 |
 Previous Article | Next Article 
Volume 17, Number 11,
Issue of June 1, 1997
pp. 4170-4179
Copyright ©1997 Society for Neuroscience
Slow-Channel Transgenic Mice: A Model of Postsynaptic Organellar
Degeneration at the Neuromuscular Junction
Christopher M. Gomez1, 2,
Ricardo Maselli3,
Jo Ellen Gundeck1,
Mary Chao3,
John W. Day1,
Shiori Tamamizu4,
Jose A. Lasalde4,
Mark McNamee4, and
Robert L. Wollmann5
1 Department of Neurology,
2 Institute of Human Genetics, University of Minnesota,
Minneapolis, Minnesota 55455, Sections of 3 Neuroscience
and 4 Molecular and Cellular Biology, University of
California, Davis, California, and 5 Section of
Neuropathology, University of Chicago, Chicago, Illinois 60637
 ABSTRACT
- INTRODUCTION
- MATERIALS AND METHODS
- RESULTS
- DISCUSSION
- FOOTNOTES
- REFERENCES
ABSTRACT 
The slow-channel congenital myasthenic syndrome (SCCMS) is a
dominantly inherited disorder of neuromuscular transmission
characterized by delayed closure of the skeletal muscle acetylcholine
receptor (AChR) ion channel and degeneration of the neuromuscular
junction. The identification of a series of AChR subunit mutations in
the SCCMS supports the hypothesis that the altered kinetics of the endplate currents in this disease are attributable to inherited abnormalities of the AChR. To investigate the role of these mutant AChR
subunits in the development of the synaptic degeneration seen in the
SCCMS, we have studied the properties of the AChR mutation,  L269F,
found in a family with SCCMS, using both in vitro and
in vivo expression systems. The mutation causes a sixfold increase in the open time of AChRs expressed in vitro,
similar to the phenotype of other reported mutants. Transgenic mice
expressing this mutant develop a syndrome that is highly reminiscent of
the SCCMS. Mice have fatigability of limb muscles, electrophysiological evidence of slow AChR ion channels, and defective neuromuscular transmission. Pathologically, the motor endplates show focal
accumulation of calcium and striking ultrastructural changes, including
enlargement and degeneration of the subsynaptic mitochondria and
nuclei. These findings clearly demonstrate the role of this mutation in
the spectrum of abnormalities associated with the SCCMS and point to
the subsynaptic organelles as principal targets in this disease. These
transgenic mice provide a useful model for the study of excitotoxic
synaptic degeneration.
Key words:
transgenic mice;
neuromuscular junction;
slow-channel
congenital myasthenic syndrome;
acetylcholine receptor;
point mutation;
calcium overload;
excitotoxicity;
synaptic degeneration
INTRODUCTION
The slow-channel congenital myasthenic
syndrome (SCCMS) is a dominantly inherited disorder of neuromuscular
transmission in which electrophysiological features of abnormal
acetylcholine receptor (AChR) ion channel kinetics have been associated
with point mutations in the genes encoding the subunits of the AChR (Engel et al., 1982 ; Oosterhuis et al., 1987; Gomez and Gammack, 1995;
Sine et al., 1995; Ohno et al., 1995; Engel et al., 1996; Gomez et al.,
1996b). It is the first of what may be a family of disorders of altered
or exaggerated synaptic receptor function that underlie poorly
understood neurodegenerative, convulsive, or psychiatric disorders
(Shiang et al., 1993; Steinlein et al., 1995; Treinin and Chalfie,
1995; Ophoff et al., 1996). Several studies have correlated the changes
in the kinetics of endplate currents with the in vitro
phenotype of the mutant subunits. In this study we seek to associate
the changes in ion channel properties with the striking clinical and
pathological features of this disease.
The SCCMS is characterized clinically by fatigability and progressive
weakness and atrophy of the skeletal muscles, especially of the
extraocular, face, and forelimb (Engel et al., 1982; Oosterhuis, et
al., 1987). The distinctive pathological features are found at the
electron microscopic level (Engel et al., 1982; Oosterhuis et al.,
1987; Gomez et al., 1996b) and consist of striking degenerative changes
involving the basement membrane, postsynaptic membrane, and subsynaptic
organelles of the neuromuscular junction. One possible explanation for
these changes is that the slowed channels, normally permeable to sodium
and calcium ions (Decker and Dani, 1990), allow entry of excessive
amounts of these cations, which activate any of several degradative
(Engel et al., 1982; Salpeter et al., 1982) or second-messenger
pathways (Bygrave and Roberts, 1995; Ghosh and Greenberg, 1995).
Confirmation of this hypothesis and elucidation of the factors and
processes responsible for the synaptic degeneration in SCCMS may aid in
understanding the basis for the excitotoxic neuronal damage in
conditions such as stroke, epilepsy, and neurodegenerative diseases.
Moreover, identification of the functional differences among distinct
AChR mutations that account for observed differences in clinical,
pathological, and electrophysiological features of the SCCMS will
greatly expand our understanding of excitotoxicity and of excitatory
synaptic transmission.
Recently we reported the occurrence of a mutation, L269F (Gomez and
Gammack, 1995), in the sequence encoding the channel-lining domain of
the subunit in the three affected members of a kindred with SCCMS.
To investigate the role of this mutant subunit in the pathogenesis
of the constellation of electrophysiological and pathological features
constituting the SCCMS, we studied its properties in vitro
and in vivo. Our findings demonstrate that this mutation is
responsible for the kinetic abnormality of the AChR observed in these
patients and for the development of Ca2+ overload and
endplate degeneration in the SCCMS.
MATERIALS AND METHODS
In vitro expression studies. Site-directed
mutagenesis of mouse subunit cDNA to generate the homologous L269F
mutation was performed using single-stranded plasmid generated from the
vector pSelect (Promega, Madison, WI). The mutation was confirmed by dideoxy nucleotide sequence determination. Mutant and wild-type AChR
subunit mRNAs (Lapolla et al., 1984; Boulter et al., 1985; Gardner,
1990) were transcribed in vitro from linearized plasmids using T7 polymerase. Plasmids containing the cDNAs for the  ,  ,
 , and subunits of mouse AChR were linearized by restriction enzyme digestion and were used as templates for T7 RNA
polymerase-mediated in vitro transcription. Transcription
was performed in the presence of the cap analog
m7G95 )ppp(5)G,5-7-methyl guanosine. Purification of RNA transcripts
was performed using Select-D(RF) columns (5 Prime-3 Prime, Boulder,
CO). Ovarian lobes were obtained from female Xenopus laevis.
Follicle cell layers were removed by incubation of the oocytes in
Ca2+-free OR2 buffer containing 82.5 mM NaCl,
2.5 mM KCl, 1 mM MgCl2, 1 mM Na2HPO4, and 5 mM
HEPES, pH 7.6, plus 2 mg/ml collagenase (Type 1A, Sigma, St. Louis, MO)
for 20 min at room temperature under slow agitation (80-100 rpm)
followed by manual defolliculation. Oocytes of stage V and VI were
chosen for injection. Fifty nanoliters of mRNA subunit transcripts of
mouse AChR (at a concentration of 0.4 ng/nl) were injected into
the cytoplasm of Xenopus oocytes at an ::: ratio
of 2:1:1:1.
Generation and screening of transgenic lines. The design for
the transgene construct, MCKL269F, was analogous to previous transgenes (Gomez et al., 1996a). In brief, the cDNA encoding the
mutant subunit, L269F, was joined to the 3 end of a 3.3 kb
portion of the mouse creatine kinase promoter (Johnson et al., 1989).
The 3 untranslated end of the L269F cDNA was removed and replaced
with the 3 untranslated region of the bacterial neomycin resistance
gene (NEO) and the SV40 small-t intron to allow distinction of
transgene mRNA. The nucleotide sequence of the junction points and
L269F cDNA were confirmed by dideoxy sequence analysis.
