Epileptic Channelopathy Syndromes

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A first expectation, based on earlier studies of channel defects in hyperexcitability disorders of skeletal muscle (e.g., myotonia) and heart (long QT syndrome), would be that channel mutations leading to epilepsy would be associated with increases in Na+ or Ca2+ channel activity (since such activity leads to membrane depolarization) or with reductions in K+ or Cl~ channel activity (since these currents prevent membrane depolarization) (13, 14). As will be discussed subsequently, we have learned that this expectation is often, but not always, fulfilled. To facilitate further reading, we have provided the listing for each disorder in the National Library of Medicine's Online Mendelian Inheritance in Man (OMIM) database (http://www.ncbi.nlm.nih.gov/omim/), which is frequently updated.

Channel Mutations in Epilepsies with Onset in Neonates and Infants

Benign Familial Neonatal Seizures (BFNS; OMIM 121200). Benign familial neonatal seizures (BFNS, also termed benign familial neonatal convulsions) is a syndrome characterized by recurrent, brief generalized or partial seizures that begin on about the fourth day of life and remit after 1-3 months (15). In the classic syndrome, first described by Rett and Teubel (15), infants otherwise grow and develop normally, without subsequent epilepsy, although affected persons carry a 10-16% risk of experiencing one or more seizures again later in life (16, 17).In 1989, Leppert et al. linked the disease geneto chromosome 20 (18). Later, investigators identified additional families linked to chromosome 8, a first example of genetic heterogeneity in epilepsy (19, 20).

The two BFNS genes on chromosome 20 and 8 encode highly homologous potassium channel subunits, KCNQ2 and KCNQ3 (21-23). These neuronal channel subunits are relatives of a cardiac channel, KCNQ1, which previously was shown to be mutated in both domi-nantly and recessively inherited forms of the long QT syndrome (24, 25). Four KCNQ1 subunits combine to form the channels in the heart. In neurons, some channels are KCNQ2 tetramers, but many are composed of a combination of KCNQ2 and KCNQ3 subunits (26-31). Some disease-causing missense mutations in KCNQ2 and KCNQ3 are associated with only modest reductions (20-30%) in current magnitude (32). This may imply the critical dependence of neuronal excitability on the absolute magnitude of KCNQ2/KCNQ3 potassium channel activity, especially during the neonatal period. KCNQ channels underlie an extensively studied potassium channel, found in central and autonomic neurons, known as the M-channel (26). The name "M-channel" comes from the fact that these channels are important effectors for acetylcholine acting via muscarinic receptors. Indeed, many neurotransmitters that activate intracellular G proteins increase neuronal excitability by reducing the openings of KCNQ channels (33). These channels open occasionally at the resting membrane potential and are slowly activated by membrane depolarization. Because of their slow kinetics, M-channel activation causes a period of membrane hyperpolarization after a cell receives excitatory input. As a result, neurons expressing M-channels tend to fire one action potential after receiving excitatory input, then become transiently quiescent. Inhibition of the M-channel by neurotransmitter receptors causes these neurons to become slightly depolarized and to fire multiple action potentials rhythmically after receiving excitatory inputs. Thus, M-channels may serve as a general brake on excess neuronal excitation, a brake that can be selectively removed when the cell receives input from neurotransmitters such as acetylcholine.

KCNQ2 subunits are widely expressed in circuits implicated in generation of epileptic seizures (30, 34, 35). Interestingly, one conspicuous localization of KCNQ2 expression is in inhibitory neurons important for synchronization of the firing of excitatory neurons, including the reticular thalamic neurons and several populations of interneurons in the hippocampus and cortex (34). A second important localization of KCNQ2 and KCNQ3 is at spike initiation zones at the beginning of axons, including those of pyramidal cells in the hippocampus and neocor-tex (30, 36). This localization may allow them to powerfully control the neuronal threshold, since each neuronal action potential originates in the proximal axon (37). In transfection experiments, some BFNS mutations strongly disrupt proper traffic at the axonal initial segment; if this disruption occurs in vivo, it could be the main contributing factor in the BFNS phenotype (38).

Recent studies have identified patients in BFNS pedigrees with KCNQ2 mutations and more severe epilepsy, impaired cognition (39-42) and myokymia (43, 44). KCNQ2 and KCNQ3 have been found at nodes of Ranvier of peripheral nerves and at the axonal initial segments of spinal motor neurons, and loss of their activity likely causes the aberrant impulse initiation in the nerve that is manifest as myokymia (30, 31, 36). It is not clear whether environmental or genetic factors underlie the more severe cognitive, motor, and epileptic phenotypes seen in some BFNS patients.

