insights from mouse models of absence epilepsy into ca2+ channel physiology and disease etiology

P1: FHD/LOV P2: FZN/GIR QC:

Cellular and Molecular Neurobiology [cemn] pp552-cemn-377486 August 19, 2002 12:44 Style file version Oct 23, 2000

Cellular and Molecular Neurobiology, Vol. 22, No. 2, April 2002 ( C© 2002)

Review

Insights From Mouse Models of Absence EpilepsyInto Ca2+ Channel Physiology and Disease Etiology

Ricardo Felix1,2

Received February 14, 2002; accepted February 24, 2002

SUMMARY

1. Changes in intracellular Ca2+ ([Ca2+]i) levels provide signals that allow neuronsto respond to a host of external stimuli. A major mechanism for elevating [Ca2+]i is theinflux of extracellular Ca2+ through voltage-gated channels (CaV) in the plasma membrane.Malfunction in CaV due to mutations in genes encoding channel proteins are increasinglybeing implicated in causing disease conditions, termed channelopathies.

2. Seven spontaneous mutations with cerebellar ataxia and generalized absence epilepsyhave been identified in mice (tottering, leaner, rolling Nagoya, rocker, lethargic, ducky, andstargazer), and these overlapping phenotypes are directly related to mutations in genes en-coding the four separate subunits that together form the multimeric neuronal CaV complex.

3. The discovery and systematic analysis of these animal models is helping to clarifyhow different mutations affect channel function and how altered channel function producesdisease.

KEY WORDS: Ca2+ channel; channelopathies; absence epilepsy; ataxia; mutant mice.

INTRODUCTION

Voltage-gated Ca2+ (CaV) channels are a large family of related heterooligome-ric membrane proteins that couple cell excitability to intracellular signaling. Throughspatial and temporal control of free intracellular Ca2+ concentration ([Ca2+]i), CaV

channels regulate a host of neuronal processes, including neurotransmitter release,electrical activity, cytoskeletal function, cell metabolism and proliferation, as well asgene expression. Recent studies on epileptic mice carrying CaV channel mutationshave provided important information about the roles carried out by these molecules

1 Department of Physiology, Biophysics and Neuroscience, Center for Research and Advanced Studiesof the National Polytechnic Institute, Cinvestav-IPN, Mexico.

2 To whom correspondence should be addressed at Departamento de Fisiologıa Biofısica yNeurociencias, Cinvestav-IPN, Avenida IPN #2508, Colonia Zacatenco, Mexico DF, CP 07000; e-mail:[email protected].

103

0272-4340/02/0400-0103/0 C© 2002 Plenum Publishing Corporation



104 Felix

in excitable cells. From the systematic analysis of these animal models new andexciting information has emerged regarding the molecular structure of CaV channelsand how these proteins contribute to regulate brain function.

STRUCTURE AND FUNCTION OF CaV CHANNELS

Ca2+ currents are diverse functionally and have been well characterized in termsof biophysical and pharmacological properties. Six different types of CaV channelshave thus far been defined and named T, L, N, P, Q, and R. These channel types can beseparated into two classes based on threshold of voltage-activation: low voltage- andhigh voltage-activated channels (LVA and HVA, respectively). HVA channels canbe further subdivided into L-type, which are sensitive to dihydropyridines (DHPs),and the various DHP-insensitive (collectively known as non-L-type) channels. BothLVA and HVA channels include a membrane-spanning ion-conducting (α1) subunit,but HVA channels also contain several ancillary subunits (Walker and De Waard,1998) that have not, as yet, been identified in LVA channels (Lacinova et al., 2000;Perez-Reyes, 1999). Molecular cloning has revealed 10 different CaV channel α1

genes (Ertel et al., 2000; Muth et al., 2001). cDNA of these α1 subunits has beenisolated and its functional expression could be correlated with a previously definedCa2+ current subtype. Hence, four L-type CaV subunits (CaV1.1 through CaV1.4) gateDHP-sensitive currents, while three CaV subunits that can be further distinguishedby their responses to various peptide toxins (Catterall, 2000; De Waard et al., 1996;Uchitel, 1997) code the non-L-type channels: CaV2.1 gates the P/Q-type currentthat is blocked by the marine snail toxin ω-conotoxin MVIIC and the spider toxinω-agatoxin IVA, whereas CaV2.2 is responsible for the N-type current that is sensitiveto ω-conotoxin GVIA. A third non-L-type channel, CaV2.3, gates the toxin-resistantR-type current. LVA pore-forming subunits have recently been cloned, and threesubtypes of channels are already known: CaV3.1 through CaV3.3 (Lacinova et al.,2000; Perez-Reyes, 1999).

The structure of the principal (α1) subunit of CaV channels (Fig. 1) is based onfour homologous transmembrane domains (I through IV) that contain six transmem-brane alpha helices denoted S1–S6 surrounding a central pore. The N- and C-terminaldomains of the α1 protein are positioned intracellularly. One transmembrane seg-ment (S4) in each repeated domain contains a positively charged amino acid everythird or fourth position and has been proposed as the voltage sensor of the channels.The region separating segments S5 and S6 of each domain may contain two additionaltransmembrane segments (known also as SS1–SS2 or P region) that together formthe ion-conducting pathway of the channel. Within the pore region of theα1subunit, aglutamate residue is found at a conserved position. In HVA channels, four glutamates(EEEE) form a selectivity gate (Yang et al., 1993), while the pore of LVA α1 subunitsdiffers slightly, with aspartates in place of glutamates at two sites (EEDD) (Talaveraet al., 2001). The intracellular domains of α1 also contain highly conserved regionsthat are implicated in protein interaction. The CaVβ subunit binds to a specific sitebetween domains I and II (Pragnell et al., 1994) termed the alpha interaction domain(AID). A portion of the loop connecting domains II and III of the α1 subunit inCaV1.1 channels is implicated in excitation–contraction coupling (Nakai et al., 1998),



Mutant Mice and Ca2+ Channel Physiology 105

Fig. 1. Subunit structure of the neuronal P/Q-type CaV channel and localizations of the mutations asso-ciated with absence epilepsy in mice. P/Q-type channels are complexes of a pore-forming CaV2.1 subunit,an intracellular β subunit, and transmembrane α2δ and γ subunits. CaV2.1 (∼200 kDa) is composed offour homologous membrane-spanning internal domains, each with six transmembrane-helixes and a pore-forming reentrant P loop. Carboxyl and amino termini as well as linkers between domains I-II, II-III, andIII-IV are all cytosolic. The CaVα2δ complex (∼170 kDa) derives from a common precursor protein thatis proteolytically processed to yield separate CaVα2 and CaVδ proteins that remain linked by disulfidebonds. CaVβ is a cytoplasmic protein (∼70 kDa) while CaVγ is a ∼36,000-Da glycosylated hydrophobicprotein that is predicted to contain four transmembrane domains. Indicated are the mutations associatedwith absence epilepsy in mice. References for these mutations are given in the text.

and a region of this same loop in the CaV2 channel subfamily interacts with vari-ous proteins implicated in excitation–secretion coupling (Jarvis and Zamponi, 2001;Seagar et al., 1999). Likewise, a segment of the I–II loop of non-L-type CaVα1 sub-units binds the G-protein βγ complex (De Waard et al., 1997; Zamponi and Snutch,1998), while a site in the proximal carboxyl tail mediates calmodulin Ca2+-dependentinactivation in both L- and non-L-type channels (Lee et al., 1999; Zuhlke et al., 1999).The α1subunits also contain recognition sites for channel-specific drugs and toxins(De Waard et al., 1996; Uchitel, 1997; Varadi et al., 1995).

In addition to the main pore-forming α1 subunit, HVA CaV channels are com-posed of at least three ancillary elements (Fig. 1): the regulatory β, α2δ, and γ sub-units (Catterall, 2000; Muth et al., 2001). The CaVβ subunit does not cross the plasmamembrane, but it can directly interact with the α1 subunit (Pragnell et al., 1994) andappears to be important for membrane targeting, as well as for shaping the kineticcharacteristics of the ionic currents (Birnbaumer et al., 1998). The CaVα2δ subunit,is a transmembrane complex subunit derived from a common precursor protein thatis proteolytically processed to yield two separate peptides (α2 and δ) that remainlinked by disulfide bonds. This regulatory subunit can also associate directly withthe α1 pore-forming subunit and alter its biophysical and binding properties (Felix,1999). Lastly, the CaVγ subunit is a membrane-spanning protein that may modulate



106 Felix

Ca2+ currents by shifting the steady-state inactivation of the channels (Freise et al.,2000; Letts et al., 1998).