Transgenic mice bearing MCKL269F were generated by microinjection of
single-cell mouse embryos of the strain FVB, as described by Hogan et
al., (1986). The genetic background was assumed not to be critical for
these experiments. Genomic DNA extracted from mouse tail tips by
standard methods (Sambrook et al., 1989) was screened for the presence
of the transgene by the PCR (Saiki et al., 1988), using primers
corresponding to sequences the subunit (Gardner, 1990) and the NEO
gene (Gorman et al., 1982).
Analysis of transgene expression. Ten micrograms total RNA
extracted from hindlimb skeletal muscle (Chomczynski and Sacchi, 1987)
of F1 mice of each of the four propagated lines and control mouse was
size-selected by agarose gel electrophoresis in 6% formaldehyde, transferred to nylon membranes, and stabilized by UV cross-linking. DNA
probes for hybridization (Feinberg and Vogelstein, 1984; Sambrook et
al., 1989) consisted of a 450 bp Pst 1 fragment from the 3 portion of
the transgene, containing both AChR and transgene-specific NEO
sequences and the 300 bp 3 untranslated region of the mouse MCK gene
(MCK 3) (Buskin et al., 1985), as a probe for comparable loading of
lanes.
Immunolocalization of AChR subunits. Transverse sections of
fresh frozen mouse forelimb muscle (8-20 µm) were collected on silanized glass slides and allowed to air dry. Sections were incubated 3-5 min in 50 mM ethanolamine hydrochloride, pH 11, to
remove cytoskeletal proteins, rinsed three times for 10 min each in
PBS, pH 7.2 then immersed in PBS containing 4% normal donkey serum (D-PBS) × 30 min. Sections were next immersed in D-PBS containing either a 1:200 dilution of affinity-purified rabbit anti- -subunit peptide (anti-485, gift of Z. Hall, National Institute of
Neurological Disorders and Stroke) (Gu and Hall, 1988) or 50 µg/ml of one of four subunit-specific monoclonal antibodies
(mAbs), 132A (rat anti- subunit) (Gomez and Richman, 1985), 111 (rat
anti- subunit, gift of J. Lindstrom, University of Pennsylvania)
(Tzartos and Lindstrom, 1980), 88B (mouse anti-- subunit, gift of
S. Froehner, University of North Carolina, Chapel Hill) (Froehner et
al., 1983) or 168 (rat anti- subunit, gift of J. Lindstrom) (Nelson
et al., 1992) for 1 hr. To remove unbound antibody, sections were then rinsed in PBS as described above. Sections were then immersed in D-PBS
containing either Cy2-conjugated -bungarotoxin (Cy2-BT, 5 µg/ml; Molecular Probes, Eugene, OR) together with
Cy3-conjugated goat anti-mouse (or-rat) IgG (1:200 dilution) or Texas
red-conjugated -bungarotoxin (Tx-BT, 5 µg/ml; Molecular
Probes) together with Cy2-conjugated donkey anti-rabbit IgG (1:200
dilution). Denervated muscle was used as a positive control for the
anti- subunit peptide antisera. Stained, washed sections were
viewed, and images were digitized using a Bio-Rad MRL 1024 confocal
head Richmond, CA and Olympus BX 60 microscope. Images from each label
were merged using a graphics program (Photoshop).
Clinical evaluation of muscle strength. Two apparatuses were
used to assess forelimb muscle strength. The first consisted of a
6-mm-diameter wooden dowel suspended fixed over a height of 35 cm. Mice
were placed on top of the dowel and observed for periods of up to 60 sec. The possible outcomes consisted of escape or persistence on the
dowel for the entire period (score of 100%) or falling off before 1 min is reached (scored as the fraction of 1 min). This test evaluates
more complex motor skills as well as strength and endurance. The second
test consisted of a taut wire suspended in place of the dowel. For
testing, mice were placed with front claws gripping the wire and
observed for periods of up to 60 sec. Possible outcomes and scoring
were identical. This test more strictly evaluated strength and
endurance alone. For each test, five sets of three repetitions were
conducted. The mean and SE for 15 tests were calculated using a
statistical program (Excel, Microsoft). Mice were tested blindly using
littermate controls.
Electromyography. Compound muscle action potentials (CMAPs)
were studied as described by Gomez et al., (1996a). For hindlimb recordings, animals were anesthetized with pentobarbital or Avertin (Hogan et al., 1986), and the sciatic nerves were exposed. The responses from both the gastrocnemius and intrinsic hindpaw muscles were recorded separately during direct sciatic nerve stimulation. For
the intrinsic hindpaw, muscle recording electrodes were placed at the
wrist and at the base of the third digit. Wounds were closed with
stainless steel wound clips. For forelimb recordings, the stimulus
electrode was placed into the brachial plexus, and a stable EMG
response was sought. Responses were recorded and displayed using a
clinical electromyography apparatus (Dantec Counterpoint). The
fractional decrease in amplitude of the CMAP (decrement) was calculated
using the amplitude (peak positive to peak negative) of the 1st and
10th responses. The mean and SE of the decrement at 5 Hz stimulation
for six transgenic and five control mice were calculated using a
statistical program (Excel, Microsoft).
Voltage-clamp. Recordings of miniature endplate currents
(MEPCs) from the diaphragm were performed using electrophysiologic techniques described previously (Maselli et al., 1989, 1991). All the
microelectrode recordings were performed in the most posterior aspect
of the left hemidiaphragm muscle. This part of the muscle is very thin
(one or two layers of muscle fibers), allowing excellent endplate
visualization (Dow Corning, Arlington, TN). After dissection the
muscles were pinned to SYLGARD (Dow Corning, Arlington, TN) on the
bottom of a Plexiglas chamber. The chamber was subsequently placed on
the stage of an upright microscope (Leitz Laborlux 12) equipped with
Hoffman interference contrast optics (magnification, 250×). The
preparations were bathed continuously in Tyrode's solution having the
following ionic composition (in mM): NaCl 140, KCl 2.6, MgCl2 0.4, CaCl2 2.5, and
NaH2PO4, pH 7.4. The solution was bubbled with
a 95% O2, 5% CO2 gas mixture, and the pH was
maintained between 7.3 and 7.4. Experiments were performed at room
temperature (26°C) using an Axoclamp 2A (Axon Instruments, Foster
City, CA). The two electrode regional voltage-current electrodes were
filled with 3M KCl and had resistances in the range of
8-12 M for the voltage electrode and 2-4 M for the current
electrode. Extensive ground shielding between the two electrodes was
necessary to minimize coupling capacitance. The output of the recording
instrument was amplified, filtered, and sampled at 10 Khz by a 12 bit
analog to digital converter (Data Translation 2818, Marlboro, MA). All electrical signals were acquired on-line and stored on a personal computer for subsequent analysis. Exponential fits to the MEPC decays
were performed using a nonlinear least square fitting routine.
Single-channel studies using the patch-clamp technique. For
oocyte experiments, the oocyte vitelline membrane was removed manually
after incubation in hypertonic solution composed of 150 mM
NaCl, 2 mM KCl, 3% sucrose, and 5 mM HEPES, pH
7.6. The oocytes were placed in a recording chamber containing bath
solution (100 mM KCl, 1 mM MgCl2,
and 10 mM HEPES, pH 7.2) at 18°C. The patch pipettes were
made of thick-walled borosilicate glass (Sutter Instruments, Novato,
CA) exhibiting resistances of 8-12 M. The pipette solution
contained 100 mM KCl, 10 mM HEPES, 10 mM EGTA, pH 7.2, and 4 µM ACh. All
experiments were performed in a cell-attached configuration.
Single-channel currents were recorded using a Dagan 3900 amplifier
(Dagan, Minneapolis, MN), filtered at 5kHz (Frequency Devices,
Haverhill, MA) and stored on VHS tapes using a digital data recorder
(VR-10B, Instrutech, Mineola, NY). The data traces were played back
into an IBM-compatible computer through DigiData 1200 interface (Axon
Instruments) and digitized at 50 µsec. Single-channel currents were
detected with a half-amplitude crossing algorithm (IPROC 3), and data
analysis was performed using pCL/AMP 6 (Axon Instruments).
