Brain (2000), 123, 463–471
Abnormal transmitter release at neuromuscular
junctions of mice carrying the tottering α1A Ca2⫹
channel mutation
Jaap J. Plomp,1,2 Monique N. Vergouwe,3 Arn M. Van den Maagdenberg,3 Michel D. Ferrari,2
Rune R. Frants3 and Peter C. Molenaar1
Departments of 1Physiology, 2Neurology and 3Human
Genetics, Leiden University Medical Centre, Leiden,
The Netherlands
Summary
Neurotransmitter release at many synapses is regulated
by P/Q-type Ca2⍣ channels containing the α1A poreforming subunit. Mutations in α1A cause cerebral
disorders including familial hemiplegic migraine (FHM)
and ataxia in humans. Tottering (tg) α1A mutant mice
display ataxia and epilepsy. It is not known whether α1A
mutations induce impairment of synaptic function, which
could underlie the symptoms of these cerebral disorders.
To assess whether α1A mutations influence neurotransmitter release, we studied P-type Ca2⍣ channelmediated acetylcholine (ACh) release at tg neuromuscular
junctions (NMJs) with micro-electrode measurements of
Correspondence to: Dr P.C. Molenaar, Department of
Physiology, Leiden University Medical Centre,
Wassenaarseweg 62, PO Box 9604, 2300 RC Leiden,
The Netherlands
E-mail: [email protected]
synaptic potentials. We found a Ca2⍣-, Mg2⍣- and K⍣dependent increase of spontaneous ACh release at both
homo- and heterozygote tg NMJs. Furthermore, there
was increased run-down of high-rate evoked release at
homozygous tg NMJs. In isotonic contraction experiments
this led to block of synaptic transmission at lower
concentrations of the ACh antagonist tubocurarine than
were needed in wild-type muscles. Our results suggest
that in tg motor nerve terminals there is increased influx
of Ca2⍣ under resting conditions. This study shows that
functional consequences of α1A mutations causing cerebral
disorders can be characterized at the NMJ.
Keywords: acetylcholine release; migraine; P/Q-type calcium channel; neuromuscular junction; tottering mouse
Abbreviations: ACh ⫽ acetylcholine; EPP ⫽ endplate potential; FHM ⫽ familial hemiplegic migraine; MEPP ⫽ miniature
endplate potential; NMJ ⫽ neuromuscular junction; PCR ⫽ polymerase chain reaction; tg ⫽ tottering
Introduction
Voltage-gated Ca2⫹ channels in nerve, muscle and secretory
cells consist of low voltage-activated T-type channels and
high voltage-activated channels, which can be subdivided on a
pharmacological basis into P/Q-, N-, L- and R-type (Catterall,
1995; Perez-Reyes and Schneider, 1995). Ca2⫹ channel
variants are formed by complexes of pore-forming α1 and
auxiliary β, α2δ and γ subunits (Mori et al., 1991; Isom
et al., 1994; Stea et al., 1994; Catterall, 1995).
P- and Q-type channels are splice variants of the same
gene (Bourinet et al., 1999) and are discriminated by the
block of Ca2⫹ current by ω-agatoxin-IVA or ω-conotoxinMVIIC in the nanomolar range (Stea et al., 1994; Randall
and Tsien, 1995; Hilaire et al., 1996; Mori et al., 1996;
Berrow et al., 1997). P/Q-type channels are involved in
transmitter secretion from nerve terminals, at which synaptic
vesicles undergo exocytosis following the action of Ca2⫹
© Oxford University Press 2000
influx on the release machinery (Takahashi and Momiyama,
1993; Geppert et al., 1994; Wheeler et al., 1994; Augustine
et al., 1996).
The α1A ion-conducting pore of P/Q-type channels is
encoded by the CACNA1A gene, which is widely expressed
in the CNS (Mori et al., 1991; Stea et al., 1994; Diriong
et al., 1995; Westenbroek et al., 1995; Fletcher et al.,
1996). In the mammalian peripheral nervous system, P/Qtype channels are relatively rare and mainly present at
the neuromuscular junction (NMJ) where P-type channels,
probably encoded by the CACNA1A gene, regulate transmitter
release (Uchitel et al., 1992; Bowersox et al., 1995; Hong
and Chang, 1995; Day et al., 1997; Lin and
Lin-Shiau, 1997).
