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The Journal of Neuroscience, October 1, 1998, 18(19):7687-7699
Whole-Cell and Single-Channel Analysis of P-Type Calcium Currents
in Cerebellar Purkinje Cells of Leaner Mutant Mice
Leonard S.
Dove1,
Louise C.
Abbott2, and
William H.
Griffith1
1 Department of Medical Pharmacology and Toxicology,
College of Medicine, Texas A&M University Health Science Center,
College Station, Texas 77843-1114, and 2 Department of
Veterinary Anatomy and Public Health, College of Veterinary Medicine,
Texas A&M University, College Station, Texas 77843-4458
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ABSTRACT |
The leaner (tgla) mutation in mice results in
severe ataxia and an overt neurodegeneration of the cerebellum.
Positional cloning has revealed that the tgla
mutation occurs in a gene encoding the voltage-activated calcium channel  1A subunit. The 1A subunit is
highly expressed in the cerebellum and is thought to be the
pore-forming subunit of P- and Q-type calcium channels. In this study
we used both whole-cell and single-channel patch-clamp recordings to
examine the functional consequences of the tgla
mutation on P-type calcium currents. High-voltage-activated (HVA) calcium currents were recorded from acutely dissociated cerebellar Purkinje cells of homozygous leaner
(tgla/tgla) and age-matched
wild-type (+/+) mice. In whole cell recordings, we observed a marked
reduction of peak current density in
tgla/tgla Purkinje cells
( 35.0 ± 1.8 pA/pF) relative to that in +/+ (103.1 ± 5.9 pA/pF). The reduced whole-cell current in
tgla/tgla cells was accompanied
by little to no alteration in the voltage dependence of channel gating.
In both genotypes, HVA currents were predominantly of the
 -agatoxin-IVA-sensitive P-type. Cell-attached patch-clamp
recordings revealed no differences in single-channel conductance
between the two genotypes and confirmed the presence of three distinct
conductance levels (9, 13-14, and 17-18 pS) in cerebellar Purkinje
cells. Analysis of patch open-probability (NPo) revealed a threefold
reduction in the open-probability of channels in
tgla/tgla patches (0.04 ± 0.01) relative to that in +/+ (0.13 ± 0.02), which may account
for the reduced whole-cell current in
tgla/tgla Purkinje cells. These
results suggest that the tgla mutation can alter
native P-type calcium channels at the single-channel level and that
these alterations may contribute to the neuropathology of the leaner
phenotype.
Key words:
Purkinje cell; calcium channel; single channel; leaner; neurological mutant; mouse
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INTRODUCTION |
The leaner (tgla)
mutation in mice results in a severe ataxia and an overt
neurodegeneration of the cerebellum. It has recently been identified
that both tgla and the allelic mutation tottering
(tg) occur in a gene encoding the calcium channel 1A
subunit (Fletcher et al., 1996 ). The 1A protein is one
of five pore-forming 1 subunits (A-E) that make up
neuronal voltage-activated calcium channels (Tsien et al., 1991;
Perez-Reyes and Schneider, 1994). The 1A subunit is
widely expressed throughout the CNS with the greatest expression
observed in the cerebellum (Mori et al., 1991; Starr et al., 1991;
Westenbroek et al., 1995; Fletcher et al., 1996). Although considerable
debate continues on the subject (Berrow et al., 1997; Moreno et al., 1997), electrophysiological experiments suggest that 1A
is the pore-forming subunit of P-type (Stea et al., 1994; Gillard et al., 1997) and/or Q-type (Sather et al., 1993; Zhang et al., 1993) calcium channels.
Fletcher et al. (1996) have revealed that the tg mutation
results in a single amino acid substitution in the extracellular region
linking the domains IIS5 and IIS6 of the 1A subunit. The tgla mutation occurs in a splice donor
consensus sequence and results in an out-of-frame splicing event. The
aberrant splicing produces two distinct transcripts, both predicted to
yield novel 1A subunits with truncated C-terminal tails
(Fletcher et al., 1996). It has recently been shown that neither
1A mRNA expression, assessed via in situ
hybridization, nor 1A protein expression, assessed via
immunohistochemistry, is altered in the cerebella of homozygous tgla/tgla mice (Lau et al.,
1998). However, it remains unknown what effect, if any, the
tgla mutation has on P- and Q-type calcium channel
function. It has been proposed that truncated 1A
proteins such as those predicted from the tgla
defect may not form functional channels or may form channels displaying
a pathological increase in function (Miller, 1997).
Cerebellar Purkinje cells represent an ideal environment in which to
examine the function of 1A-containing calcium channels. High-voltage-activated (HVA) calcium currents in Purkinje cells are
primarily of the -agatoxin-IVA- (Aga-IVA) sensitive P-type (Mintz
et al., 1992a,b). Calcium currents recorded in Purkinje cells are
nearly insensitive to both -conotoxin-GVIA (CTX-GVIA) and the
dihydropyridines, suggesting little contribution of N- and L-type
calcium channels to the whole-cell current (Regan, 1991; Regan et al.,
1991). Here we use both whole-cell and cell-attached patch-clamp
recordings to assess the effects of the tgla
mutation on the function of 1A-containing calcium
channels in acutely dissociated Purkinje cells of homozygous
tgla/tgla mice. The present study
represents a unique opportunity to examine the functional consequences
of a calcium channel mutation in native cells.
