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The role of calcium channel mutations in human epilepsy
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The role of calcium channel mutations in human epilepsy
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Antonio Gambardella1, Angelo Labate Institute of Neurology, Department of Medical Sciences, University Magna Graecia, Catanzaro, Italy 1 Corresponding author: Tel.: +39-0961-3647270; Fax: +39-0961-3697177, e-mail address: [email protected]
Abstract Molecular insights into monogenic idiopathic epilepsies have illustrated the central role of channelopathies in their etiology. Among ion channels, both high- and low-voltage-activated calcium channels and their ancillary subunits Cav2.1 (P/Q-type) calcium channels support a number of dynamic processes in neurons at both presynaptic and postsynaptic levels being critical determinants of neuronal excitability. Therefore, their alterations in the expression or biophysical properties may have a central role in the pathogenesis of epilepsy phenotypes. Indeed, low-voltage-activated (T-type) calcium channels are critically involved in normal burst firing in the thalamocortical circuitry recruited in the spike-wave discharges underlying absence seizures. Moreover, gain-of-function mutations have been identified in several calcium channel genes in both epilepsy patients and animal models of epilepsy, further underpinning the role of calcium channels in epilepsy pathophysiology. Thus, the selective pharmacological blockade of calcium channel subtypes may provide attractive targets for the development of antiepileptic therapies.
Keywords calcium channel, P/Q-type channels, T-type channels, epilepsy, seizures
1 INTRODUCTION The international classification of epilepsies divides both focal and generalized epilepsies into idiopathic syndromes where the cause is believed to be genetic and cryptogenic/symptomatic syndromes where acquired factors are thought to predominate (ILAE, 1989). It has become increasingly clear, however, that many, and perhaps most epilepsies, display a complex pattern of inheritance, and that various genetic inputs on the one hand, and various acquired factors on the other, act in a different way in each patient (Berkovic et al., 2006). So far, success in identifying single genes Progress in Brain Research, Volume 213, ISSN 0079-6123, http://dx.doi.org/10.1016/B978-0-444-63326-2.00004-1 © 2014 Elsevier B.V. All rights reserved.
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has come from rare monogenic idiopathic epilepsy syndromes, and the prospective will be on extending these findings to the most common polygenic epilepsies with complex inheritance. Most of the established major mutations are in genes that encode for ion channels or their accessory subunits (Graves, 2006). This has led to the concept that idiopathic epilepsies are usually a family of channelopathies, even though the link between molecular deficit and clinical phenotype remains blurred. Moreover, it is particularly challenging to understand the large phenotypic variability observed in these single gene epilepsies that range from mild phenotypes to very severe ones. It is reasonable to hypothesize that, even in such monogenic epilepsies, a more complex interaction between genetic and acquired factors modulates disease severity of a produced phenotype. These challenges are even greater in complex epilepsies in which it also remains open to discussion whether ion channels are strong functional candidates (Cavalleri et al., 2007). It is also important to highlight that almost all ion channel epilepsy mutations express themselves in a heterozygous manner, which is evident only in one allele. The pathological impact of each mutation is therefore either due to a dominant effect of the mutant protein within neurons (or glia) or a loss of function due to haploinsufficiency (i.e., a loss of ability to generate sufficient protein from the one “working” allele). Among these ion channels, both high- and low-voltage-activated calcium channels and their ancillary subunits Cav2.1 (P/Q-type) calcium channels support a number of dynamic processes in neurons with both presynaptic and postsynaptic Ca2+ channels being critical determinants of neuronal excitability (Catterall, 2011). Therefore, mutations in genes encoding calcium channels are excellent candidates in the context of epilepsy phenotypes (Cain and Snutch, 2012; Zamponi et al., 2010). The remainder of this chapter addresses the state of knowledge on the roles of voltage-gated calcium channels, their mutations, and how they might contribute to the pathophysiology of human epilepsies. Table 1 summarizes these findings.
2 CALCIUM CHANNEL NOMENCLATURE AND BIOPHYSICAL PROPERTIES Voltage-gated Ca2+ channels reside in the plasma membrane of excitable cells, and open in response to membrane depolarization, thereby allowing calcium ion entry to cells (Catterall, 2011). They are broadly classified into high-voltage-activated (HVA) or low-voltage activated (LVA) calcium channels (Fig. 1), with HVA channels requiring larger membrane depolarizations for activation (Catterall, 2011; Ertel et al., 2000). HVA channels are made up of one or more pore-forming a1 subunit that coassembles with ancillary subunits and can be further subdivided into L-, N-, P/Q(Cav2.1), N- (Cav2.2), and R- (Cav2.3) types (Fig. 1), by virtue of their distinct functional and pharmacological profiles (Catterall, 2011). Conversely, LVA channels, also referred to as “T-type channels,” are thought to be a1 subunit monomers and are further classified according to their a1 subunit composition into Cav3.1, Cav3.2, and Cav3.3 isoforms (Fig. 1), each with unique biophysical,
2 Calcium channel nomenclature and biophysical properties
Table 1 Genes and diseases for inherited calcium channelopathies Calcium channela
Current type
Principal physiological functions
Gene and chromosome
Inherited diseases
Cav1.1
L
CACNA1S; 1q31-32
HPP
Cav1.2
L
CACNA1C; 12p13.3
TS
Cav1.3
L
CACNA1D; 3p14.3
Not reported
Cav1.4
L
Excitation–contraction coupling in skeletal muscle, regulation of transcription Excitation–contraction coupling in cardiac and smooth muscle, endocrine secretion, neurons, regulation of enzyme activity, regulation of transcription Endocrine secretion, cardiac pacemaking, neurons, auditory transduction Visual transduction
SNB
Cav2.1
P/Q
Cav2.2
N
Cav2.3
R
Cav3.1
T
Cav3.2
T
Cav3.3
T
CACNA1F; Xp11.23 CACNA1A; 19p13 CACNA1B; 9q34 CACNA1E; 1q25 CACNA1G; 17q22 CACNA1H; 16p13.3 CACNA1I; 22q12.3
Neurotransmitter release, dendritic Ca2+ transients Neurotransmitter release, dendritic Ca2+ transients Neurotransmitter release, dendritic Ca2+ transients Pacemaking and repetitive firing Pacemaking and repetitive firing Pacemaking and repetitive firing
EA, FHM, SCA6
AS Not reported
AS, absence seizures; CACNA, a1 subunit gene encoding voltage-dependent Ca2+ channels; EA, episodic ataxia; FHM, familial hemiplegic migraine; HPP, hypokalemic periodic paralysis; SNB, stationary night blindness; SCA6, cerebellar ataxia type 6; TS, Timothy syndrome (cardiac arrhythmia with developmental abnormalities and autism spectrum disorders). a The cloned voltage-gated calcium channels a1-subunits are presented following the proposed nomenclature (Ertel et al., 2000).