Retigabine is a novel anticonvulsant, initially identified in the National Institute of Neurological Disease and Stroke (NINDS) antiepileptic (AED) drug screening program, that is effective in preventing seizures induced by electrical shock or by a broad range of chemical convul-sants (pentylenetetrazole, N-methyl-D-aspartate [NMDA], 4-aminopyridine, and picrotoxin, but not bicuculline or strychnine) (45). BMS-204352 is another drug initially developed as a potential therapy for acute stroke (46). Remarkably, recent studies have revealed that both these agents are potent activators of neuronal KCNQ channels (47-50). The discover of retigabine's novel mode of action has contributed to new interest in its potential clinical usefulness, and it is currently undergoing stage III trials for adult partial epilepsy (50). Although these agents have not yet been shown to be of clinical usefulness, it is clear that the cloning of the KCNQ channels has revealed an important potential new target for drug screening studies.

Benign Familial Neonatal-infantile Seizures (BFNIS; OMIM 607745). Kaplan and Lacey (51) and Shevell et al. (52) first described families in which nonfebrile seizures, like those seen in BFNS but with a somewhat later onset ranging from the first week of life to 3.5 months, were inherited in an autosomal dominant pattern. This delayed onset led to the suspicion of a distinctive etiology, which was affirmed when the two BFNS loci on chromosomes 8 and 20 were excluded by genetic linkage analysis, and another location on chromosome 2, harboring a cluster of genes encoding several Na+ channel proteins, was implicated (53-55). Screening of the chromosome 2 genes in BFNIS patients revealed mutations in SCN2A, encoding the Na+ channel subunit, Nav1.2.

Cell biologic studies using rodents indicate that Nav1.2 channels play an important, transient role on neuronal axons in the developing brain and peripheral nerve (56). In rats and mice, Nav1.2 is at birth the predominant sodium channel at the spike initiation zone in the proximal axon (i.e., the axonal initial segment), but it is largely replaced at this location during the second and third postnatal week. A similar Nav1.2 to Nav1.6 switch occurs at the nodes of Ranvier of many newly myelinating fibers. Based on studies of other proteins, it seems likely that similar subunit switches occur in humans, but over a slower time period of several months (57). The observation that the channels mutated in BFNS and BFNIS are expressed in similar axonal locations during early development is certainly intriguing—but available data do not yet explain why KCNQ2/3 mutations would produce an earlier onset than SCN2A mutations.

Generalized Epilepsy with Febrile Seizures Plus (GEFS+; OMIM 604233), Severe Myoclonic Epilepsy of Infancy (SMEI; OMIM 607208), Intractable Childhood Epilepsy with GeneralizedTonic-clonicSeizures (ICEGTCS;OMIM 607208). Febrile seizures occur in 3-5% of all children. When febrile seizures occur in isolation, between age 3 months and 5 years, in children who are otherwise developing normally, the risk of later epilepsy is low (see Chapter 19). For a minority of cases, however, febrile seizures herald epilepsy of a variety of forms and degrees of severity.

In a seminal clinical study, Scheffer and Berkovic (58) described a exceptionally large multigenerational family in which typical febrile seizures of infancy and early childhood were common, but a variety of nonfebrile seizure types also occurred. This spectrum of other seizure types represented in the pedigree included nonfebrile convulsions, absences, myoclonic seizures, atonic seizures, and severe myoclonic-astatic epilepsy with developmental delay. Scheffer and Berkovic argued that this heterogeneity represented variable phenotypes associated with the dominantly inherited trait and introduced the term "generalized epilepsy with febrile seizures plus" (GEFS +) to describe the new seizure syndrome. They further noted that such phenotypic heterogeneity would make this syndrome difficult to detect in smaller families, obscuring its frequency in the clinic. They concluded that molecular studies of large pedigrees such as the one they had analyzed might reveal the underlying genetic mechanism(s). Follow-up studies by the authors and others have confirmed all these predictions.

Mutations in four channel genes have thus far been identified in patients with GEFS + . By far the largest numbers of them are in SCN1A, a close relative of SCN2A, also encoding a voltage-gated Na+ channel pore-forming subunit. In addition, a few mutations have been found in SCN2A, SCN1B (encoding Nav |1, a voltage-gated Na+ channel single-transmembrane accessory subunit) and GABRG2 and GABRD (encoding gamma-aminobu-tyric acid [GABA] receptor subunits).

The single mutation identified in Nav beta1, C121W, has been described in two Anglo-Australian pedigrees (59, 60). C121 is located in the extracellular domain of

Nav beta1, where it forms a disulfide bridge that plays a critical role in the proper assembly of the extracellular domain into an immunoglobulin-like folding pattern (61). Nav beta1 subunits have multiple roles, enhancing surface expression of the pore-forming alpha subunits, participating in protein-protein interactions within the extracellular space that are important for channel clustering at key sites such as the nodes of Ranvier, and enhancing rates of channel inactivation (62). Careful biochemical and electrophysiologic studies using coexpression with neuronal alpha subunits in mammalian cells have revealed two rather subtle effects of C121W (63). The mutation prevents protein-protein interactions mediated by the extracellular domain of Nav beta1 and shifts inactiva-tion to hyperpolarized potentials, thereby increasing the number of channels available for opening.