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MATERIALS AND METHODS |
Animals. Male and female control (+/+), heterozygous
tottering (tg/+), and heterozygous leaner (tgla/+)
mice, on the C57BL/6J background, were originally obtained from the The
Jackson Laboratory (Bar Harbor, ME) and bred to produce either control
(+/+), homozygous tottering (tg/tg), or homozygous leaner
(tgla/tgla) offspring. All mice
were housed in a constant temperature (23-24°C), constant humidity
(45-50%) room, with a 12 hr light/dark cycle, and were allowed access
to food (Wayne rodent chow) and water ad libitum.
tgla/tgla mice become extremely
ataxic beginning at postnatal day 10. All tgla/tgla mice were supplemented
with hand feeding of human infant formula four times a day, starting at
postnatal day 15 or 16, and were hand fed until postnatal day 50. In
addition to hand feeding, the leaner mice had constant access to water
(via a water bottle and sipper tube located very close to the bottom of
the cage) and rodent chow (both dry and moistened) on the bottom of the cage. The moistened rodent chow was changed twice a day.
Isolated cells. Mice were anesthetized with isofluorane and
decapitated. Cerebella were removed and placed in an icy sucrose solution similar to that described by Reynolds et al. (1992). The
solution contained (in mM): 248 sucrose, 26 NaHCO3, 10 glucose, 5 KCl, 2 MgCl2, 1 CaCl2 and 1 Na-pyruvate, pH
7.4. Parasagittal cerebellar slices (450 µm) were cut on a McIlwain
tissue chopper and were held at room temperature in oxygenated sucrose
solution. Slices were enzymatically treated in the sucrose solution
with 0.4-0.5 mg/ml Protease type XXIII (Sigma, St. Louis, MO) for 20 min at 35-36°C.
Cerebellar slices were transferred to DMEM (Life Technologies,
Gaithersburg, MD) and were mechanically triturated through a series of
fire-polished Pasteur pipettes to isolate individual Purkinje cells.
Isolated cells were then dispersed onto the floor of a recording
chamber pretreated with 0.1% Alcian blue solution to facilitate cell
adhesion. Purkinje cells were identified morphologically by their large
somata and single stumps of apical dendrite (Regan, 1991).
Data acquisition and analysis. Whole-cell and cell-attached
patch-clamp recordings (Hamill et al., 1981) were performed with an
Axopatch 200A amplifier (Axon Instruments, Foster City, CA). For both
recordings, patch electrodes were pulled from borosilicate glass
(#7052; Garner Glass Company, Claremont, CA) on a Flaming/Brown micropipette puller (Sutter Instruments, Novato, CA). Electrodes were
coated with wax to reduce stray capacitance and were fire polished to
final resistances of 2-5 M .
For whole-cell recordings, cell capacitance was used as an indicator of
cell size and was read directly from the potentiometer after capacity
transients were nullified. Cell capacitance and series resistance were
adjusted as necessary throughout the recordings, with series resistance
generally compensated >80%. Data were low-pass filtered at 2 kHz and
acquired at a sampling rate of 5-10 kHz.
All whole-cell current measurements were made relative to baseline. In
most cases, currents were normalized to current density (pA/pF) by
dividing by cell capacitance. In time-course experiments, currents were
normalized to I/Imax, where
Imax is the largest current in the experiment.
To measure voltage-dependent inactivation, we used a double-pulse
protocol similar to that described previously (Murchison and Griffith,
1996). To analyze voltage-dependent inactivation, we fit
current-voltage plots with a single Boltzmann distribution. Voltage of
half-inactivation (V1/2) was determined for
individual cells and averaged.
For cell-attached single-channel recordings, all currents were low-pass
filtered at 2 kHz and acquired at a sampling rate of 10 kHz. Currents
were evoked with 300 msec voltage steps to the indicated potentials
delivered at 4 sec intervals. Capacitive transients were partially
nullified during current recordings. The remaining capacitive currents
and leak currents were removed by subtracting the average of blank
sweeps. Single-channel records were analyzed using Pclamp6 software
(Axon Instruments). Unitary current amplitudes were calculated as the
arithmetic mean of cursor measurements when the number of openings was
low. For kinetic analysis, open and closed events were determined with
a 50% threshold-crossing method in which a minimal resolvable duration
of 200 µsec was required for openings and closings. This duration was
set at 1.25× the rise time calculated for a 2 kHz filter (Colquhoun
and Sigworth, 1983). Patch open-probability was calculated as
NPo where N refers to the
number of channels in the patch. Only patches in which one channel was
apparent were included in our analysis, with the absence of
simultaneous channel openings used as an indicator of single-channel
activity. NPo was calculated as
(To)/(To + Tc), where To
equals total open time during a sweep and
Tc equals total closed time during a
sweep. Open-time histograms were fitted with a single exponential using
a Levenberg-Marquardt chi-square minimization method. All statistical
comparisons used an independent Student's t test.
Statistical significance was based on p < 0.05, with
all averaged values reported as mean ± SE.
Solutions and drugs. Solutions were designed to maximize
Ba2+ current through calcium channels while
minimizing contaminating conductances. For whole-cell recordings, cells
in the recording chamber were continuously perfused with an external
solution containing (in mM): 140 NaCl, 3 KCl, 2 CaCl2, 1.2 MgCl2, 10 HEPES, and 33 D-glucose, pH 7.4 with NaOH (310-330
mOsm). Before whole-cell recordings, the external solution was
exchanged for a modified recording solution containing (in
mM): 132 NaCl, 2 BaCl2, 2 MgCl2, 10 HEPES, 33 D-glucose, 10 tetraethylammonium chloride (TEA-Cl), and 0.0005 tetrodotoxin
(Calbiochem, La Jolla, CA), pH 7.4 with NaOH (310-330 mOsm). For
cell-attached recordings, the membrane potential was zeroed with a high
K+ solution containing (in mM): 140 K-aspartate, 5 MgCl2, 20 HEPES, 10 D-glucose, 0.5 EGTA, and 0.0005 tetrodotoxin, pH 7.4 with KOH (310-330 mOsm).