pharmacological, and regulatory properties (Catterall, 2011). Additional structural and functional variants of each Cav subtype can be generated by alternative splicing to produce a large number of different “splice variants” and therefore increase the repertoire and complexity of calcium channel properties (Catterall, 2011). Calcium channels are usually slower at opening (activation) and closing (deactivation) than other voltage-activated channels. In detail, HVA channels display slower activation and faster deactivation than LVA channels. Moreover, HVA channels usually inactivate much more slowly than LVA channels. All together, these
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FIGURE 1 Voltage-gated calcium channels. Schematic representation of the high-voltage-activated calcium channel complex showing the main pore-forming a1 subunit and ancillary, b-, g-, and a2-d-subunits. Low-voltage-activated calcium channels may consist of only the a1-subunit. (Modified, with permission, from Physiological Reviews; Khosravani and Zamponi, 2006, Vol. 86, Issue 3, The American Physiological Society)
properties make HVA channels generate longer lasting calcium influxes upon sustained depolarizations, while T-type channels conduct more rapid and shorter calcium influxes under both brief and sustained depolarizations (Catterall, 2011). In the nervous system, the three T-type calcium channels are often expressed differently in the same cell types; thus, a wide repertoire of diverse net LVA calcium currents can be generated depending on the relative contribution from each subtype. Moreover, the spectrum of T-type calcium channel types is made more complex by the occurrence of multiple alternatively spliced isoforms, adding further functional complexity to the overall spectrum of T-type currents (Catterall, 2011). Then, distinct T-type channel isoforms exhibit a differential distribution across somatic, dendritic, and axonal compartments, indicating that compartmental factors also contribute to excitability and cellular output. HVA calcium channels, P/Q-type, N-type, and, to some extent, R-type channels, are expressed highly at presynaptic nerve terminals where their activities evoke neurotransmitter release (Rajakulendran et al., 2012). The different subtypes support a variety of biological processes, including gene expression, neuronal architecture development, signal propagation, and neurotransmitter release from nerve terminals. Among the different channel subtypes, the related subfamily of P/Q channels (Cav2.1), N channels (Cav2.2), and R channels (Cav2.3) are commonly known as the neuronal channels. N and P/Q channels are the principal subtypes involved in neurotransmission. P/Q channels are thought to be predominantly located at central synapses, whereas N channels are more prominent in peripheral nerve terminals, particularly those involved in sensory and autonomic function. The P/Q channels are widely expressed throughout the CNS, especially in Purkinje and granule cells of the cerebellum, where they also have important postsynaptic roles
3 Calcium channels in epilepsy
in calcium signaling. By linking calcium influx to activation of AMPA (a-amino-3hydroxy-5-methyl-4-isoxazolepropionic acid) receptors, these channels are thought to have a role in bidirectional cerebellar plasticity (Catterall, 2011).
