Mutations of the α1A calcium channel subunit have been shown to cause such human neurological diseases as familial hemiplegic migraine, episodic ataxia-2, and spinocerebellar ataxia 6 and also to cause the murine neurological phenotypes oftottering and leaner. Theleaner phenotype is recessive and characterized by ataxia with cortical spike and wave discharges (similar to absence epilepsy in humans) and a gradual degeneration of cerebellar Purkinje and granule cells. The mutation responsible is a single-base substitution that produces truncation of the normal open reading frame beyond repeat IV and expression of a novel C-terminal sequence. Here, we have used whole-cell recordings to determine whether theleaner mutation alters calcium channel currents in cerebellar Purkinje cells, both because these cells are profoundly affected in leaner mice and because they normally express high levels of α1A. In Purkinje cells from normal mice, 82% of the whole-cell current was blocked by 100 nmω-agatoxin-IVA. In Purkinje cells from homozygousleaner mice, this ω-agatoxin-IVA-sensitive current was 65% smaller than in control cells. Although attenuated, the ω-agatoxin-IVA-sensitive current in homozygous leanercells had properties indistinguishable from that of normal Purkinje neurons. Additionally, the ω-agatoxin-IVA-insensitive current was unaffected in homozygous leaner mice. Thus, theleaner mutation selectively reduces P-type currents in Purkinje cells, and the α1A subunit and P-type current appear to be essential for normal cerebellar function.
Recently, mutations in the α1A calcium channel subunit have been shown to produce the human neurological disorders of autosomal dominant spinocerebellar ataxia (Zhuchenko et al., 1997), familial hemiplegic migraine, and episodic ataxia type-2 (Ophoff et al., 1996). In each of these diseases, alterations of a single gene result in a host of aberrant neurological phenotypes including abnormal cerebellar function and cerebellar atrophy. Defects in the gene encoding the α1Acalcium channel subunit are also responsible for the phenotypes of tottering (tg) and leaner (tg la) mutant mice. The tg la mutation has been identified as a single-base pair substitution in a splice donor consensus sequence of the gene for the α1A subunit, which results in a truncation in the normal open reading frame beyond repeat IV and expression of a novel C-terminal sequence (Fletcher et al., 1996; Doyle et al., 1997).
The tg la mutation is inherited as a recessive trait, and homozygous mutants are severely ataxic with cortical spike and wave discharges similar to absence epilepsy in humans (Noebels, 1984). The tg la mutation is also associated with cerebellar atrophy resulting from a gradual degeneration of cerebellar Purkinje and granule cells. The cerebella oftg la/tg la mice contain 80% fewer Purkinje cells compared with those of normal mice (Herrup and Wilczynski, 1982), and the surviving Purkinje cells exhibit aberrant morphology of the dendritic tree and axonal swellings (Heckroth and Abbott, 1994).
Normally, α1A channels are highly expressed in cerebellar Purkinje cells (Mori et al., 1991; Starr et al., 1991; Stea et al., 1994; Westenbroek et al., 1995). This, together with the observation that low concentrations of ω-agatoxin-IVA block ∼90% of the calcium channel current in Purkinje cells (Mintz et al., 1992b), has been the basis for the assumption that α1A encodes P-type channels (which are defined by block with low concentrations of ω-agatoxin-IVA). However, heterologous expression of the α1A subunit produces a current that (1) is not blocked by low ω-agatoxin-IVA and (2) physiologically resembles the Q-type current recorded from cerebellar granule cells (Sather et al., 1993;Stea et al., 1994). These findings have led to the suggestion that α1A encodes Q-type channels (in addition to its presumed encoding of P-channels). Here we have used whole-cell recordings to determine whether the tg la mutation nonspecifically alters calcium channel currents in cerebellar Purkinje cells or affects only P-type current as defined by sensitivity to ω-agatoxin-IVA.
We report here that the tg la mutation causes a marked reduction of P-type current in cerebellar Purkinje cells, and that non-P-type currents are unaffected by thetg la mutation. Thus, α1A most likely does encode P-type channels. It remains to be determined why a decrease in P-type calcium current ultimately causes the disease phenotype.