MUTATIONS IN THE α1 ION-CONDUCTING SUBUNIT

The influx of Ca2+ ions through CaV channels contributes to neuronal excitabi-lity and, as we shall see later, is an important feature of epileptogenesis (Steinlein andNoebels, 2000). Recently, genetic studies have linked Ca2+ channels to several animalseizure models and human neurological disorders collectively known as Ca2+ chan-nelopathies (Felix, 2000; Lorenzon and Beam, 2000). The first hereditary diseasesrecognized as being caused by spontaneous mutations affecting CaV channels, famil-ial hemiplegic migraine (FHM), and episodic ataxia type-2 (EA-2), were identified asdefects in the human CaV2.1 subunit (Ophoff et al., 1996). With this finding, the studyof Ca2+ channels entered to a new and exciting phase where evidence for missensemutations, truncated and expanded versions of the CaV2.1 subunit has been linked toseveral diseases of mice and humans. Attention has been directed to epileptic strainsof mice that exhibit absence seizures with electrographic firing patterns, onset, local-ization, and drug response similar to petit mal epilepsy in humans. Simultaneous withthe discovery of the FHM/EA-2 gene defects, the mutations underlying the tottering(Cacna1atg , formerly tg) and leaner (Cacna1atg−la , formerly la and tgla) mouse phe-notypes were identified as defects in the murine CaV2.1 subunit (Fletcher et al., 1996).The study of these mouse mutants has been instrumental in understanding some im-portant aspects of the molecular physiology and pathophysiology of Ca2+ channels.

The Tottering Mutation

Mutations at the mouse tottering locus on mouse chromosome 8 cause a delayed-onset, recessive neurological disorder resulting in ataxia, an intermittent motordysfunction best described as paroxysmal dystonia, polyspike (cortical spike-wave)discharges and behavioral absence seizures resembling absence (petit mal) epilepsyin humans. By positional cloning, the CaV2.1 subunit was identified as the gene thatis mutated in tottering (Fletcher et al., 1996). Sequence analysis showed that the mu-tation causes substitution of proline for leucine at a position close to the conservedpore-lining region in the extracellular segment in the II repeat of the CaV2.1 sub-unit (Fletcher et al., 1996) (Fig. 1). Although the replacement of proline for leucine(P601L) might conceivably cause a substantial conformational change and alter theion-conducting pathway of the channels, the electrophysiological characterization ofthe CaV2.1 subunit in neurons dissociated from tottering mice, and their comparisonwith the properties of wild type and mutant Cacna1atg channels recombinantly ex-pressed in an heterologous system, showed a marked reduction in current densitynot associated to changes in the single-channel conductance of the mutant channels(Wakamori et al., 1998). In addition, because the Cacna1atg mutation is a singlenucleotide substitution, it seems that a posttranslational mechanism that impairedchannel activity rather than an alteration in the efficiency of the transcription andtranslation of the CaV2.1 subunit, may account for the decreased Ca2+ currents ob-served in the mutant neurons (Wakamori et al., 1998).




The reduction in CaV2.1 channel current density in the Cacna1atg mouseappeared to be associated with changes in other current components relative to wildtype animals, including an increase of L-type CaV channels in cerebellar Purkinje cells(Campbell and Hess, 1999). Interestingly, this increase was related with the ability ofL-type channel blockers to prevent the intermittent dystonic episodes and suggestedthat these channels may play a role in the abnormal motor phenotype expressed byCacna1atg mutant mice (Campbell and Hess, 1999). More importantly however, theabnormal functional expression of CaV2.1 may help to understand the endogenousneural mechanisms that regulate network excitability and synchronization. CaV2.1-containig channels reside on somatodendritic membranes and presynaptic terminalsof many different neuron types and are predominantly expressed in cerebellar gran-ule and Purkinje cells, and are important regulators of neural excitability, integration,and Ca2+-dependent neurotransmitter release. Therefore, changes in any of the func-tions due to an inherited Ca2+ channel disorder may significantly alter both synapticand nonsynaptic signaling. For example, it has been suggested that glutamate releaseis attenuated in the tottering (as well as in the leaner) mutant, and that there is araise in the threshold for initiating the cortical spreading depression (CSD), a waveof depolarization that spreads in the neocortex (Ayata et al., 2000). Hence, once thecortical wave is initiated in the tottering brain by exposure to high K+ levels (10-foldhigher than in the wild type) it travels more slowly and propagation frequently failscompletely. These changes may be due, at least in part, to a depression of exci-tatory neurotransmission during the wave of CSD (Ayata et al., 2000), suggestingthat CaV2.1 channel mutations may decrease transmitter release under conditions ofsustained cortical depolarization.

In addition, experimental evidence indicates that the spike-wave discharges inthe tottering mice, which are generated when a physiological tonic mode of neuronalfiring shifts to a burst-firing mode (Steriade and Llinas, 1988), are produced by aber-rant thalamocortical oscillations involving neocortical pyramidal neurons, thalamicrelay neurons, and GABAergic neurons of the nucleus reticularis thalami (Snead,1995). T-type Ca2+ currents in thalamic neurons are important in the generation ofnormal thalamocortical rhythms (Coulter et al., 1989; Wang et al., 1991) and P/Q-typeCa2+ channels have an important role in presynaptic release of neurotransmitters(Hong and Chang, 1995; Mintz et al., 1995; Qian and Noebels, 2001), providing twopotential mechanistic links between Ca2+ currents and thalamocortical circuits. Asa matter of fact, a reduction of excitatory synaptic transmission in the thalamus oftottering (as well as in lethargic) mice has been documented (Caddick et al., 1999)and it was proposed that a net enhanced GABAergic input in thalamocortical neu-rons might synchronize them into a burst-firing mode. However, additional work isrequired to elucidate the mechanism of spike-wave discharges generation in totteringand lethargic mice, given that compensatory mechanisms in the regulation of presy-naptic Ca2+ entry and hippocampal neurotransmitter release have been found alsoin these mutant animals (Qian and Noebels, 2000).

Lastly, the expression pattern of tyrosine hydroxylase (TH) is also altered inPurkinje cells of adult tottering mutant mice (Hess and Wilson, 1991). TH expre-ssion was first observed on postnatal age P21 and persisted throughout adulthood,in contrast to Purkinje cells of normal mice that synthesize it transiently during



108 Felix

development (from P21 to P35). Thus, tottering mice are deficient in suppressing thenormal transient expression of TH in developing Purkinje cells, suggesting that theprotein encoded by the tottering locus, CaV2.1, may play a crucial role in neuronal de-velopment. Comparison of adult and young preseizure mutant mice showed Purkinjecells densely labeled for TH mRNA at both ages, indicating that TH gene expressionin Purkinje cells is independent of the onset of seizures (Austin et al., 1992).

The Leaner Mutation

The leaner mutant allele (Cacna1ala) produces animals severely ataxic with cor-tical spike-wave discharges. The Cacna1ala mutation is also associated with cerebellaratrophy resulting from a gradual degeneration of granule, Purkinje, and Golgi cells.The cerebella of leaner mice contain∼80% fewer Purkinje cells compared with thoseof normal mice (Herrup and Wilczynski, 1982), and the surviving Purkinje cells ex-hibit aberrant morphology of the dendritic tree and axonal swellings (Heckroth andAbbott, 1994). Molecular analysis showed that the Cacna1ala allele contains a muta-tion of a splice donor consensus that results in truncation of the normal open readingframe beyond repeat IV at amino acid positions 1922 or 1968 and expression of twonovel CaV2.1 subunits with truncated carboxyl tails (Doyle et al., 1997; Fletcher et al.,1996) (Fig. 1). Functional studies have shown that this mutation specifically reducesthe amplitude of Ca2+ current through CaV2.1 without affecting its macroscopic prop-erties and antagonist sensitivity (Dove et al., 1998; Lorenzon et al., 1998; Wakamoriet al., 1998). Because no differences in either mRNA or protein expression of theCaV2.1 subunit has been reported (Lau et al., 1998), it was suggested that the leanermutation causes an alteration in Ca2+ channel function. Based on electrophysiologi-cal recordings of normal and mutant Purkinje cells it was found that open-probability(Po) was∼3-fold lower in leaner cell-attached patches. Interestingly, the reduction inPo was not reflected in a reduction of channel mean open time. Rather, it seemed toresult from a lower frequency of channel opening. Therefore, it was speculated thatthe truncated C-tails resulting from the leaner mutation could interfere with essen-tial cytoplasmic interactions of the C-terminal responsible for normal channel gatingand channel availability, thus leading to reduced Po (Dove et al., 1998). In addition,the comparison of the properties of native and recombinant mutant channels showedthat the expression of one form of the two possible Cacna1ala aberrant splicing prod-ucts, Cacna1ala “short” channel, induced a significant reduction in current density,while the other form, Cacna1ala “long” channel, had a current density comparable tothe control, though a shift in the voltage dependence of activation and inactivationwas observed (Wakamori et al., 1998).