Mutations in the α1A gene have been found to be
responsible for the human episodic neurological disorders
464
J. J. Plomp et al.
familial hemiplegic migraine (FHM) and episodic ataxia type
2 and also for chronic spinocerebellar ataxia type 6 (Ophoff
et al., 1996; Zhuchenko et al., 1997). The α1A gene is likely
to be also involved in (non-hemiplegic) typical migraine
(Terwindt et al., 1997, 1998; Nyholt et al., 1998). Likewise,
natural α1A mutations have been reported for the tottering
(tg) and leaner (tgla) mice, of which the homozygous animals
exhibit symptoms of ataxia and epilepsy (Fletcher et al.,
1996; Doyle et al., 1997). The location of the tg mutation
P601L (Fletcher et al., 1996) corresponds to proline at
position 647 in the human sequence in the extracellular loop
between transmembrane domains S5 and S6 of repeat II of
the α1A subunit. This is just before the pore-forming loop in
IIS5S6 (position 651–675) where one of the FHM mutations
(T666M) is located (Ophoff et al., 1996). The tgla mutation
is a splice-site mutation leading to a truncated carboxyterminus (Fletcher et al., 1996).
Each of the four known FHM mutations seems to induce
specific changes in Ca2⫹ current density and/or kinetics. This
follows from studies in which rabbit and human α1A genes
with introduced FHM mutations were expressed in,
respectively, Xenopus oocytes and human embryonic kidney
cells (Kraus et al., 1998; Hans et al., 1999). The tg and tgla
mutations appear to decrease whole-cell Ca2⫹ current in
Purkinje cells, whereas the single Ca2⫹ channel conductances
are unchanged (Dove et al., 1998; Lorenzon et al., 1998;
Wakamori et al., 1998). Thus, either the number of tg Ca2⫹
channels is decreased or a channel parameter, such as the
likelihood of channel opening, is changed. Glutamatergic
synaptic potentials in tg/tg mouse thalamus slices are
decreased by 40% (Caddick et al., 1999).
We hypothesized that pathological α1A gene mutations will
change neurotransmitter release at NMJs from tg mice,
because mammalian motor nerve terminals are endowed with
P-type channels, supposedly encoded by the CACNA1A gene.
Quantal acetylcholine (ACh) release at the NMJ can be
studied electrophysiologically with relative ease and great
precision. From a clinical point of view it is also of interest
whether there is disturbed ACh release in skeletal muscles
in inherited α1A cerebral disorders. For instance, reduced
ACh release could decrease the safety factor of neuromuscular
transmission, rendering patients abnormally sensitive to
muscle relaxants.
Here we describe profound changes in ACh release at
NMJs from both homo- and heterozygous tg mice.
Material and methods
Mice
Breeding pairs of heterozygous C57BL/6J-tg mice were
obtained from the Jackson Laboratory, Bar Harbor, Me.,
USA. Litters were genotyped after weaning. The experiments
were carried out under permission of the Leiden Animal
Ethics Committee, approval number 97008.
Genotyping
Genomic DNA was isolated from 4–6 mm portions of the
tail. These were rotated overnight at 55°C in a solution
containing 50 mM Tris pH 8.9, 0.45% Nonidet P-40 (Sigma
Chemical Co., St Louis, Mo., USA) and 100 µg proteinase
K (Boehringer Mannheim, Mannheim, Germany) in a total
volume of 250 µl. After incubation, proteinase K was
inactivated for 10 min at 95°C. A polymerase chain reaction
(PCR), spanning the predicted intron 14 and the tg mutation
in exon 15 (Fletcher et al., 1996), was performed on 1 µl
genomic tail DNA (diluted 1 : 20 in H2O) in a total volume
of 30 µl containing 60 mM Tris–HCl, pH 8.5; 15 mM
(NH4)2SO4; 2.5 mM MgCl2; 200 µM of each dNTP; 15 pmol
of each primer (1729f: 5⬘-CTGTTCATCGTTGTCTTTGC3⬘, 1831r: 5⬘-CTGCTGGAAAAGTGTCGAAG-3⬘); 0.6 U
AmpliTaq (Perkin Elmer Cetus, Foster City, Calif., USA)
and 3 µg bovine serum albumin (Pharmacia Biotech, Uppsala,
Sweden). After an initial denaturation step of 3 min at 94°C,
the products were amplified for 34 cycles (30 s at 94°C,
30 s at 56°C, 45 s at 72°C) followed by a final elongation
step of 5 min at 72°C. Subsequently, the 650 bp PCR product
was cloned into the pCR® 2.1-TOPO vector of the TOPO
TA Cloning kit (Invitrogen Co., Carlsbad, Calif., USA)
according to the manufacturer. Several clones were subjected
to the above-mentioned PCR and clones containing the
product were sequenced by dideoxy sequence analysis
(T7 sequencing kit, Pharmacia Biotech, Uppsala, Sweden),
allowing the design of primer int1778f.