Standard cell-attached patch-clamp techniques (Brehm and
Kullberg, 1987) were used to record ACh-gated single channels. The flexor digitorum brevis (fdb) muscles from mice were dissociated acutely. The muscles were treated with 0.2% collagenase (type 1A,
Sigma), followed by gentle trituration. Patch-clamp recordings were
performed on single fibers bathed in a physiological saline solution
(120 mM NaCl, 1 mM KCl, 1 mM
CaCl2, 10 mM HEPES buffer, pH 7.2) at room
temperature. The patch-clamp electrodes were fabricated from
borosilicate glass using a two-stage Narishige puller (Narishige 9-28
Kasuya 4-chrome, Setagaya-ku, Tokyo, Japan) to obtain a diameter of 1 µm. The electrodes were coated with SYLGARD to reduce capacitance and
recording noise. Finally, the electrodes were fire-polished with
Narishige forge and backfilled with 200-400 nM ACh diluted in HEPES buffer. Single-channel currents were recorded and amplified with an Axopatch 200 amplifier (Axon Instruments). Single currents were
filtered with a low-pass Bessel filter (80 dB/decade) set at 2 kKz and
input to a TL-1 interface for digital conversion. The currents were
recorded on-line using a personal computer loaded with pClamp software
(Axon Instruments). All data analysis, including open-time and
close-time histogram were done using pClamp 6 software.
Pathology. Fresh frozen sections of forelimb flexors and
extensor muscles, biceps, triceps, tibialis anterior, gastrocnemius, soleus, and diaphragm were examined for light microscopic studies. The
Karnovsky and Roots cholinesterase method was used to localize endplates (Namba et al., 1967) and the glyoxal bis-(2-hydroxyanil) (GBHA; Kashiwa, 1970) or von Kossa (Rungby et al., 1993) methods were
used to localize Ca2+. Acid phosphatase was localized using
an azo-dye method (Barka, 1960).
For electron microscopy mice were perfused with 2% glutaraldehyde by
cardiac puncture. Forelimb flexor muscles were then removed and fixed
overnight in fresh glutaraldehyde, rinsed in 0.1 M
phosphate buffer. The muscle bellies were teased into 1 mm bundles,
osmicated, and embedded in Epon.
RESULTS
Effect of the L269F mutation on AChRs expressed in
Xenopus oocytes
The L269F mutation is present in the three affected members of
a family with the SCCMS (Gomez and Gammack, 1995). To confirm that this
mutation was not a silent polymorphism in the AChR subunit gene, we
generated the identical mutation in the mouse subunit in
Xenopus oocytes. Oocytes injected with mouse wild-type ,
, , and subunit cRNAs exhibit channel openings typical of the
adult AChR (Mishina et al., 1986). As shown in Figure 1 and Table 1, the open-time distribution of wild-type
AChR (top) shows two components,  0, 0.75 msec
(f = 0.158), and 1, 3.63 msec
(f = 0.842), where f refers to the
fraction of the total events with the indicated open time. The L269F
mutation increases the AChR single-channel, open-time significantly.
The open-time distribution of L269F (bottom panels) shows
three components at  100 mV and 18°C: 0, 4.21 msec
(f = 0.161); msec 1, 19.95 msec
(f = 0.452), and 2, 45.44 msec
(f = 0.387), indicating a pronounced slowing of
channel closure.
Fig. 1.
The L269F mutation causes increased AChR
channel open times. Single-channel currents (left) and
open time histogram (right) for wild-type mouse AChRs
(top) and AChRs expressing the L269F mutation
(bottom) expressed in Xenopus oocytes.
Recordings were performed at 100 mV and 4.0 µM ACh. The
L269F mutation shows three open time components: 0,
4.21 msec (f = 0.161); 1, 19.95 msec (f = 0.452), and 2, 45.44 msec (f = 0.387), total detected events (2, 061). The wild type only show two main components: 0,
0.75 msec (f = 0.158) and 1, 3.63 msec (f = 0.842), total detected events (1995).
The analysis was performed using the Marquardt least squares fitting in
pClamp6.
[View Larger Version of this Image (27K GIF file)]
Generation and characterization of transgenic lines
Of five founder mice bearing the transgene MCKL269F, four
(5, 9, 12, and 14) were successfully propagated by
breeding. The founder 4 did not breed, although electrophysiological
and histological studies indicated that it was severely affected. Southern blot analysis of genomic digests indicated that in each line
the transgene was integrated head to tail at a single site, with line
9 having 2 copies, lines 5 and 14 having 5 copies, and line
12 having 25 copies (data not shown).
Expression studies in vivo
Figure 2 shows the results of an RNA blot analysis
of skeletal muscle RNA from control and four lines of
L269F-transgenic mice. Mice from all four lines studied have
detectable steady state levels of the 2.6 kb transgene mRNA. The level
of expression ranges approximately eightfold with line 9 having the
lowest level of expression, lines 5 and 12 approximately
threefold over that of line 9, and line 14 approximately
eightfold over that of line 9. Because, at this level of sensitivity
the mRNA from the wild-type subunit is undetectable (Merlie and
Sanes, 1985), the transgene mRNA level exceeds the abundance of
endogenous subunit. The two smaller RNA species could result either
from aberrant splicing or from differential polyadenylation.
Fig. 2.
RNA blot analysis of L269F transgene expression
in muscle. Total RNA (10 µg) from skeletal muscle of control mouse
and individuals from 4 L269F-transgenic lines was hybridized with
the transgene probe NEO (top panel, 24 hr exposure) or
the MCK probe (bottom panel, 4 hr exposure). Lane
1, 5; lane 2, 9; lane 3,
12; lane 4, control; lane 5, 14. The ratios
of steady state transgene mRNA levels are 1(9):3(5,
12):8(14). Although it is difficult to compare these levels with
those of the endogenous mRNA [expressed only in the subsynaptic
nuclei (Merlie and Sanes, 1985)], the abundance of the transgene mRNA
seems much greater than the wild type.
[View Larger Version of this Image (111K GIF file)]
Marked overexpression of the L269F transgene could theoretically
alter the subunit composition of endplate AChRs. We used AChR
subunit-specific antibodies to test for the presence of the normal AChR
subunits. Figure 3A demonstrates the binding
of the --specific monoclonal antibody, 88B, to transgenic mouse
endplates. The yellow-stained endplates demonstrate that there is
colocalization of the green-staining Cy2-BT with the red-staining
Cy-3-labeled - epitope. Figure 3B demonstrates the
absence of binding of the anti- antibody to the endplates on a
serial section, which stain only with Tx-BT. This confirms that mAb 88B
readily detects the -subunit in the L269F-endplates. As a
positive control for the peptide-specific antiserum, Figure 3B,
inset, shows the binding of the anti- antisera to endplates in
neonatal muscle in the same experiment. Similar endplate staining was
seen with mAbs specific for , , and subunits. Therefore, the
overexpression of the mouse subunit mutant L269F does not
prevent the formation of AChRs with the normal AChR subunit
composition.
Fig. 3.
L269F-transgenic endplates express subunits, but not subunits. A, The
--subunit-specific mAb 88B (Froehner et al., 1983), stained with
Cy3-conjugated anti-mouse antibody, colocalizes with Cy2-conjugated bungarotoxin (double-stained endplates are yellow) on
L269F-transgenic mouse endplates. Identical patterns were seen with
mAbs specific for , , and subunits. B, Section from the same muscle and L269F-transgenic mouse showing binding of
Texas Red-conjugated bungarotoxin (Tx-BT, red), but no
binding of antibody to -subunit peptide (Gu and Hall, 1988), stained with Cy2-conjugated anti-rabbit antibody (double-stained endplates should be yellow) but staining with Texas Red-conjugated bungarotoxin (Tx-BT, red). As a positive control for the
peptide-specific antiserum, the inset shows neonatal control
muscle stained with anti- antisera and Cy2-conjugated second
antibody (green) along with Tx-BT
(red, double-stained endplates are yellow).
Calibration bar, 25 µM.
[View Larger Version of this Image (85K GIF file)]
Clinical studies
By 6-8 weeks of age all transgenic mice from lines 5, 12,
and 14, the three high-expressing lines, are distinguished from their littermate controls by the presence of a faster respiratory rate.
