated families with startle disease.122 Mutations in this position transform the natural agonists taurine and β-alanine into competitive antagonists and affect the chloride conductance of the receptor, most likely causing startle disease by impairing glycine inhibition. The treatment of choice is clonazepam, a drug that acts as a γaminobutyric acid agonist and increases GABAergic inhibition. It is therefore suited to counterbalance the loss of inhibition caused by glycine receptor mutations. In most patients with startle disease, the benzodiazepine drug clonazepam is able to reduce generalized muscular hypertonia and other symptoms that often threaten to interfere with the young patient’s psychomotor development.123−125

9. ACQUIRED CHANNELOPATHIES Not all channelopathies are caused by inherited or newly occurred mutations or genetic polymorphisms within the coding region or in regulatory sequences of an ion channel gene. There are two additional groups of channelopathies, one that is caused by the patient’s immune system that produces autoantibodies against the patients own ion channels and another one that results from the aberrant transcription of an ion channel gene.126

9.2. Transcriptional Channelopathies

In transcriptional channelopathies aberrant expression of an otherwise normal, nonmutated ion channel gene is observed. These channelopathies might be secondary to noninfectious central nervous system inflammatory disorders such as multiple sclerosis (encephalomyelitis disseminata) or caused by brain or peripheral nerve injury. In cerebellum the neuronal demyelination that is typical for multiple sclerosis has been shown in rat models to cause an increased expression of the sodium channel SCN1OA (NaV1.8) compared to control mice.132 Dysregulated expression of SCN1OA was also found in cerebellar Purkinje cells in tissue obtained from multiple sclerosis patients at autopsy. The SCN1OA channel is a socalled “slow” sodium channel that is characterized by slower activation and inactivation kinetics and more rapid recovery from inactivation than traditional “fast” sodium channels such as SCN1A. Its aberrant expression can be predicted to alter the pattern of impulses that neurons produce in response to different stimuli, a mechanism that might contribute to the cerebellar ataxia and other signs of cerebellar dysfunction that often affect patients with multiple sclerosis.133 Nerve injuries that cause axonal transection have been shown to trigger the transcriptional downregulation of some previously activated sodium channel genes, while at least one previously silent sodium channel gene (SCN3A, coding for Nav1.3) was upregulated. The Nav1.3 protein is usually not detectable in normal nerves but is most likely responsible for abnormal impulse generation in injured nerves. The sodium channel produces rapidly repriming sodium currents that display a fast recovery from inactivation. Electrophysiologial recordings showed abnormal repetitive action potential activity arising from prolonged depolarization. These functional changes can result in hyperexcitability of the injured neurons and are likely to be the cause of the paresthesia and neuropathic pain that often accompany chronic nerve injuries.134,135

9.1. Channelopathies Caused by Autoantibodies

Examples for aquired neuronal channelopathies caused by autoantibodies are the Lambert−Eaton−Rooke syndrome, neuromyotonia, Rasmussen’s encephalitis, and myasthenia gravis. The Lambert−Eaton−Rooke syndrome (also named Lambert−Eaton myasthenic syndrome), a typical example of an autoimmune channelopathy, presents with proximal muscle weakness and areflexia. Facial weakness, eye muscle complaints, bulbar muscular weakness, and distal pareses are frequent symptoms. The syndrome usually occurs after the age of 40 years, and in about 50−70% of the patients it is associated with cancer, mostly small cell lung cancer. In approximately 85% of patients with Lambert−Eaton−Rooke syndrome, high-specificity autoantibodies are detectable that attack voltage-gated calcium channels located in the cell membrane of the presynaptic motor nerve terminal. Neuromyotonia (Isaac’s syndrome) is another neurological disorder that is often caused by ion channel autoantibodies.127 About 80% of the cases are suspected to be due to autoantibodies against the neuromuscular end plate. The peripheral motor nerve hyperactivity that characterizes this disorder causes painful muscle cramps and pseudomyotonia, often associated with excessive sweating. Within affected tissues autoantibodies against voltage-gated potassium channels can be detected by immunoprecipitation or immunostaining, suggesting that neuromyotonia is an immune-mediated voltage-gated potassium channelopathy. A third example for a channelopathy caused by autoantibodies is Rasmussen’s encephalitis or chronic focal encephalitis, a childhood disorder characterized by severe seizures, loss of motor skills and speech, hemiparesis, and encephalitis. Typically, one cerebral hemisphere shows chronic inflammation with infiltration of T lymphocytes. Rasmussen’s encephalitis shows severe progression, resulting in hemiatrophy of the brain accompanied by profound intellectual deterioration.128 First it was suggested that Rasmussen’s encephalitis is caused by autoantibodies raised against either the AMPA receptor subunit GluR3.129 More recent studies described the presence anti-

10. EPILEPSY: ARE ION CHANNELS THE WHOLE STORY? Several of the preceding sections discussed the role different ion channels have in the pathogenesis of epilepsy. Nevertheless, many questions concerning the contribution of genes to the pathogenesis of seizure disorders have not been answered yet. 6347