For mutation analysis, 1 µl of tail genomic DNA (diluted
1 : 20 in H2O) was subjected to PCR in a total volume of
15 µl containing 60 mM Tris–HCl, pH 8.5, 15 mM
(NH4)2SO4, 1.5 mM MgCl2, 200 µM dTTP, dATP, dGTP,
dCTP, 100 ng of each primer (int1778f: 5⬘-TTCTGGGTACCAGATACAGG-3⬘, 1831r: 5⬘- CTGCTGGAAAAGTGTCGAAG-3⬘), 0.3 U AmpliTaq and 1.5 µg bovine
serum albumin. The initial denaturation step (3 min at 94°C)
was followed by 33 cycles of amplification (30 s at 94°C,
30 s at 57°C, 60 s at 72°C) and a final elongation step of
5 min at 72°C.
Subsequently, 10 µl of PCR product was digested with
0.5 µl restriction enzyme AciI (5 U/µl, New England Biolabs,
Beverly, Mass., USA) for at least 1 h at 37°C according to
the recommendations of the manufacturer. Upon digestion,
the PCR fragments were separated on a 1.5% agarose gel
for genotyping. Fragment lengths of 150/28 or 178 were
obtained for a wild-type (⫹) or tg allele, respectively. Figure
1 shows the position of the tg mutation (proline to leucine
mutation as a result of a base-pair substitution at position
1802) and an example of the tg and wild-type bands on
agarose gel.
In vitro electrophysiology of the neuromuscular
junction
The mice were killed by ether inhalation. The left phrenic
nerve-hemidiaphragm was dissected and mounted in a 2 ml
ACh release at tottering neuromuscular junctions
465
the phrenic nerve was stimulated supramaximally at either
0.3 or 40 Hz. The amplitudes of MEPPs and EPPs were
normalized to –75 mV, assuming 0 mV as the reversal
potential for ACh-induced current (Magleby and Stevens,
1972). The normalized EPPs were corrected for non-linear
summation with the formula of McLachlan and Martin (1981)
using a value for f of 0.8. The quantal content was calculated
by dividing the normalized and corrected EPP amplitude by
the normalized MEPP amplitude.
ω-Agatoxin-IVA (Scientific Market Associates) was
dissolved to a concentration of 20 µM in distilled water
containing 0.1 mg/ml bovine serum albumin. Aliquots of 10
µl were stored at –70°C until further dilution to 200 nM in
Ringer’s solution just before the experiment. In the
experiments where the reducing effect of ω-agatoxin-IVA on
quantal contents was measured after 1 h incubation of nerve–
muscle preparations, the effect partially waned during the
subsequent 45 min measuring period in spite of the continuous
presence of the toxin, as described by others (Hong and
Chang, 1995), and possibly due to instability of the toxin or
its binding to non-specific sites.
Contraction experiments
Fig. 1 Detection of the tg mutation in the mouse Cacna1a gene.