By ~5 months of age they seem to have less spontaneous exploratory activity than controls. They are also more susceptible than controls to
fatal complications during pentobarbital anesthesia, probably attributable to respiratory muscle weakness. Formal strength testing of
lines 5 and 14 revealed that these mice readily fatigued in
comparison to their littermates. Figure 4 displays the
results with two strength measures. In nearly all cases transgenic mice were able to grip either the wire or dowel at least briefly but tended
to fatigue and drop off the apparatus well before 60 sec. Control mice
on average either remained hanging the full minute or climbed off.
Transgenic mice were not able to climb off the apparatus.
Fig. 4.
L269F-transgenic mice have skeletal muscle
weakness. Transgenic mice (dark bars) performed
significantly more poorly than control mice (light
bars) on the wire-hang test (61.5%, compared with 86.7% for
control) and on the dowel perch test (44.1%, compared with 93.4% for
control; n = 15; p < 0.001). Perfect
score (100%) = 1 min hang or escape.
[View Larger Version of this Image (42K GIF file)]
Electromyography
In four of the lines of L269F-transgenic mice, single stimuli
to peripheral nerves evoked repetitive CMAP in at least two of the
three muscle groups tested (Table 1). The size and number of repetitive
CMAP varied depending on the muscle groups tested, but were similar for
the same muscle groups for different animals. The repetitive CMAP was
always prominent in the gastrocnemius but was absent or diminished in
the intrinsic hindpaw muscles. Figure 5A
shows a CMAP recorded from the gastrocnemius after a single stimulus to
the sciatic nerve. Two additional action potentials follow the initial
action potential. This repetitive firing of action potentials, which is
similar to that seen in SCCMS, is caused by the prolongation of the
endplate potential beyond the muscle fiber refractory period. Evoked
CMAPs recorded in control mice were always single responses (Fig.
5B, Table 1).
Fig. 5.
Abnormal neuromuscular transmission in
L269F-transgenic mice. A, L269F-transgenic mouse. Repetitive
compound muscle action potentials (CMAP) recorded over gastrocnemius,
evoked by a single stimulus to sciatic nerve. The predominant
repetitive CMAP occurs at an interval of 3.6 msec. B,
Control mouse. CMAP recorded over gastrocnemius is a single action
potential. C, L269F-transgenic mouse. Decremental CMAP
recorded over intrinsic muscles of foot, evoked by stimulation of
sciatic nerve at 10 hz. The amplitude of the CMAP decays by 18% by the
10th response. D, Control mouse. CMAP recorded over
intrinsic muscles of foot, evoked by repetitive stimulation at 10 hz
have no decrement in amplitude. E, Miniature endplate
current (MEPC) recorded from hemidiaphragm of L269F-transgenic mouse. The amplitude is 2 nA and the decay time constant of the predominant decay component is 26.6 msec. F, MEPC recorded
from control mouse, with an amplitude of 2.8 nA and principal decay time constant of 1.4 msec. G-J, Single-channel currents
(G, H) and open time histograms (I,
J) recorded for endplate AChRs in dissociated flexor
digitorum brevis (fdb) muscle fibers of L269F-transgenic mouse
(G, I) and control mouse (H,
J). Recordings were made at 50 mV holding potential.
Calibration bars: A, B, 10 mV, 2 msec; C, D, 5 mV, 1 msec; E, F, 1nA, 15 msec; G, H, 20 pA, 50 msec.
[View Larger Version of this Image (26K GIF file)]
The effect of repetitive nerve stimulation on the CMAP in
the L269F-transgenic mice also depended on the muscle type.
Responses evoked in gastrocnemius with repetitive stimulation at rates
from 2 to 10 Hz were of constant amplitude to at least the 10th
response in both transgenic and control. In comparison, stimulation at rates from 2 to 10 Hz while recording over the intrinsic muscles of the
hindpaws of mice from three transgenic lines elicited decremental CMAP
(Fig. 5C, D, Table 1). The mean decrement at 5 Hz
stimulation for the hindpaw was 13.6 ± 1.6% (n = 6) compared with a control decrement of 3.0 ± 0.9%
(n = 5; p < 0.001). Decremental CMAPs of 25.6% during 5 Hz stimulation were also recorded over the forelimb flexor muscles of transgenic founder 4-0 that did not breed. This
decremental response, a pattern typical of myasthenic disorders, indicates the presence of a reduced safety factor of neuromuscular transmission in these muscles. Variable involvement of different muscle
groups with prominent involvement of small muscles is also typical of
SCCMS.
Voltage-clamp analysis of endplate currents
The altered muscle responses in L269F-transgenic mice indicated
abnormalities in both kinetics and amplitudes of the MEPCs. We studied
MEPCs using the classic two-electrode voltage clamp and hemidiaphragm
preparation (Glaminovic, 1979) in lines 5 and 14. The kinetics of
channel openings of junctional AChRs in the L269F-transgenic mice
determine the time course of the spontaneous MEPCs, with the decay
phase of MEPCs depending on the mean open duration of the AChR channels
(Magleby and Stevens, 1972). MEPC decay phases in L269F-transgenic
mice were predominantly biphasic and had significantly prolonged decay
phases (Fig. 5E, F, Table 1). The first component, which
represented ~60% of the decay in lines 5 and 14, had a mean
time constant (1) of 0.81 ± 0.12 msec
(n = 105), whereas the second component, representing
~40% of the decay, had a much slower exponential course, with a mean time constant (2) of 26.6 ± 2.34 msec
(n = 105). In contrast, a biexponential decay phase
could be detected in only 4 of the 33 control fibers with a
1 of 1.23 ± 0.08 msec and a 2 of
3.47 ± 3.18 (n = 4). Whereas the transgenic
1 was slightly less than either the control
1 or the time constant of the predominantly monophasic
decays (s), 1.44 ± 0.05 msec (n = 33), the transgenic 2 was nearly eightfold longer than
the control 2. The presence of biphasic decay phases in
MEPCs with one normal component and one slow component suggests either
the presence of two populations of AChRs within the endplate or a
single population with two predominant open states.
The decremental CMAP responses to repetitive stimulation in the hindpaw
muscle of L269F-transgenic mice suggest a critical reduction in the
safety factor of neuromuscular transmission. The amplitude of endplate
currents is a principal determinant of the safety factor. We found that
the quantally evoked MEPCs recorded from diaphragm muscle of
the L269F-transgenic mice had reduced amplitude, 2.0 ± 0.18 mV, (p < 0.001; n = 105, 8 animals) compared with 2.9 ± 0.26, (n = 33, 4 animals) (Table 1). This reduction of 31% in amplitude of the
diaphragm MEPCs indicates a significant impairment in neuromuscular
transmission, but probably would not be sufficient to lead to
decremental CMAP responses in this muscle. In fact, a greater
impairment of neuromuscular transmission in the diaphragm might cause
lethal respiratory insufficiency. The foot muscles or forelimb flexors,
where the decremental CMAP responses were recorded, were not studied by
voltage-clamp analysis.
Patch-clamp
Patch-clamp analysis of ACh-induced single channels in
L269F-transgenic endplates revealed channels with prolonged open
durations. At a 50 mV holding potential, in the presence of 400 nM ACh, muscle fibers from transgenic and control mice
exhibited channel events of both short and long duration (Fig. 5,
G-J, Table 1). The short duration events were similar in
duration for the transgenic (1 = 0.52 ± 0.08 msec)
vs control (1 = 0.31 ± 0.09). The long duration events of the L269F-transgenic mice (2 = 13.2 ± 2.55, n = 6) were more than threefold longer than the
corresponding events recorded from control mice (2 = 3.85 ± 0.35; n = 4, p < 0.02). These two kinetic states, which probably represent events evoked by
single-and double-ligand occupancy of the AChRs, were similar to the
findings of patch-clamp analysis of an SCCMS patient that bears the
L269F mutation (Table 1) (Engel et al., 1996), and can account for
the prolonged decay phases of the transgenic MEPCs. There was no
difference in single-channel conductance between transgenic (76.5 ± 13.1 pS) and control endplate AChRs (75.1 ± 16 pS).