For whole-cell recordings, the internal pipette solution contained (in
mM): 110 Cs-acetate, 15 CsCl, 10 TEA-Cl, 2 MgCl2, 20 HEPES, 10 EGTA, 4 Mg-ATP, and 0.1 Na-GTP,
pH 7.2 with CsOH (290-310 mOsm). For cell-attached recordings, the
internal pipette solution contained (in mM): 110 BaCl2, 15 CsCl, 15 TEA-Cl, and 10 HEPES, pH 7.4 with
CsOH (320 mOsm).
In whole-cell experiments, calcium channel antagonists
-conotoxin-GVIA (Alamone Labs, Jerusalem, Israel), nifedipine
(dissolved in 0.2% ethanol and 0.1% ethylene glycol), and cadmium
chloride were bath applied in the recording solution as noted. The
nifedipine vehicle had no effect on HVA calcium currents in either
phenotype. -Agatoxin-IVA (Calbiochem) was dissolved in recording
solution and applied through a drug-barrel pipette positioned adjacent to the recording electrode. For whole-cell experiments involving -conotoxin-GVIA and -agatoxin-IVA, 0.1 mg/ml cytochrome C was added to the recording solution to saturate nonspecific peptide-binding sites. For cell-attached recordings, nifedipine (10 µM)
was included in the internal pipette solution. Salts and other
chemicals were obtained from Sigma except as noted.
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RESULTS |
Whole-cell currents in leaner and wild-type mice
Whole-cell barium currents through HVA calcium channels
were recorded from acutely dissociated Purkinje cells from leaner (tgla/tgla) and wild-type (+/+)
mice. Purkinje cells were identified morphologically by their
characteristic appearance after dissociation (see Materials and
Methods). After establishment of the whole-cell voltage clamp, currents
were elicited by voltage steps to 10 mV delivered at 12 sec
intervals. To minimize any low-voltage-activated (LVA) current that
might be present (Kaneda et al., 1990; Mouginot et al., 1997), we held
cells at 60 mV, a potential known to inactivate fully the LVA
currents in Purkinje cells (Regan, 1991). Because the phenotypic
abnormalities in tgla/tgla mice
are not apparent before 10 d of age (Herrup and Wilczynski, 1982),
we limited our investigation to cells from mice between 18 and 35 d of age. Figure 1A
shows typical current traces recorded from
tgla/tgla and +/+ Purkinje cells
of similar capacitance. The current is notably reduced in amplitude for
the tgla/tgla Purkinje cell.
There was a statistically significant reduction in HVA currents in
tgla/tgla cells (511 ± 30 pA; n = 46) compared with that in +/+ cells (1602 ± 96 pA; n = 45; p < 0.001). This reduction in current observed in
tgla/tgla did not result from
reduced cell size, because there was no statistical difference in cell
capacitance between tgla/tgla
(14.7 ± 0.4 pF; n = 46) and +/+ (15.6 ± 0.3 pF; n = 45) cells. Figure 1B shows
peak currents of the two genotypes normalized to current density
(pA/pF) and displayed across a range of ages. There was a statistically
significant reduction in peak current density in
tgla/tgla cells (35.0 ± 1.8 pA/pF; n = 46) relative to that in +/+ cells (103.1 ± 5.9 pA/pF; n = 45; p < 0.001). These data were collected from Purkinje cells of 26 leaner
mice (17 litters) and 27 wild-type mice (19 litters). The current
reduction in tgla/tgla Purkinje
cells persisted past 40 d of age, when neurodegeneration of
Purkinje cells is expected to begin in
tgla/tgla mice (Herrup and
Wilczynski, 1982). The inset provides a box plot summary of
the distribution of peak current densities (pA/pF) in
tgla/tgla and +/+ mice between 18 and 35 d of age.
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Figure 1.
HVA calcium currents recorded from wild-type (+/+)
and leaner (tgla/tgla) Purkinje
cells. A, Currents were elicited by a depolarizing test
pulse to 10 mV (40 msec) from Vh = 60 mV. Voltage
protocol is shown at top. B, Peak
currents from +/+ and tgla/tgla
Purkinje cells were divided by cell capacitance and reported as current
densities (pA/pF). Peak current densities for individual cells were
plotted versus days of age. Inset, Box
plot summary for current densities in +/+
(shaded) and
tgla/tgla (open)
Purkinje cells is shown. Box boundaries represent 25th
and 75th percentiles. The horizontal line within each
box represents the median percentile, and
extended lines represent 10th and 90th percentiles. The
filled square within the box represents
the mean, and asterisks and filled
diamonds (below and above extended
lines) represent 1st and 99th percentiles, respectively. There
is a statistically significant difference (p < 0.001) between the means for +/+ (103.1 ± 5.9 pA/pF;
n = 45 cells) and
tgla/tgla (35.0 ± 1.8 pA/pF; n = 46 cells) Purkinje cells.
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To determine whether a current reduction was unique to
tgla/tgla Purkinje cells, we also
recorded HVA currents from Purkinje cells of tg/tg mice.