(A) The exonic region of the gene harbouring the tg mutation is
depicted in capital letters and the corresponding amino acid
sequence is also given. The region of the AciI restriction site is
shown in large capital letters with the base pair (bp) change
resulting from the tg mutation in bold. (B) Samples illustrated are
of ⫹/⫹, tg/⫹ and tg/tg genotypes together with a 100 bp size
marker (lane under M in the gel). The PCR product of the wildtype (⫹) allele is cut by restriction enzyme AciI, resulting in
fragments of 150 bp and 28 bp (not visible), whereas the tg allele
is not digested by the same enzyme.
bath with Ringer’s medium at 26–28°C. The Ringer’s solution
contained (in mM): NaCl 116; KCl 4.5; MgSO4 1; CaCl2
2; NaH2PO4 1; NaHCO3 23; glucose 11, pH 7.4. The
concentrations of Ca2⫹, Mg2⫹ and K⫹ were varied as
indicated.
Intracellular recordings of miniature endplate potentials
(MEPPs) and endplate potentials (EPPs) were made from the
endplate region, using standard micro-electrode equipment
as described previously (Plomp et al., 1992). At least 25
MEPPs and EPPs were recorded from each fibre. To be able
to measure EPPs, muscle action potentials were blocked by
treatment with 2.3 µM µ-conotoxin GIIIB (Scientific Market
Associates, Barnet, UK) which blocks Na⫹ channels of
mouse muscle but not those of nerve and does not influence
the quantal release of ACh or its effect on ACh receptors
(Cruz et al., 1985; Di Gregorio et al., 1989; Hong and Chang,
1989; Plomp et al., 1992). For the generation of the EPPs
In right phrenic nerve-hemidiaphragms of mice, isotonic
contractions were recorded with a force transducer. The
phrenic nerve was stimulated supramaximally once every
5 min with a train of 150 stimuli at 50 Hz. The muscles were
incubated in Ringer’s solution to which tubocurarine was
added in increasing concentrations (1 h incubation at each
concentration). The amplitude of the contractions was
measured after 100 stimuli (i.e. 2 s) after the onset of
each train.
Statistics
The data are presented as the grand muscle mean ⫾ standard
error of the mean. Possible statistical differences were
analysed with a paired or unpaired Student’s t-test, wherever
appropriate. To correct for multiple testing in Table 1 and
Figs 2, 3 and 6, a P value of ⬍0.01 was considered to be
significant in all other cases P ⬍ 0.05 was considered to be
significant.
Results
High-rate evoked ACh release is depressed at
tg/tg NMJs
The EPP is the postsynaptic response resulting from
simultaneous exocytosis of a number of ACh quanta (quantal
content) from the motor nerve terminal in response to a
single nerve action potential. The MEPP is the response
resulting from spontaneous exocytosis of a single ACh
quantum. We made micro-electrode recordings of MEPPs
and EPPs at NMJs in µ-conotoxin-paralysed diaphragm-
466
J. J. Plomp et al.
Table 1 Electrophysiological parameters at NMJs of tg mice
tg/tg
n (muscles)
EPP amplitude at 0.3 Hz (mV)
MEPP amplitude (mV)
Quantal content at 0.3 Hz
MEPP frequency (s–1)
EPP run-down at 40 Hz (% amplitude first EPP)
5
25.7
0.86
43.5
1.91
73.2
⫹/⫹
tg/⫹
⫾
⫾
⫾
⫾
⫾
0.9
0.01
1.9
0.12*,‡
0.6†
5
25.3
0.80
47.3
1.28
81.0
⫾
⫾
⫾
⫾
⫾
1.1
0.06
3.1
0.12*
0.9
8
25.3
0.84
44.8
0.93
81.1
⫾
⫾
⫾
⫾
⫾
0.8
0.02
2.0
0.05
0.9
Measurements were made at NJMs of diaphragms from five tg/tg, five tg/⫹ and eight ⫹/⫹ mice. Data
represent the grand means ⫾ standard error of the mean of the number of muscles. Fifteen NMJs were
sampled in each muscle. EPP amplitudes are not yet corrected for non-linear summation. Amplitudes of
EPPs and MEPPs have been normalized to a resting membrane potential of –75 mV. For EPP run-down
the depression level of the EPP amplitude was determined by calculating the mean from the amplitudes
of the 10–25th EPP in each train. * P ⬍ 0.01, †P ⬍ 0.001, different from ⫹/⫹; ‡P ⬍ 0.01, different
from tg/⫹, Student’s t-test.
