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The Journal of Neuroscience, June 15, 1998, 18(12):4482-4489
Altered Calcium Channel Currents in Purkinje Cells of the
Neurological Mutant Mouse leaner
Nancy M.
Lorenzon1,
Cathleen M.
Lutz2,
Wayne N.
Frankel2, and
Kurt G.
Beam1
1 Department of Anatomy and Neurobiology, Colorado
State University, Fort Collins, Colorado 80523, and
2 The Jackson Laboratory, Bar Harbor, Maine 04609
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ABSTRACT |
Mutations of the  1A 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 of
tottering and leaner. The
leaner 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 the
leaner 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 1A. In Purkinje cells from normal
mice, 82% of the whole-cell current was blocked by 100 nM
 -agatoxin-IVA. In Purkinje cells from homozygous
leaner mice, this -agatoxin-IVA-sensitive current was
65% smaller than in control cells. Although attenuated, the -agatoxin-IVA-sensitive current in homozygous leaner
cells had properties indistinguishable from that of normal Purkinje
neurons. Additionally, the -agatoxin-IVA-insensitive current was
unaffected in homozygous leaner mice. Thus, the
leaner mutation selectively reduces P-type currents in
Purkinje cells, and the 1A subunit and P-type current
appear to be essential for normal cerebellar function.
Key words:
calcium channel; cerebellum; Purkinje cell; neurological
disorders; mutant mice; -agatoxin-IVA
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INTRODUCTION |
Recently, mutations in the
1A 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 1A
calcium channel subunit are also responsible for the phenotypes of
tottering (tg) and leaner (tgla)
mutant mice. The tgla mutation has been
identified as a single-base pair substitution in a splice donor
consensus sequence of the gene for the 1A 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 tgla 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 tgla mutation is also associated
with cerebellar atrophy resulting from a gradual degeneration of
cerebellar Purkinje and granule cells. The cerebella of
tgla/tgla 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, 1A 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 -agatoxin-IVA block ~90% of the
calcium channel current in Purkinje cells (Mintz et al., 1992b), has
been the basis for the assumption that 1A encodes P-type
channels (which are defined by block with low concentrations of
-agatoxin-IVA). However, heterologous expression of the
1A subunit produces a current that (1) is not blocked by
low -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
1A encodes Q-type channels (in addition to its presumed
encoding of P-channels). Here we have used whole-cell recordings to
determine whether the tgla mutation
nonspecifically alters calcium channel currents in cerebellar Purkinje
cells or affects only P-type current as defined by sensitivity to
-agatoxin-IVA.
We report here that the tgla mutation causes
a marked reduction of P-type current in cerebellar Purkinje cells, and
that non-P-type currents are unaffected by the
tgla mutation. Thus, 1A 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.
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MATERIALS AND METHODS |
Mutant mice. C57BL/6J mice (breeding pairs of
tgla/+, +/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 tgla. Heterozygous
Os/+ mice, identified by the oligosyndactylism, were
inferred to be heterozygous for the tgla
mutation. Mice with no oligosyndactylism were inferred to be homozygous
for the tgla mutation. Because homozygous
Os/Os is embryonic lethal, we used pups from
C57BL/6J wild-type matings as normal controls (without the
tgla 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 mM
glucose, 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. -Agatoxin-IVA (-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 t
test.
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RESULTS |
Barium currents were recorded from cerebellar Purkinje cells of
homozygous leaner
(tgla/tgla),
heterozygous leaner (tgla/+), and wild-type
mice. Barium currents from
tgla/tgla mice
appeared qualitatively similar to those from
tgla/+ 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 mM
Ba2+ 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 in
tgla/tgla,
tgla/+, and wild-type cells. A small
reduction in the slope of the conductance-voltage (G-V)
curve was observed in
tgla/tgla cells
compared with tgla/+ and wild-type cells
(Table 1). However, the most striking alteration in the calcium channel currents of
tgla/tgla cells was
a marked reduction in the peak current density as compared with
tgla/+ and wild-type cells (Table 1).