Light microscopy
In transverse sections through endplate regions, transgenic muscle
fibers demonstrated increased eosinophilic staining over either the
entire muscle fiber or in the portion closest to the endplate region,
suggesting a localized hypercontracture (Fig. 6A). This was supported by teased
fiber studies showing localized contractures at the endplate (data not
shown). These changes tended to be the principal light microscopic
changes in younger animals (2-4 months). In older transgenic animals
(>4 months) within the endplate region, there were scattered angulated
or atrophic fibers and some degenerating fibers. Fibers undergoing
splitting and fibers with increased central nuclei were common (Fig.
6C). There was intense staining for acid phosphatase at most
motor endplates and patchy acid phosphatase activity throughout many
fibers in the endplate region (Fig. 6D), indicating
increased lysosomal activity, as commonly seen in myopathies. A similar
picture was present in all muscle groups tested.
Fig. 6.
L269F-mice have segmental myopathy and
Ca2+-overloaded endplates. Serial transverse sections of
forelimb flexors of 4-month-old mouse (A, B) and of
triceps muscle of 10-month-old mouse (C, D) were stained for
cholinesterase (brown stain, hematoxylin and eosin
counterstain) to localize motor endplates (A, C), glyoxal bis (2-hydroxyanil) (GBHA, red stain, methylene blue
counterstain) to localize accumulation of ionized calcium
(B), and for acid phosphatase (red stain, hematoxlin
counterstain) to localize lysosomal activity
(D). In A, four of the muscle fibers have
increased eosinophilic staining at or near endplate regions
(arrowheads), suggesting either early degeneration or
localized contracture. In B, the adjacent section, three of
these fibers stain intensely for GBHA (red) in a focal
distribution on the periphery of the fiber, suggesting accumulation of
Ca2+ in the subsynaptic regions. In older muscle
(C) myopathic features are prevalent in the endplate
regions. The splitting fibers (arrowheads), degenerating
fibers (black arrow), atrophic fibers (white
arrow), and central nuclei (asterisk) seen here are
typical of multiple fields and demonstrate the combined neurogenic and
myopathic changes that are the classical features of the slow-channel
congenital myasthenic syndrome. The fiber splitting occurs at the
endplate regions. In D, the adjacent section, the necrotic
fiber in C stains intensely for acid phosphatase. Both
normal-appearing fibers (arrows) and splitting fibers
(arrowheads) have focal acid phosphatase staining near the
endplate regions. At the endplate region of one fiber
(asterisk) there is intense staining for acid phosphatase centrally. Calibration bar, 50 µM.
[View Larger Version of this Image (97K GIF file)]
Histochemical methods that detect deposits of ionized Ca2+
stained many endplate regions intensely in each of the four
L269F-transgenic lines (Fig. 6B). The
Ca2+ stain is deposited focally along the perimeter of the
muscle fiber, over regions that colocalize with acetylcholinesterase, indicating the presence of a neuromuscular synapse (Fig.
6A). Similar intense staining near the endplate
region using the GBHA method has been reported for the SCCMS patient
bearing the L269F mutation (Engel et al., 1982; Gomez and Gammack,
1995). In contrast to the myopathic features, which seemed to increase
with age, the frequency of endplates with GBHA staining decreased with
age.
Electron microscopy
Forelimb neuromuscular junctions of mice from two of the
transgenic lines were examined by electron microscopy (Fig.
7). Nearly every junction examined showed subsarcolemmal
vacuoles of a range of sizes (Fig. 7A, B) having the
appearance of dilated sarcoplasmic reticulum. Most of these appeared
empty, but some contained electron-dense granular material and/or
membranous debris. The vacuoles were confined entirely to the
junctional regions of the muscle fibers. Most junctions also contained
lysosomes filled with membranous debris and degenerating subsarcolemmal
organelles. Postsynaptic membrane folds appeared simplified with
shallow secondary synaptic clefts but showed no signs of degeneration.
The primary synaptic clefts were not significantly widened. Junctional
basal laminae were neither duplicated nor thickened (Fig.
7A,B).
Fig. 7.
L269F-mice have degeneration of the
neuromuscular synapse. Neuromuscular junctions from forelimb flexor
muscles of 4-month-old L269F-transgenic mice. To facilitate
orientation all nerve termini are indicated using white
arrowheads. In all three views the postsynaptic folds are
simplified, whereas the adjacent the nerve termini (white arrowheads) seem normal. A, large vacuoles containing
membranous or granular debris fill the junctional sarcoplasm. The
composition of the vacuoles can be seen more clearly in the enlargement
from the boxed section (B). The mitochondria in
the muscle fiber in A and in B (inset)
are greatly enlarged compared with those in the nerve termini.
Black arrows indicate what seem to be two degenerating mitochondria. Three mitochondria are massively enlarged and have densely packed cristae (black arrowheads). C, A
degenerating myonucleus (black arrow) filled with
autophagic debris and cytoplasmic contents lies immediately beneath a
neuromuscular junction and adjacent to a relatively normal-appearing
nucleus (white arrow). Calibration bars: A,
20 nM; B, 10 nM; C, 15 nM.
[View Larger Version of this Image (66K GIF file)]
Many of the mitochondria in the subjunctional sarcoplasm were markedly
enlarged, >10 times the size of the mitochondria in the adjacent nerve
terminus (Fig. 7B). The cristae in these giant mitochondria
stained heavily with osmium and contained minute osmiophilic granules.
Some mitochondria were frankly degenerating (Fig. 7A,B).
Degenerating nuclei with crenated membranes, condensed chromatin,
cytoplasmic inclusions, and myelinoid membranous debris were
occasionally seen (Fig. 7C). Except for the absence of an acutely degenerating postsynaptic membrane, these ultrastructural abnormalities in 4-month-old L269F-transgenic mice are identical to
the endplate myopathy described in the SCCMS (Engel et al., 1982).
DISCUSSION
The slow-channel syndrome is a prototype for a disorder of
hereditary excitotoxicity. The AChR subunit point mutation L269F is one of several mutations that have been identified in the SCCMS (Gomez and Gammack, 1995; Engel et al., 1996). In the present study we
demonstrate that the L269F mutation greatly prolongs the single
opening events using the oocyte expression system. The predominant
opening events have durations of either 19.95 or 45.44 msec, compared
with the control events of 3.63 msec. A similar study has shown that
the AChRs with the L269F mutation, expressed in human embryonic
kidney (HEK), fibroblasts contain three open time components (Engel et
al., 1996) that differ from the values that we obtained in
Xenopus oocytes. In the HEK cells a 50.6 msec time constant
seems to be similar to the 45 msec component in the oocyte. The 20 msec
component seen in oocytes, however, was not present in the HEK cells,
in which faster openings (0.04 and 0.28 msec) were the most common. The
absence of these fast components in the oocytes may be attributable to
technical reasons, such as lower recording temperature, but this result
is in accord with a recent finding that AChR channel kinetics can be
influenced by the expression system (Zanello et al., 1996).
Interestingly, the endplate AChRs in acutely dissociated muscle fibers
of one L269F patient had single-channel open times of 7.21 msec and bursts of up to 16.67 msec (at 80 mV) (Engel et al., 1996) in close
agreement with our findings.
Whereas the above in vitro findings correlate the identified
mutation with the electrophysiological findings in the SCCMS, they do
not provide a basis for the progressive myopathy and synaptic degeneration seen in this syndrome. Targeted expression in transgenic mouse muscle of L269F mRNA induced focal Ca2+ deposition
and accumulation of vacuoles, lysosomes, and degenerating organelles in
the postsynaptic region of the neuromuscular junction. The mice develop
electrical evidence of impaired neuromuscular transmission and
clinically evident fatigability of the forelimb muscles as in the
SCCMS. The kinetics of the MEPCs and single channels recorded from
L269F-transgenic muscle fibers indicate that the properties of the
AChR channels expressed in these mice resemble those of the
L269F-AChRs expressed in oocytes. These observations strongly
suggest a role for the L269F mutation in the pathogenesis of the
spectrum of abnormalities in this disorder.