Currents were recorded from Purkinje cells of tg/tg mice between 28 and
35 d of age when the phenotype is readily identifiable. Using the
same voltage protocol mentioned above [step depolarizations from a
holding potential (Vh) of 60 to 10 mV
delivered at 12 sec intervals], we found no significant difference in
current density between +/+ (103.1 ± 5.9 pA/pF; n = 45 cells) and tg/tg (88.0 ± 5.1 pA/pF;
n = 12 cells, 9 mice, 6 litters) Purkinje cells (data
not shown). Apparently, the tg mutation and resultant single amino acid
substitution on the extracellular IIS5-IIS6 linking region of the
1A subunit are not reflected in a reduced current
density. Thus it seems likely that the tgla mutation
altering the cytoplasmic C-terminal region of 1A leads to the distinct current reduction observed in
tgla/tgla Purkinje cells.
Voltage-dependent activation
A possible explanation for the difference in HVA currents between
the wild-type and leaner genotypes may be alterations in the voltage
dependence of channel gating. Therefore, we sought to characterize the
voltage dependence of current activation in tgla/tgla and +/+ Purkinje cells.
Current-voltage relationships for
tgla/tgla and +/+ Purkinje cells
were examined using both voltage-step and voltage-ramp protocols.
Figure 2A shows
superimposed current traces from
tgla/tgla and +/+ cells generated
by a series of voltage steps delivered at 8 sec intervals. Currents
were normalized to current densities and plotted against membrane
potential in Figure 2B. Averaged HVA current
densities in cells from tgla/tgla
and +/+ mice both reached a maximum after voltage steps to 10 mV. We
observed no significant difference in the voltage of half-activation (V1/2) in
tgla/tgla cells
(V1/2, 16.3 ± 0.8 mV; n = 16) relative to that in +/+ cells (V1/2, 18.3 ± 0.7 mV; n = 15; p > 0.05). However,
a slight alteration in the slope of the activation curve was observed
for tgla/tgla cells
(k, 5.5 ± 0.3 mV; n = 16) relative to
that for +/+ cells (k, 4.5 ± 0.2 mV; n = 15; p < 0.05). Because the reduction in current
density in leaner cells persists across nearly all sampled membrane
potentials, it is unlikely the current reduction in Figure 1 results
from incomplete channel activation. Because HVA currents may display
current- and/or voltage-dependent inactivation during the course of the
step protocol, current-voltage relationships were also constructed
with 400 msec voltage ramps. Figure 2C shows current traces
generated by voltage ramps in
tgla/tgla and +/+ cells of
similar size. Similar to the voltage-step data in Figure
2B, the averaged-ramp data in Figure
2D shows a reduced current-density in
tgla/tgla relative to that in
+/+, with very little change in voltage of activation.
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Figure 2.
Current-voltage relationships in +/+ and
tgla/tgla Purkinje cells.
A, Representative current traces are
shown for +/+ and tgla/tgla
Purkinje cells. Currents were elicited by voltage steps from 80 to
+30 mV (200 msec) delivered at 8 sec intervals with Vh = 60 mV. Voltage protocol is shown at top.
B, Currents were divided by cell capacitance and
reported as current densities (pA/pF). Current density ± SE was
plotted versus test voltage. There are statistically significant
differences (p < 0.002) in current
densities between +/+ (n = 15 cells, 10 mice, 8 litters) and tgla/tgla
(n = 16 cells, 8 mice, 7 litters) Purkinje cells
from 30 to +30 mV. Inset, The scaled activation curve
for tgla/tgla cells is
superimposed on the +/+ activation curve. C,
Representative current traces are shown for +/+ and
tgla/tgla Purkinje cells.
Currents were elicited by voltage ramps from 80 to +30 mV (400 msec)
with Vh = 60 mV. Voltage protocol is shown at
top. D, Currents were divided by cell
capacitance and reported as current densities (pA/pF). Current
densities were averaged and plotted versus membrane voltage. SEs
are shown at peak current densities.
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Voltage-dependent inactivation
A characteristic of voltage-activated calcium channels is
voltage-dependent inactivation. Because the reduced current amplitude in leaner Purkinje cells could be explained by a change in
voltage-dependent inactivation, we investigated whether this property
was altered in tgla/tgla relative
to +/+ Purkinje cells. To compare voltage dependence of inactivation,
we used a double-pulse protocol similar to that described previously
(Murchison and Griffith, 1996). Figure
3A shows 24 superimposed
current traces for tgla/tgla and
+/+ cells of similar capacitance. Currents were normalized to the
maximum current in each episode
(I/Imax) and plotted versus conditioning pulse potential. The curves were fit with a single Boltzmann distribution. Figure 3B shows averaged
inactivation curves for tgla/tgla
and +/+ Purkinje cells. There was no significant difference in V1/2 for
tgla/tgla cells
(V1/2, 18.8 ± 1.3 mV; n = 7)
relative to that for +/+ cells (V1/2, 21.8 ± 0.7 mV; n = 6; p > 0.05). Likewise,
there was no significant difference in the slope factor of the
inactivation curves for tgla/tgla
cells (k, 10.3 ± 0.7 mV; n = 7)
relative to that for +/+ cells (k, 13.3 ± 2.0 mV;
n = 6; p > 0.05). It should be noted
that during the course of the inactivating protocol, there appeared to
be some current-dependent inactivation as described previously for Purkinje cells (Regan, 1991). This was more apparent for wild type than
for leaner, because the currents were always larger in amplitude in +/+
cells. The current inactivated by the depolarizing conditioning pulses
never fully recovered, even after returning to a hyperpolarized resting
potential.
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Figure 3.
Voltage-dependent inactivation in +/+ and
tgla/tgla Purkinje cells.