The discovery of a second GEFS+ locus on chromosome 2q (64, 65), a region that harbors a cluster of three genes encoding sodium channel alpha subunits, led to a search for mutations within these genes in GEFS+ pedigrees. Sequencing of patient DNA revealed many different mutations in SCN1A and a single mutation in SCN2A, all resulting in single amino acid substitutions (missense mutations) in the resulting alpha subunit polypeptides (66-70). Interestingly, these studies have revealed a high frequency of partial seizures in members of some pedigrees that otherwise fit the original GEFS+ profile (60, 69, 70). The electrophysiologic properties of many of the mutant alpha subunits have now been studied fairly intensively in heterologous cells. A subset of the mutations show defective inactivation mechanisms, as observed first in studies of the C121W mutation in beta1 discussed in the preceding paragraph. For example, Lossin et al. found three mutations that exhibited a persistent current during prolonged depolarizations due to an apparent failure of inactivation gating (71). However, other mutations seem to show reduced current amplitudes or changes in gating that are predicted to reduce current flow, without a defect in inactivation (72, 73). Thus, only some GEFS + mutations appear to be associated with enhanced channel activity similar to the mutations in heart and skeletal muscle Na+ channels that cause disorders of hyperex-citability and excessive synchrony (myotonia, long QT syndrome) in those tissues (74, 75).

This issue has been further complicated by genetic and clinical evidence that an additional epilepsy syndrome, severe myoclonic epilepsy of infancy (SMEI), represents a severe form of the GEFS+ spectrum (76-78). SMEI, first described by Dravet in 1978, is a progressive syndrome of refractory seizures and cognitive decline. After a normal early infancy, patients present with prolonged generalized or unilateral febrile seizures at 5-12 months of age. Other seizure types, including myoclonic, absence, partial, and atonic seizures, begin between 1 and 4 years of age. Psychomotor slowing becomes apparent during the second year of life, and the patient may also experience obtunded states associated with myoclonus and develop ataxia, pyramidal dysfunction, and refractory generalized tonic-clonic and partial seizures. Intellectual outcome is generally poor. Although a family history of epilepsy had been previously recognized as common in SMEI, the nature of the seizure disorder in family members had not been systematically explored until recently. Singh et al. (76) described 12 SMEI pedigrees within which other family members exhibited forms of epilepsy consistent with the GEFS+ spectrum. Subsequent screening of additional SMEI patients for mutations in SCN1A has identified more than 100 additional mutations. The majority of SMEI mutations are de novo frame-shift, nonsense, or splice-donor mutations predicted to result in markedly aberrant, truncated forms of the channel protein. In contrast to the modest changes in gating seen with GEFS + mutations that cause single amino acid changes, the SMEI truncations would be expected to produce proteins incapable of conducting ions. Most recently, an additional clinical syndrome, intractable childhood epilepsy with generalized tonic-clonic seizures (ICEGTCS), which is quite similar to SMEI except that patients do not show myoclonic seizures, has also been shown to result from mutations in SCN1A (79).

These clinical and genetic observations are perplexing and lead to many questions concerning genotype-phenotype relations in the GEFS+/SMEI patients. How could gain-of-function mutations (i.e., the impaired inactivation seen in some GEFS+ patients) cause a mild syndrome, if loss-of-function mutations cause a severe phenotype (in SMEI and ICEGTCS). Cell biologic studies are beginning to shed light on this. In early studies, SCN1A was described as widely expressed by excitatory and inhibitory neurons, with Nav1.1 proteins observed on neuronal somata and proximal dendrites (80). Very recent experiments are revising this view, however. In hippocampus, both the protein and mRNA are reportedly much more expressed in inhibitory neurons than in excitatory pyramidal cells, leading to the suggestion that the severe phenotype of null mutations reflects a simple loss of inhibition (81). Furthermore, careful immunohistochemical studies have revealed Nav1.1 channels clustered in the initial segments of some cortical interneurons (82).

In short, the localization and relative importance of various subpopulations of Na+ channels is incompletely understood but is rapidly being addressed by current studies. Studies using transgenic mouse models that allow mutant channels to be studied in vivo are critical to future success, as are correlative studies of channel distribution in human tissue.