The situation in the L269F-transgenic mice differs from that of the
human disease, which is autosomal dominant, in that the mutation is
overexpressed, not co-expressed to the same level with the wild-type
subunit. This may have several implications. First, because of
overexpression of the transgene, the predominant subunit species is
probably L269F, and the transgenic mice may approach a phenotype
equivalent to the human homozygous mutant state. This would explain the
more severe disease. Second, because of overexpression of the transgene
it is conceivable that the L269F subunit might displace one of the
other wild-type subunits, such as the subunit, and thereby alter
the normal AChR subunit stoichiometry. This is unlikely, because the
L269F-transgenic endplates stain intensely with the - specific
monoclonal antibody, 88B (Froehner et al., 1983) (Fig. 3), whereas they
do not stain with a peptide-specific anti- polyclonal antibody (Gu
and Hall, 1988) nor is the mRNA detected by northern blot (data not
shown). This indicates that there is abundant -subunit protein in
the transgenic endplates. Also, the channel properties of -less
channels have been studied and have much lower conductances and shorter open times than the channels measured in the transgenic muscle fibers
(Kullberg et al., 1990). Finally, we cannot rule out a role of other
transgene actions that would not be present in the SCCMS, such as a
dominant negative effect of overexpressed subunit, that alters AChR
assembly or the effect of possible splicing artifacts seen in the RNA
blot. Nevertheless, the similarity of the electrophysiological and
pathological changes seen in these mice, from whole animal to the
single channel to the changes seen in oocytes and in patients argues
that the principal properties of the mutant are reproduced in these
mice.
Altered electrophysiology
Each of the electrophysiological abnormalities in the
L269F-transgenic mice can now be ascribed to the effects of a single point mutation. Prolonged MEPC decay phases are caused by the long
duration activation events of the slow L269F-AChR channels in
oocytes and muscle fibers. The repetitive CMAP that follow single nerve
stimuli result directly from the prolonged endplate currents, because
the resultant prolonged endplate potentials outlast the preceding
muscle fiber refractory period. The decremental CMAP responses arise
from the diminished MEPC amplitudes, which cause a reduced
neuromuscular safety factor of neuromuscular transmission. The reduced
MEPCs are unrelated to the single-channel conductance of the
L269F-AChRs, because this is normal, but may have several explanations: (1) the density of AChRs may be reduced; (2) the endplate
architecture may be altered by the destructive changes occurring in the
junctional sarcoplasm; (3) the sodium driving force for the MEPCs may
be reduced by an increase in the local sodium concentration of the
junctional sarcoplasm, because of the cation overload; and (4) the
endplate AChRs may exhibit a greater tendency to desensitize, which
could reduce the responsiveness of the postsynaptic membrane.
Calcium-induced transsynaptic degeneration
Intracellular Ca2+ accumulation through glutamate and
nicotinic receptors plays a critical role in several experimental
models of excitotoxicity (Kawabuchi, 1982; Salpeter et al., 1982; Choi, 1985). The normal AChR channel is highly permeable to Ca2+
(Decker and Dani, 1990). Preliminary data suggest that L269F-AChRs have no difference in Ca2+ permeability (Gomez et al.,
unpublished observations). Although the GBHA histochemical stain shows
focal Ca2+ accumulation at the motor endplate, it does not
permit subcellular localization of the excess Ca2+ (Evans,
1974; Kawabuchi, 1982). Other approaches have demonstrated an
increase in Ca2+ in cytoplasmic,
mitochondrial, or endoplasmic reticulum in neurons after excitation
(Andrews et al., 1988; Werth and Thayer, 1994; White and Reynolds,
1995). In the L269F mice, the ultrastructural findings at the motor
endplate suggest that sarcoplasmic reticulum and mitochondria
participate in Ca2+ buffering. The myriads of vacuoles in
the junctional sarcoplasm, which are also seen in the SCCMS (Engel et
al., 1982; Gomez et al., 1996b), seem to arise in part from the
sarcoplasmic reticulum. Many are filled with electron-dense granules
suggestive of Ca2+ precipitates.
The junctional subsarcolemmal mitochondria in the L269F
transgenic mice demonstrate two types of abnormalities, suggesting a
progression of changes. Many are massively enlarged compared with the
presynaptic nerve terminal mitochondria and intermyofibrillar mitochondria. Abnormally enlarged and proliferating mitochondria are
associated with inherited defects in mitochondrial oxidative metabolism
and in aging tissue (Wilson and Franks, 1975). Because mitochondria use
oxidative metabolism to buffer large Ca2+ loads, the
hypertrophic changes in these mice may reflect the response of the
organelle to the increased energy demands. A smaller number of
mitochondria appear dilated or degenerating, as has been seen in SCCMS
(Engel et al., 1982).
In addition to enlarged and degenerating mitochondria, the junctional
sarcoplasm of L269F-transgenic mice contains degenerating nuclei,
which along with the other organellar changes, occurs in the absence of
frank degeneration of postsynaptic folds. Degenerating nuclei and
mitochondria are seen in nearly all reported cases of SCCMS, whereas
the type and degree of changes in the synaptic cleft and postsynaptic
membrane are quite variable (Engel et al., 1982; Oosterhuis et al.,
1987; Gomez et al., 1996b). Together with the findings in the
transgenic mice, this suggests that, in this complicated
ultrastructural picture, focal organellar degeneration at the
neuromuscular junction is the earliest change, and that the
abnormalities at the basement membrane and postsynaptic membrane
represent a more chronic process.
The organellar degeneration associated with prolonged synaptic currents
is most likely a consequence of transsynaptic
Ca2+-overload. It will be critical to identify the pathways
responsible for this process because they may be similar to those
involved in stroke, epilepsy, and certain neurodegenerative diseases
(Choi, 1992). Possibilities include the following: (1) intracellular damage from calcium-activated enzymes, such as calpain, DNase, or
phospholipase (Salpeter et al., 1982; Choi, 1992); (2) depletion of
energy stores in the muscle fiber because of the metabolic demands
caused by the cation overload; (3) free radical-mediated damage of
subcellular components. Excessive production of free radicals could
arise from at least two sources. First, the neuronal form of nitric
oxide synthase, which is stimulated by Ca2+, forms a tight
noncovalent linkage with the syntrophin moiety of the dystrophin
complex in the neuromuscular junction (Brenman et al., 1995). Excessive
Ca2+ entry might give rise to overproduction of nitric
oxide that could lead to oxidative damage (Dawson et al., 1991).
Second, under normal resting conditions mitochondria are the greatest cellular source of reactive oxygen species (ROS) (Beal, 1995). Their
production of ROS increases with metabolic load (Dykens, 1994). The
increase in mitochondrial size and buffering activity in the muscle
fiber would lead to a further increases in ROS production.
This animal model of a human disorder of synaptic transmission
demonstrates the connection between a mutation resulting in a kinetic
disturbance in ion channel function and a progressive degenerative
disease. Furthermore, it suggests a pathological sequence of events and
establishes a means of investigating the pathways leading to
excitotoxic synaptic degeneration that are likely to have counterparts
within other forms of excitotoxicity.
FOOTNOTES
Received Jan. 14, 1997; revised March 17, 1997; accepted March 21, 1997.
This work was supported by National Institutes of Health Clinical
Investigator Development Award K08NS01540 and Muscular Dystrophy Association Grant R01 NS33202.
Correspondence should be addressed to Dr. Christopher M. Gomez,
Department of Neurology, P.O. Box 295, University of Minnesota, 420 Delaware Street SE, Minneapolis, MN 55455.
Dr. Lasalde's present address: University of Puerto Rico, Department
of Biology, P.O. Box 23360, San Juan, PR
00931-3360.
REFERENCES
-
Andrews SB,
Leapman RD,
Landis DM,
Reese TS
(1988)
Activity-dependent accumulation of calcium in Purkinje cell dendritic spines.
Proc Natl Acad Sci USA
85:1682-1685[Abstract/Free Full Text].
-
Barka T
(1960)
A simple azo-dye method for histochemical demonstration of acid phosphatase.