A, Inactivation records show 24 superimposed
traces of currents elicited by a depolarizing test pulse
to 10 mV (120 msec). Before the test pulse, a 2 sec conditioning
pulse varying in 5 mV increments between 110 and +5 mV was given.
B, Voltage-dependent inactivation curves in +/+ and
tgla/tgla Purkinje cells are
shown. Currents were measured near the end of the pulse and were
normalized to the maximum current in each record. Normalized
currents ± SE were plotted versus conditioning voltage and fit
with a Boltzmann distribution. Inset, Normalized
activation curves are superimposed for +/+ and
tgla/tgla Purkinje cells.
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Although this analysis suggests voltage-dependent inactivation is
unlikely to account for the generalized current reduction in
tgla/tgla Purkinje cells, it is
possible that a holding potential of 60 mV may introduce steady-state
inactivation that is not alleviated by the 2 sec conditioning pulse. An
increased degree of steady-state inactivation of
tgla/tgla calcium channels would
leave them less fully available for opening and could account for the
current reduction. To address this issue, we compared the degree of
steady-state inactivation relieved after hyperpolarizing the holding
potential of tgla/tgla and +/+
Purkinje cells from 60 to 80 mV. HVA currents were elicited
from a holding potential of 60 mV as described above, the holding
potential was then hyperpolarized to 80 mV, and HVA currents were
elicited. In both genotypes, HVA currents increased after
hyperpolarization, with a maximum relief of inactivation usually
observed by 1 min. Figure 4 shows the
time course of relief of steady-state inactivation for a +/+ (Fig.
4A) and a
tgla/tgla (Fig.
4B) Purkinje cell. After maximal relief of
steady-state inactivation, the relative increase in HVA current
densities was comparable. In eight +/+ cells, average HVA current
density increased from 102.5 ± 14.8 to 133.5 ± 17.8 pA/pF (30% increase). In nine tgla/tgla cells, average HVA
current density increased from 35.4 ± 4.8 to 44.7 ± 4.9 pA/pF (26% increase). These results indicate a similar degree of
steady-state inactivation of
tgla/tgla and +/+ calcium
channels when held at 60 mV and suggest this is unlikely to account
for the generalized reduction in HVA currents.
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Figure 4.
Relief of steady-state inactivation in +/+ and
tgla/tgla Purkinje cells.
Currents were elicited by a depolarizing test pulse to 10 mV from the
indicated Vh. Currents were divided by cell capacitance and
reported as current density. A, Time course of relief of
steady-state inactivation after changing Vh from 60 to
80 mV is shown for a +/+ Purkinje cell. B, Time course
of relief of steady-state inactivation after changing Vh
from 60 to 80 mV is shown for a
tgla/tgla Purkinje cell.
Insets, Superimposed current traces
elicited from holding potentials of 60 and 80 mV are shown.
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Pharmacology of HVA currents
HVA currents in Purkinje cells are predominately of the
Aga-IVA-sensitive P-type. Approximately 90% of the HVA current
in Purkinje cells is insensitive to either dihydropyridines or
CTX-GVIA, suggesting very little contribution of L-and N-type
channels (Regan, 1991; Regan et al., 1991; Mintz et al., 1992b). We
investigated the pharmacological profile of HVA currents in
tgla/tgla and +/+ Purkinje cells
by applying nifedipine, CTX-GVIA, and Aga-IVA, antagonists of L-, N-,
and P- and Q-type calcium channels, respectively.
As in previous experiments, HVA currents were elicited by test pulses
to 10 mV. To ascertain the time course of block by calcium channel
antagonists, currents were elicited continuously at 12 sec intervals. A
baseline recording of 10 min was taken before bath application of
nifedipine or CTX-GVIA to distinguish between current rundown and
antagonist blockade. For both
tgla/tgla (n = 9)
and +/+ (n = 7) cells, the effect of CTX-GVIA was
indistinguishable from the current rundown observed during the
experiment, suggesting neither genotype exhibits a notable amount of
N-type current. To ensure the concentration of CTX-GVIA used possessed
reasonable activity, we applied the toxin to medial septum/diagonal
band neurons on the same days of use. CTX-GVIA (500 nM)
rapidly blocked ~40% of HVA current in these neurons (data not
shown), consistent with previous findings (Murchison and Griffith,
1996).
Unlike the results with CTX-GVIA, modest block by nifedipine was
noticeable in both genotypes. There was no difference in either the
development or magnitude of nifedipine block between tgla/tgla (6.2 ± 2.1%;
n = 9) and +/+ (7.2 ± 2.4%; n = 6) cells. For both genotypes, the block was not fully reversible and in
some cells could not be clearly distinguished from current rundown.
Figure 5C summarizes the
effects of the N- and L-type calcium channel antagonists. Bar graphs
represent the current fraction remaining after application of the
antagonists, with block by CTX-GVIA nominally 5%. Taken together,
these data suggest that ~90% of the HVA current in both genotypes is
of the non-N-, non-L-type. Thus, it seems unlikely that
tgla/tgla Purkinje cells develop
additional N- or L-type calcium channels to compensate for the decrease
in 1A-mediated current.
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Figure 5.
Antagonist block of HVA current in +/+ and
tgla/tgla Purkinje cells.
Currents were elicited by depolarizing test pulses to 10 mV (40 msec)
from Vh = 60 mV at 12 sec intervals. A,
Time course of block by Aga-IVA is shown for a +/+ Purkinje cell.