The leaner mutation seems to have an impact also on Ca2+ homeostatic mech-anisms in cerebellar neurons. It has been shown that leaner Purkinje cells have adiminished Ca2+ buffering ability, which has been attributed to reduced uptake bythe endoplasmic reticulum and decreased expression of Ca2+-binding proteins (Doveet al., 2000). The reduction in Ca2+ buffering in the mutant cells has been interpretedas a compensatory homeostatic effort to maintain normal Ca2+ signaling functiondespite reduced Ca2+ influx through CaV channels. However, this reduced bufferingability may be also a part of the continuum of delayed physiological maturation that




is presumably mediated by decreased P/Q-type Ca2+ channel activity in early devel-opment. This alternative hypothesis is supported by morphological data such as thepersistence of multiple synaptic contacts on dendritic spines and altered spinogene-sis of leaner Purkinje cells that are reminiscent of the situation in immature normalPurkinje cells (Rhyu et al., 1999a). In addition, TH, which is known to be transientlyexpressed in early stages of development in normal Purkinje neurons is persistentlyexpressed in the mutant cells (Austin et al., 1992; Hess and Wilson, 1991).

Although the functional properties of CaV2.1 appear significantly altered inCacna1ala Purkinje cells, less obvious is how these alterations lead to the ataxia anddegeneration of cerebellar neurons observed in the leaner mouse. Contrary to initialexpectations, the reduction and apoptosis of Purkinje cells observed in histologicalstudies (Heckroth and Abbott, 1994) appear not to be caused by Ca2+ overload asa result of prolonged opening of CaV channels due to alterations in the inactivationprocess. Conversely, experimental evidence shows that Ca2+ conductance of Purkinjecells is markedly reduced in the leaner mutant mice. Interestingly, a mutation nearthe P region of the I repeat of CaV2.1 has been associated to autosomal dominantprogressive ataxia in humans (Yue et al., 1997) presumably by reducing channelconductance due to the electrorepulsive force between Ca2+ ions and the positivelycharged residue introduced by the mutation (a substitution of glycine by arginine atcodon 293). Taken as a whole, these observations suggest that insufficient Ca2+ influxmight lead to shrinkage and apoptosis of Purkinje cells and ultimately to cerebellaratrophy (Wakamori et al., 1998).

Recent behavioral analyses in the leaner mouse revealed reduced responses tomechanical stimuli and enhanced responses to heat stimuli suggesting that the P/Q-type Ca2+ channel participates in the modulation of acute nociceptive responses(Ogasawara et al., 2001). Electrophysiological studies showed that leaner mice had asignificantly reduced ability to evoke dorsal root potentials, suggestive of a functionaldeficit in the spinal dorsal horn local circuitry responsible for presynaptic inhibition ofprimary sensory fibers (Ogasawara et al., 2001). Because the C-terminal in the CaV2.1subunit has been implicated in several functions including proper subunit associationand channel localization (Maximov et al., 1999; Sandoz et al., 2001), the possibilityexists that the lower level of P/Q-type current observed in leaner Purkinje cells (asa result of the presence of aberrant C-terminal sequences), may be also presentin spinal neurons expressing this type of channels. By comparing mechanical andthermal nociceptive responses in leaner with those of CaV2.2 and CaV2.3 null mice,Tanabe and colleagues have shown that CaV channels may participate differentiallyin the control of pain behaviors by spinal and supraspinal mechanisms (Ogasawaraet al., 2001; Saegusa et al., 2000, 2001).

The Rolling Nagoya Mutation

Mice carrying the rolling Nagoya allele (Cacna1atg−rol , formerly rol and tgrol)present an intermediate phenotype: ataxia is to some extent more severe than withtottering, but motor seizures do not occur. Compared with wild type mice, the num-ber of granule cells is decreased in the anterior lobe of the Cacna1atg−rol cerebellumand this cell loss is reminiscent of that observed in the leaner cerebellum (Rhyu



110 Felix

et al., 1999a). However, as mentioned earlier, in the leaner mouse cerebellum notonly granule cells are lost, but Purkinje and Golgi neurons also degenerate. In ad-dition, similar to leaner, TH is expressed persistently in rolling Purkinje cells andgiven that the TH promoter is activated by Ca2+, it has been speculated that TH ex-pression in the mutant Purkinje cells and neuronal dysfunction might be caused byalterations in Ca2+ currents (Sawada et al., 1999). Even more, considering the reportsthat CaV2.1 was the location of the leaner mutation, this subunit was also expectedto be the candidate gene carrying the rolling mutation (Rhyu et al., 1999b). Recentexperimental evidence has revealed this to be true. The Cacna1atg−rol mutation wasidentified through cloning of mouse CaV2.1 subunit cDNA from the rolling Nagoyamouse brain by RT-PCR. Cacna1atg−rol contains a cytosine to thymidine change atnucleotide position 3784, which results in an arginine to glycine substitution at aminoacid position 1262 (Mori et al., 2000). Notably, R1262 is near the C-terminal of theS4 segment in repeat III (Fig. 1), being altered from the characteristic arrangementof the positively charged amino acids located at every third or fourth position in thevoltage-sensing region of the channels.

The electrophysiological characterization of native P/Q-type Ca2+ channels hasshown that the mean amplitude of Ba2+ currents through these channels is signifi-cantly smaller (about 30%) for Cacna1atg−rol mice than that for normal mice (Moriet al., 2000). In addition, the activation curve displays a shift of ∼8 mV in the de-polarizing direction in the rolling Purkinje cells. Qualitatively similar results havebeen obtained with the rolling mutation introduced in recombinant CaV2.1/α2δ/β1b

channels transiently expressed in the mammalian BHK cell line, suggesting thatthe Cacna1atg−rol mutation leads to an alteration in the voltage-sensing function ofboth native and recombinant P/Q-type Ca2+ channels (Mori et al., 2000). Likewise,current-clamp recordings performed using brain slices showed similar firing patternsin Purkinje neurons in response to small amounts of depolarizing current in eithernormal and homozygous rolling Nagoya mice. However, when larger amounts ofcurrent were injected, bursts of Na+ action potentials were evoked in wild type andno response was recorded in rolling Purkinje neurons. Interestingly, a similar blockof Na+-dependent action potentials has been observed in normal Purkinje cells whenthe Ca2+ currents are blocked with Cd2+, which has been attributed to the fact thatduring Na+ bursts the membrane potential between action potentials remains rela-tively polarized because of the activation of Ca2+-activated K+ channels (Llinas andSugimori, 1980; Raman and Bean, 1999), preventing Na+ channels from completeinactivation. Since these channels are known to be present in both the somatic anddendritic regions of the neurons and play an important role in spike repolariza-tion (Gruol et al., 1991), it has been suggested that one of the consequences of theCacna1atg−rol mutation could be a failure in the activation of Ca2+-activated K+

channels in the rolling Purkinje cells as a result of reduced Ca2+ influx, leading todepolarization block of Na+-dependent action potentials (Mori et al., 2000).

The Rocker Mutation

A new Cacna1a mutant allele, rocker (Cacna1arkr ), that represents a slightlydifferent neurological mutant phenotype has been recently described. Similar to the




original tottering mutation, rocker mutant mice display an ataxic gait initiated byan action tremor typical of cerebellar dysfunction. The age of onset of the rockerbehavioral abnormalities is P21–P28 and the electroencephalographic studies re-veal frequent, spontaneous seizures, and spike-wave discharges (Zwingman et al.,2001). However, important differences between rocker and the previously charac-terized alleles of this locus include the absence of aberrant TH expression in Purkinjecells and the separation of the spike-wave discharges from the paroxysmal dyskine-sia phenotype. Nevertheless, the analysis of the cytoarchitecture of the adult rockerbrain reveals some abnormalities such as degeneration of axons, reduction of branch-ing in the Purkinje cell dendritic arbor, and a “weeping willow” appearance of thesecondary branches (Zwingman et al., 2001). Genetic analysis revealed that rockercontains a cytosine to adenosine change at nucleotide position 3929 resulting in anamino acid change (threonine to lysine) at position 1310 between transmembraneregions S5 and S6 in the III homologous domain of CaV2.1 (Fig. 1). It remains to bedetermined how this mutation affects neuronal CaV channel activity.