Screening of large clinical populations suggests, however, that amino acid coding mutations in SCN1A, SCN2A, and SCN1B account for only about 20% of familial cases of GEFS+ and a far lower percentage of cases of idiopathic generalized epilepsy (67, 68). This implies that other genes may be involved, and the search has begun. Recently, mutations in the GABRG2 and GABRD genes, which encode the gamma2 and delta subunits of the GABAA receptor, were also identified as causes of GEFS+ (83, 84). GABAA receptors are ligand-gated Cl~ channels (85). GABA released at inhibitory synapses binds to sites on the extracellular side of the channel, leading to channel opening and Cl~ influx and generating a fast inhibitory postsynaptic potential (IPSP). There are 16 human genes for subunits of GABA receptors: alpha1-6, beta1-4, gamma1-3, delta, epsilon, and theta. Each GABA receptor is a pentameric complex consisting of two alpha subunits, two beta subunits, and single subunit of class gamma, delta, epsilon, or theta. Although receptors containing a variety of different subunit combinations are represented in vivo, the most abundant and widely distributed form of the receptor is formed by alpha1, beta2, and gamma2 subunits. The known gamma2 subunit mutations range widely in the severity of their effects on channel function and the associated clinical phenotype. One mutation (R43N), found in a pedigree with a relatively mild seizure phenotypes (febrile seizures and childhood absence epilepsy), introduces a point mutation in the site for channel modulation by benzodiazepines such as diazepam and lorazepam (86). R43N mutant channels expressed in Xenopus oocytes exhibited only 10% reductions in GABA current amplitudes, though responsiveness to benzodiazepines was abolished completely. A second missense mutation (K289M), in an extracellular loop between the M2 and M3 transmembrane segments, was associated with a ~90% reduction in GABA currents and a more severe phenotype of febrile seizures and afebrile generalized tonic-clonic seizures (83). Finally, a truncation mutation in GABRG2 was found in a patient with SMEI within a branch of a large GEFS+ pedigree with bilineal inheritance of epilepsy (87). When expressed in oocytes or cultured mammalian cells, this truncation mutation (Q351X) acted as a potent dominant negative, coassembling with normal GABA receptor subunits and preventing them from leaving the endoplasmic reticulum (87). Beyond these direct effects on GABA receptor functioning, three of the GEFS+ mutations has been shown to impair channel transport to the membrane when expressed in cells maintained at elevated temperature (40°C); by contrast, wild-type channels did not show temperature dependence (88).

Very recently, a fourth GABRG2 mutation was found in a small pedigree with febrile seizures and no other forms of epilepsy (89). The mutation described (R139G) was located in the extracellular portion of the subunit, near the location where benzodiazepines bind and thereby enhance channel openings. R139G had a slight effect on function, speeding the rate of channel closing after an opening elicited by GABA. Although the number of families is very small, so far there is a direct correlation between the severity of epilepsy and the severity of the effects on channel gating observed in the four known GABRG2 mutations.

A single GEFS+ pedigree with four affected patients has been has been linked to a mutation in GABRD. Channels including the delta subunit encoded by GABRD are located at nonsynaptic sites on the soma and dendrites (90). Although delta-containing receptors are present at low density, they make important contributions to regulation of neuronal responsiveness because of their long-lasting openings (91).

Recessive Cortical Dysplasia—Focal Epilepsy Syndrome (CDFE; OMIM 610042). An Amish family was described in which 9 children were affected by a syndrome of cortical dysplasia, focal epilepsy, relative macrocephaly, and diminished deep-tendon reflexes (92). Intractable focal seizures began at 14-20 months of age. Subsequently, autism-like features, including language regression, hyperactivity, impulsive behavior, and mental retardation, developed in all children. Attempts to treat the refractory epilepsy by surgical removal of focal dys-plasic cortical regions did not prevent the recurrence of seizures. Pathologic studies of dysplasias showed abnormal neurons, altered neuronal migration, and gliosis. All affected children were found to be homozygous for a mutation in CNTNAP2, encoding the protein contactin-associated protein-like 2 (Caspr2). Caspr2 is a single transmembrane protein possessing a large extracellular domain with multiple protein-protein interaction modules. Remarkably, Caspr2 is localized on axonal membranes at the axon initial segments and the nodes of Ranvier (93, 94). Caspr2 is required for targeting the Kv1 subclass of voltage-gated potassium channels to these important axonal subdomains (95-97).

The Kv1 channel pore-forming subunits are encoded by KCNA genes. Six genes, KCNA1-6, are known to be expressed in the CNS, produced by gene duplication from a single ancient, ancestral gene. Indeed these genes are the homologs of a single fly gene, called Shaker. Flies with mutations in Shaker are hyperactive when undisturbed and, if exposed to low levels of anesthetic vapors, develop rapid shaking movements of their wings, legs, and abdomen that prevent normal behavior (98). Recordings from Shaker flies show failure of action potentials propagated along neuronal axons to repolarize normally (99). In mammals, the role played by Kv1 channels on axons undergoes a developmental switch during myelination (96, 100). Kv1 channels arrive near the node as it begins to form, and they play a key role in axonal action potential repolarization during the early stages of myelination. Once myelination is complete, Kv1 channels have a diminished role at nodes, because they are sequestered beneath the myelin sheath. However, they are also present on unmyelinated portions of the axon both proximally (i.e., the initial segments) and distally (at presynaptic terminals), and in these locations they contribute to control of initiation and neurotransmitter release (93, 101). The basis for epilepsy based on recessive inheritance of Caspr2 mutations is not established, but the connection with axonal excitability is particularly provocative in view of the better-established axonal roles of Nav and KCNQ subunits mutated in neonatal and infantile forms of epilepsy. This family study is also the first human example of recessive inheritance of epilepsy involving channel regulation. A large number of recessive epileptic channelopathies have been identified in inbred stains of laboratory mice (102); the relative lack of such mutations detected in human epilepsy reflects the uncommonness of consanguinity required for the rare pathogenic mutations to become symptomatic, and the difficulty of identifying mutations in small families.