3 CALCIUM CHANNELS IN EPILEPSY 3.1 T-TYPE CALCIUM CHANNEL MUTATIONS IN EPILEPSY T-type calcium channels are critically involved in normal burst firing in the thalamocortical circuitry recruited in the spike-wave discharges that underlie absence seizures (Heron et al., 2007) and in the intrinsic burst firing of hippocampal pyramidal neurons in temporal lobe epilepsy (TLE) (Cain and Snutch, 2012). The three T-type calcium channels (Cav3.1, Cav3.2, and Cav3.3) are widely but differentially expressed in the thalamocortical circuitry implicated in absence seizures. The role of T-type channels in absence epilepsies is also highlighted by the anticonvulsive effects of ethosuximide, an inhibitor of T-type Ca2+ currents, in the treatment of absence seizures (Coulter et al., 1989). It is therefore not unexpected that variants in the T-type calcium channel gene CACNA1H which encodes a low-threshold T-type Ca2+ channel were initially associated with childhood absence epilepsy (CAE) and have since been described in other epilepsy phenotypes (Chen et al., 2003; Heron et al., 2007; Khosravani et al., 2005; Vitko et al., 2005). Initial studies by Chen et al. (2003) found 12 rare missense mutations in 14 of 118 patients suffering CAE, suggesting that it may be a susceptibility gene involved in the pathogenesis of this disease. Nonetheless, evaluation of 44 pedigrees and 220 unrelated patients with CAE of Caucasian European origin failed to find evidence for mutations in CACNA1H (Chioza et al., 2006), suggesting that some genetic variants contribute to disease predisposition in an apparently populationspecific manner. Some of the alterations in Cav3.2 have been shown to induce altered biophysical properties or increase channel expression when examined in exogenous expression systems. However, some have no apparent effect, potentially reflecting the polygenic nature of idiopathic generalized epilepsies, and/or that a subset of the changes represent single nucleotide polymorphisms (Heron et al., 2007). There are also a large volume of data from experiments on animal models of both genetic generalized epilepsy (GGE) and acquired TLE that have implicated these channels as key players in regulating neuronal excitability (Cain and Snutch, 2012; Zamponi et al., 2010). In the kindling model of TLE, T-type calcium currents were significantly larger in CA1 pyramidal cells of animals that had experienced kindled seizures compared to controls, and these current changes persisted up to 6 weeks after the cessation of kindling (Cain and Snutch, 2012; Zamponi et al., 2010). Moreover, several studies investigating the role of T-type calcium channels during epileptogenesis after pilocarpine-induced status epilepticus found that there was a selective and transient increase in Cav3.2 mRNA expression in CA1 pyramidal neurons coupled with an upregulation of T-type calcium currents (Cain and Snutch, 2012; Zamponi et al., 2010). Of note, hippocampal sclerosis and mossy fiber sprouting, histopathological hallmarks of TLE in humans and animal models, were absent in
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Cav3.2 knockout mice, and these mice were resistant to the development of chronic seizures induced by pilocarpine (Cain and Snutch, 2012; Zamponi et al., 2010). There is also substantial evidence in the literature from human and animal studies linking T-type calcium channels to the pathogenesis of GGEs. Indeed, a mutation in the rat Cav3.2 gene (R1584P) has been identified in the GAERS animal model of GGE with absence seizures. It has been also illustrated that Cav3.1 knockout mice lacked the burst firing mode of action potentials in thalamocortical neurons, seen during absence seizures, whereas the normal tonic mode of firing was unaffected (Cain and Snutch, 2012; Zamponi et al., 2010). Conversely, transgenic mice overexpressing Cav3.1 showed increased functional thalamic T-type calcium currents and frequent bilateral rhythmic SWDs that could be blocked by treating with ethosuximide (Cain and Snutch, 2012; Zamponi et al., 2010). All these experimental data further illustrated the role of T-type calcium channels in the pathophysiology of GGE; nonetheless, the contribution of Cav3.1 T-type calcium currents to the generation of absence seizures is still matter of discussion. Increased Cav3.1 T-type calcium currents have been reported to be sufficient to induce pure absence epilepsy, while in a study utilizing Cav2.1 mutant mice double-crossed with Cav3.1 mutant mice it was found that increased thalamic Cav3.1 T-type calcium currents are not essential for the generation of absence seizures (Cain and Snutch, 2012; Zamponi et al., 2010). Increased mRNA expression of T-type calcium channels are also evident in animal models of GGE compared to controls, especially in the lateral geniculate nucleus and centrolateral nucleus of the intralaminar nuclei in WAG/Rij, and in the ventral posterior thalamic relay nuclei of adult GAERS, as well as in the thalamus in both GAERS and WAG/Rij rats. All these data indicate that increases in any of the T-type calcium channels are important factors in contributing to GGE phenotype in these animal models (Cain and Snutch, 2012; Zamponi et al., 2010).
3.2 P/Q-TYPE CALCIUM CHANNEL MUTATIONS IN EPILEPSY The CACNA1A gene on chromosome 19 encodes both P-type and Q-type channels, also known as Cav2.1 P/Q-type calcium channels subunit, through an alternate splicing mechanism. These channels are highly expressed presynaptically where they are critically involved in neurotransmission and synaptic efficacy and therefore have a great influence on neuronal excitability. This aspect is reflected by a number of mutations in the Cav2.1 gene identified in patients suffering from severe neurological disorders including ataxias and congenital migraine (Rajakulendran et al., 2012). So far, three autosomal dominant allelic diseases are associated with mutations of CACNA1A gene (Rajakulendran et al., 2012): (1) familial hemiplegic migraine (FHM) that is a rare and severe variant of migraine with aura, characterized by disabling attacks of hemiparesis and hemisensory disturbance, which can last from hours to days, and visual disturbance. (2) Episodic ataxia type 2 (EA2), whose clinical features include intermittent ataxia, headache, and vertigo, and acetazolamide is an effective treatment. (3) Spinocerebellar ataxia type 6 (SCA6), which is a late-onset, progressive, cerebellar syndrome characterized by impaired balance, limb
3 Calcium channels in epilepsy