Inset, Superimposed current records show
traces before and after application of Aga-IVA (100 nM). B, Time course of block by Aga-IVA is
shown for a tgla/tgla Purkinje
cell. Inset, Superimposed current records show
traces before and after application of Aga-IVA (100 nM). C, Current fractions in +/+
(shaded) and
tgla/tgla (open)
Purkinje cells are shown. Bar graphs with error bars show current
remaining after maximum blockade. Percent blockade was corrected for
gradual current rundown. The effect of CTX-GVIA was indistinguishable
from current rundown in both +/+ (n = 7 cells) and
tgla/tgla (n = 9 cells) Purkinje cells (nominally 5%). There were no significant
differences in the amount of current blockade by nifedipine in +/+
(n = 6 cells) and
tgla/tgla (n = 9 cells) cells or by Aga-IVA in +/+ (n = 4) and
tgla/tgla (n = 5 cells) cells.
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We used Aga-IVA to confirm that the HVA current recorded in
tgla/tgla and +/+ Purkinje cells
was mediated by 1A-containing channels. We applied 100 nM Aga-IVA via a single-barrel drug pipette positioned adjacent to the test cell. Although this concentration is too high to
differentiate P-type from Q-type channels (Birnbaumer et al., 1994),
Purkinje cells are thought to possess the former (Llinas et al., 1989,
1992; Regan, 1991; Usowicz et al., 1992). Figure 5, A and
B, shows the time course of block by Aga-IVA in +/+ and
tgla/tgla cells, respectively.
Neither the development nor the magnitude of block by Aga-IVA appeared
to differ between the two genotypes. The insets in Figure 5,
A and B, show superimposed current traces recorded before and after application of 100 nM Aga-IVA.
This concentration of Aga-IVA (100 nM) routinely blocked
80-95% of the HVA current in both genotypes. The average amount of
block by Aga-IVA was not different in +/+ (85.9 ± 2.9%;
n = 4) and
tgla/tgla (85.5 ± 2.1%;
n = 5) Purkinje cells (Fig. 5C). For both
genotypes, maximal blockade was consistent with that reported
previously (Mintz et al., 1992a,b), in which Aga-IVA concentrations
between 60 and 200 nM blocked 85-100% of the HVA current
in rat Purkinje cells. In both genotypes, Aga-IVA at concentrations as
low as 20 nM was sufficient to block 75-80% of the HVA
current over a longer time course (data not shown). Cadmium (100 µM) blocked the remaining current in both genotypes.
These results suggest little difference in sensitivity to Aga-IVA and
confirm the presence of P-type calcium current in both
tgla/tgla and +/+ Purkinje
cells.
Single-channel conductance
Unitary current recordings were made from cell-attached patches of
tgla/tgla and +/+ Purkinje cells
to examine properties of single-channel currents. The patch potential
was maintained at 60 mV, and currents were elicited by 300 msec
voltage steps to the indicated potentials. We first sought to compare
the single-channel conductance of the HVA channels in
tgla/tgla cells with that in +/+
cells, because an alteration in this property might account for the
reduced whole-cell current. Figure 6
shows examples of current traces recorded in a
tgla/tgla (Fig.
6A) and a +/+ (Fig. 6C) patch. With 110 Ba2+ as charge carrier and 10 µM
nifedipine included in the patch pipette, we observed current openings
to three distinct amplitude levels. Consistent with a previous study,
these three current levels were present throughout the 300 msec voltage
step and were apparent when the holding potential varied from 60 to
90 mV (Usowicz et al., 1992). When plotted against test potential,
currents from 12 +/+ patches yielded slope conductances of 9, 14, and
17 pS (Fig. 6B) with unitary currents of 0.65, 1.14, and 1.62 pA at 10 mV. For eight
tgla/tgla patches, slope
conductances were 9, 13, and 18 pS (Fig. 6D) with unitary currents of 0.64, 1.13, and 1.62 pA at 10 mV. This suggests that no difference in slope conductance exists between +/+ and tgla/tgla channels. For both
genotypes, the slope conductances are in excellent agreement with the
9, 14, and 19 pS conductances previously ascribed to native P-type
channels in cerebellar Purkinje cells (Usowicz et al., 1992).
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Figure 6.
Conductance of single calcium channels in +/+ and
tgla/tgla Purkinje cells.
Single-channel currents were recorded from cell-attached patches with
110 mM Ba2+ in the patch pipette.
Currents were elicited by 300 msec steps to the indicated potential
from a holding potential of 60 mV at 4 sec intervals. Currents were
low-pass filtered at 2 kHz and digitally acquired at 10 kHz.
A, Single-channel current records from a +/+ patch are
shown. Three open levels are indicated on the bottom
trace. B, Single-channel
I-V plot yields three distinct slope
conductances as labeled. C, Single-channel current
records from a tgla/tgla patch
are shown. Open levels are indicated on the bottom
trace. D, Single-channel
I-V plot yields three distinct slope
conductances as labeled.
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Because the patch potential in the above analysis was 60 mV, it is
highly unlikely the lowest conductance level represents a T-type
channel. Additionally, the currents recorded showed little to no
time-dependent inactivation, further arguing against the presence of
T-type channels. Although conductances of 18-19 pS are in the range
reported for L-type channels (Bean, 1989), the presence of 10 µM nifedipine in the patch pipette eliminates their presence. The unitary conductances are also in the range reported for
N-type channels (Fox et al., 1987). However, the virtual insensitivity of whole-cell currents to 500 nM CTX-GVIA suggests that the
mouse Purkinje cells in our study lack any substantial N-type current. Although we observed patches from both genotypes with superimposed openings indicative of the presence of multiple channels, we limited our analysis to patches in which only a single channel was apparent. Therefore it seems probable that the three conductance levels we
observed represent subconductance levels of a single type of calcium channel, most likely the P-type channel as described previously (Usowicz et al., 1992).