Axonal Hyperexcitability: An Emerging Theme in the Hereditary Epilepsies of Neotates and Infants. From the foregoing discussion, it is clear that recent evidence has linked KCNQ channels, Nav1.2, Nav1.1, and Caspr2 mutations to forms of epilepsy involving neonates and/or infants. In parallel, cell biologic studies have revealed that all of these proteins have major roles in regulation of intrinsic axonal excitability in the developing nervous system. It seems clear that in these early-onset forms of epilepsy, disturbance of the ion channels that mediate the actual initiation and firing of action potentials is centrally important. It is not yet known why, for example, mutations in KCNQ2 should cause onset of seizures at an earlier age than mutations in Nav1.2. Further experiments should help to clarify the specific age-dependent phenotypes associated with various mutations and, in the process, the precise functions played by these channel types.

Channel Mutations in Epilepsies with Onset In Childhood and Adolescence

Autosomal Dominant Nocturnal Frontal Lobe Epilepsy (ADNFLE; OMIM 600513). In 1994, Scheffer et al. described a new epilepsy syndrome characterized by clusters of brief nocturnal motor seizures (103, 104). The attacks began in childhood (median age 8 years) and persisted throughout life, often misdiagnosed as night terrors, nightmares, hysteria, or paroxysmal nocturnal dystonia. Video-EEG recordings revealed that the nocturnal attacks were epileptic seizures heralded by frontally predominant sharp- and slow-wave activity (104), usually arising from stage 2 non-REM sleep. In spite of improved understanding of the disorder, clinical differentiation from parasomnias and other paroxysmal disorders of sleep can be challenging and may require video-EEG monitoring and polysomnography (105, 106).

Initial analysis of ADNFLE pedigrees suggested autosomal dominant inheritance with incomplete (~ 70%) penetrance. Subsequent molecular genetic studies have revealed mutations in genes for three subunits of neuronal nicotinic acetylcholine receptors (nAchRs) in affected individuals: CHRNA4 on chromosome 20, CNRNB2 on chromosome 1, and CHRNA2 on chromosome 8 (107-111).

Central nAchRs are pentameric cation channels, closely related to the well-studied nAchRs at skeletal muscle endplates, but are composed of alpha and beta subunits expressed only (or at least, primarily) by neurons. Binding of acetylcholine to sites on alpha subunits leads to channel opening and membrane depolarization. Although there are distinct genes for seven neuronal nAchR alpha subunits and three beta subunits, hetero-meric channels formed by coassembly of alpha4 and beta2 subunits appear to be the most abundant form expressed in brain. The subunits share a common basic structure, with a large, extracellular amino-terminal domain and four transmembrane alpha-helices. The second (M2) helix lines the transmembrane ion pathway and is believed to undergo a twisting conformational change after acetyl-choline binding that allows ions to flow.

The ADNFLE mutations in alpha4 or beta2 described so far all result in single amino acid changes at key positions within the pore-lining helices and might therefore be expected to cause changes in gating or ion conductance properties. Indeed, electrophysiologic experiments using the mutant subunits expressed in experimental cells such as Xenopus oocytes or mammalian cultured cells have revealed marked, albeit complex, changes in these properties (108, 112, 113). The a4 S248F mutant AchR responses showed faster desensitization, slower recovery from desensitization, less inward rectification, and virtually no Ca2+ permeability as compared with wild-type a4/p2 AChRs (112). Although these effects would all tend to reduce channel activity, this mutation also caused use-dependent up-regulation of activity and was more strongly activated by low levels of agonist, effects that might result in enhanced activity in vivo. Studies of the two known CHRNB2 mutants show differing effects: The V287M mutation shows normal kinetics but 10-fold increased Ach sensitivity (110), whereas the V287L mutation shows normal Ach sensitivity but 30-fold slower kinetics (109). The alpha2 mutation, I279N, is in the first transmembrane segment (M1) that links the extracellular ligand-binding domain to the pore-lining segment (M1). Recordings of transfected cells expressing either the mutant or the wild-type receptor showed that I279N markedly increases the receptor sensitivity to acetylcholine (109).

Immunolocalization studies show that the alpha4 subunit is widely expressed and is localized on neuronal somata and dendrites in cortical and subcortical structures, and it is particularly highly expressed by dopaminergic neurons in the substantia nigra (114). Labarca et al generated CHRNA mutant mice harboring a point mutation in the pore near the position of the ADNFLE mutations (115). This mutation caused 30-fold increased Ach sensitivity and is partially activated by choline at levels that are normally present in cerebrospinal fluid. Mice exhibited early lethality and loss of nigral dopaminergic neurons. More recently, investigators generated two lines of trans-genic mice expressing different ADNFLE mutations and found, paradoxically, a 20-fold increase in nicotine-induced GABAergic inhibitory postsynaptic currents in cortical neurons, with no change in excitatory currents (116).