MATERIALS AND METHODS
Mutant mice. C57BL/6J mice (breeding pairs oftg la/+, +/Os, and +/+ mice) were provided from colonies at The Jackson Laboratory (Bar Harbor, ME).Os is a tightly linked, semidominant skeletal mutation carried in repulsion with tg la. HeterozygousOs/+ mice, identified by the oligosyndactylism, were inferred to be heterozygous for the tg lamutation. Mice with no oligosyndactylism were inferred to be homozygous for the tg la mutation. Because homozygousOs/Os is embryonic lethal, we used pups from C57BL/6J wild-type matings as normal controls (without thetg la or Os mutation). Cells were isolated from 1-week-old mice (P7–P9). This age precedes Purkinje cell death [marked Purkinje cell loss is observed at approximately postnatal day 40 (P40); granule cell death begins at approximately P10 (Herrup and Wilczynski, 1982)] but is a stage when acutely dissociated Purkinje cells can be easily identified morphologically.
Dissociation of neurons. Mouse cerebellar Purkinje cells were acutely isolated using a procedure modified from Mintz et al. (1992b). Briefly, cerebella were removed from P7–P9 mice in an ice-cold solution containing (in mm): 82 Na2SO4, 30 K2SO4, 5 MgCl2, 10 HEPES, and 10 glucose, pH 7.4. The tissue was cut into small pieces and then placed into the same high-Na/high-K solution with enzyme (protease type XXIII; Sigma, St. Louis, MO; 3.0 mg/ml at 37°C). After 7–8 min, the tissue was washed two times in the high-Na/high-K solution without enzyme. The tissue was triturated with a fire-polished Pasteur pipette in minimum essential medium (Life Technologies, Grand Island, NY) containing 1 mg/ml trypsin inhibitor type II-0 (Sigma), 1 mg/ml bovine serum albumin (Sigma), 15 mm HEPES, and 10 mmglucose, pH 7.4. The cell suspension was transferred to poly-l-lysine-coated plastic Petri dishes and remained in the trituration solution at room temperature until just before electrophysiological recording. Purkinje cells were identified by the morphological characteristics of relatively large size and apical dendritic stump (Regan, 1991; Mintz et al., 1992b).
Recording solutions and pharmacological agents. Calcium channel currents were isolated using an external solution that included (in mm): 160 TEA-Cl, 5 BaCl2, and 10 HEPES, pH 7.4, and an internal solution that consisted of (in mm): 108 Cs-methanesulfonate, 4 MgCl2, 9 HEPES, 9 EGTA, 14 phosphocreatine, 0.3 GTP, and 4 ATP, pH 7.4. ω-Agatoxin-IVA (ω-AgTx-IVA; a gift from Pfizer, Groton, CT; or purchased from Peptides International, Louisville, KY) was prepared as a concentrated stock in distilled water with 1 mg/ml bovine serum albumin and then stored as aliquots at −80°C. The calcium channel antagonist was diluted in the external recording solution on the day of the experiment. Drugs were delivered with a multibarrel array (“sewer pipe”) of glass capillary tubing (150 μm outer diameter) mounted on a micromanipulator.
Whole-cell patch-clamp recordings. Whole-cell recordings were obtained using conventional techniques (Hamill et al., 1981) at room temperature with a Dagan 3900 patch-clamp amplifier and a 3911 whole-cell expander. Electrodes (1–3 m) were pulled from soda lime glass with a Brown-Flaming puller (Sutter) and coated with wax to reduce capacitance. After attainment of a gigaohm seal and entry into whole-cell mode, series resistance was compensated electronically. Linear leak and capacitative currents were removed by digital subtraction of scaled control currents (evoked by a 20 mV hyperpolarizing voltage step from the holding potential). Data acquisition and analysis were performed with software programs written in BASIC-23. Current densities were calculated by dividing the current amplitude by the linear capacitance of the cell. All summary data are presented as mean ± SEM (number of cells), and population data were compared with the use of an unpaired Student’s ttest.
Barium currents were recorded from cerebellar Purkinje cells of homozygous leaner (tg la/tg la), heterozygous leaner (tg la/+), and wild-type mice. Barium currents fromtg la/tg la mice appeared qualitatively similar to those fromtg la/+ and wild-type mice. Thus, in neurons of all three genotypes, a sustained inward current was activated by depolarizing voltage steps from a holding potential of −80 mV (Fig.1). With 5 mmBa2+ as the charge carrier, the whole-cell current first activated at approximately −45 mV and reached maximum amplitude at approximately −10 mV. The half-activation voltages of barium currents were also similar intg la/tg la,tg la/+, and wild-type cells. A small reduction in the slope of the conductance–voltage (G–V) curve was observed intg la/tg la cells compared with tg la/+ and wild-type cells (Table 1). However, the most striking alteration in the calcium channel currents oftg la/tg la cells was a marked reduction in the peak current density as compared withtg la/+ and wild-type cells (Table 1). Specifically, the current density intg la/tg la cells was only about half that in tg la/+ and wild-type cells (51 and 44%, respectively).