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subunits could be used as molecular targets for therapies that combine effectiveness with a minimum of unwanted side effects. However, today’s technical possibilities are not yet fully up to this challenge. The existing gene-silencing strategies still have limitations that prevent their widespread use in therapeutic aims such as the target-orientated knockdown of a specific ion channel subunit gene. This is demonstrated by one of the most up to date gene silencing approaches, RNA interference (RNAi) method. The RNAi method uses short, double-stranded RNA molecules (small interfering RNAs (siRNAs) and microRNAs (miRNAs)) that are incorporated into the RNA induced silencing complex (RISC). The RISC contains key proteins for the processing and functioning of double-stranded RNAs. The processed siRNAs and miRNAs can bind to mRNAs and induce their cleavage and subsequent degeneration, thereby preventing translation of these target mRNAs at the ribosomes. At a first glance such methods seemed to be ideally suited for therapeutic silencing approaches in transcriptional or inherited channelopathies. However, practice tests have demonstrated that both methods need to be optimized before they can become really useful for clinical approaches. The main problems that still wait to be solved are the nonspecific effects triggered by these small RNA molecules. These problems have their origin in the basic mechanisms by which small siRNA and miRNA molecules control gene function. They were shaped during evolution to manipulate not only single genes but entire gene networks. To do this miRNAs developed two different mechanisms, one that is based on perfect base pairing with the target RNA, followed by its destruction by the protein Argonaute in a concentration-independent manner, and one that attacks target mRNAs with binding sequences that are less than perfect. The silencing effect on these latter target mRNAs is concentration-dependent, repressing at low concentrations of target mRNA but having a much weaker effect when the target RNA concentration is low. In the latter situation the effect of miRNAs is progressively weakening with decreasing concentrations of target mRNA. It is the ability to affect a large range of target RNAs with not perfectly homologous binding sequences and regulate them in a concentration-dependent manner that creates one of the major problems if miRNAs or the closely related siRNAs are used for gene therapy approaches. Designing an artificial miRNA or siRNA molecule that targets specifically a certain mRNA species has turned out to be anything but trivial. More often than not the experimental introduction of such a molecule into cultured cells or model animals triggers nonspecific effects caused by unintended offtargeting, a problem that can render these approaches unsuitable for clinical trials.143,44 Another problem that has quickly become obvious with the first in vivo tests was toxicity that is most likely due to saturation of the endogenous RNAi pathway. Both exogenous and endogenous miRNAs and siRNAs are stabilized by the same protein, Exportin-5, which is expressed only at low levels and is therefore a limiting factor for miRNA function. One resolution to this problem could be the use of delivery systems with weak promoters that keep the transcription of the therapeutic miRNA or siRNA at a minimal level. This might prevent the saturation of Exportin-5 and minimize disturbances in endogenous miRNA regulatory networks. Another solution would be the design of siRNAs that do not depend on Exportin-5 for their transport to the cytoplasm. This would prevent competition with endogenous miRNAs and the toxic

Important issues remain, mostly regarding the genetics of common forms of epilepsy such as juvenile myoclonic epilepsy, childhood and juvenile absence epilepsies, or grand mal epilepsy on awakening. These seizure disorders are representative for a group of idiopathic epilepsies that arise on a multifactorial rather than a monogenic background. They are not caused by mutations in single genes but by a combination of several (or many?) genetic susceptibility factors and unknown environmental factors. At a first glance, ion channel genes seem to be the perfect choice for researchers selecting promising candidate genes for their studies on common idiopathic epilepsies. They fulfill all the important criteria apparently required for genes that cause episodic seizures. Characterized by super fast reaction times, they exercise a powerful yet highly flexible control over diverse neuronal functions. Their embedment within the vast network of the super family of ion channels creates a reliable redundancy, and the multigene structures displayed by most ion gene families provide a safety net that buffers the damages or failures caused by mutations in single subunits. The interconnected structure of ion channels most likely explains why many mutations only episodically lead to clinical symptoms such as seizures. This is best demonstrated by the above-described mutations with dominant negative effects on KCNQ2/KCNQ3 protein function that in most patients cause rather benign and selflimiting seizures, and not the severe type of disorder one would expect from a functional defect in such an important regulator of neuronal excitability.136 The often benign outcome is thought to be due to some kind of functional compensation, probably provided by other members of the KCNQ gene family. Nevertheless, ion channels are not the only genetic factors that have to be taken into consideration in epilepsy research. There are several examples that demonstrate that it does not always need fast-acting ion channels to cause epilepsy, at least not as a primary cause. The most obvious examples are epilepsies that are due to inborn changes in brain function and/ or structure, such as abnormal neuronal migration in disorders such as tuberous sclerosis and lissencephaly or acquired structural conditions including brain tumors and injuries.137,138 Furthermore, the first nonion channel genes that cause autosomal dominant forms of epilepsy have been described. These include the LGI1 gene, which leads to autosomal dominant lateral temporal lobe epilepsy and is involved in synapse transmission, and EFHC1, a gene of unknown function for which mutations have been described in rare families with familial juvenile myoclonic epilepsy.139−142 Both epilepsy genes, LGI1 and EFHC1, demonstrate that, although ion channels will remain an important cause for seizure disorders, the possibility of alternative pathomechanisms has to be kept in mind.