Specifically, the current density in
tgla/tgla cells was
only about half that in tgla/+ and wild-type
cells (51 and 44%, respectively).
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Figure 1.
Barium currents in Purkinje cells of
wild-type and leaner mice. A1-A3, Inward
currents elicited by depolarizing voltage steps from a holding
potential of 80 mV in wild-type (A1),
tgla/+ (A2), and
tgla/tgla
(A3) cells. Note the difference in current densities
between cell types (Ipk for
wild-type = 134.3 pA/pF, tgla/+ = 132.7 pA/pF, and
tgla/tgla = 70.4 pA/pF). B1-B3, Conductance versus voltage curves
plotted from currents in A1-A3. The data were
fitted with a single Boltzmann function. The G-V curves
had similar half-activation voltages (for wild-type cell,
V1/2 = 25.3 mV;
tgla/+ cell,
V1/2 = 23.9 mV;
tgla/tgla
cell, V1/2 = 23.2 mV). The slope factor of
the
tgla/tgla
curve (6.2) was greater than that for the
tgla/+ (5.7) and wild-type cells
(3.9).
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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 between
tgla/tgla 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 in
tgla/tgla Purkinje
cells (Fig. 2, Table 1). This could indicate a difference in kinetic
properties of the mutant channels. Alternately, it could indicate that
the tgla mutation decreased the fractional
contribution of 1A channels to the total current, so
that the whole-cell current became dominated by current through
non-1A channels with a greater ratio of peak to 2 sec
current amplitude.
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Figure 2.
Inactivation of barium currents in wild-type and
leaner cells. A1-A3, Currents elicited
by a two-pulse protocol: 2 sec depolarizing prepulses (110 to 30 mV)
followed by a test pulse to 10 mV with a 10 sec time interval between
measurements. Peak current density during the test pulse was used to
plot inactivation curves
(I/Imax vs prepulse voltage;
data were fit with a single Boltzmann function). The half-inactivation
voltages of the inactivation curves were 46.2 mV (slope factor, 18.8)
for the wild-type cell (A1), 52.3 mV (slope factor,
18.4) for the tgla/+ cell
(A2), and 50.7 mV (slope factor, 19.1) for the
tgla/tgla
cell (A3). B1-B3, Barium currents
elicited by a long 2 sec depolarizing voltage step to 10 mV. Percent
inactivation was 69% in the wild-type cell (B1), 55%
in the tgla/+ cell (B2),
and 46% in the
tgla/tgla
cell (B3). The same cells were used in A
and B.
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The tgla mutation strongly
attenuates the P-type current in Purkinje cells
To determine the specific effects of the 1A
mutation on P-type currents, barium currents in
tgla/tgla and
control Purkinje cells were recorded before and after the application
of -AgTx-IVA, and the difference currents were analyzed. -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 -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 -AgTx-IVA was significantly
greater in wild-type and tgla/+ cells
compared with
tgla/tgla cells
(Table 1). The current remaining after application of -AgTx-IVA had
similar kinetics and density in
tgla/tgla and
control cells, suggesting that the reduction of the 1A
current does not affect the remaining, non-P/Q current types in
Purkinje cells (Figs. 3A,
4). Thus, the -AgTx-IVA-sensitive
current (determined by subtraction of the currents recorded after
application of -AgTx-IVA from control records obtained before
-AgTx-IVA) is selectively reduced by the
tgla mutation (Figs. 3B, 4).
Despite the difference in amplitude, the -AgTx-IVA-sensitive current
exhibited a similar time course in
tgla/tgla and
control cells (Fig. 3B). When the -AgTx-IVA-sensitive
currents in the tgla/+ and
tgla/tgla cells
were scaled by current amplitude, the traces closely superimposed (Fig.
3D).
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Figure 3.
Effects of -AgTx-IVA on barium currents in
wild-type and leaner Purkinje cells. A1,
A2, Barium currents before and after application of 100 nM -AgTx-IVA. -AgTx-IVA blocked 82% of the current
in the tgla/+ cell (A1)
and 63% in the
tgla/tgla
cell (A2). Note the similarities of the currents
resistant to -AgTx-IVA in A1 and A2.