Nature
187:248-249[Medline].
-
Beal MF
(1995)
Aging, energy, and oxidative stress in neurodegenerative diseases.
Ann Neurol
38:357-366[Web of Science][Medline].
-
Boulter J,
Luyten W,
Evans K,
Mason P,
Ballivet M,
Goldman D,
Stengelin S,
Martin G,
Heinemann S,
Patrick J
(1985)
Isolation of a clone coding for the alpha-subunit of a mouse acetylcholine receptor.
J Neurosci
5:2545-2452[Abstract].
-
Brehm P,
Kullberg R
(1987)
Acetylcholine receptor channels on adult mouse skeletal muscle are functionally identical in synaptic and nonsynaptic membrane.
Proc Natl Acad Sci USA
84:2550-2554[Abstract/Free Full Text].
-
Brenman JE,
Chao DS,
Xia HH,
Aldape K,
Bredt DS
(1995)
Nitric oxide synthase complexed with dystrophin and absent from skeletal muscle sarcolemma in Duchenne muscular dystrophy.
Cell
82:743-752[Web of Science][Medline].
-
Buskin JN,
Jaynes JB,
Chamberlain JS,
Hauschka SD
(1985)
The mouse muscle creatine kinase cDNA and deduced amino acid sequences: comparison to evolutionarily related enzymes.
J Mol Evol
22:334-341[Web of Science][Medline].
-
Bygrave FL,
Roberts HR
(1995)
Regulation of cellular calcium through signaling cross-talk involves an intricate interplay between the actions of receptors, G-proteins, and second messengers.
FASEB J
9:1297-1303[Abstract].
-
Choi DW
(1985)
Glutamate neurotoxicity in cortical cell culture is calcium dependent.
Neurosci Lett
58:293-297[Web of Science][Medline].
-
Choi DW
(1992)
Excitotoxic cell death.
J Neurobiol
23:1261-1276[Web of Science][Medline].
-
Chomczynski P,
Sacchi N
(1987)
Single-step method of RNA isolation by acid guanidinium thiocyanate-phenol-chloroform extraction.
Anal Biochem
162:156-159[Web of Science][Medline].
-
Dawson VL,
Dawson TM,
London ED,
Bredt DS,
Snyder SH
(1991)
Nitric oxide mediates glutamate neurotoxicity in primary cortical cultures.
Proc Natl Acad Sci USA
88:6368-6371[Abstract/Free Full Text].
-
Decker ER,
Dani JA
(1990)
Calcium permeability of the nicotinic acetylcholine receptor: the single-channel calcium influx is significant.
J Neurosci
10:3413-3420[Abstract].
-
Dykens JA
(1994)
Isolated cerebral and cerebellar mitochondria produce free radicals when exposed to elevated Ca2+ and Na+: implications for neurodegeneration.
J Neurochem
63:584-591[Web of Science][Medline].
-
Engel AG,
Lambert EH,
Mulder DM,
Torres CF,
Sahashi K,
Bertorini TE,
Whitaker JN
(1982)
A newly recognized congenital myasthenic syndrome attributed to a prolonged open time of the acetylcholine-induced ion channel.
Ann Neurol
11:553-569[Web of Science][Medline].
-
Engel AG,
Ohno K,
Milone M,
Wang HL,
Nakano S,
Bouzat C,
Pruitt JN,
Hutchinson DO,
Brengman JM,
Bren N
(1996)
New mutations in acetylcholine receptor subunit genes reveal heterogeneity in the slow-channel congenital myasthenic syndrome.
Hum Mol Genet
5:1217-1227[Abstract/Free Full Text].
-
Evans RH
(1974)
The entry of labeled calcium into the innervated region of the mouse diaphragm.
J Physiol (Lond)
240:517-533[Abstract/Free Full Text].
-
Feinberg AP,
Vogelstein B
(1984)
A technique for radiolabeling DNA restriction endonuclease fragments to high specific activity. Addendum.
Anal Biochem
137:266-267[Web of Science][Medline].
-
Froehner SC,
Douville K,
Klink S,
Culp WJ
(1983)
Monoclonal antibodies to cytoplasmic domains of the acetylcholine receptor.
J Biol Chem
258:7112-7120[Abstract/Free Full Text].
-
Gardner PD
(1990)
Nucleotide sequence of the epsilon-subunit of the mouse muscle nicotinic acetylcholine receptor.
Nucleic Acids Res
18:6714[Free Full Text].
-
Ghosh A,
Greenberg ME
(1995)
Calcium signaling in neurons: molecular mechanisms and cellular consequences.
Science
268:239-247[Abstract/Free Full Text].
-
Glaminovic MI
(1979)
Voltage clamping of unparalysed cut diaphragm for the study of transmitter release.
J Physiol (Lond)
290:467-480[Abstract/Free Full Text].
-
Gomez CM,
Richman DP
(1985)
Monoclonal anti-acetylcholine receptor antibodies with differing capacities to induce experimental autoimmune myasthenia gravis.
J Immunol
135:234-241[Abstract].
-
Gomez CM,
Gammack JT
(1995)
A leucine-to-phenylalanine substitution in the acetylcholine receptor ion channel in a family with the slow-channel syndrome.
Neurology
45:982-985[Abstract].
-
Gomez CM,
Bhattacharyya BB,
Charnet P,
Day JW,
Labarca C,
Wollmann RW,
Lambert EH
(1996a)
A transgenic mouse model of the slow-channel syndrome.
Muscle Nerve
19:79-87[Web of Science][Medline].
-
Gomez CM,
Ricardo Maselli BS,
Lasalde J,
Tamamizu S,
Cornblath DR,
Lehar M,
McNamee M,
Kuncl RW,
Pestronk A
(1996b)
A subunit mutation in the acetylcholine receptor channel gate causes severe slow-channel syndrome.
Ann Neurol
39:717-723.
-
Gorman CM,
Moffat LF,
Howard BH
(1982)
Recombinant genomes which express chloramphenicol acetyltransferase in mammalian cells.
Mol Cell Biol
2:1044-1051[Abstract/Free Full Text].
-
Gu Y,
Hall ZW
(1988)
Immunological evidence for a change in subunits of the acetylcholine receptor in developing and denervated rat muscle.
Neuron
1:117-125[Web of Science][Medline].
-
Hogan R,
Costantini F,
Lacey E
(1986)
In: Manipulating the mouse embryo: a laboratory manual. Cold Spring Harbor, NY: Cold Spring Harbor Laboratory.
-
Johnson JE,
Wold BJ,
Hauschka SD
(1989)
Muscle creatine kinase sequence elements regulating skeletal and cardiac muscle expression in transgenic mice.
Mol Cell Biol
9:3393-3399[Abstract/Free Full Text].
-
Kashiwa HK
(1970)
Calcium phosphate in osteogenic cells. A critique of the glyoxal bis(2-hydroxyanil) and the dilute silver acetate methods.
Clin Orthop Rel Res
70:200-211.[Medline]
-
Kawabuchi M
(1982)
Neostigmine myopathy is a calcium ion-mediated myopathy initially affecting the motor end-plate.
J Neuropathol Exp Neurol
41:298-314[Web of Science][Medline].
-
Kullberg R,
Owens JL,
Camacho P,
Mandel G,
Brehm P
(1990)
Multiple conductance classes of mouse nicotinic acetylcholine receptors expressed in Xenopus oocytes.
Proc Natl Acad Sci USA
87:2067-2071[Abstract/Free Full Text].
-
Lapolla RJ,
Mayne KM,
Davidson N
(1984)
Mouse muscle nicotinic acetylcholine receptor subunit: cDNA sequence and gene expression.
Proc Natl Acad Sci USA
81:7970-7974[Abstract/Free Full Text].
-
Magleby KL,
Stevens CF
(1972)
A quantitative description of end-plate currents.
J Physiol (Lond)
233:173-197.
-
Maselli R,
Mass D,
Distad B,
Richman D
(1991)
Anconeus muscle: a human preparation suitable for in vitro microelectrode studies.
Muscle Nerve
14:1189-1192[Web of Science][Medline].