Open-probability
Another possible explanation for the reduced whole-cell current
observed in tgla/tgla Purkinje
cells is a reduction in channel open-probability. We sought to compare
patch NPo for
tgla/tgla and +/+ Purkinje cells.
From a patch potential of 60 mV, currents were elicited with 300 msec
voltage steps to 10 mV. Routinely, >40 individual current sweeps
were averaged to determine NPo for a
patch. For nine +/+ patches, the mean NPo
equaled 0.13 ± 0.02. For nine
tgla/tgla patches, we observed a
significant reduction in NPo (0.04 ± 0.01; p < 0.001). Figure
7 shows 10 consecutive sweeps for a +/+
patch in which NPo = 0.10 (Fig.
7A) and for a
tgla/tgla patch in which
NPo = 0.02 (Fig. 7C). For both
patches shown in Figure 7, A and C,
NPo is plotted against sweep episode in
Figure 7, B and D, respectively. Figure
7D demonstrates that the general reduction in
NPo observed in
tgla/tgla patches is not
attributable to a preponderance of blank sweeps. For the 45 sweeps
displayed for the tgla/tgla patch
in Figure 7D, three blank sweeps were observed. For the 45 sweeps displayed for the +/+ patch in Figure 7B, no blank
sweeps were observed. This is consistent with the other patches in the analysis, in which the occurrence of blank sweeps was slightly higher
for tgla/tgla (7%) than for +/+
(2%) patches.
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Figure 7.
Patch open-probability
(NPo) for cell-attached patches of +/+ and
tgla/tgla Purkinje cells.
Currents were elicited by 300 msec steps to 10 mV from a holding
potential of 60 mV at 4 sec intervals. Currents were low-pass
filtered at 2 kHz and digitally acquired at 10 kHz. Currents are
displayed here with a 1 kHz filter imposed. A, Ten
consecutive current traces recorded from a +/+ patch are
shown. B, For the +/+ patch in A,
NPo has been calculated for individual
sweeps. NPo is displayed for 45 consecutive sweeps in which the total patch
NPo is 0.10. C, Ten
consecutive current traces recorded from a
tgla/tgla patch are shown.
D, For the
tgla/tgla patch in
C, NPo is displayed for 45 consecutive sweeps in which the total patch
NPo is 0.02.
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The mean open time of the channels in
tgla/tgla and +/+ patches was
assessed by plotting the open-time histograms. For construction of
open-time histograms, openings to all conductance levels were included.
The distributions were generally well described by a single
exponential. Figure 8 shows current
traces for a +/+ (Fig. 8A) and a
tgla/tgla (Fig. 8C)
patch. The open-time histograms for the patches in Figure 8,
A and C, are presented in Figure 8, B
and D, respectively. Note the difference in number of events
for the tgla/tgla patch relative
to that for the +/+ patch. For the +/+ patch in Figure
8B, the fit of the open-time histogram yields a time
constant of 0.19 msec. For the
tgla/tgla patch in Figure
8D, a time constant of 0.15 msec was calculated. These fits were representative of the two genotypes, with the average
time constant for +/+ patches equal to 0.18 ± 0.01 and that for
tgla/tgla patches equal to
0.15 ± 0.01. These open times are consistent with previous
examination (Usowicz et al., 1992). Although mean open times do not
appear substantially different for the two genotypes, NPo is markedly reduced in cell-attached
patches from tgla/tgla Purkinje
cells. This generalized reduction in NPo
may explain the significant diminution in whole-cell current observed
in tgla/tgla Purkinje cells.
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Figure 8.
Mean open times of calcium channels from +/+ and
tgla/tgla Purkinje cells. Calcium
currents were elicited by 300 msec steps to 10 mV from a holding
potential of 60 mV at 4 sec intervals. Currents were low-pass
filtered at 2 kHz and digitally acquired at 10 kHz. A,
Single-channel current records from a +/+ patch. B,
Open-time histogram for the +/+ patch shown in A, fitted
with a single exponential. C, Single-channel current
records from a tgla/tgla patch.
D, Open-time histogram for the
tgla/tgla patch shown in
C, fitted with a single exponential.
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DISCUSSION |
The tgla mutation occurs in a gene encoding the
1A calcium channel subunit (Fletcher et al., 1996).
Based on electrophysiological recordings (Sather et al., 1993; Zhang et
al., 1993; Stea et al., 1994; Gillard et al., 1997) and anatomical
localization (Stea et al., 1994; Westenbroek et al., 1995),
1A is thought to be the pore-forming subunit of P-
and/or Q-type voltage-activated calcium channels. The
tgla mutation results in an out-of-frame splicing
event and is predicted to yield novel 1A subunits with
truncated C-terminal tails. Recent evidence shows that neither
1A mRNA expression nor 1A protein expression are altered in the cerebella of homozygous
tgla/tgla mice (Lau et al.,
1998). In this study we demonstrate the effects of the
tgla mutation on P-type calcium channel function in
cerebellar Purkinje cells.
The tgla mutation causes a distinct reduction in
whole-cell P-type calcium current in Purkinje cells of 18-35-d-old
tgla/tgla mice, an age range when
the leaner phenotype is readily identifiable. A recent report has shown
that whole-cell calcium currents are reduced in Purkinje cells of
7-9-d-old tgla/tgla mice
(Lorenzon et al., 1998), an age range that precedes the cerebellar cell
loss and ataxia characteristic of the leaner phenotype. The reduced
current we observed is accompanied by little to no effect on the
voltage dependence of channel gating or on antagonist sensitivity.