Fig. 3 Effect of increasing concentrations of tubocurarine on
tetanic contraction of diaphragms from tg/tg and ⫹/⫹ mice. The
nerve was stimulated at 50 Hz and contraction was measured
from the paper chart recording at 2 s after the start of the
stimulation. Data are plotted as the percentage of the contraction
of the muscle at the beginning of the experiment in the absence
of tubocurarine. The temperature was 20–22°C. Means ⫾
standard error of the mean of observations from three mice. The
differences in contraction between tg/tg and ⫹/⫹ muscles at 100,
200 and 400 nM tubocurarine were tested with Student’s t-test;
P values were 0.06, 0.01 and 0.04, respectively.
Fig. 2 Depression of EPP amplitude during stimulation of the
phrenic nerve at 40 Hz in diaphragms from tg/tg, tg/⫹ and ⫹/⫹
mice. (A) Mean run-down profiles. Each EPP amplitude in a train
was expressed as the percentage of the first EPP in the train
before the means were calculated. Grand means ⫾ standard error
of the mean of the muscles from five tg/tg, five tg/⫹ and eight
⫹/⫹ mice, 15 NMJs sampled per muscle. (B) Typical examples
of 40 Hz EPP run-down at ⫹/⫹ and tg/tg NMJs.
phrenic nerve preparations (Table 1). When supramaximal
electric stimulations were delivered to the nerve at a low rate
(0.3 Hz), the mean amplitude of EPPs was ~25 mV, and did
not differ between tg/tg, tg/⫹ and wild-type mice. Also, the
mean amplitude of MEPPs (~0.8 mV) did not differ between
these groups. The quantal content, derived from the corrected
and normalized mean amplitudes of EPP and MEPP, was
~45 in all groups. However, the amplitude of EPPs during
40 Hz nerve stimulation showed a run-down at tg/tg NMJs
to a level of ~71% of the amplitude of the first EPP, which
was greater than the EPP run-down at tg/⫹ and ⫹/⫹ NMJs
(to, in each case, ~81%, P ⬍ 0.001; Fig. 2). Enlarged EPP
run-down in tg/tg NMJs predicts a smaller safety factor of
neuromuscular transmission (this factor being the ratio of the
EPP size and the depolarization at which the muscle fibre
ACh release at tottering neuromuscular junctions
467
Fig. 4 Effect of 200 nM ω-agatoxin-IVA on MEPP frequency of
diaphragms from tg/tg and wild-type mice. Grand means ⫾
standard error of the mean of the muscles from four tg/tg and
four ⫹/⫹ mice, 15 NMJs sampled per muscle. Statistical
significance (paired Student’s t-test): P ⬍ 0.02 in the ⫹/⫹ group,
P ⬍ 0.01 in the tg/tg group.
starts firing). Changes of the safety factor can be tested for
by measuring nerve stimulation-evoked muscle contraction
at increasing concentrations of tubocurarine, a blocker of
nicotinic ACh receptors. Indeed, contraction experiments
showed that diaphragms from tg/tg muscles became paralysed
at lower concentrations of tubocurarine than those of ⫹/⫹
mice when measured at 50 Hz nervous stimulation (Fig. 3;
tg/⫹ muscles were not tested).
At both tg/tg and ⫹/⫹ NMJs, the quantal content at 0.3
Hz nerve stimulation was reduced by ~70% when measured
after 1 h incubation with 200 nM ω-agatoxin-IVA. The
quantal contents decreased from 43.6 ⫾ 1.9 to 14.3 ⫾ 2.4
in a tg/tg muscle (15 NMJs sampled) and from 40.7 ⫾ 3.0
to 11.5 ⫾ 3.4 in a ⫹/⫹ muscle (10 NMJs sampled).
Increased spontaneous quantal ACh release at
tg/tg and tg/⍣ NMJs
We measured spontaneous quantal release of ACh as the
frequency of MEPPs (Table 1). The mean MEPP frequency
at NMJs of tg/tg mice was more than twice that found in
⫹/⫹ mice. The MEPP frequency at tg/⫹ mice was in
between that of tg/tg and ⫹/⫹ mice.