How could increased inhibition lead to seizures? Because cortical GABAergic interneurons project widely to large numbers of neighboring pyramidal cells, they are powerful synchronizers of cortical output. Neurons in basal cholinergic nuclei project to the thalamocortical circuits that mediate transitions between sleep and wakeful-ness; acetylcholine release in these circuits contributes to arousal and alertness (117). Excessive cholinergic responsiveness during a critical stage of desynchronization of this circuit appears to be sufficient to cause seizures. The frontal lobe predominance of the semiology and epileptic discharges in ADNFLE patients is not explained by the distribution patterns of the receptors, which are widespread, but may reflect the inportance of the physiologic contribution made by the frontal lobes to the critical arousal transition that is disrupted in the disorder.

Episodic Ataxia with Myokymia (and Partial Epilepsy) (EA-1; OMIM 160120). EA-1 is a dominantly inherited disorder involving both the brain and peripheral nerves (118-120). Patients experience recurrent attacks of unsteady gait and loss of limb coordination lasting minutes to hours. This phenotype suggests an intermittent derangement of cerebellar function. In some cases, transient cognitive deficits accompany the motor symptoms. Attacks may be provoked by a sudden stress or startle. Myokymia is continuously present in many EA-1 patients. This myokymia is unaffected by pharmacologic block at the proximal portion of peripheral nerves but is reduced by distal nerve block and abolished by inhibition of neuro-muscular transmission. This observation suggests that, unlike myotonia or epilepsy, myokymia results from abnormal hyperactivity in the peripheral nerve. However, patients with EA-1 have about a 10-fold greater risk of epileptic seizures compared to the general population (118, 121).

In 1994, Browne et al linked EA-1 to missense mutations in KCNA1 (120). KCNA1, one of the mammalian Shaker homologs, encodes voltage-gated potassium Kv1.1 channels that, as discussed previously in connection with the severe CDFE syndrome, are targeted to axons by interaction with Caspr2. Extensive further work, including physiologic studies of the mutant channels, anatomical studies of KCNA channel localization in the central nervous system and peripheral nerve, and studies of knockout and knockin mice, gives useful clues for understanding the basis of the EA-1 phenotype in the brain and peripheral nerve (14). Studies in Xenopus oocytes indicate that the mutations causing EA-1 result in reductions in KCNA1 expression and current magnitude (121-123). In some cases, EA-1 mutant channels in heterologous cells exhibit clear "dominant negative" effects, interacting with coex-pressed normal channels and preventing them from functioning normally or trafficking to the cell surface. Other EA-1 mutants exhibit simple loss-of-function properties in vitro. As discussed previously, Kv1 family channels have been localized along axons and on presynaptic terminals (101, 124-126). Smart et al. generated knockout mice lacking KCNA1 (127). Interestingly, mice heterozygous for the knockout allele appear normal; this is in contrast to the dominantly inherited effects of EA-1 mutations in humans. Mice that are homozygous for the knockout allele have subtle defects in cerebellar function (128), but the most obvious aspects of their phenotype are very frequent generalized epileptic seizures and associated premature death (127). It has proved difficult to identify the alterations in cellular physiology that cause frequent spontaneous seizures in the Kv1.1 knockout mice; hippocampal slices prepared from the knockouts exhibit remarkably normal intrinsic neuronal and synaptic properties (127). A possible explanations for this was that channels containing the subunit are often localized to small axons and presynaptic termini, which are difficult to study directly using currently available physiologic methods (126). An interesting clue to the pathophysiology of EA-1 is the responsiveness of the condition in some but not all pedigrees to treatment with the carbonic anhydrase inhibitor acetazolamide (129). To further understand the EA-1 phenotype, Herson et al generated knockin mice bearing a human EA-1 point mutation (130). Heterozygous mutant mice exhibited stress-induced loss of coordination that was ameliorated by pretreatment with acetazolamide. Recordings from brain slices suggest that expression of the mutant subunits results in increased neurotransmitter release at presynaptic terminals after invasion by action potentials. Seizures were not reported in the knockin heterozygous mutants, and the homozygous state was lethal early in embryonic development.