incoordination, and dysarthria. The prevalence of SCA6 seems to be highest in Japan, and the mean age of onset is in the fifth decade of life, although onset in patients in their twenties has been described. Although these three diseases are conventionally described as distinct, they exhibit considerable overlap in clinical features (Rajakulendran et al., 2012). Moreover, kindreds have been reported in which different affected members appear to suffer from different syndromes within this spectrum. Furthermore, a small proportion of patients with FHM and with underlying mutations in the Cav2.1 channel also display both generalized and complex partial seizures (Rajakulendran et al., 2012). In detail, a heterozygous mutation in CACNA1A (P/Q-type Ca2+ channel) has been described in one individual with a complex phenotype that included generalized epilepsy ( Jouvenceau et al., 2001). Further, a small family exhibiting absence epilepsy combined with ataxia has a mutation in CACNA1A (Imbrici et al., 2004). Functional studies implicate a loss of P/Q-type Ca2+ channel function in both cases (Imbrici et al., 2004; Jouvenceau et al., 2001). Similarly, a mutation in CACNB4 (b4 subunit) described in one patient with JME resulted in a loss of protein function (Escayg et al., 2000). Regarding the genotype–phenotype relationship, there is evidence that FHM, EA2, and SCA6 are generally caused by distinct defects of CACNA1A (Rajakulendran et al., 2012). FHM is associated with several missense mutations affecting conserved residues located throughout the a1 subunit. These mutations are thought to confer a gain of function on the P/Q channel. Supportive evidence was provided by a study of knock-in mouse models that demonstrated an increase in calcium current density and neurotransmission at the neuromuscular junction, with a susceptibility to cortical spreading depression, which is considered to be the physiological correlate of the aura in migraine (Rajakulendran et al., 2012). In contrast, EA2 is generally associated with premature stop codons and splice site mutations, predicted to result in a truncated peptide, which cause a profound or total loss of P/Q channel function (as measured by calcium current density; Rajakulendran et al., 2012). Moreover, some evidence initially pointed to haploinsufficiency as the underlying disease-causing mechanism. Nonetheless, later studies have illustrated that mutant calcium channels might instead exert a dominantnegative effect by interfering with correct folding and trafficking of wild-type channels, causing them to be retained in the endoplasmic reticulum (Rajakulendran et al., 2012). SCA6 is unique among the channelopathies in that it is caused by an expansion of a polyglutamine repeat in the intracellular C-terminus of the channel and symptoms are typical not episodic, but rather slowly progressive. Current pathophysiological hypothesis is in favor of a direct toxic effect of the polyglutamine expansion in Purkinje cells, which degenerate in SCA6, particularly in the cerebellar midline. There is also evidence that genetic variation in the P/Q channel might be associated with a predisposition to seizure disorders (Rajakulendran et al., 2012). Absence seizures are generally thought to arise as a result of abnormal reverberation in thalamocortical loops, as opposed to an increase in the ratio of excitatory
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(glutamatergic) versus inhibitory (GABAergic) signaling. Accordingly, a relatively selective decrease in glutamate release from thalamic synapses has been reported in one of the mutant mouse strains (Rajakulendran et al., 2012). Moreover, EEG recordings of tottering, leaner, rocker, and tg-4J strains of mice carrying spontaneous recessive mutations in the CACNA1A gene displayed generalized spike-and-wave discharges at a 5–7 Hz frequency, in combination with cerebellar ataxia and degeneration (Rajakulendran et al., 2012). Furthermore, although absence seizures are not a consistent feature of the known human CACNA1A mutations, EEG abnormalities have been reported in patients with acetazolamide-responsive ataxia (Rajakulendran et al., 2012), and polymorphisms in the CACNA1A gene are in linkage disequilibrium with primary generalized epilepsy (Chioza et al., 2001). A combination of seizures and episodic ataxia has also been tentatively linked to the accessory b4 calcium channel subunit (Escayg et al., 2000). Despite these findings, the functional consequence of this mutation and the broader role of Cav2.1 in human absence epilepsy remain to be determined.
3.3 ANCILLARY SUBUNITS OF VOLTAGE-GATED CALCIUM CHANNELS IN SEIZURE DISORDERS Ancillary calcium channel subunits are important regulators of HVA calcium channel function. So far, mutations in either g- or a2-d-subunits have so far not been linked to epilepsy in humans; however, there are several mouse phenotypes associated with these subunits (Cain and Snutch, 2012; Zamponi et al., 2010). The b, a2-d, and g ancillary calcium channel subunits that modulate the biophysical properties and expression of the HVA a1 subunits have been implicated in animal models of absence epilepsy and ataxia (Cain and Snutch, 2012; Zamponi et al., 2010). There is also some evidence for changes in calcium channel function via altered b-subunit expression patterns in TLE (Cain and Snutch, 2012; Zamponi et al., 2010). A role of HVA calcium channel interacting proteins is also exemplified in the case of juvenile myoclonic epilepsy patients that carry a mutation in the calciumbinding protein EFHC1 that under normal conditions enhances R-type channel activity, an effect that is abolished by the mutations in this protein (Cain and Snutch, 2012; Zamponi et al., 2010). However, mice deficient of R-type channels do not display seizure activity, and hence, it is not clear if the clinical manifestation of the EFHC1 mutation is directly related to the activity of R-type channels. On this basis, the effects of mutations in ancillary subunits appear to cause an inhibition of P/Q-type channel activity, although it is not proven that exclusively these channels mediate such physiological effects.
4 CONCLUSION Calcium ion channels are critical for normal neuronal excitability, so it is not surprising that the majority of heritable defects so far identified in human epilepsies code for ion channel subunits. Nonetheless, there is no unifying mechanism that can explain
References
how the spectrum of the observed functional effects of epileptogenic mutations relates to the epilepsy syndromes seen in patients. There is indeed functional heterogeneity among mutant channels, revealing a complex relationship between clinical and biophysical phenotypes. Because of this, a genetic classification of clinical epilepsies is currently elusive. It is also becoming increasingly clear that, even in Mendelian epilepsies, specific phenotypes probably result from the cumulative effects or interactions of a few or several genes, of which the identified one is only a player. Moreover, recent studies provided evidence that defective protein trafficking and protein–protein interactions may modulate the effect of mutation and, so, underlie phenotypic variation in such epilepsies related to channelopathies (Rusconi et al., 2007). The diversity of mutations that can cause a similar phenotype argues for points of physiological convergence that can give rise to a definite network hyperexcitability able to selectively cause specific phenotypes. This may be the case not only for Mendelian epilepsies but also in epilepsies with complex inheritance where gene discovery is just beginning. The development of animal models and multielectrode array experiments will probably be useful in investigating this point. Identifying such specific epileptogenic networks is essential for deeper understanding of mechanisms of epileptogenesis and for designing therapeutic strategies.