The effect of the P-type Ca2⫹ channel blocker ω-agatoxinIVA (continuous presence of 200 nM toxin during the 30 min
measurement period, starting after 15 min incubation) on
spontaneous ACh release was studied at tg/tg and ⫹/⫹
NMJs. The toxin depressed the MEPP frequency in both
groups by ~55%, from 2.26 ⫾ 0.10/s to 0.94 ⫾ 0.07/s and
from 1.13 ⫾ 0.14/s to 0.55 ⫾ 0.04/s in the tg/tg and ⫹/⫹
group, respectively (Fig. 4). The observation that ω-agatoxinIVA reduced MEPP frequency at wild-type NMJs conflicts
for unknown reasons with studies that failed to demonstrate
an effect of ω-agatoxin-IVA on MEPP frequency at normal
mouse and rat NMJs (Protti and Uchitel, 1993; Hong and
Chang, 1995; Losavio and Muchnik, 1997).
Fig. 5 Effect on MEPP frequency of incubation of diaphragms
from tg/tg and wild-type mice with 10 mM K⫹ Ringer’s solution
and the effect of 200 nM ω-agatoxin-IVA thereupon. (A) Grand
means ⫾ standard error of the mean of the muscles from three
tg/tg and three ⫹/⫹ mice, 15 NMJs sampled per muscle. The
difference in relative increase of mean MEPP frequency at
10 mM K⫹ compared with 4.5 mM K⫹ was tested with Student’s
t-test. The increase was more pronounced in the tg/tg group
(9-fold) than in the ⫹/⫹ group (5-fold); P ⬍ 0.02.
(B) Typical examples of MEPP recordings in tg/tg and ⫹/⫹
NMJs at 10 mM K⫹.
The MEPP frequency was also measured in a medium
with elevated (10 mM) K⫹ concentration, which should
depolarize the motor nerve terminal to about –65 mV and
induce a 4-fold increase of the MEPP frequency at mouse
NMJs (Protti and Uchitel, 1993). High K⫹ increased the
MEPP frequency at tg/tg NMJs 9-fold, which was
significantly more than the 5-fold increase found at ⫹/⫹
NMJs (Fig. 5; P ⬍ 0.02). Upon subsequent incubation of
the 10 mM K⫹-depolarized muscles with 200 nM ω-agatoxinIVA, the MEPP frequencies at both tg/tg and ⫹/⫹ NMJs
decreased by ~75%, approaching the MEPP frequencies
measured in standard 4.5 mM K⫹ medium (Fig. 5), as
observed by others at normal NMJs at high K⫹ concentration
(Protti and Uchitel, 1993; Hong and Chang, 1995; Losavio
and Muchnik, 1997).
Effect of Ca2⍣ and Mg2⍣ on MEPP frequency
Spontaneous quantal release of ACh at the NMJ is mainly
dependent on the concentration of Ca2⫹ in the medium. We
468
J. J. Plomp et al.
Evoked ACh release in tg/tg mice
Fig. 6 MEPP frequency at diaphragms of tg/tg and ⫹/⫹ mice as
a function of the concentration of Ca2⫹ (A) and Mg2⫹ (B) in the
medium. Grand means ⫾ standard error of the mean of the
muscles from three tg/tg and three ⫹/⫹ mice, 15 NMJs sampled
per muscle. Differences between grand mean MEPP frequency of
tg/tg and ⫹/⫹ group were tested at 2 mM Ca2⫹/1 mM Mg2⫹
(P ⫽ 0.004), 5 mM Ca2⫹/1 mM Mg2⫹ (P ⫽ 0.014) and 2 mM
Ca2⫹/0.2 mM Mg2⫹ (P⫽ 0.026) with Student’s t-test.
measured the MEPP frequency at tg/tg and ⫹/⫹ NMJs at
low (0.2 mM), standard (2 mM), and high (5 mM) Ca2⫹
concentration. The difference in MEPP frequency between
tg/tg and ⫹/⫹ varied with the Ca2⫹ concentration; it
disappeared at 0.2 mM Ca2⫹ (Fig. 6A).