MUTATIONS IN THE ANCILLARY SUBUNITS OF THE CaV

CHANNEL COMPLEX

Direct implication of CaV channel auxiliary subunits in genetic disease hasemerged recently. The mouse mutant lethargic (lh) exhibits ataxia, and spike-waveepilepsy due to a mutation that deletes the α1 subunit interaction domain of theCaVβ4 subunit (Burgess et al., 1997). Likewise, a gene containing the mouse mutantducky (du) has been recently cloned and characterized (Barclay et al., 2001). Thisgene encodes the novel C2Vα2δ-2 subunit (Gao et al., 2000). Lastly, the stargazer (stg)mouse mutation causes absence seizures, ataxic gait, and vestibular problems. In thiscase, the genetic defect resides in an altered expression of the gene encoding theCaVγ2 subunit (Letts et al., 1998).

The Lethargic Mutation

The mouse mutant lethargic (lh) exhibits severe neurological defects inclu-ding ataxia, episodic dyskinesia, and generalized spike-wave epilepsy associated toa mutation that deletes the α1 subunit interaction domain of the CaVβ4 subunit.Specifically, the lh mutation causes a four base pair insertion into a splice donor sitewithin the Cacnb4 gene on chromosome 2 (Burgess et al., 1997). The mutation re-sults in aberrant pre-mRNA splicing and translational frameshift, and is predictedto encode a severely truncated CaVβ4 protein missing 60% of the C-terminal rel-ative to wild type including the essential α1-β interaction domain (Fig. 1). Notably,recombinant CaVβ subunits expressed with deletions of this domain lose their abilityto modulate the function of the α1 subunit in vitro (De Waard et al., 1994). It hasbeen documented that neither full-length nor truncated β4 proteins are expressed asa result of the lh mutation (McEnery et al., 1998a), though the immunolocalization ofthe neuronal CaV2.1 and CaV2.2 proteins in lethargic brain is indistinguishable fromwild type by bright-field microscopy (Burgess et al., 1999). In addition, the expression



112 Felix

of the CaVβ4 protein increases ∼10-fold during ontogeny in the rat brain beginningat the time (P7–P14) of cerebellar maturation (Vance et al., 1998). This increase par-allels the expression of the CaVβ4 mRNA in the rat cerebellum as determined byin situ hybridization (Tanaka et al., 1995). It is also worth noting that the use of specificantibodies has shown that a significant proportion (25–30%) of CaVβ4 is associatedto purified neuronal N-type Ca2+ channels (Scott et al., 1996; Vance et al., 1998) andhas identified this CaV channel auxiliary subunit as an important component of theP/Q-type channels (Liu et al., 1996).

Taken as a whole, the above mentioned findings suggested that the increasedexcitability and susceptibility to seizures observed in the lethargic mouse might arisefrom inappropriate expression of an immature population of CaV channels through-out neuronal development due to the absence of the CaVβ4 subunit (McEnery et al.,1998a,b). Unexpectedly however, biochemical evidence showed an important com-pensatory up-regulation in the expression of the CaVβ1b subunit in the lethargic mice,with a consequent increase in its incorporation into neuronal CaV channel complexesrelative to wild type animals (McEnery et al., 1998a). Even more, functional stud-ies showed that current amplitude through P/Q-type Ca2+ channels in dissociatedlethargic Purkinje neurons showed that these CaV2.1-containing channels were notaffected by the CaVβ4 subunit mutation and retained regulation by other β isoforms,suggesting that some properties of the CaV2.1 subunit are not uniquely regulated byCaVβ4 in vivo and may be rescued by other CaVβ subunits by a process termed“reshuffling” (Burgess et al., 1999). Subunit reshuffling, however, does not rule outthe presence of functional Ca2+ defects in lh brain. It is likely that the abnormalcurrents are preferentially localized within dendrites or synaptic terminals and arenot easily detected in electrophysiological recordings of dissociated neurons. Alter-natively, novel CaVβ associations might introduce subtle alterations in kinetic ormodulatory properties of the channels that are not apparent also under some exper-imental conditions (Burgess and Noebels, 1999).

Lastly, the lh mutation offered also the opportunity to evaluate the contributionof LVA channels to the lethargic phenotype. Though T-type Ca2+ channels have beenimplicated in the initiation of thalamic seizures in absence epilepsies in other mutantmice, a consensus binding domain for the CaVβ subunits in the three α1 isoformscoding T-type channels (CaV3.1 through CaV3.3) has not been identified (Lacinovaet al., 2000; Perez-Reyes, 1999). In addition, experimental evidence indicates thatblockade of CaVβ synthesis does not alter the biophysical properties of the T-typecurrents in cranial sensory neurons (Lambert et al., 1997). Together, these findingsimply that (i) LVA channels might not be directly related to the molecular mecha-nisms underlying epilepsy in the lethargic mouse, and (ii) T-type Ca2+ channel subunitdevelopmental expression, unlike HVA channels (at least those of the CaV2.1 andCaV2.2 type) may not be directly regulated by CaVβ auxiliary subunits.

The Ducky Mutation

Recently, a novel auxiliary C2Vα2δ-2 subunit (CACNA2D2) that shares ∼56%amino acid identity with the first cloned α2δ-1 subunit was positionally cloned andcharacterized. When coexpressed in the Xenopus oocyte system, α2δ-2 affected the




properties of neuronal recombinant Ca2+ channels suggesting that the α2δ-2 geneencodes indeed a functional auxiliary subunit of CaV channels. Interestingly, thehomologous location on mouse chromosome 9 is also the site of the neurologicmutant ducky (du) mouse, and thus, CACNA2D2 was proposed as a candidate genefor this inherited channelopathy (Gao et al., 2000). Homozygous ataxic du mutantmice have atrophy of the cerebellum, medulla oblongata, and spinal cord (Meier,1968) and develops a spike-wave phenotype in the electroencephalogram (Noebelset al., 1997).

Additional molecular studies have shown that wild type Cacna2d2 transcript isabsent from the brain of the original du strain and in a newly identified strain (du2J).Hence, in ducky mice, a genomic rearrangement disrupts Cacna2d2 and duplicates anonfunctional open reading frame by an unknown mechanism. Mutant transcripts arepresent at very low levels and encode proteins that are unlikely to function normally.One of the mutant transcripts would lack most of the extracellular α2 domain of theprotein as well as the transmembrane δ domain (Fig. 1), whereas the other mutanttranscript is unlikely to be targeted to membrane correctly (Barclay et al., 2001). Indu2J mice, a 2 bp deletion in exon 9 of Cacna2d2 would result in a truncated proteinlacking >800 amino acids, including the δ transmembrane domain (Barclay et al.,2001). By using site-directed antipeptide antibodies, evidence for the expression ofthe protein corresponding to the mutant predicted transcripts in the du cerebellumand in Purkinje cells was obtained recently (Brodbeck et al., 2002).

Functional studies showed that the macroscopic Ca2+ current in acutely dis-sociated ducky Purkinje cells was reduced ∼35%, with no appreciable changes insingle-channel conductance. In contrast, no effect on Ca2+ currents was seen in cere-bellar granule cells, which is consistent with the high level of expression of Cacna2d2in Purkinje, but not granule neurons (Barclay et al., 2001). In addition, the possiblepathological role of the duckyα2δ-2 has been studied using recombinant channels. Ex-perimental evidence indicates that the intranuclear injection of the cDNA encodinga mutant α2δ-2 protein induced a significant reduction (∼50%) in peak Ba2+ cur-rent through CaV2.1/β4 recombinant channels expressed in Xenopus oocytes (Brod-beck et al., 2002). Likewise, the analysis of the single channel parameters betweencell-attached patches of COS-7 cells transfected with the cDNAs coding CaV2.1/β4channels showed that neither the conductance nor the amplitude of the current, aswell as other biophysical properties of the channels were significantly affected by co-expression of the ducky α2δ-2 truncated protein. In all these experiments, however,the presence of the full-length α2δ-2 subunit caused a ∼3-fold increase in whole-cellcurrent amplitude. Consequently, though the individual channels remain unchanged,the modulation by α2δ-2 must involve an alteration in the number of active channelsin the plasma membrane. Therefore, the α2δ-2 subunit of CaV channels probably hasits main effect on the lifetime of the channel complex in the membrane, either byenhancing trafficking or reducing turnover.