11. GENE THERAPY APPROACHES FOR CHANNELOPATHIES: TOO EARLY? Transcriptional channelopathies not only offer insights into the molecular pathomechanisms of some of the more common neurological disorders but might also provide an important key for their treatment. The ion channel subunits that are dysregulated in these disorders often display a very restricted expression pattern and are being preferentially located on specific groups of neurons. The same is true for several of the above-described inherited channelopathies that are caused by single-gene mutations in some of the less ubiquitously expressed ion channel genes. These mutated ion channel 6348


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method will lead to a type of personalized medicine that combines preventive and therapeutic strategies customized for each patient individually. Unraveling the genetic background of multifactorial diseases promises to lead to new therapeutic approaches, including second-generation RNA-based gene therapy strategies. The involvement of ion channel subunits in many multifactorial disorders might turn out to be advantageous, not at least because of the redundancy potential in their multisubunit gene families. Each ion channel family has subunits that are widely expressed, but also those that display an expression pattern that is restricted to certain organs or tissues. Such a restricted expression pattern would allow for targeted knockout strategies. These would promise to have a minimum of unwanted side effects not only because the target has a restricted expression pattern but also because of the possibility that other subunits from the same ion channel family might (at least in part) take over some of the function of the subunit knocked out by the gene therapy. Gene therapy-induced gene silencing would also be helpful to further elucidate the numerous functions ion channels have in brain and non-neuronal tissue and to increase our knowledge of their role in physiological processes and disease.

effects that result from the premature depletion of these endogenous RNAs.143,144 Once these initial problems are overcome, the RNAi technology should have the potential to become a valuable therapeutic tool in the treatment of channelopathies and other genetic disorders. The primary causes (i.e., the ion channel subunits) in these disorders are well-characterized and present perfect targets for therapeutic strategies that aim to block the production of disease-causing proteins. Furthermore, the RNAi technology can be used to explore the function of single-ion channel subunits in vivo. In most ion channel subfamilies there are one or more subunits that still play an orphan role with regard to research. An example would be CHRNA5, a nAChR subunit for which it is not even known if its protein is capable to assemble with β subunits without the help of other α subunits. Knockdown approaches in model animals by RNAi strategies could help to better understand the functional role of α5 and other orphan ion channel subunits.

12. CONCLUSIONS AND FUTURE PERSPECTIVES Genomic research has discovered several different ion channel families, each composed of many major and auxiliary subunits. They are encoded by a large number of ion channel subunit genes, and many of these genes are known to be associated with human disorders. Some of these disorders are rare and inherited in an autosomal-dominant or, less often, autosomalrecessive manner. The mutations that cause these disorders often profoundly affect the function of the channel but rarely shut them completely down. Compared to other classes of genetic disorders, gain-of-function mutations are surprisingly frequent. This might very well be due to the fact that within the highly balanced networks of ion channels both loss-of-function and gain-of-function mutations can have adverse effects that cause clinical symptoms. Other disorders are common and multifactorial in origin; thus, ion channels in these disorders act as susceptibility factors rather than as disease-causing genes. The mechanisms that link specific ion channels to common disorders are mostly unknown, which is not surprising because researchers have to hunt for genetic variants within ion channel genes that, most likely, exhibit rather small functional effects. These ion channel variants probably only become relevant for the pathogenesis of disease if an individual inherits them by chance together with susceptibility variants in many other genes, and if certain environmental factors exist that further trigger the manifestation of a specific disease. Such susceptibility variants are notoriously difficult to identify because they are able to hide in plain view within the huge number of genetic variants present in every individual genome. Even the most cost-intensive genome-wide association study (GWAS) that includes thousands of patients is only able to point to a possible location rather than to actually identify a certain genetic risk variant. Most variants selected by GWAS are likely to be functionally neutral but might cosegregate with susceptibility variants located in their vicinity. Thus, cost- and labor-intensive functional studies are needed to separate these innocent bystanders from the true culprits. Nevertheless, in the long term the efforts put into such projects are likely to prove fruitful for future research strategies. Common multifactorial disorders such as heart diseases, diabetes mellitus, or mental health disorders cause significant costs to the health systems and are also a social and economic burden. It is therefore hoped that the introduction of whole-genome sequencing as a routine

AUTHOR INFORMATION Corresponding Author

*E-mail: [email protected]. Phone: (+49)89-5160-4468. Fax: (+49)89-5160-4470. Notes

The authors declare no competing financial interest. Biography

Ortrud K. Steinlein studied medicine at the University of Mainz, Germany, where she received her M.D. in 1987. Subsequently she worked as a postdoc and later as a senior researcher at the Institutes of Human Genetics at the Universities of Heidelberg and Bonn, Germany. In addition to her research, she qualified as a specialist in human genetics and works as a genetic counselor. In 2004 she became head of the Institute of Human Genetics at the University of Munich, Germany. Ion channels and the genetic disorders caused by these have long been a main focus of her research.

ACKNOWLEDGMENTS This work was supported by the DFG (Grant STE16511-2). 6349


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