Mg2⫹ is a reversible Ca2⫹ channel blocker. It antagonizes
Ca2⫹-induced neurotransmitter release at the NMJ (Jenkinson,
1957). We measured MEPP frequency at tg/tg and ⫹/⫹
NMJs at low (0.2 mM), standard (1 mM), and high (10 mM)
Mg2⫹ concentration. The mean MEPP frequency decreased
with increasing concentration of Mg2⫹, but not to the same
extent in tg/tg and ⫹/⫹ NMJs. The difference between mean
MEPP frequency of the groups disappeared at 10 mM Mg2⫹
(Fig. 6B).
Discussion
This study shows that the tg α1A gene mutation can influence
neurotransmitter release. We observed two types of changes
at tg NMJs. First, an increase of run-down of ACh release
during high frequency nerve stimulation, and secondly, an
increase in rate of spontaneous quantal release, the extent of
which was dependent on the concentrations of Ca2⫹, Mg2⫹
and K⫹ in the extracellular medium. This strongly suggests
that the Ca2⫹ channel involved in ACh release at mouse
motor nerve terminals is encoded by the α1A gene, as was
predicted on pharmacological grounds (see Introduction).
Thus, if the CACNA1A gene encodes endplate P/Q-type Ca2⫹
channels it would follow that subclinical abnormalities at the
NMJ may exist in cerebral diseases such as FHM, possibly
leading to an altered sensitivity for non-depolarizing muscle
relaxants. Neuromuscular transmission has a substantial
safety factor, preventing small decreases in ACh output
from affecting the all-or-none process of muscle activation.
However, blockade of some of the ACh receptors may unveil
subclinical defects at the NMJ, as demonstrated in this study
by the effect of tubocurarine in tg/tg muscles.
P/Q-type Ca2⫹ channel expression or the likelihood of Ca2⫹
channel opening is decreased in tg Purkinje cells (see
Introduction). It came as a surprise, therefore, that the quantal
content at low-rate (0.3 Hz) nerve stimulation in tg/tg NMJs
was unchanged. This suggests that there is no reduced
expression of mutant P-type Ca2⫹ channels at tg motor nerve
terminals and that the density of mutant P-type channels, or
their subunit composition, in the NMJ is regulated in a
different manner, as in Purkinje cell bodies. However, it
cannot be excluded that a decrease of P/Q-type Ca2⫹ channels
was masked in NMJs of tg mice by a compensatory increase
of the sensitivity to Ca2⫹ of the ACh release process.
Yet, at high-rate nerve stimulation the quantal content at
tg/tg NMJs fell below the level of controls; there was
increased run-down of EPPs. This may be caused by a
decrease in Ca2⫹ channel availability due to slowed recovery
from inactivation, as has been shown for the nearby T666M
FHM mutation (Kraus et al., 1998; Hans et al., 1999).
Another factor that could add to extra EPP run-down is
reduced availability of ACh quanta at tg/tg NMJs, since it is
possible that tg mutant P-type channels have abnormal
interactions with active zone proteins involved in recruiting
and docking of synaptic vesicles.
The fact that we did not observe increased EPP run-down
at tg/⫹ NMJs indicates that this is a threshold phenomenon
and a recessive phenotype with loss of physiological function,
i.e. increased EPP rundown requires more than half of the
population of P-type channels at the NMJ to be tg mutated.
Spontaneous ACh release and low-voltageactivated Ca2⍣ channels
MEPP frequency was increased by ~100% in tg/tg and by
40% in tg/⫹ mice. The increase in MEPP frequency in the
tottering mouse was therefore a dominant trait with gain-of(physiological) function. The difference between tg/tg and
control MEPP frequency was abolished at low (0.2 mM)
Ca2⫹ or high (10 mM) Mg2⫹ in the medium. In tg mice
there is upregulation of L-type Ca2⫹ channels in Purkinje
cells (Campbell and Hess, 1999). The question arises as to
whether the increased MEPP frequency was due to expression
of L-type channels in motor nerve terminals or some other,
unrelated secondary adaptive reaction. However, the fact that
heterozygous tg mice have no epileptic/ataxic symptoms, but
show increased MEPP frequency, seems to argue against this
possibility. Furthermore, MEPP frequencies at tg/tg and
⫹/⫹ NMJs with standard (4.5 mM) as well as elevated
(10 mM) K⫹ concentration in the medium were equally
sensitive to ω-agatoxin-IVA. These findings indicate that
P-type Ca2⫹ channels are responsible for ~50% of the MEPP
frequency at wild-type NMJs and for most of the MEPP
increase at tg NMJs.