As in other mouse models of absence epilepsy, a central role for disturbed neu-ronal CaV channel function can be invoked to explain the ducky phenotype. In thiscase, such a hypothesis is reinforced by the finding that the CaVα2δ subunit can sup-port pharmacological interactions with the antiepileptic drug gabapentin (Felix, 1999;Kelly, 1998). Moreover, the persistence of Purkinje neurons with an immature and



114 Felix

grossly abnormal morphology including multiple primary dendrites and a reductionin the size of the dendritic tree (Brodbeck et al., 2002) may provide the anatomicalbasis for the presumed cerebellar dysfunction underlying ataxia in the ducky mouse.The mechanism of the altered Purkinje cells morphology in the du mutant may resulteither from the reduced Ca2+ currents (Barclay et al., 2001) or more directly fromthe loss of the CaVα2δ subunit (Brodbeck et al., 2002).

The Stargazer Mutation

The stargazer (stg) mutation causes absence seizures that are more prolongedand frequent than any other petit mal mouse model (Noebels et al., 1990) and areprevented by the antiepileptic drug ethosuximide (Aizawa et al., 1997). Stargazermice also have an ataxic gait and vestibular alterations, including a distinctive head-tossing motion. Although synaptic changes in cerebellar granule cells, hippocampalCA3/mossy fibers, and cortical neurons in layer V that presumably lead to ataxiaand seizures, no major cytoarchitectural abnormalities are apparent in the stargazermice cerebellum (Qiao et al., 1996, 1998; Qiao and Noebels, 1993). Electrophysi-ological field recordings in neocortical slices from stg mice revealed spontaneoussynchronous network discharges not present in the wild type. Likewise, intracellularand whole-cell recordings showed altered intrinsic active properties and a decreasein threshold favoring repetitive action potential firing. Interestingly, a twofold in-crease in a hyperpolarization-activated Cs+-sensitive inward current (Ih) has beenalso observed, suggesting that enhanced Ih activity in stg mice may modify an intrin-sic component of the burst-induced afterhyperpolarizations of pyramidal neuronscontributing to the hyperexcitability of the network (Di Pasquale et al., 1997).

In addition to these functional alterations, the expression levels of brain-derivedneurotrophic factor (BDNF) in stg do not follow the developmental increase foundin wild type mice. In fact, stargazer mutant mouse shows a selective and almost com-plete reduction of BDNF mRNA expression in cerebellar granule cells (Qiao et al.,1996), and a ∼70% reduction in BDNF mRNA in the whole cerebellum. This hasbeen observed at P15, coincident with the onset of ataxia. Unexpectedly, BDNF ex-pression defect in stg mice is not associated with any changes in mRNA and proteinlevels of the BDNF receptor (TrkB), though a ligand-induced tyrosine phosphoryla-tion of several TrkB-activated signal transduction molecules is significantly reduced(Qiao et al., 1996, 1998). These data in conjunction with the persistency of immaturegranule cells in the adult stg cerebellum (Qiao et al., 1998) are indicative of retardedcytodifferentiation and suggest that an early failure of cerebellar BDNF expressionmight be related to the ataxic phenotype.

The stargazer locus has been mapped on mouse chromosome 15 betweenD15Mit30 and the parvalbumin gene, and a novel gene whose expression is dis-rupted in two stargazer alleles (stg and waggler) has been characterized (Chen et al.,1999; Letts et al., 1997). This gene, Cacng2, was shown to encode a∼36 KDa protein(stargazing) homologous to the γ subunit of muscle CaV channels and suggested thatstargazin represented a novel neuronal CaV channel subunit gene (hence termed γ 2)(Letts et al., 1997) (Fig. 1). The mutation in the CaVγ 2 gene was determined to bethe insertion of a 6-kb early transposon into the intron between exon 2 and exon 3.




Northern blot analysis suggested that the mRNA encoding CaVγ 2 in the stg mousewas decreased and could be distinguished from the wild type message both in its sizeand absence of the 3′ downstream UTR (Letts et al., 1997). Consistent with this, it hasbeen reported that the genetic defect in stargazer results in the loss of CaVγ 2 proteinexpression with no compensatory changes in the expression of other nonmutatedCaVγ proteins (Sharp et al., 2001). Functional studies have shown that transienttransfection of CaVγ 2 in BHK cells stably expressing recombinant CaV2.1/α2δ/β1

decreased the availability of the channels, as indicated by a negative shift in theinactivation curve at the steady-state (Letts et al., 1997).

Despite the fact that native CaV channel activity seemed relatively unaffected instg cerebellar neurons (Chen et al., 2000), the idea that the CaVγ 2 subunit is indeed animportant constituent of the neuronal Ca2+channel complex has been recently rein-forced by biochemical data showing cosedimentation and coimmunoprecipitation ofCaVγ 2 with other purified neuronal Ca2+ channel subunits (Kang et al., 2001; Sharpet al., 2001). In parallel, electrophysiological analyses showed that CaVγ 2 coexpres-sion caused a significant change in current amplitude through CaV2.1 and CaV2.2recombinant channels expressed in Xenopus oocytes (Kang et al., 2001). Inasmuchas CaV channels are major mediators of neurotransmitter release at the presynapticterminals (Hong and Chang, 1995; Mintz et al., 1995; Qian and Noebels, 2001), it wasspeculated that CaVγ 2 could inhibit presynaptic Ca2+ entry in stg mutant mice, caus-ing an inappropriate modulation of Ih that normally governs excitability in thalamicneurons (Di Pasquale et al., 1997; Luthi and McCormick, 1998). In this manner, theup-regulation of Ih in stg would be a link between the mutation and the epilepticphenotype. Moreover, the scope of the involvement of neuronal CaVγ subunits tonormal cellular events has been greatly extended by the recent identification of ad-ditional CaVγ isoforms (Black and Lennon, 1999; Burgess et al., 2000; Chen et al.,1999; Chu et al., 2001; Klugbauer et al., 2000; Sharp et al., 2001).

It is worth mentioning that the immunostaining pattern of CaVγ 2 resemblesclosely that of the GluR1 AMPA receptor with enrichment in the cerebellar molecu-lar layer, the hippocampus and olfactory nuclei (Petralia and Wenthold, 1992; Sharpet al., 2001), suggesting a possible convergence of CaV channel and AMPA receptorfunction. This interesting possibility was first suggested by distinct cerebellar granulecell defects present in stargazer due to an almost complete loss of AMPA receptorsynaptic responses, despite normal expression of AMPA receptor mRNA and pro-tein (Hashimoto et al., 1999). More recently, Chen et al. (2000) reported that cellsurface expression and synaptic clustering of AMPA receptors is abolished in thecerebellar granule cells of stg mice, and that this mutant phenotype can be rescuedby transfecting wild type CaVγ 2 into granule cells in vitro. In addition, deleting thecytoplasmic C-terminal of CaVγ 2 (which binds to the PDZ domains of PSD-95, apostsynaptic anchoring protein) still allowed rescue of the surface expression, butnot the synaptic localization, of AMPA receptors (Chen et al., 2000). These data, to-gether with the observation that CaVγ 2 influences the functional expression (Kanget al., 2001) and the voltage dependence of inactivation of recombinant CaV channels(Klugbauer et al., 2000; Letts et al., 1998), suggest that CaVγ 2 serves to couple someaspect of CaV channel signaling to the expression and/or recruitment of functionalAMPA receptors.



116 Felix

CONCLUDING REMARKS

Progress made in the complementary fields of molecular genetics and cellularelectrophysiology revealed that some paroxysmal disorders including epilepsy, arelinked to CaV channel abnormalities. Recent studies showed that the gene encodingthe CaV2.1 subunit (of the P/Q-type Ca2+ channels) is altered in the mutant micetottering, leaner, rolling Nagoya, and rocker. Phenotypic alterations of these miceinclude cerebellar dysfunction and a spike-wave pattern (absence epilepsy) in theelectroencephalogram. The same is true for mouse models lethargic, ducky, andstargazer that harbor mutations in the ancillary subunits of the CaV channel complex.These mutant mice have offered a unique opportunity to elucidate the molecular,developmental, and physiological mechanisms underlying CaV channel activity inthe brain, and have provided the possibility to link specific CaV channel subunits tocellular disease processes, including altered excitability, synaptic signaling, and celldeath.