Whole-cell current I is given by the product of
single-channel unitary current i, the number of functional
channels N, and channel open-probability
Po: I = iNPo. We examined properties of single channels to determine whether an alteration in one of the these parameters might underlie the reduced whole-cell current in
tgla/tgla Purkinje cells. We
observed no difference in single-channel conductances between the two
genotypes. Our observations confirm the presence of three distinct
conductance levels in cerebellar Purkinje cells, with the three levels
being consistent with the previously described subconductance levels of
native P-type channels (Usowicz et al., 1992). Amplitude histograms
(data not shown) reveal that all three conductance levels appear in
approximately the same proportions in the two genotypes, with the lower
two levels predominating. Thus, it is highly unlikely a change in
unitary current accounts for the reduced whole-cell current in
tgla/tgla Purkinje cells.
Analysis of patch NPo with cell-attached
recordings revealed marked differences for +/+ and
tgla/tgla Purkinje cells.
Relative to +/+, there was a threefold lower probability of opening
observed in tgla/tgla patches.
Interestingly, the observance of lower probability was not reflected in
a reduction of channel mean open time. Rather, the reduction in
NPo seems to result from a lower frequency
of channel opening in tgla/tgla
patches. It should be noted that because of the extremely low probability of channel opening observed, it is difficult to know with
certainty the number of channels within individual patches (Colquhoun
and Hawkes, 1990; Horn, 1991). However, because it was possible to
clearly discern patches with multiple channels, we are confident our
analysis of open-probability is a reasonable reflection of
single-channel activity. Thus, these results suggest that a lower
channel open-probability is likely to underlie the reduction of
whole-cell currents recorded in
tgla/tgla Purkinje cells. We have
not, however, unequivocally eliminated the possibility that a reduction
in the number of functional channels may contribute to both the reduced
whole-cell current and the reduced patch open-probability. An
interesting observation was the higher frequency of inactive patches
from tgla/tgla Purkinje cells. Of
41 tgla/tgla patches examined,
slightly more than half lacked activity or displayed activity so low as
to not be distinguishable from random noise. This lack of activity
prevailed even at extremely hyperpolarized patch potentials (negative
to 90 mV). By contrast, only 3 of 24 +/+ patches lacked activity. The
absence of activity did not obviously correlate with the positioning of
the patch pipette on the cell soma. Although the differential
observation of inactive patches may be accounted for by subtle
differences in pipette tip diameter and pipette positioning, it does
raise the possibility of reduced functional channels on the membrane of
tgla/tgla Purkinje cells.
The functional consequences of the leaner mutation may help elucidate
the putative role of the C-terminal tail in modulating calcium channel
function. Discrete structural motifs of 1 proteins play
vital roles in influencing channel gating, ion selectivity, and
antagonist binding (Catterall and Striessnig, 1992; Mori et al., 1996).
The functional significance of the C terminal has only recently been
explored, however. In cardiac L-type channels, the C-terminal region
seems to play an important regulatory role in both channel gating and
channel availability (Wei et al., 1994; Klockner et al., 1995; Soldatov
et al., 1997). Amino acid deletions in the C terminal from rabbit
cardiac 1C result in a four- to sixfold increase in
current density without changing the number of functional channels (Wei
et al., 1994). The increase in current density has been attributed to
an increased Po. A similar increase in
current density as well as a slight alteration in the voltage dependence of inactivation results from removal of part of the C
terminal from the human cardiac 1C subunit (Klockner et
al., 1995). In contrast to these results, our analysis reveals that truncations in the 1A subunit result in a marked
decrease in current density because of a reduced
Po. It should be noted, however, that the
truncated tails resulting from the tgla mutation are
also predicted to have novel amino acid sequences. Such alterations
could interfere with essential cytoplasmic interactions of the C
terminal responsible for normal channel gating and channel availability, thus leading to a reduced
Po.
The present study demonstrates in native cells the functional
consequences of tgla, a mutation in the
1A calcium channel subunit. Although the functional
properties of 1A appear significantly altered in
tgla/tgla Purkinje cells, less
obvious is how these alterations ultimately lead to the severe
cerebellar ataxia and degeneration of cerebellar neurons observed in
tgla/tgla mice (Herrup and
Wilczynski, 1982). Interestingly, there seems to be a close association
between mutations in 1A and the presence of cerebellar
ataxia. In humans, separate mutations in 1A lead to the
disorders autosomal dominant cerebellar ataxia (Zhuchenko et
al., 1997), episodic ataxia type-2, and familial hemoplegic migraine
(Ophoff et al., 1996). Whether any of these mutations effect calcium
channel function in a manner similar to that of tgla
remains uncertain. In any regard, our results represent an important first step in determining the functional consequences of mutations in
the 1A subunit and suggest these neurological mutations
can alter native P-type calcium channels at the single-channel
level.
| |
FOOTNOTES |
Received June 5, 1998; revised July 16, 1998; accepted July 17, 1998.
This work was supported in part by National Institutes of Health Grants
AG07805 (W.H.G.) and NS01681 (L.C.A.) and by Texas A&M University
Interdisciplinary Research Initiatives (W.H.G. and L.C.A.).
Correspondence should be addressed to Dr. William H. Griffith,
Department of Medical Pharmacology and Toxicology, College of Medicine,
Texas A&M University Health Science Center, College Station, TX
77843-1114.
| |
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Copyright © 1998 Society for Neuroscience 0270-6474/98/18197687-13$05.00/0
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Top
Abstract
Introduction