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The role of calcium channel mutations in human epilepsy
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Antonio Gambardella1, Angelo Labate Institute of Neurology, Department of Medical Sciences, University Magna Graecia, Catanzaro, Italy 1 Corresponding author: Tel.: +39-0961-3647270; Fax: +39-0961-3697177, e-mail address: [email protected]
Abstract Molecular insights into monogenic idiopathic epilepsies have illustrated the central role of channelopathies in their etiology. Among ion channels, both high- and low-voltage-activated calcium channels and their ancillary subunits Cav2.1 (P/Q-type) calcium channels support a number of dynamic processes in neurons at both presynaptic and postsynaptic levels being critical determinants of neuronal excitability. Therefore, their alterations in the expression or biophysical properties may have a central role in the pathogenesis of epilepsy phenotypes. Indeed, low-voltage-activated (T-type) calcium channels are critically involved in normal burst firing in the thalamocortical circuitry recruited in the spike-wave discharges underlying absence seizures. Moreover, gain-of-function mutations have been identified in several calcium channel genes in both epilepsy patients and animal models of epilepsy, further underpinning the role of calcium channels in epilepsy pathophysiology. Thus, the selective pharmacological blockade of calcium channel subtypes may provide attractive targets for the development of antiepileptic therapies.
Keywords calcium channel, P/Q-type channels, T-type channels, epilepsy, seizures
1 INTRODUCTION The international classification of epilepsies divides both focal and generalized epilepsies into idiopathic syndromes where the cause is believed to be genetic and cryptogenic/symptomatic syndromes where acquired factors are thought to predominate (ILAE, 1989). It has become increasingly clear, however, that many, and perhaps most epilepsies, display a complex pattern of inheritance, and that various genetic inputs on the one hand, and various acquired factors on the other, act in a different way in each patient (Berkovic et al., 2006). So far, success in identifying single genes Progress in Brain Research, Volume 213, ISSN 0079-6123, http://dx.doi.org/10.1016/B978-0-444-63326-2.00004-1 © 2014 Elsevier B.V. All rights reserved.
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has come from rare monogenic idiopathic epilepsy syndromes, and the prospective will be on extending these findings to the most common polygenic epilepsies with complex inheritance. Most of the established major mutations are in genes that encode for ion channels or their accessory subunits (Graves, 2006). This has led to the concept that idiopathic epilepsies are usually a family of channelopathies, even though the link between molecular deficit and clinical phenotype remains blurred. Moreover, it is particularly challenging to understand the large phenotypic variability observed in these single gene epilepsies that range from mild phenotypes to very severe ones. It is reasonable to hypothesize that, even in such monogenic epilepsies, a more complex interaction between genetic and acquired factors modulates disease severity of a produced phenotype. These challenges are even greater in complex epilepsies in which it also remains open to discussion whether ion channels are strong functional candidates (Cavalleri et al., 2007). It is also important to highlight that almost all ion channel epilepsy mutations express themselves in a heterozygous manner, which is evident only in one allele. The pathological impact of each mutation is therefore either due to a dominant effect of the mutant protein within neurons (or glia) or a loss of function due to haploinsufficiency (i.e., a loss of ability to generate sufficient protein from the one “working” allele). Among these ion channels, both high- and low-voltage-activated calcium channels and their ancillary subunits Cav2.1 (P/Q-type) calcium channels support a number of dynamic processes in neurons with both presynaptic and postsynaptic Ca2+ channels being critical determinants of neuronal excitability (Catterall, 2011). Therefore, mutations in genes encoding calcium channels are excellent candidates in the context of epilepsy phenotypes (Cain and Snutch, 2012; Zamponi et al., 2010). The remainder of this chapter addresses the state of knowledge on the roles of voltage-gated calcium channels, their mutations, and how they might contribute to the pathophysiology of human epilepsies. Table 1 summarizes these findings.
2 CALCIUM CHANNEL NOMENCLATURE AND BIOPHYSICAL PROPERTIES Voltage-gated Ca2+ channels reside in the plasma membrane of excitable cells, and open in response to membrane depolarization, thereby allowing calcium ion entry to cells (Catterall, 2011). They are broadly classified into high-voltage-activated (HVA) or low-voltage activated (LVA) calcium channels (Fig. 1), with HVA channels requiring larger membrane depolarizations for activation (Catterall, 2011; Ertel et al., 2000). HVA channels are made up of one or more pore-forming a1 subunit that coassembles with ancillary subunits and can be further subdivided into L-, N-, P/Q(Cav2.1), N- (Cav2.2), and R- (Cav2.3) types (Fig. 1), by virtue of their distinct functional and pharmacological profiles (Catterall, 2011). Conversely, LVA channels, also referred to as “T-type channels,” are thought to be a1 subunit monomers and are further classified according to their a1 subunit composition into Cav3.1, Cav3.2, and Cav3.3 isoforms (Fig. 1), each with unique biophysical,
2 Calcium channel nomenclature and biophysical properties
Table 1 Genes and diseases for inherited calcium channelopathies Calcium channela
Current type
Principal physiological functions
Gene and chromosome
Inherited diseases
Cav1.1
L
CACNA1S; 1q31-32
HPP
Cav1.2
L
CACNA1C; 12p13.3
TS
Cav1.3
L
CACNA1D; 3p14.3
Not reported
Cav1.4
L
Excitation–contraction coupling in skeletal muscle, regulation of transcription Excitation–contraction coupling in cardiac and smooth muscle, endocrine secretion, neurons, regulation of enzyme activity, regulation of transcription Endocrine secretion, cardiac pacemaking, neurons, auditory transduction Visual transduction
SNB
Cav2.1
P/Q
Cav2.2
N
Cav2.3
R
Cav3.1
T
Cav3.2
T
Cav3.3
T
CACNA1F; Xp11.23 CACNA1A; 19p13 CACNA1B; 9q34 CACNA1E; 1q25 CACNA1G; 17q22 CACNA1H; 16p13.3 CACNA1I; 22q12.3
Neurotransmitter release, dendritic Ca2+ transients Neurotransmitter release, dendritic Ca2+ transients Neurotransmitter release, dendritic Ca2+ transients Pacemaking and repetitive firing Pacemaking and repetitive firing Pacemaking and repetitive firing
EA, FHM, SCA6
AS Not reported
AS, absence seizures; CACNA, a1 subunit gene encoding voltage-dependent Ca2+ channels; EA, episodic ataxia; FHM, familial hemiplegic migraine; HPP, hypokalemic periodic paralysis; SNB, stationary night blindness; SCA6, cerebellar ataxia type 6; TS, Timothy syndrome (cardiac arrhythmia with developmental abnormalities and autism spectrum disorders). a The cloned voltage-gated calcium channels a1-subunits are presented following the proposed nomenclature (Ertel et al., 2000).