The current densities of the -AgTx-IVA-resistant current were 24.2
pA/pF for the tgla/+ cell and 25.2
pA/pF for the
tgla/tgla
cell. The current remaining after -AgTx-IVA block represents
non-P-type currents. B1, B2, -AgTx-IVA-sensitive
currents were derived by subtracting the control and -AgTx-IVA
traces in A1 and A2. The
-AgTx-IVA-sensitive current from the
tgla/+ cell had a density of 107.1
pA/pF (B1), and that from the
tgla/tgla
cell was 37.5 pA/pF (B2). C, Plot of
the peak current density versus time for the cells in A1
and A2. Gray circles represent data from
the tgla/+ cell in A1, and
the black circles were from the
tgla/tgla
cell in A2. D, -AgTx-IVA-sensitive
currents from B1 and B2 were scaled and
superimposed.
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Figure 4.
The bar graph depicts the current density of the
barium currents in wild-type and leaner Purkinje cells.
The total barium current densities in
tgla/tgla
cells were reduced compared with tgla/+
cells (gray bars; p < 0.01).
There was no significant difference between the current densities of
the -AgTx-IVA-resistant currents (the current remaining after
-AgTx-IVA block; black bars) in
tgla/+ and
tgla/tgla
cells. The mean current density of the -AgTx-IVA-sensitive current
(striped bars) in
tgla/tgla
cells was 65% smaller than that in
tgla/+ cells (p
 0.0001).
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To determine the effects of the tgla mutation
more directly, we characterized the barium current flowing through the
1A channels in isolation (-AgTx-IVA-sensitive
currents). The -AgTx-IVA-sensitive currents in
tgla/+ and
tgla/tgla cells
exhibited no differences in the voltage dependence of activation (Fig.
5). The half-activation voltage of the -AgTx-IVA-sensitive current
was 22.7 ± 1.9 mV (n = 7) in
tgla/+ cells and 22.5 ± 1.9 mV
(n = 6) in
tgla/tgla cells.
The slopes of the G-V curve for
tgla/+ and
tgla/tgla currents
were 4.3 ± 0.2 (n = 7) and 4.5 ± 0.1 (n = 6), respectively. Thus, the decreased slope of the
G-V curve for the whole-cell currents in
tgla/tgla cells was
not a reflection of altered biophysical properties of the
1A channels associated with the
tgla mutation.
The inactivation characteristics of the -AgTx-IVA-sensitive currents
also appeared similar in
tgla/tgla 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 for
tgla/+ cells; vs 0.51 ± 0.06;
n = 4 for
tgla/tgla 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 of
tgla/tgla 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.
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Figure 5.
Activation of -AgTx-IVA-sensitive currents
in tgla/+ and
tgla/tgla
cells. A1, A2, Barium currents elicited by depolarizing
voltage steps from a 80 mV holding potential. -AgTx-IVA-sensitive
currents were derived by subtracting currents recorded after
-AgTx-IVA block from control currents. Current density was markedly
attenuated in the
tgla/tgla
cell (Ipk = 17.3 pA/pF) compared with the
tgla/+ cell
(Ipk = 121.3 pA/pF). B1,
B2, G-V curves for the -AgTx-IVA-sensitive
currents in A1 and A2. Voltage dependence
of activation was similar for the tgla/+
currents (V1/2 = 21.9 mV; k = 5.0)
and the
tgla/tgla
currents (V1/2 = 26.6 mV; k = 4.7).
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Figure 6.
Inactivation properties of -AgTx-IVA-sensitive
currents in tgla/+ and
tgla/tgla
cells. A1, A2, Voltage dependence of inactivation was
described using a Boltzmann function. The
tgla/+ currents exhibited a
half-inactivation voltage of 41.2 mV (k = 13.2), and the
tgla/tgla
currents were half-maximally inactivated at 55.6 mV (k = 17.4).
B1, B2, Percent inactivation was 51% for the
tgla/+ current and 67% for the
tgla/tgla
current.