To examine the voltage dependence of inactivation, a two-pulse protocol was applied; 2 sec depolarizing prepulses to various potentials (−110 to +30 mV) were followed by a voltage step to the test potential eliciting maximal current in that cell (with a 10 sec interval between inactivation measurements; Fig. 2). The peak current amplitude during the test pulse was measured, and inactivation curves were constructed. The voltage dependence of inactivation did not differ significantly betweentg la/tg la and control currents (Table 1). However, the ratio of the peak current amplitude to the current amplitude at the end of a 2 sec pulse was greater intg la/tg la Purkinje cells (Fig. 2, Table 1). This could indicate a difference in kinetic properties of the mutant channels. Alternately, it could indicate that the tg la mutation decreased the fractional contribution of α1A channels to the total current, so that the whole-cell current became dominated by current through non-α1A channels with a greater ratio of peak to 2 sec current amplitude.
The tgla mutation strongly attenuates the P-type current in Purkinje cells
To determine the specific effects of the α1Amutation on P-type currents, barium currents intg la/tg la and control Purkinje cells were recorded before and after the application of ω-AgTx-IVA, and the difference currents were analyzed. ω-AgTx-IVA was applied to the cells at a concentration of 100 nm to ensure that all P-type channels were blocked, because previous studies have shown that 50 nm ω-AgTx-IVA produces maximal block of all non-L-type and non-N-type currents in rat cerebellar Purkinje cells, with no further inhibition by 200 nm toxin (Mintz et al., 1992b). The percentage of the current blocked by 100 nm ω-AgTx-IVA was significantly greater in wild-type and tg la/+ cells compared withtg la/tg la cells (Table 1). The current remaining after application of ω-AgTx-IVA had similar kinetics and density intg la/tg la and control cells, suggesting that the reduction of the α1Acurrent does not affect the remaining, non-P/Q current types in Purkinje cells (Figs. 3 A,4). Thus, the ω-AgTx-IVA-sensitive current (determined by subtraction of the currents recorded after application of ω-AgTx-IVA from control records obtained before ω-AgTx-IVA) is selectively reduced by thetg la mutation (Figs. 3 B, 4). Despite the difference in amplitude, the ω-AgTx-IVA-sensitive current exhibited a similar time course intg la/tg la and control cells (Fig. 3 B). When the ω-AgTx-IVA-sensitive currents in the tg la/+ andtg la/tg la cells were scaled by current amplitude, the traces closely superimposed (Fig.3 D).
To determine the effects of the tg la mutation more directly, we characterized the barium current flowing through the α1A channels in isolation (ω-AgTx-IVA-sensitive currents). The ω-AgTx-IVA-sensitive currents intg la/+ andtg la/tg la cells exhibited no differences in the voltage dependence of activation (Fig.5). The half-activation voltage of the ω-AgTx-IVA-sensitive current was −22.7 ± 1.9 mV (n = 7) intg la/+ cells and −22.5 ± 1.9 mV (n = 6) intg la/tg la cells. The slopes of the G–V curve fortg la/+ andtg la/tg la currents were 4.3 ± 0.2 (n = 7) and 4.5 ± 0.1 (n = 6), respectively. Thus, the decreased slope of theG–V curve for the whole-cell currents intg la/tg la cells was not a reflection of altered biophysical properties of the α1A channels associated with thetg la mutation.
The inactivation characteristics of the ω-AgTx-IVA-sensitive currents also appeared similar intg la/tg la and control cells, as indicated both by the voltage dependence of inactivation (Fig.6) and by the ratio of peak current amplitude to current amplitude at 2 sec (0.56 ± 0.04; n = 4 fortg la/+ cells; vs 0.51 ± 0.06;n = 4 fortg la/tg la cells). Therefore, the only alteration in the calcium channel current directly caused by the mutation appeared to be the substantial reduction in P-type current density. The small differences in the whole-cell calcium channel current oftg la/tg la cells (reduced G–V slope and altered current time course during a 2 sec pulse) evidently resulted from the greater relative proportion of non-P-type calcium channel currents in these cells.