pharmacological, and regulatory properties (Catterall, 2011). Additional structural and functional variants of each Cav subtype can be generated by alternative splicing to produce a large number of different “splice variants” and therefore increase the repertoire and complexity of calcium channel properties (Catterall, 2011). Calcium channels are usually slower at opening (activation) and closing (deactivation) than other voltage-activated channels. In detail, HVA channels display slower activation and faster deactivation than LVA channels. Moreover, HVA channels usually inactivate much more slowly than LVA channels. All together, these
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FIGURE 1 Voltage-gated calcium channels. Schematic representation of the high-voltage-activated calcium channel complex showing the main pore-forming a1 subunit and ancillary, b-, g-, and a2-d-subunits. Low-voltage-activated calcium channels may consist of only the a1-subunit. (Modified, with permission, from Physiological Reviews; Khosravani and Zamponi, 2006, Vol. 86, Issue 3, The American Physiological Society)
properties make HVA channels generate longer lasting calcium influxes upon sustained depolarizations, while T-type channels conduct more rapid and shorter calcium influxes under both brief and sustained depolarizations (Catterall, 2011). In the nervous system, the three T-type calcium channels are often expressed differently in the same cell types; thus, a wide repertoire of diverse net LVA calcium currents can be generated depending on the relative contribution from each subtype. Moreover, the spectrum of T-type calcium channel types is made more complex by the occurrence of multiple alternatively spliced isoforms, adding further functional complexity to the overall spectrum of T-type currents (Catterall, 2011). Then, distinct T-type channel isoforms exhibit a differential distribution across somatic, dendritic, and axonal compartments, indicating that compartmental factors also contribute to excitability and cellular output. HVA calcium channels, P/Q-type, N-type, and, to some extent, R-type channels, are expressed highly at presynaptic nerve terminals where their activities evoke neurotransmitter release (Rajakulendran et al., 2012). The different subtypes support a variety of biological processes, including gene expression, neuronal architecture development, signal propagation, and neurotransmitter release from nerve terminals. Among the different channel subtypes, the related subfamily of P/Q channels (Cav2.1), N channels (Cav2.2), and R channels (Cav2.3) are commonly known as the neuronal channels. N and P/Q channels are the principal subtypes involved in neurotransmission. P/Q channels are thought to be predominantly located at central synapses, whereas N channels are more prominent in peripheral nerve terminals, particularly those involved in sensory and autonomic function. The P/Q channels are widely expressed throughout the CNS, especially in Purkinje and granule cells of the cerebellum, where they also have important postsynaptic roles
3 Calcium channels in epilepsy
in calcium signaling. By linking calcium influx to activation of AMPA (a-amino-3hydroxy-5-methyl-4-isoxazolepropionic acid) receptors, these channels are thought to have a role in bidirectional cerebellar plasticity (Catterall, 2011).