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DISCUSSION |
Calcium channel currents in
tgla/tgla mice
We have compared calcium channel currents in Purkinje cells from
wild-type mice and mice homozygous for the
tgla mutation of the 1A gene.
This tgla 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 tgla mutation causes reduced expression
of 1A. This hypothesis is supported by the observation
that cerebella of
tgla/tgla mice
express reduced levels of 1A mRNA at all ages tested
(embryonic day 10.5 to adult; Doyle et al., 1997). Western blot
analysis would reveal whether this reduction in 1A
transcript results in reduced 1A protein. In addition to
an altered level of channel expression, it might also be that the
tgla mutation results in altered subcellular
distribution, a possibility which could be examined with
immunohistochemistry.
Relationship of P/Q current to the 1A 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 -conotoxin-GVIA but is
blocked by funnel web spider venom or low concentrations of a component
of the venom, -AgTx-IVA (Llinas et al., 1989; Mintz et al., 1992a).
Q-type current is defined on the basis of block by high concentrations
of -AgTx-IVA or -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 1A subunit was originally thought to encode a P-type
channel (Mori et al., 1991), but when expressed in Xenopus,
the 1A 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 1 genes, alternatively spliced forms of the
same gene, or the same 1A 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 1A
subunit. Our experiments on the
tgla/tgla mouse
support the argument that 1A channels are responsible for P-type current in these cells. To determine whether
1A channels also carry Q-type current, future
investigation of the calcium channel currents in cerebellar granule
cells from
tgla/tgla 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 the
tgla/tgla mouse is
restricted to alternating sagittal compartments of the cerebellar
cortex. The subpopulation of Purkinje cells that survive in
tgla/tgla 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 the
tgla/tgla
cerebellum 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 1A channels are important for dendritic growth and
development in Purkinje cells, thus possibly explaining the abnormal
morphological characteristics of Purkinje cells in tgla/tgla mice.
Immunolocalization in normal mice reveals pronounced 1A
staining 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 in
tgla/tgla 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
1A channels are additionally distributed along dendrites and in the cell body. 1A 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 in
tgla/tgla 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 1A
subunit mutations
Tottering (tg) is another recessively inherited
1A mutation described in the mouse. The tg
mutation is a single-nucleotide change resulting in a substitution of
leucine for proline in the IIS5-IIS6 linker of the 1A
subunit (Fletcher et al., 1996; Doyle et al., 1997). The
tg/tg mouse resembles the
tgla/tgla mouse in
having absence seizures. However, these two mutant mice differ in that
tg/tg mice exhibit only mild ataxia, intermittent focal motor seizures (which are not seen in the
tgla/tgla mouse;
Noebels, 1984), and minor diffuse loss of cerebellar granule and
Purkinje cells. As in
tgla/tgla mice, the
Purkinje cells in tg/tg mice aberrantly express
tyrosine hydroxylase (Hess and Wilson, 1991).
Several human disorders have been associated with 1A
subunit 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
1A 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 1A 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 1A
subunit gene but rather in the gene encoding the  4
auxiliary subunit that associates with the 1 subunit. In
heterologous expression studies, subunits have been shown to
augment and modulate 1 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 1-binding site (Burgess et al., 1997). This
mutation would cause a functional loss of 4,
which could ultimately result in altered 1 calcium
channel currents.
In summary, the present study demonstrates that 1A
expression and P-type current in cerebellar Purkinje cells are critical for normal cerebellar function. Currently, it is unclear how the tgla mutation of the 1A
channel subunit and subsequent decrease in current density cause the
mutant phenotypes. This 1A 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
1A diseases may suggest hypotheses regarding the etiology of these diseases and methods for treatment.
| |
FOOTNOTES |
Received Jan. 27, 1998; revised March 30, 1998; accepted April 1, 1998.
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 -agatoxin-IVA.
Correspondence should be addressed to Kurt G. Beam, Department of
Anatomy and Neurobiology, Colorado State University, Fort Collins, CO
80523.
| |
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Abstract
Introduction