Calcium channel currents intgla/tgla mice
We have compared calcium channel currents in Purkinje cells from wild-type mice and mice homozygous for thetg la mutation of the α1A gene. This tg la mutation specifically reduces the amplitude of P-type current without changing its macroscopic properties. Non-P-type currents are unaffected by the mutation.
Although our electrophysiological measurements cannot determine the specific cause of the attenuated P-type current, we hypothesize that the tg la mutation causes reduced expression of α1A. This hypothesis is supported by the observation that cerebella oftg la/tg la mice express reduced levels of α1A mRNA at all ages tested (embryonic day 10.5 to adult; Doyle et al., 1997). Western blot analysis would reveal whether this reduction in α1Atranscript results in reduced α1A protein. In addition to an altered level of channel expression, it might also be that thetg la mutation results in altered subcellular distribution, a possibility which could be examined with immunohistochemistry.
Relationship of P/Q current to the α1A subunit
P- and Q-type calcium currents are differentiated mainly on the basis of pharmacology. Calcium channel current through P-type channels is not sensitive to dihydropyridines or ω-conotoxin-GVIA but is blocked by funnel web spider venom or low concentrations of a component of the venom, ω-AgTx-IVA (Llinas et al., 1989; Mintz et al., 1992a). Q-type current is defined on the basis of block by high concentrations of ω-AgTx-IVA or ω-conotoxin-MVIIC (Hillyard et al., 1992; Sather et al., 1993; Randall and Tsien, 1995). However, the molecular identity of the channels that carry P- and Q-type currents has been questioned. The α1A subunit was originally thought to encode a P-type channel (Mori et al., 1991), but when expressed in Xenopus, the α1A channel has different characteristics (Sather et al., 1993; Stea et al., 1994) resembling the Q-type current recorded in cerebellar granule cells (Randall and Tsien, 1995). It is currently unclear whether the P- and Q-type currents actually arise from different α1 genes, alternatively spliced forms of the same gene, or the same α1A subunit that is modulated differently by auxiliary subunits. In a recent study using antisense oligonucleotides, Gillard et al. (1997) suggested that P-type current in cerebellar Purkinje cells is encoded by the α1Asubunit. Our experiments on thetg la/tg la mouse support the argument that α1A channels are responsible for P-type current in these cells. To determine whether α1A channels also carry Q-type current, future investigation of the calcium channel currents in cerebellar granule cells fromtg la/tg la mice would be valuable, because Q-type current is a high percentage of the total calcium channel current in this cell type.
Effects of the tgla mutation on cellular function
With regard to the pathological consequences of the channel mutation, the question arises: why does a decrease in calcium current density ultimately result in cell death? Several studies have suggested that not only high intracellular calcium concentrations, but also low intracellular calcium levels can result in cell death (Koike et al., 1989; McCaslin and Smith, 1990; Koh and Cotman, 1992). This explanation is insufficient, however, to explain why the Purkinje cell loss in thetg la/tg la mouse is restricted to alternating sagittal compartments of the cerebellar cortex. The subpopulation of Purkinje cells that survive intg la/tg la mice appears to be the same population of neurons expressing Zebrin II and displaying abnormal persistent expression of tyrosine hydroxylase (normally Purkinje cells only express tyrosine hydroxylase from the third to fifth postnatal weeks; Heckroth and Abbott, 1994). What allows these cells to be spared is currently not known. The Purkinje cells that are spared in thetg la/tg lacerebellum have abnormal morphology of their dendritic trees and axonal swellings. Because voltage-gated calcium channels are important for growth cone function in neurons (Williams et al., 1992), it is likely that α1A channels are important for dendritic growth and development in Purkinje cells, thus possibly explaining the abnormal morphological characteristics of Purkinje cells intg la/tg la mice.
Immunolocalization in normal mice reveals pronounced α1Astaining in terminals along dendrites of cerebellar Purkinje cells, hippocampal neurons, and cortical pyramidal neurons (Westenbroek et al., 1995). The pharmacological block of P/Q currents inhibits transmission at various synapses in the central and peripheral nervous systems (Luebke et al., 1993; Regehr and Mintz 1994; Wheeler et al., 1994; Bowersox et al., 1995). Thus, a decrease in calcium current density intg la/tg la mice would most likely attenuate synaptic transmission at synapses between many cell types.