Wild-type P-type Ca2⫹ channels are high-voltage-activated
with minute probability of opening at a membrane potential
ACh release at tottering neuromuscular junctions
range of –80 to –60 mV, according to the Boltzmann equation
using the constants of rabbit or mouse α1A channels (Kraus
et al., 1998; Wakamori et al., 1998). Thus, in spontaneous
quantal ACh release at wild-type NMJs, an underlying Ca2⫹
channel has the ω-agatoxin-IVA sensitivity but not the gating
characteristics of a P-type channel. Interestingly, low-voltageactivated ‘T-type’ channels can be transformed by an as yet
unknown mechanism from high-voltage-activated N-, L- and
R-type channels (Meir and Dolphin, 1998). Hence, P/Q-type
channels may also exhibit dual forms with different activation
characteristics, and MEPP frequency may be determined by
a fraction of the channels expressed in a low-voltage-activated
form (perhaps too low in number to be recognized in wholecell I–V relationships).
The increase in MEPP frequency at tg NMJs could, in
principle, be caused by a shift of activation voltage of highvoltage-activated P-type channels to values in the direction
of the resting membrane potential. However, no significant
changes of the Boltzmann constants have been found in tg
mice and at any rate it would require a much greater change
than reported for FHM mutations in rabbit and human α1A
(Kraus et al., 1998; Wakamori et al., 1998; Hans et al.,
1999). A better explanation seems to be that the tg mutation
is shifting activation voltage of the putative α1A gene-encoded
low-voltage-activated P-type Ca2⫹ channel in the negative
direction. The observation that slight depolarization by 10 mM
K⫹ increased MEPP frequency more at tg/tg than at ⫹/⫹
NMJs supports this hypothesis. It has previously been
suggested that the α1A gene might in addition encode a lowthreshold isoform, or that the tg mutation has an indirect
effect on T-type Ca2⫹ channel function (Fletcher et al., 1996).
It has been suggested that Mg2⫹ is involved in the
pathogenesis of migraine (Welch and Ramadan, 1995). Our
observation that high Mg2⫹ in the medium abolishes the
difference in the rate of spontaneous ACh release between
tg/tg and ⫹/⫹ NMJs is therefore of interest in relation to
migraine.
469
The four known FHM mutations cause different effects on
kinetic parameters of whole-cell Ca2⫹ currents in expression
systems (Kraus et al., 1998; Hans et al., 1999). Accordingly,
it should be realized that different mutations in the α1A gene
may lead to different ‘synaptic phenotypes’, even if the
mutations give rise to very similar clinical phenotypes.
CNS synaptic dysfunction may also play a role in
progression of symptoms and cerebral atrophy in α1A diseases.
Synaptic activity influences gene expression in the
postsynaptic neuron, thus regulating protein synthesis for
maintenance of synaptic integrity and structural changes
accompanying functional synaptic plasticity (Bito et al.,
1997; Bito, 1998). Furthermore, increased presynaptic Ca2⫹
influx via mutated α1A subunits and the overstimulation
of postsynaptic receptors by the consequent increase in
spontaneous transmitter release may result in pre- and/or
postsynaptic Ca2⫹ overload, triggering apoptotic mechanisms.
In conclusion, the present study strongly suggests that
P/Q-type channels in the NMJ are encoded by the same gene
as that encoding cerebral P/Q-type Ca2⫹ channels involved
in FHM and episodic ataxia type 2. Accordingly, the NMJ,
being relatively easy to study as compared with synapses in
the CNS, can be used to study in detail the changes in
neurotransmitter release resulting from P/Q-type Ca2⫹
channel mutations.
Acknowledgements
The authors wish to thank Mr J. A. van der Zwet for breeding
the tg mice and Drs A. R. Wintzen, J. J. G. M. Verschuuren,
R. J. van den Berg and D. L. Ypey for valuable discussions.
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Received July 2, 1999. Revised August 27, 1999.
Accepted September 28, 1999