As a larger number of patients with EA-1 have been identified, it has become clear that they differ in the severity of their symptoms over a broad spectrum and that more severe forms of the disorder are often associated with epilepsy. Zuberi et al described a family containing five patients with episodic ataxia and myokymia, two of which additionally had partial complex seizures (121). Eunson et al described a second family with this combination of symptoms, as well as families with severe and acetazolamide-resistant ataxia, or isolated myokymia (131). When studied in heterolo-gous cells, the mutations associated with severe ataxia and epilepsy caused severe reductions in channel activity (132). In some cases, the EA-1 plus epilepsy mutant subunits were capable of dominant-negative interactions with wild-type subunits; that is, they were capable of coassembling with the wild-types, but the resulting channels were hypofunctional because of failure of intracel-lular trafficking or lack of intrinsic channel activity (132). Trafficking defects are likely to be particularly critical in neurons, because the KCNA channel proteins are normally localized to sites along myelinated and nonmyelin-ated axons and at presynaptic terminals, which are very distant from their sites of translation and assembly in the endoplasmic reticulum (125, 126, 133).

Autosomal Dominant Juvenile Myoclonic Epilepsy (JME; OMIM 606904). JME is characterized by the onset, usually during adolescence, of bilateral single or repetitive myoclonic jerks, occurring most commonly upon morning awakening (134). A majority of patients have at least one generalized tonic-clonic seizure, and about 15% have absences. A characteristic EEG finding is frontally predominant bilateral 4-6 Hz polyspike-wave discharges. JME is not uncommon and represents approximately 4-6% of all patients with epilepsy (134). Nevertheless, identifying genes for JME has been particularly difficult, likely as a result of the genetic heterogeneity and complexity underlying the syndrome. Recently, mutations in genes for a GABA receptor subunit and a subtype of voltage-gated Cl~ channel were identified.

Cossette et al. ascertained a large family in which epilepsy, with characteristic clinical and electroencepha-lographic features of common JME, was inherited in an autosomal dominant pattern (135). A missense mutation was found in GABRA1, resulting in an A322D change in a conserved site in the third transmembrane helix of the GABA receptor alpha1 subunit. Two alpha subunits are present in each pentameric GABA receptor; each of these subunits contributes both to a binding site for GABA and to the transmembrane pore. Peak currents mediated by receptors that included the A322D GABA alpha1 mutants were only 10% of those of wild-type receptors, and this weak response required 200-fold higher concentrations of GABA. Accordingly, the epileptic phenotype would be expected to be associated with greatly reduced responsiveness to inhibitory neurotransmission. It is unclear why this mutation results in epilepsy of the JME type.

A genomewide linkage analysis of 130 families with idiopathic generalized epilepsy (IGE) identified a susceptibility locus on chromosome 3q (135). The gene CLCN2, encoding a voltage-gated Cl~ channel protein

(called ClC-2 or CLC-2), was identified within the linked region and sequenced for mutations in samples from 46 families (137). Three mutations were identified among these families, one in a small family with 5 patients with JME. CLC-2, like other members of the chloride channel family, forms dimeric channel complexes. Hereti-cally, each chloride channel subunit possesses its own transmembrane ion path, and gating of the two pores within each channel is regulated by allosteric interactions within the protein (10). The JME mutation identified in the single family produced a truncated form of the protein that was unable to conduct ions itself or coassemble with wild-type subunits.

Epilepsy with Grand Mal Seizures on Awakening (EGMA, OMIM 607628) and Childhood Absence Epilepsy (CAE; OMIM 600131). JME overlaps with another syndrome, termed "awakening epilepsy" or "epilepsy with grand mal seizures on awakening." Patients with EGMA have difficulty falling asleep, abnormal sleep architecture, and the vast majority of their seizures occur clustered within the first minutes after awakening from sleep (irrespective of the time of day) (138). A CLCN2 mutation encoding a nonfunctional channel was identified in a large family with EGMA (137).

A subset of JME and EGMA patients experience absence spells. A third CLCN2 mutation was found to segregate in a small family in which three children had typical childhood absence epilepsy (CAE) (137). This CAE-linked mutation resulted in a single amino acid change (G715E); mutant channels were reported to be functional but were reported to exhibit altered voltage dependence. This finding has been contradicted by two subsequent investigations showing activity of the mutant indistinguishable from wild type, further weakening the modest genetic evidence provided by the small family studied (10). A more recent survey of 55 families with IGE failed to identify additional CLCN2 mutations (138). At present, the strongest evidence linking CLCN2 to generalized epilepsy is provided by the large EGMA pedigree.

Autosomal Dominant Partial Epilepsy with Auditory Features (ADPEAF, OMIM 600512). In 1995 Ottman et al described a large family in which partial seizures associated with unusual auditory auras were inherited in an autosomal dominant pattern linked to a gene on chromosome 10 (140). Several additional families with this distinctive syndrome were subsequently identified (141, 142). The age of seizure onset was typically at 9 to 12 years of age. Seizures are heralded by a variety of stereotyped, but in nearly all cases nonverbal, auditory hallucinations (141). Mutations were subsequently identified in the gene Lgi1, a gene of unknown function previously shown to be disrupted in some patients with primary brain tumors (143). This set in motion efforts to understand the function of the Lgi1 protein in brain development, auditory processing, and epileptogenesis.