3 CALCIUM CHANNELS IN EPILEPSY 3.1 T-TYPE CALCIUM CHANNEL MUTATIONS IN EPILEPSY T-type calcium channels are critically involved in normal burst firing in the thalamocortical circuitry recruited in the spike-wave discharges that underlie absence seizures (Heron et al., 2007) and in the intrinsic burst firing of hippocampal pyramidal neurons in temporal lobe epilepsy (TLE) (Cain and Snutch, 2012). The three T-type calcium channels (Cav3.1, Cav3.2, and Cav3.3) are widely but differentially expressed in the thalamocortical circuitry implicated in absence seizures. The role of T-type channels in absence epilepsies is also highlighted by the anticonvulsive effects of ethosuximide, an inhibitor of T-type Ca2+ currents, in the treatment of absence seizures (Coulter et al., 1989). It is therefore not unexpected that variants in the T-type calcium channel gene CACNA1H which encodes a low-threshold T-type Ca2+ channel were initially associated with childhood absence epilepsy (CAE) and have since been described in other epilepsy phenotypes (Chen et al., 2003; Heron et al., 2007; Khosravani et al., 2005; Vitko et al., 2005). Initial studies by Chen et al. (2003) found 12 rare missense mutations in 14 of 118 patients suffering CAE, suggesting that it may be a susceptibility gene involved in the pathogenesis of this disease. Nonetheless, evaluation of 44 pedigrees and 220 unrelated patients with CAE of Caucasian European origin failed to find evidence for mutations in CACNA1H (Chioza et al., 2006), suggesting that some genetic variants contribute to disease predisposition in an apparently populationspecific manner. Some of the alterations in Cav3.2 have been shown to induce altered biophysical properties or increase channel expression when examined in exogenous expression systems. However, some have no apparent effect, potentially reflecting the polygenic nature of idiopathic generalized epilepsies, and/or that a subset of the changes represent single nucleotide polymorphisms (Heron et al., 2007). There are also a large volume of data from experiments on animal models of both genetic generalized epilepsy (GGE) and acquired TLE that have implicated these channels as key players in regulating neuronal excitability (Cain and Snutch, 2012; Zamponi et al., 2010). In the kindling model of TLE, T-type calcium currents were significantly larger in CA1 pyramidal cells of animals that had experienced kindled seizures compared to controls, and these current changes persisted up to 6 weeks after the cessation of kindling (Cain and Snutch, 2012; Zamponi et al., 2010). Moreover, several studies investigating the role of T-type calcium channels during epileptogenesis after pilocarpine-induced status epilepticus found that there was a selective and transient increase in Cav3.2 mRNA expression in CA1 pyramidal neurons coupled with an upregulation of T-type calcium currents (Cain and Snutch, 2012; Zamponi et al., 2010). Of note, hippocampal sclerosis and mossy fiber sprouting, histopathological hallmarks of TLE in humans and animal models, were absent in
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Cav3.2 knockout mice, and these mice were resistant to the development of chronic seizures induced by pilocarpine (Cain and Snutch, 2012; Zamponi et al., 2010). There is also substantial evidence in the literature from human and animal studies linking T-type calcium channels to the pathogenesis of GGEs. Indeed, a mutation in the rat Cav3.2 gene (R1584P) has been identified in the GAERS animal model of GGE with absence seizures. It has been also illustrated that Cav3.1 knockout mice lacked the burst firing mode of action potentials in thalamocortical neurons, seen during absence seizures, whereas the normal tonic mode of firing was unaffected (Cain and Snutch, 2012; Zamponi et al., 2010). Conversely, transgenic mice overexpressing Cav3.1 showed increased functional thalamic T-type calcium currents and frequent bilateral rhythmic SWDs that could be blocked by treating with ethosuximide (Cain and Snutch, 2012; Zamponi et al., 2010). All these experimental data further illustrated the role of T-type calcium channels in the pathophysiology of GGE; nonetheless, the contribution of Cav3.1 T-type calcium currents to the generation of absence seizures is still matter of discussion. Increased Cav3.1 T-type calcium currents have been reported to be sufficient to induce pure absence epilepsy, while in a study utilizing Cav2.1 mutant mice double-crossed with Cav3.1 mutant mice it was found that increased thalamic Cav3.1 T-type calcium currents are not essential for the generation of absence seizures (Cain and Snutch, 2012; Zamponi et al., 2010). Increased mRNA expression of T-type calcium channels are also evident in animal models of GGE compared to controls, especially in the lateral geniculate nucleus and centrolateral nucleus of the intralaminar nuclei in WAG/Rij, and in the ventral posterior thalamic relay nuclei of adult GAERS, as well as in the thalamus in both GAERS and WAG/Rij rats. All these data indicate that increases in any of the T-type calcium channels are important factors in contributing to GGE phenotype in these animal models (Cain and Snutch, 2012; Zamponi et al., 2010).
3.2 P/Q-TYPE CALCIUM CHANNEL MUTATIONS IN EPILEPSY The CACNA1A gene on chromosome 19 encodes both P-type and Q-type channels, also known as Cav2.1 P/Q-type calcium channels subunit, through an alternate splicing mechanism. These channels are highly expressed presynaptically where they are critically involved in neurotransmission and synaptic efficacy and therefore have a great influence on neuronal excitability. This aspect is reflected by a number of mutations in the Cav2.1 gene identified in patients suffering from severe neurological disorders including ataxias and congenital migraine (Rajakulendran et al., 2012). So far, three autosomal dominant allelic diseases are associated with mutations of CACNA1A gene (Rajakulendran et al., 2012): (1) familial hemiplegic migraine (FHM) that is a rare and severe variant of migraine with aura, characterized by disabling attacks of hemiparesis and hemisensory disturbance, which can last from hours to days, and visual disturbance. (2) Episodic ataxia type 2 (EA2), whose clinical features include intermittent ataxia, headache, and vertigo, and acetazolamide is an effective treatment. (3) Spinocerebellar ataxia type 6 (SCA6), which is a late-onset, progressive, cerebellar syndrome characterized by impaired balance, limb
3 Calcium channels in epilepsy