The attenuation of calcium channel current would also affect other cellular functions, especially in cerebellar Purkinje cells, where α1A channels are additionally distributed along dendrites and in the cell body. α1A channels expressed in Purkinje cell dendrites may play an important role in integration and propagation of synaptic signals (Llinas et al., 1992). However, it is also possible that alterations in calcium current density affect nervous system function via a more indirect route. The aberrant tyrosine hydroxylase expression intg la/tg la Purkinje cells suggests that the mutation ultimately causes abnormal developmental signaling such that the mutant cells cannot recognize the signal that downregulates tyrosine hydroxylase expression (Hess and Wilson, 1991). Although L-type channels have been associated with gene regulation in many neurons (Bading et al., 1993), P-type channels might assume this role in Purkinje cells. This difference can be rationalized on the basis that L-type channels dominate somatically in most CNS neurons, but not in Purkinje cells where the majority of the voltage-gated calcium channels in the soma are P-type.
Other disorders associated with α1Asubunit mutations
Tottering (tg) is another recessively inherited α1A mutation described in the mouse. The tgmutation is a single-nucleotide change resulting in a substitution of leucine for proline in the IIS5-IIS6 linker of the α1Asubunit (Fletcher et al., 1996; Doyle et al., 1997). Thetg/tg mouse resembles thetg la/tg la mouse in having absence seizures. However, these two mutant mice differ in thattg/tg mice exhibit only mild ataxia, intermittent focal motor seizures (which are not seen in thetg la/tg la mouse;Noebels, 1984), and minor diffuse loss of cerebellar granule and Purkinje cells. As intg la/tg la mice, the Purkinje cells in tg/tg mice aberrantly express tyrosine hydroxylase (Hess and Wilson, 1991).
Several human disorders have been associated with α1Asubunit mutations. One of these diseases, autosomal dominant spinocerebellar ataxia (SCA6), is characterized by slowly progressive ataxia, nystagmus, dysarthria, and cerebellar atrophy (with a severe loss of Purkinje cells, moderate loss of granule cells and dentate nucleus neurons, and mild to moderate cell loss in the inferior olive). Genetic analysis of patients with SCA6 disclosed an expansion of a CAG repeat resulting in a polyglutamine expansion (Zhuchenko et al., 1997). Another human neurological disorder associated with an α1A channel mutation is familial hemiplegic migraine (FHM), which presents as ictal hemiparesis, ataxia, nystagmus, and, in some families, cerebellar atrophy. FHM is associated with four different missense mutations in conserved functional domains (Ophoff et al., 1996). A third disorder, episodic ataxia type-2, is associated with cerebellar ataxia, migraine-like symptoms, interictal nystagmus, and cerebellar atrophy and is correlated with two mutations that disrupt the reading frame and cause premature stops so that the channel protein contains only repeats I, II, and part of III (Ophoff et al., 1996).
Another mutant mouse exhibiting some disease traits that overlap with those previously described for α1A human and murine diseases (ataxia and spontaneous focal motor and absence seizures) is the lethargic (lh) mouse. However, no pathological changes are observed in the brains of these mutants (Dung and Swigart, 1972). The lh mutation is not located in the α1Asubunit gene but rather in the gene encoding the β4auxiliary subunit that associates with the α1 subunit. In heterologous expression studies, β subunits have been shown to augment and modulate α1 calcium channel currents (Hullin et al., 1992; Castellano et al., 1993). The lh mutation is a four-nucleotide insertion into a splice donor sequence that results in exon skipping, translational frame shift, and protein truncation with loss of the α1-binding site (Burgess et al., 1997). This mutation would cause a functional loss of β4, which could ultimately result in altered α1 calcium channel currents.
In summary, the present study demonstrates that α1Aexpression and P-type current in cerebellar Purkinje cells are critical for normal cerebellar function. Currently, it is unclear how thetg la mutation of the α1Achannel subunit and subsequent decrease in current density cause the mutant phenotypes. This α1A mutation ultimately results in abnormal cellular morphology and function and cell death. Additionally, the mutation most likely causes attenuation of synaptic transmission, alters dendritic integration, and results in abnormal developmental signaling in Purkinje cells. Comparing calcium channel function and the neurological characteristics in different α1A diseases may suggest hypotheses regarding the etiology of these diseases and methods for treatment.
This work was supported by National Institutes of Health Grant NS24444 to K.G.B. We gratefully acknowledge the excellent technical assistance of K. Lopez-Jones and K. Parsons. We are grateful to Pfizer, Inc. for a gift of ω-agatoxin-IVA.
Correspondence should be addressed to Kurt G. Beam, Department of Anatomy and Neurobiology, Colorado State University, Fort Collins, CO 80523.