At present, evidence supporting two conflicting models has been described. One set of experiments shows that the Lgi1 protein is physically associated with and enhances the activity of Kv1 family voltage-gated potassium channels whose pore-forming subunits are encoded by the KCNA genes (144). In this model, Lgi1 is a cyto-solic protein that binds to the intracellular portion of channels that contain both pore-forming subunits and previously described channel intracellular accessory units, called beta subunits. The beta1 subunit causes Kv1 channels to close after a period of milliseconds of membrane depolarization. This time- and voltage-dependent closing, called inactivation, is similar to the fast inactivation of sodium channels that helps terminate each action potential. However, inactivation of K+ channels will generally tend to increase neuronal excitability. Wild-type Lgi1 proteins antagonize this inactivation, prolonging channel opening. Mutations derived from ADPEAF patients, which produce truncated proteins, all abolish the ability of the Lgi1 protein to prolong the K+ channel opening. This model is consistent with previously discussed findings that loss-of-function KCNA1 mutations that cause epilepsy in some patients with EA-1, and mutations in Caspr2 that disrupt Kv1 channel targeting in the recessive focal epilepsy.

The alternative, and very different, model is based on the recent report that Lgi1 protein is packaged in secretory vesicles and released into the synaptic cleft, where it binds tightly to a cluster of proteins linked to postsynaptic glutamate receptors (145, 146). Lgi1 binds directly to ADAM22, and is thereby linked to PSD-95 and stargazin, all of which are components of the excitatory postsynaptic density. Application of soluble Lgi1 proteins to brain slices in vitro results in enhancement of alpha-amino-3-hydroxy-5-methylisoxazole-4-propionic acid (AMPA)-type glutamate receptor currents. Lgi1 applied to neurons grown in dissociated culture increases the number of AMPA receptors at the cell surface. Mutations in stargazin and ADAM22 that are capable of disrupting the protein complex are associated with epilepsy in mice (although human mutations in these genes are unknown) (146-148). Effects of Lgi1 mutations on this complex and the implications for receptor function are incompletely known, but the implication is that mutations would tend to reduce postsynaptic excitatory currents.

How can the two proposed functional roles of Lgi1 be reconciled? Immunoblots of proteins separated by sodium dodecyl sulfate (SDS) gel electrophoresis reveal two forms of Lgi1 that differ by 3-5 kDa, indicating that the protein is either alternatively spliced or post-translationally processed (150). Moreover, the larger form localizes to the cytoplasm, whereas the smaller is found in a micro-somal fraction containing secretory vesicles. Although it is not clear which form is most important for epilepsy, hippocampal and cerebellar cortex (areas with particularly high densities of AMPA-type glutamatergic synapses) showed predominantly the smaller, presumed secreted form. The highest percentage of the cytosolic, high-molecular-weight form was present in temporal neo-cortex (150). This result favors the idea that the cytoplas-mic interaction with potassium channel contributes to the auditory auras and seizures in ADPEAF, but additional studies are needed.

Generalized Epilepsy with Paroxysmal Dyskinesias (OMIM 609446). Clinical studies have noted patients, sometimes in familial groups, with a combination of generalized epilepsy and paroxysmal dyskinesia (151, 152). Du et al. described an unusual family with 16 affected individuals in five consecutive generations, strongly indicating dominant inheritance. Seven had paroxysmal non-kinesogenic dyskinesias, four developed epilepsy only, and five exhibited both symptoms. The symptom onset age ranged broadly from 3 to 15 years, with one patient developing seizures before 6 months. After establishing linkage to chromosome 10, the investigators identified a mutation in KCNMA1, which encodes a pore-forming alpha subunit of a type of calcium-activated potassium channel called BK ("Big-K"; also "Maxi-K") because these channels pass ions at higher rates than other K+ channel types. BK channels are activated synergistically by membrane depolarization and elevated intracellular Ca2 + (1). Their opening rates are very fast, enabling them to speed repolarization of action potentials, shortening the sodium channel refractory period and thereby facilitating rapid neuronal bursting. The pathogenic mutation in BK identified increases channel activity (153). Interestingly, a knockout mutation in a BK accessory subunit has also recently been shown to cause epilepsy in transgenic mice (154). Careful electrophysiologic analysis of these mice indicates that this mutation also increases the activity of BK, leading to increased neuronal firing. The detailed biophysical mechanism whereby excitability is increased in the mice is intriguingly indirect. First, the enhanced BK activity and associated shortened action potential duration causes a reduction in Ca2+ entry during each spike. This diminishes the activation of a second type of Ca2+-activated K+channel, known as SK ("small-conductance K+ channel"). Despite their name, SK channels very potently limit excitability by means of their long-lasting openings. So preventing SK activation (by increasing BK openings) leads to markedly increased cellular excitability. In effect, the cell must balance BK and SK activity precisely to maintain the desired overall level of excitability. This elegant electrophysiologic analysis again illustrates the importance of transgenic mouse models for current studies of the epileptic channelopathies, and it begins to hint at how very indirect the effects of a mutation can be.

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