-
Maselli R,
Nelson D,
Richman D
(1989)
Effects of a monoclonal anti-acetylcholine receptor antibody on the avian end-plate.
J Physiol (Lond)
411:271-283[Abstract/Free Full Text].
-
Merlie JP,
Sanes JR
(1985)
Concentration of acetylcholine receptor mRNA in synaptic regions of adult muscle fibres.
Nature
317:66-68[Medline].
-
Mishina M,
Takai T,
Imoto K,
Noda M,
Takahashi T,
Numa S,
Methfessel C,
Sakmann B
(1986)
Molecular distinction between fetal and adult forms of muscle acetylcholine receptor.
Nature
321:406-411[Medline].
-
Namba T,
Nakamura T,
Grob D
(1967)
Staining for nerve fiber and cholinesterase activity in fresh frozen sections.
Am J Clin Pathol
7:74-77.
-
Nelson S,
Shelton GD,
Lei S,
Lindstrom JM,
Conti TB
(1992)
Epitope mapping of monoclona antibodies to Torpedo acetylcholine receptor gamma subunits, which specifically recognize the epsilon subunit of mammalian muscle acetylcholine receptor.
J Neuroimmunol
36:13-27[Web of Science][Medline].
-
Ohno K,
Hutchinson D,
Milone M,
Brengman J,
Bouzat C,
Sine S,
Engel A
(1995)
Congenital myasthenic syndrome caused by prolonged acetylcholine receptor channel openings due to a mutation in the M2 domain of the subunit.
Proc Natl Acad Sci USA
92:758-762[Abstract/Free Full Text].
-
Oosterhuis HJ,
Newsom-Davis J,
Wokke JH,
Molenaar PC,
Weerden TV,
Oen BS,
Jennekens FG,
Veldman H,
Vincent A,
Wray DW
(1987)
The slow channel syndrome: two new cases.
Brain
110:1061-1079[Abstract/Free Full Text].
-
Ophoff RA,
Terwindt GM,
Vergouwe MN,
Vaneijk R,
Oefner PJ,
Hoffman S,
Lamerdin JE,
Mohrenweiser HW,
Bulman DE,
Ferrari M
(1996)
Familial hemiplegic migraine and episodic ataxia type-2 are caused by mutations in the Ca2+ channel gene cacnl1a4.
Cell
87:543-552[Web of Science][Medline].
-
Rungby J,
Kassem M,
Eriksen EF,
Danscher G
(1993)
The von Kossa reaction for calcium deposits: silver lactate staining increases sensitivity and reduces background.
Histochem J
25:446-451[Web of Science][Medline].
-
Saiki RK,
Gelfand DH,
Stoffel S,
Scharf SJ,
Higuchi R,
Horn GT,
Mullis KB,
Erlich HA
(1988)
Primer-directed enzymatic amplification of DNA with a thermostable DNA polymerase.
Science
239:487-491[Abstract/Free Full Text].
-
Salpeter MM,
Leonard JP,
Kasprzak H
(1982)
Agonist-induced postsynaptic myopathy.
Neurosci Comment
1:73-83.
-
Sambrook J,
Fritsch FE,
Maniatis T
(1989)
In: Molecular cloning: a laboratory manual, 2nd Ed. Cold Spring Harbor, NY: Cold Spring Harbor.
-
Shiang R,
Ryan SG,
Zhu YZ,
Hahn AF,
O'Connell P,
Wasmuth JJ
(1993)
Mutations in the alpha 1 subunit of the inhibitory glycine receptor cause the dominant neurologic disorder, hyperekplexia.
Nat Genet
5:351-358[Web of Science][Medline].
-
Sine SM,
Ohno K,
Bouzat C,
Auerbach A,
Milone M,
Pruitt JN,
Engel AG
(1995)
Mutation of the acetylcholine receptor a subunit causes a slow-channel myasthenic syndrome by enhancing agonist binding affinity.
Neuron
15:229-239[Web of Science][Medline].
-
Steinlein OK,
Mulley JC,
Propping P,
Wallace RH,
Phillips HA,
Sutherland GR,
Scheffer IE,
Berkovic SF
(1995)
A missense mutation in the neuronal nicotinic acetylcholine receptor alpha 4 subunit is associated with autosomal dominant nocturnal frontal lobe epilepsy.
Nat Genet
11:201-203[Web of Science][Medline].
-
Treinin M,
Chalfie M
(1995)
A mutated acetylcholine receptor subunit causes neuronal degeneration in C. elegans.
Neuron
14:1-20[Web of Science][Medline].
-
Tzartos SJ,
Lindstrom JM
(1980)
Monoclonal antibodies used to probe acetylcholine receptor structure: localization of the main immunogenic region and detection of similarities between subunits.
Proc Natl Acad Sci USA
77:755-759[Abstract/Free Full Text].
-
Werth JL,
Thayer SA
(1994)
Mitochondria buffer physiological calcium loads in cultured rat dorsal root ganglion neurons.
J Neurosci
14:348-356[Abstract].
-
White RJ,
Reynolds IJ
(1995)
Mitochondria and Na+/Ca2+ exchange buffer glutamate-induced calcium loads in cultured cortical neurons.
J Neurosci
15:1318-1328[Abstract].
-
Wilson P,
Franks L
(1975)
The effect of age on mitochondrial ultrastructure and enzymes.
Adv Exp Med Biol
53:171-183[Medline].
-
Zanello LP,
Aztiria E,
Antonelli S,
Barrantes FJ
(1996)
Nicotinic acetylcholine receptors channels are influenced by the physical state of their membrane environment.
Biophys J
70:2155-2164[Web of Science][Medline].
This article has been cited by other articles:
|
|
|
|
 
S. Elenes, M. Decker, G. D. Cymes, and C. Grosman
Decremental Response to High-Frequency Trains of Acetylcholine Pulses but Unaltered Fractional Ca2+ Currents in a Panel of "Slow-Channel Syndrome" Nicotinic Receptor Mutants
J. Gen. Physiol.,
February 1, 2009;
133(2):
151 - 169.
[Abstract]
[Full Text]
[PDF]
|
|
|
|
|
|
|
D. M. Jones-Davis, L. Song, M. J. Gallagher, and R. L. Macdonald
Structural Determinants of Benzodiazepine Allosteric Regulation of GABAA Receptor Currents
J. Neurosci.,
August 31, 2005;
25(35):
8056 - 8065.
[Abstract]
[Full Text]
[PDF]
|
|
|
|
|
|
|
J. L. Lefebvre, F. Ono, C. Puglielli, G. Seidner, C. Franzini-Armstrong, P. Brehm, and M. Granato
Increased neuromuscular activity causes axonal defects and muscular degeneration
Development,
June 1, 2004;
131(11):
2605 - 2618.
[Abstract]
[Full Text]
[PDF]
|
|
|
|
|
|
|
C. M. Gomez, R. A. Maselli, J. Groshong, R. Zayas, R. L. Wollmann, T. Cens, and P. Charnet
Active Calcium Accumulation Underlies Severe Weakness in a Panel of Mice with Slow-Channel Syndrome
J. Neurosci.,
August 1, 2002;
22(15):
6447 - 6457.
[Abstract]
[Full Text]
[PDF]
|
|
|
|
|
|
|
M. T. Bianchi, L. Song, H. Zhang, and R. L. Macdonald
Two Different Mechanisms of Disinhibition Produced by GABAA Receptor Mutations Linked to Epilepsy in Humans
J. Neurosci.,
July 1, 2002;
22(13):
5321 - 5327.
[Abstract]
[Full Text]
[PDF]
|
|
|
|
|
|
|
S. Restituito, R. M. Thompson, J. Eliet, R. S. Raike, M. Riedl, P. Charnet, and C. M. Gomez
The Polyglutamine Expansion in Spinocerebellar Ataxia Type 6 Causes a beta Subunit-Specific Enhanced Activation of P/Q-Type Calcium Channels in Xenopus Oocytes
J. Neurosci.,
September 1, 2000;
20(17):
6394 - 6403.
[Abstract]
[Full Text]
[PDF]
|
|
|
|