Likewise, the possibility of understanding the cellular and molecular basis ofepilepsy has been greatly enhanced by the use of the mutant mice. However, therole of CaV channels in generating the absence epilepsy phenotype has proved to becomplex, because CaV channels are involved in numerous cellular functions, includ-ing proliferation and differentiation, membrane excitability, neurite outgrowth andsynaptogenesis, signal transduction, and gene expression. This is even more challeng-ing because experimental studies and clinical observations indicate a central role ofthalamocortical circuits, that comprise multiple neuronal populations, in the genesisof absence seizures. Nevertheless, a comprehensive analysis of CaV channel activityand neural excitability patterns in mutant mice brain should be useful in identifyingthe common pathophysiological elements involved in absence epilepsy.

ACKNOWLEDGMENTS

I am grateful to Drs R. Alvarado, E. Garcıa, and C. L. Trevino for critical readingof the manuscript. Work in the author’s laboratory is supported by the NationalCouncil for Science and Technology (Conacyt, Mexico) Grant No. 31735-N.

REFERENCES

Aizawa, M., Ito, Y., and Fukuda, H. (1997). Pharmacological profiles of generalized absence seizures inlethargic, stargazer and gamma-hydroxybutyrate-treated model mice. Neurosci. Res. 29:17–25.

Austin, M. C., Schultzberg, M., Abbott, L. C., Montpied, P., Evers, J. R., Paul, S. M., and Crawley, J. N.(1992). Expression of tyrosine hydroxylase in cerebellar Purkinje neurons of the mutant tottering andleaner mouse. Brain Res. Mol. Brain Res. 15:227–240.

Ayata, C., Shimizu-Sasamata, M., Lo, E. H., Noebels, J. L., and Moskowitz, M. A. (2000). Impaired neuro-transmitter release and elevated threshold for cortical spreading depression in mice with mutationsin the α1A subunit of P/Q type calcium channels. Neuroscience 95:639–645.

Barclay, J., Balaguero, N., Mione, M., Ackerman, S. L., Letts, V. A., Brodbeck, J., Canti, C., Meir, A., Page,K. M., Kusumi, K., Perez-Reyes, E., Lander, E. S., Frankel, W. N., Gardiner, R. M., Dolphin, A. C.,and Rees, M. (2001). Ducky mouse phenotype of epilepsy and ataxia is associated with mutations inthe Cacna2d2 gene and decreased calcium channel current in cerebellar Purkinje cells. J. Neurosci.21:6095–6104.




Birnbaumer, L., Qin, N., Olcese, R., Tareilus, E., Platano, D., Costantin, J., and Stefani, E. (1998). Structuresand functions of calcium channel β subunits. J. Bioenerg. Biomembr. 30:357–375.

Black, J. L., and Lennon, V. A. (1999). Identification and cloning of putative human neuronal voltage-gatedcalcium channel γ -2 and γ -3 subunits: Neurologic implications. Mayo Clin. Proc. 74:357–361.

Brodbeck, J., Davies, A., Courtney, J. M., Meir, A., Balaguero, N., Canti, C., Moss, F. J., Page, K. M., Pratt,W. S., Hunt, S. P., Barclay, J., Rees, M., and Dolphin, A. C. (2002). The ducky mutation in Cacna2d2results in altered Purkinje cell morphology and is associated with the expression of a truncated α2δ-2protein with abnormal function. J. Biol. Chem. 277:7684–7693.

Burgess, D. L., Biddlecome, G. H., McDonough, S. I., Diaz, M. E., Zilinski, C. A., Bean, B. P., Campbell,K. P., and Noebels, J. L. (1999). β subunit reshuffling modifies N- and P/Q-type Ca2+ channel subunitcompositions in lethargic mouse brain. Mol. Cell. Neurosci. 13:293–311.

Burgess, D. L., Jones, J. M., Meisler, M. H., and Noebels, J. L. (1997). Mutation of the Ca2+ channel βsubunit gene Cchb4 is associated with ataxia and seizures in the lethargic (lh) mouse. Cell 88:385–392.

Burgess, D. L., Matsuura, T., Ashizawa, T., and Noebels, J. L. (2000). Genetic localization of the Ca2+channel gene CACNG2 near SCA10 on chromosome 22q13. Epilepsia 41:24–27.

Burgess, D. L., and Noebels, J. L. (1999). Single gene defects in mice: The role of voltage-dependentcalcium channels in absence models. Epilepsy Res. 36:111–122.

Caddick, S. J., Wang, C., Fletcher, C. F., Jenkins, N. A., Copeland, N. G., and Hosford, D. A. (1999).Excitatory but not inhibitory synaptic transmission is reduced in lethargic (Cacnβ4lh) and tottering(Cacnα1Atg) mouse thalami. J. Neurophysiol 81:2066–2074.

Campbell, D. B., and Hess, E. J. (1999). L-type calcium channels contribute to the tottering mouse dystonicepisodes. Mol. Pharmacol. 55:23–31.

Catterall, W. A. (2000). Structure and regulation of voltage-gated Ca2+ channels. Annu. Rev. Cell. Dev.Biol. 16:521–555.

Chen, L., Bao, S., Qiao, X., and Thompson, R. F. (1999). Impaired cerebellar synapse maturation inwaggler, a mutant mouse with a disrupted neuronal calcium channel γ subunit. Proc. Natl. Acad. Sci.USA 96:12132–12137.

Chen, L., Chetkovich, D. M., Petralia, R. S., Sweeney, N. T., Kawasaki, Y., Wenthold, R. J., Bredt, D. S.,and Nicoll, R. A. (2000). Stargazing regulates synaptic targeting of AMPA receptors by two distinctmechanisms. Nature 408:936–943.

Chu, P. J., Robertson, H. M., and Best, P. M. (2001). Calcium channel gamma subunits provide insightsinto the evolution of this gene family. Gene 280:37–48.

Coulter, D. A., Huguenard, J. R., and Prince, D. A. (1989). Calcium currents in rat thalamocortical relayneurones: Kinetic properties of the transient, low-threshold current. J. Physiol. 414:587–604.

De Waard, M., Gurnett, C. A., and Campbell, K. P. (1996). Structural and functional diversity of voltage-activated calcium channels. In Narahashi, T. (ed.), Ion Channels, Vol. IV, Plennum, New York, pp.41–87.

De Waard, M., Liu, H., Walker, D., Scott, V. E., Gurnett, C. A., and Campbell, K. P. (1997). Direct bindingof G-protein βγ complex to voltage-dependent calcium channels. Nature 385:446–450.

De Waard, M., Pragnell, M., and Campbell, K. P. (1994). Ca2+ channel regulation by a conserved β subunitdomain. Neuron 13:495–503.

Di Pasquale, E., Keegan, K. D., and Noebels, J. L. (1997). Increased excitability and inward rectification inlayer V cortical pyramidal neurons in the epileptic mutant mouse stargazer. J. Neurophysiol. 77:621–631.

Dove, L. S., Abbott, L. C., and Griffith, W. H. (1998). Whole-cell and single-channel analysis of P-typecalcium currents in cerebellar Purkinje cells of leaner mutant mice. J. Neurosci. 18:7687–7699.

Dove, L. S., Nahm, S. S., Murchison, D., Abbott, L. C., and Griffith, W. H. (2000). Altered calciumhomeostasis in cerebellar Purkinje cells of leaner mutant mice. J. Neurophysiol. 84:513–524.

Doyle, J., Ren, X., Lennon, G., and Stubbs, L. (1997). Mutations in the Cacnl1a4 calcium channel geneare associated with seizures, cerebellar degeneration, and ataxia in tottering and leaner mutant mice.Mamm. Genome 8:113–120.

Ertel, E. A., Campbell, K. P., Harpold, M. M., Hofmann, F., Mori, Y., Perez-Reyes, E., Schwartz, A.,Snutch, T. P., Tanabe, T., Birnbaumer, L., Tsien, R. W., and Catterall, W. A. (2000). Nomenclature ofvoltage-gated calcium channels. Neuron 25:533–535.

Felix, R. (1999). Voltage-dependent Ca2+ channelα2δ auxiliary subunit: Structure, function and regulation.Receptors Channels 6:351–362.

Felix, R. (2000). Channelopathies: Ion channel defects linked to heritable clinical disorders. J. Med. Genet.37:729–740.

Fletcher, C. F., Lutz, C. M., O’Sullivan, T. N., Shaughnessy, J. D., Jr., Hawkes, R., Frankel, W. N., Copeland,N. G., and Jenkins, N. A. (1996). Absence epilepsy in tottering mutant mice is associated with calciumchannel defects. Cell 87:607–617.