incoordination, and dysarthria. The prevalence of SCA6 seems to be highest in Japan, and the mean age of onset is in the fifth decade of life, although onset in patients in their twenties has been described. Although these three diseases are conventionally described as distinct, they exhibit considerable overlap in clinical features (Rajakulendran et al., 2012). Moreover, kindreds have been reported in which different affected members appear to suffer from different syndromes within this spectrum. Furthermore, a small proportion of patients with FHM and with underlying mutations in the Cav2.1 channel also display both generalized and complex partial seizures (Rajakulendran et al., 2012). In detail, a heterozygous mutation in CACNA1A (P/Q-type Ca2+ channel) has been described in one individual with a complex phenotype that included generalized epilepsy ( Jouvenceau et al., 2001). Further, a small family exhibiting absence epilepsy combined with ataxia has a mutation in CACNA1A (Imbrici et al., 2004). Functional studies implicate a loss of P/Q-type Ca2+ channel function in both cases (Imbrici et al., 2004; Jouvenceau et al., 2001). Similarly, a mutation in CACNB4 (b4 subunit) described in one patient with JME resulted in a loss of protein function (Escayg et al., 2000). Regarding the genotype–phenotype relationship, there is evidence that FHM, EA2, and SCA6 are generally caused by distinct defects of CACNA1A (Rajakulendran et al., 2012). FHM is associated with several missense mutations affecting conserved residues located throughout the a1 subunit. These mutations are thought to confer a gain of function on the P/Q channel. Supportive evidence was provided by a study of knock-in mouse models that demonstrated an increase in calcium current density and neurotransmission at the neuromuscular junction, with a susceptibility to cortical spreading depression, which is considered to be the physiological correlate of the aura in migraine (Rajakulendran et al., 2012). In contrast, EA2 is generally associated with premature stop codons and splice site mutations, predicted to result in a truncated peptide, which cause a profound or total loss of P/Q channel function (as measured by calcium current density; Rajakulendran et al., 2012). Moreover, some evidence initially pointed to haploinsufficiency as the underlying disease-causing mechanism. Nonetheless, later studies have illustrated that mutant calcium channels might instead exert a dominantnegative effect by interfering with correct folding and trafficking of wild-type channels, causing them to be retained in the endoplasmic reticulum (Rajakulendran et al., 2012). SCA6 is unique among the channelopathies in that it is caused by an expansion of a polyglutamine repeat in the intracellular C-terminus of the channel and symptoms are typical not episodic, but rather slowly progressive. Current pathophysiological hypothesis is in favor of a direct toxic effect of the polyglutamine expansion in Purkinje cells, which degenerate in SCA6, particularly in the cerebellar midline. There is also evidence that genetic variation in the P/Q channel might be associated with a predisposition to seizure disorders (Rajakulendran et al., 2012). Absence seizures are generally thought to arise as a result of abnormal reverberation in thalamocortical loops, as opposed to an increase in the ratio of excitatory
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(glutamatergic) versus inhibitory (GABAergic) signaling. Accordingly, a relatively selective decrease in glutamate release from thalamic synapses has been reported in one of the mutant mouse strains (Rajakulendran et al., 2012). Moreover, EEG recordings of tottering, leaner, rocker, and tg-4J strains of mice carrying spontaneous recessive mutations in the CACNA1A gene displayed generalized spike-and-wave discharges at a 5–7 Hz frequency, in combination with cerebellar ataxia and degeneration (Rajakulendran et al., 2012). Furthermore, although absence seizures are not a consistent feature of the known human CACNA1A mutations, EEG abnormalities have been reported in patients with acetazolamide-responsive ataxia (Rajakulendran et al., 2012), and polymorphisms in the CACNA1A gene are in linkage disequilibrium with primary generalized epilepsy (Chioza et al., 2001). A combination of seizures and episodic ataxia has also been tentatively linked to the accessory b4 calcium channel subunit (Escayg et al., 2000). Despite these findings, the functional consequence of this mutation and the broader role of Cav2.1 in human absence epilepsy remain to be determined.
3.3 ANCILLARY SUBUNITS OF VOLTAGE-GATED CALCIUM CHANNELS IN SEIZURE DISORDERS Ancillary calcium channel subunits are important regulators of HVA calcium channel function. So far, mutations in either g- or a2-d-subunits have so far not been linked to epilepsy in humans; however, there are several mouse phenotypes associated with these subunits (Cain and Snutch, 2012; Zamponi et al., 2010). The b, a2-d, and g ancillary calcium channel subunits that modulate the biophysical properties and expression of the HVA a1 subunits have been implicated in animal models of absence epilepsy and ataxia (Cain and Snutch, 2012; Zamponi et al., 2010). There is also some evidence for changes in calcium channel function via altered b-subunit expression patterns in TLE (Cain and Snutch, 2012; Zamponi et al., 2010). A role of HVA calcium channel interacting proteins is also exemplified in the case of juvenile myoclonic epilepsy patients that carry a mutation in the calciumbinding protein EFHC1 that under normal conditions enhances R-type channel activity, an effect that is abolished by the mutations in this protein (Cain and Snutch, 2012; Zamponi et al., 2010). However, mice deficient of R-type channels do not display seizure activity, and hence, it is not clear if the clinical manifestation of the EFHC1 mutation is directly related to the activity of R-type channels. On this basis, the effects of mutations in ancillary subunits appear to cause an inhibition of P/Q-type channel activity, although it is not proven that exclusively these channels mediate such physiological effects.
4 CONCLUSION Calcium ion channels are critical for normal neuronal excitability, so it is not surprising that the majority of heritable defects so far identified in human epilepsies code for ion channel subunits. Nonetheless, there is no unifying mechanism that can explain
References
how the spectrum of the observed functional effects of epileptogenic mutations relates to the epilepsy syndromes seen in patients. There is indeed functional heterogeneity among mutant channels, revealing a complex relationship between clinical and biophysical phenotypes. Because of this, a genetic classification of clinical epilepsies is currently elusive. It is also becoming increasingly clear that, even in Mendelian epilepsies, specific phenotypes probably result from the cumulative effects or interactions of a few or several genes, of which the identified one is only a player. Moreover, recent studies provided evidence that defective protein trafficking and protein–protein interactions may modulate the effect of mutation and, so, underlie phenotypic variation in such epilepsies related to channelopathies (Rusconi et al., 2007). The diversity of mutations that can cause a similar phenotype argues for points of physiological convergence that can give rise to a definite network hyperexcitability able to selectively cause specific phenotypes. This may be the case not only for Mendelian epilepsies but also in epilepsies with complex inheritance where gene discovery is just beginning. The development of animal models and multielectrode array experiments will probably be useful in investigating this point. Identifying such specific epileptogenic networks is essential for deeper understanding of mechanisms of epileptogenesis and for designing therapeutic strategies.
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