118 Felix

Freise, D., Held, B., Wissenbach, U., Pfeifer, A., Trost, C., Himmerkus, N., Schweig, U., Freichel, M., Biel,M., Hofmann, F., Hoth, M., and Flockerzi, V. (2000). Absence of the γ subunit of the skeletal muscledihydropyridine receptor increases L-type Ca2+ currents and alters channel inactivation properties.J. Biol. Chem. 275:14476–14481.

Gao, B., Sekido, Y., Maximov, A., Saad, M., Forgacs, E., Latif, F., Wei, M. H., Lerman, M., Lee, J. H.,Perez-Reyes, E., Bezprozvanny, I., and Minna, J. D. (2000). Functional properties of a new voltage-dependent calcium channel α2δ auxiliary subunit gene (CACNA2D2). J. Biol. Chem. 275:12237–12242.

Gruol, D. L., Jacquin, T., and Yool, A. J. (1991). Single-channel K+ currents recorded from the somaticand dendritic regions of cerebellar Purkinje neurons in culture. J. Neurosci. 11:1002–1015.

Hashimoto, K., Fukaya, M., Qiao, X., Sakimura, K., Watanabe, M., and Kano, M. (1999). Impairment ofAMPA receptor function in cerebellar granule cells of ataxic mutant mouse stargazer. J. Neurosci.19:6027–6036.

Heckroth, J. A., and Abbott, L. C. (1994). Purkinje cell loss from alternating sagittal zones in the cerebellumof leaner mutant mice. Brain Res. 658:93–104.

Herrup, K., and Wilczynski, S. L. (1982). Cerebellar cell degeneration in the leaner mutant mouse.Neuroscience 7:2185–2196.

Hess, E. J., and Wilson, M. C. (1991). Tottering and leaner mutations perturb transient developmentalexpression of tyrosine hydroxylase in embryologically distinct Purkinje cells. Neuron 6:123–132.

Hong, S. J., and Chang, C. C. (1995). Inhibition of acetylcholine release from mouse motor nerve by aP-type calcium channel blocker, omega-agatoxin IVA. J. Physiol. 482:283–290.

Jarvis, S. E., and Zamponi, G. W. (2001). Interactions between presynaptic Ca2+ channels, cytoplasmicmessengers and proteins of the synaptic vesicle release complex. Trends Pharmacol. Sci. 22:519–525.

Kang, M. G., Chen, C. C., Felix, R., Letts, V. A., Frankel, W. N., Mori, Y., and Campbell, K. P. (2001).Biochemical and biophysical evidence for γ 2 subunit association with neuronal voltage-activatedCa2+ channels. J. Biol. Chem. 276:32917–32924.

Kelly, K. M. (1998). Gabapentin. Antiepileptic mechanism of action. Neuropsychobiology. 38:139–144.Klugbauer, N., Dai, S., Specht, V., Lacinova, L., Marais, E., Bohn, G., and Hofmann, F. (2000). A family

of gamma-like calcium channel subunits. FEBS Lett. 470:189–197.Lacinova, L., Klugbauer, N., and Hofmann, F. (2000). Low voltage activated calcium channels: From genes

to function. Gen. Physiol. Biophys. 19:121–136.Lambert, R. C., Maulet, Y., Mouton, J., Beattie, R., Volsen, S., De Waard, M., and Feltz, A. (1997). T-type

Ca2+ current properties are not modified by Ca2+ channel β subunit depletion in nodosus ganglionneurons. J. Neurosci. 17:6621–6628.

Lau, F. C., Abbott, L. C., Rhyu, I. J., Kim, D. S., and Chin, H. (1998). Expression of calcium channel α1AmRNA and protein in the leaner mouse (tgla/tgla) cerebellum. Brain Res. Mol. Brain Res. 59:93–99.

Lee, A., Wong, S. T., Gallagher, D., Li, B., Storm, D. R., Scheuer, T., and Catterall, W. A. (1999).Ca2+/calmodulin binds to and modulates P/Q-type calcium channels. Nature 399:155–159.

Letts, V. A., Felix, R., Biddlecome, G. H., Arikkath, J., Mahaffey, C. L., Valenzuela, A., Bartlett, F. S., II,Mori, Y., Campbell, K. P., and Frankel, W. N. (1998). The mouse stargazer gene encodes a neuronalCa2+-channel gamma subunit. Nat. Genet. 19:340–347.

Letts, V. A., Valenzuela, A., Kirley, J. P., Sweet, H. O., Davisson, M. T., and Frankel, W. N. (1997). Geneticand physical maps of the stargazer locus on mouse chromosome 15. Genomics 43:62–68.

Liu, H., De Waard, M., Scott, V. E., Gurnett, C. A., Lennon, V. A., and Campbell, K. P. (1996). Identificationof three subunits of the high affinity omega-conotoxin MVIIC-sensitive Ca2+ channel. J. Biol. Chem.271:13804–13810.

Llinas, R., and Sugimori, M. (1980). Electrophysiological properties of in vitro Purkinje cell somata inmammalian cerebellar slices. J. Physiol. 305:171–195.

Lorenzon, N. M., and Beam, K. G. (2000). Calcium channelopathies. Kidney Int. 57:794–802.Lorenzon, N. M., Lutz, C. M., Frankel, W. N., and Beam, K. G. (1998). Altered calcium channel currents

in Purkinje cells of the neurological mutant mouse leaner. J. Neurosci. 18:4482–4489.Luthi, A., and McCormick, D. A. (1998). Periodicity of thalamic synchronized oscillations: The role of

Ca2+-mediated upregulation of Ih. Neuron 20:553–563.Maximov, A., Sudhof, T. C., and Bezprozvanny, I. (1999). Association of neuronal calcium channels with

modular adaptor proteins. J. Biol. Chem. 274:24453–24456.McEnery, M. W., Copeland, T. D., and Vance, C. L. (1998a). Altered expression and assembly of N-type

calcium channel α1B and β subunits in epileptic lethargic (lh/lh) mouse. J. Biol. Chem. 273:21435–21438.

McEnery, M. W., Vance, C. L., Begg, C. M., Lee, W. L., Choi, Y., and Dubel, S. J. (1998b). Differentialexpression and association of calcium channel subunits in development and disease. J. Bioenerg.Biomembr. 30:409–418.




Meier, H. (1968). The neuropathology of ducky, a neurological mutation of the mouse. A pathologicaland preliminary histochemical study. Acta Neuropathol. 11:15–28.

Mintz, I. M., Sabatini, B. L., and Regehr, W. G. (1995). Calcium control of transmitter release at a cerebellarsynapse. Neuron 15:675–688.

Mori, Y., Wakamori, M., Oda, S., Fletcher, C. F., Sekiguchi, N., Mori, E., Copeland, N. G., Jenkins, N. A.,Matsushita, K., Matsuyama, Z., and Imoto, K. (2000). Reduced voltage sensitivity of activation of P/Q-type Ca2+ channels is associated with the ataxic mouse mutation rolling Nagoya (tgrol ). J. Neurosci.20:5654–5662.

Muth, J. N., Varadi, G., and Schwartz, A. (2001). Use of transgenic mice to study voltage-dependent Ca2+channels. Trends Pharmacol. Sci. 22:526–532.

Nakai, J., Tanabe, T., Konno, T., Adams, B., and Beam, K. G. (1998). Localization in the II-III loop of thedihydropyridine receptor of a sequence critical for excitation–contraction coupling. J. Biol. Chem.273:24983–24986.

Noebels, J. L., Fariello, R. G., Jobe, P. C., Lasley, S. M., and Marescaux, C. (1997). Genetic models ofgeneralized epilepsy. In Pedley, J. E., and Pedley, T. A. (eds.), Epilepsy: A Comprehensive Textbook,Lippincott-Raven, Philadelphia, pp. 457–465.

Noebels, J. L., Qiao, X., Bronson, R. T., Spencer, C., and Davisson, M. T. (1990). Stargazer: A newneurological mutant on chromosome 15 in the mouse with prolonged cortical seizures. Epilepsy Res.7:129–135.

Ogasawara, M., Kurihara, T., Hu, Q., and Tanabe, T. (2001). Characterization of acute somatosensory paintransmission in P/Q-type Ca2+ channel mutant mice, leaner. FEBS Lett. 508:181–186.

Ophoff, R. A., Terwindt, G. M., Vergouwe, M. N., van Eijk, R., Oefner, P. J., Hoffman, S. M., Lamerdin,J. E., Mohrenweiser, H. W., Bulman, D. E., Ferrari, M., Haan, J., Lindhout, D., van Ommen, G. J.,Hofker, M. H., Ferrari, M. D., and Frants, R. R. (1996). Familial hemiplegic migraine and episodicataxia type-2 are caused by mutations in the Ca2+ channel gene CACNL1A4. Cell 87:543–552.

Perez-Reyes, E. (1999). Three for T: Molecular analysis of the low voltage-activated calcium channelfamily. Cell. Mol. Life Sci. 56:660–669.

Petralia, R. S., and Wenthold, R. J. (1992). Light and electron immunocytochemical localization of AMPA-selective glutamate receptors in the rat brain. J. Comp. Neurol. 318:329–354.

Pragnell, M., De Waard, M., Mori, Y., Tanabe, T., Snutch, T. P., and Campbell, K. P. (1994). Calciumchannel β-subunit binds to a conserved motif in the I-II cytoplasmic linker of the α1-subunit. Nature368:67–70.

Qian, J., and Noebels, J. L. (2000). Presynaptic Ca2+ influx at a mouse central synapse with Ca2+ channelsubunit mutations. J. Neurosci. 20:163–170.

Qian, J., and Noebels, J. L. (2001). Presynaptic Ca2+ channels and neurotransmitter release at the terminalof a mouse cortical neuron. J. Neurosci. 21:3721–3728.

Qiao, X., Chen, L., Gao, H., Bao, S., Hefti, F., Thompson, R. F., and Knusel, B. (1998). Cerebellar brain-derived neurotrophic factor-TrkB defect associated with impairment of eyeblink conditioning inStargazer mutant mice. J. Neurosci. 18:6990–6999.

Qiao, X., Hefti, F., Knusel, B., and Noebels, J. L. (1996). Selective failure of brain-derived neurotrophicfactor mRNA expression in the cerebellum of stargazer, a mutant mouse with ataxia. J. Neurosci.16:640–648.

Qiao, X., and Noebels, J. L. (1993). Developmental analysis of hippocampal mossy fiber outgrowth in amutant mouse with inherited spike-wave seizures. J. Neurosci. 13:4622–4635.

Raman, I. M., and Bean, B. P. (1999). Ionic currents underlying spontaneous action potentials in isolatedcerebellar Purkinje neurons. J. Neurosci. 19:1663–1674.

Rhyu, I. J., Abbott, L. C., Walker, D. B., and Sotelo, C. (1999a). An ultrastructural study of granulecell/Purkinje cell synapses in tottering (tg/tg), leaner (tgla /tgla) and compound heterozygous totter-ing/leaner (tg/tgla) mice. Neuroscience 90:717–728.

Rhyu, I. J., Oda, S., Uhm, C. S., Kim, H., Suh, Y. S., and Abbott, L. C. (1999b). Morphologic investigationof rolling mouse Nagoya (tgrol /tgrol ) cerebellar Purkinje cells: An ataxic mutant, revisited. Neurosci.Lett. 266:49–52.

Saegusa, H., Kurihara, T., Zong, S., Kazuno, A., Matsuda, Y., Nonaka, T., Han, W., Toriyama, H., andTanabe, T. (2001). Suppression of inflammatory and neuropathic pain symptoms in mice lacking theN-type Ca2+ channel. EMBO J. 20:2349–2356.

Saegusa, H., Kurihara, T., Zong, S., Minowa, O., Kazuno, A., Han, W., Matsuda, Y., Yamanaka, H.,Osanai, M., Noda, T., and Tanabe, T. (2000). Altered pain responses in mice lacking α1E subunit ofthe voltage-dependent Ca2+ channel. Proc. Natl. Acad. Sci. USA 97:6132–6137.

Sandoz, G., Bichet, D., Cornet, V., Mori, Y., Felix, R., and De Waard, M. (2001). Distinct properties anddifferential beta subunit regulation of two C-terminal isoforms of the P/Q-type Ca2+-channel α1Asubunit. Eur. J. Neurosci. 14:987–997.



120 Felix

Sawada, K., Komatsu, S., Haga, H., Sun, X. Z., Hisano, S., and Fukui, Y. (1999). Abnormal expressionof tyrosine hydroxylase immunoreactivity in cerebellar cortex of ataxic mutant mice. Brain Res.829:107–112.

Scott, V. E., De Waard, M., Liu, H., Gurnett, C. A., Venzke, D. P., Lennon, V. A., and Campbell, K. P.(1996). β subunit heterogeneity in N-type Ca2+ channels. J. Biol. Chem. 271:3207–3212.

Seagar, M., Leveque, C., Charvin, N., Marqueze, B., Martin-Moutot, N., Boudier, J. A., Boudier, J. L., Shoji-Kasai, Y., Sato, K., and Takahashi, M. (1999). Interactions between proteins implicated in exocytosisand voltage-gated calcium channels. Philos. Trans. R. Soc. Lond. B Biol. Sci. 354:289–297.

Sharp, A. H., Black, J. L., III, Dubel, S. J., Sundarraj, S., Shen, J. P., Yunker, A. M., Copeland, T. D.,and McEnery, M. W. (2001). Biochemical and anatomical evidence for specialized voltage-dependentcalcium channel gamma isoform expression in the epileptic and ataxic mouse, stargazer. Neuroscience105:599–617.

Snead, O. C., III. (1995). Basic mechanisms of generalized absence seizures. Ann. Neurol. 37:146–157.Steinlein, O. K., and Noebels, J. L. (2000). Ion channels and epilepsy in man and mouse. Curr. Opin. Genet.

Dev. 10:286–291.Steriade, M., and Llinas, R. F. (1988). The functional states of the thalamus and the associated neuronal

interplay. Physiol. Rev. 68:649–742.Talavera, K., Staes, M., Janssens, A., Klugbauer, N., Droogmans, G., Hofmann, F., and Nilius, B. (2001).

Aspartate residues of the Glu-Glu-Asp-Asp (EEDD) pore locus control selectivity and permeationof the T-type Ca2+ channel α1G. J. Biol. Chem. 276:45628–45635.

Tanaka, O., Sakagami, H., and Kondo, H. (1995). Localization of mRNAs of voltage-dependentCa2+-channels: Four subtypes of α1- and β-subunits in developing and mature rat brain. Brain Res.Mol. Brain Res. 30:1–16.

Uchitel, O. D. (1997). Toxins affecting calcium channels in neurons. Toxicon 35:1161–1191.Vance, C. L., Begg, C. M., Lee, W. L., Haase, H., Copeland, T. D., and McEnery, M. W. (1998). Differential

expression and association of calcium channel α1B and β subunits during rat brain ontogeny. J. Biol.Chem. 273:14495–14502.

Varadi, G., Mori, Y., Mikala, G., and Schwartz, A. (1995). Molecular determinants of Ca2+ channel functionand drug action. Trends Pharmacol. Sci. 16:43–49.

Wakamori, M., Yamazaki, K., Matsunodaira, H., Teramoto, T., Tanaka, I., Niidome, T., Sawada, K.,Nishizawa, Y., Sekiguchi, N., Mori, E., Mori, Y., and Imoto, K. (1998). Single tottering mutationsresponsible for the neuropathic phenotype of the P-type calcium channel. J. Biol. Chem. 273:34857–34867.

Walker, D., and De Waard, M. (1998). Subunit interaction sites in voltage-dependent Ca2+ channels: Rolein channel function. Trends Neurosci. 21:148–154.

Wang, X. J., Rinzel, J., and Rogawski, M. A. (1991). A model of the T-type calcium current and thelow-threshold spike in thalamic neurons. J. Neurophysiol. 66:839–850.

Yang, J., Ellinor, P. T., Sather, W. A., Zhang, J. F., and Tsien, R. W. (1993). Molecular determinants ofCa2+ selectivity and ion permeation in L-type Ca2+ channels. Nature 366:158–161.

Yue, Q., Jen, J. C., Nelson, S. F., and Baloh, R. W. (1997). Progressive ataxia due to a missense mutationin a calcium-channel gene. Am. J. Hum. Genet. 61:1078–1087.

Zamponi, G. W., and Snutch, T. P. (1998). Modulation of voltage-dependent calcium channels by Gproteins. Curr. Opin. Neurobiol. 8:351–356.

Zuhlke, R. D., Pitt, G. S., Deisseroth, K., Tsien, R. W., and Reuter, H. (1999). Calmodulin supports bothinactivation and facilitation of L-type calcium channels. Nature 399:159–162.

Zwingman, T. A., Neumann, P. E., Noebels, J. L., and Herrup, K. (2001). Rocker is a new variant of thevoltage-dependent calcium channel gene Cacna1a. J. Neurosci. 21:1169–1178.

insights from mouse models of absence epilepsy into ca2+ channel physiology and disease etiology

Documents