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The Journal of Neuroscience, August 1, 2002, 22(15):6447-6457
Active Calcium Accumulation Underlies Severe Weakness in a Panel
of Mice with Slow-Channel Syndrome
Christopher M.
Gomez1,
Ricardo A.
Maselli2,
Jason
Groshong1,
Roberto
Zayas1,
Robert L.
Wollmann3,
Thierry
Cens4, and
Pierre
Charnet4
1 Departments of Neurology and Neuroscience,
University of Minnesota, Minneapolis, Minnesota 55455, 2 Section of Neuroscience, University of California, Davis,
California 95616, 3 Section of Neuropathology, University
of Chicago, Chicago, Illinois 60637, and 4 Centre de
Recherches de Biochimie Macromoléculaire (Centre National de la
Recherche Scientifique Unité Propre de Recherche 1086),
Montpellier, France
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ABSTRACT |
Mutations affecting the gating and channel properties of ionotropic
neurotransmitter receptors in some hereditary epilepsies, in familial
hyperekplexia, and the slow-channel congenital myasthenic syndrome
(SCCMS) may perturb the kinetics of synaptic currents, leading to
significant clinical consequences. Although at least 12 acetylcholine
receptor (AChR) mutations have been identified in the SCCMS, the
altered channel properties critical for disease pathogenesis in the
SCCMS have not been identified. To approach this question, we
investigated the effect of different AChR subunit mutations on muscle
weakness and the function and viability of neuromuscular synapses in
transgenic mice. Targeted expression of distinct mutant AChR subunits
in skeletal muscle prolonged the decay phases of the miniature endplate
currents (MEPCs) over a broad range. In addition, both muscle strength
and the amplitude of MEPCs were lower in transgenic lines with
greater MEPC duration. SCCMS is associated with calcium overload of the
neuromuscular junctional sarcoplasm. We found that the extent of
calcium overload of motor endplates in the panel of transgenic mice was
influenced by the relative permeability of the mutant AChRs to calcium,
on the duration of MEPCs, and on neuromuscular activity. Finally, severe degenerative changes at the motor endplate (endplate myopathy) were apparent by electron microscopy in transgenic lines that displayed
the greatest activity-dependent calcium overload. These studies
demonstrate the importance of control of the kinetics of AChR channel
gating for the function and viability of the neuromuscular junction.
Key words:
synaptic currents; kinetics; degeneration; calcium; mutation; neuromuscular junction
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INTRODUCTION |
The importance of the gating
properties of the muscle and neuronal nicotinic acetylcholine receptor
(AChR) ion channels has recently been highlighted by the description of
several kinetic disorders of the AChR that comprise prominent subgroups
of both the epilepsies and congenital myasthenic syndromes (Engel,
1994 ; Lerche et al., 2001; Steinlein, 2001; Muley and Gomez, 2002). The
slow-channel congenital myasthenic syndrome (SCCMS) is characterized by
weakness, impaired neuromuscular transmission, AChR loss, and progressive degeneration of the neuromuscular junction (NMJ)
(endplate myopathy) associated with different missense mutations in the muscle AChR subunits. Although the pathogenesis of the SCCMS has not
been fully explained, the common effect of point mutations in the SCCMS
is to prolong AChR activation events and "slow" the channel closure
rate. In neuromuscular preparations from SCCMS patients, endplate
currents are prolonged 4- to >10-fold. Because the effect of prolonged
AChR activation events is to increase the entry of
Ca2+ and Na+
into the junctional sarcoplasm through the endplate AChRs, it has been
suggested that the disease results from a disturbance of intracellular
ionic millieu. Ca2+ overload may have
direct effects on AChR function (Siara et al., 1990) and may lead to
focal muscle contractures or activation of calcium-activated proteases,
DNases, and phospholipases resulting in subsynaptic vacuoles,
degenerating junctional nuclei and mitochondria, and postsynaptic folds
seen in the endplate myopathy (Engel et al., 1982; Choi, 1994).
Elucidation of the pathogenesis of synaptic dysfunction in SCCMS will
improve our understanding of kinetic disorders of the AChR, such as
SCCMS, epilepsies, and other ion channelopathies.
Both the clinicopathological findings (severity of weakness and
endplate myopathy) and the magnitude of change in synaptic currents in
SCCMS vary significantly among individuals bearing different AChR
mutations (Engel et al., 1996; Gomez et al., 1996b, 2002; Croxen et
al., 1997). The  subunit mutation, L269F, has been identified in
patients from at least two SCCMS kindreds and is associated with
moderate to severe weakness (Gomez and Gammack, 1995; Engel et al.,
1996). When expressed in vitro, this mutation causes a
9-fold increase in affinity for ACh and a 40-fold increase in AChR
channel burst duration (Engel et al., 1996; Gomez et al., 1997).
Single-channel currents recorded in L269F patient endplates have
burst durations similar to those recorded in endplates from SCCMS
patients bearing other AChR mutations, but there is poor correlation
between disease severity and the single AChR channel properties studied
(Engel et al., 1996; Gomez et al., 2002). As with patients, transgenic
mice expressing the L269F mutation are weak, have prolonged synaptic
currents, increased single channel open times,
Ca2+ overload of endplate regions, and
endplate myopathy (Gomez et al., 1997). Other SCCMS patients appear to
have little or no endplate myopathy or severely perturbed ion channel
kinetics, or both (Croxen et al., 1997; Gomez et al., 2002). Although
the relationship is not yet clear, it is tempting to speculate that
differences in kinetic properties of mutant AChRs in SCCMS are
responsible for the different clinical and pathological picture. The
determination of which critical changes in channel properties control
the function and integrity of the neuromuscular synapse may have
far-reaching implications for the diagnosis and treatment of ion
channel disorders. In this study we used transgenic mice expressing
different AChR subunit mutations to explore the relationship between
the kinetics of endplate currents and weakness, impaired neuromuscular
transmission, Ca2+ overload, and the
degenerative changes at the synapse. We show that weakness, diminished
miniature endplate current (MEPC) amplitudes, and endplate degeneration
occur with increasing duration of endplate currents and synaptic
Ca2+ overload.
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MATERIALS AND METHODS |
Oocyte studies. Characterization of AChR properties
in Xenopus oocytes was performed as described previously
(Charnet et al., 1992; Gomez et al., 1996a). Briefly, oocytes were
injected with cRNA synthesized from linearized plasmids (pAlter,
Promega, Madison WI) containing mutant or wild-type AChR subunit cDNAs
using T7 polymerase and an in vitro transcription kit
(mMessage mMachine, Ambion, Austin, TX). Fifty nanoliters of mRNA of
mouse AChR subunit (at a concentration of 0.4 ng/nl) were injected into
the cytoplasm of oocytes at an  / / / ratio of 2:1:1:1. After
3 d, oocytes were placed individually in a 50 µl recording
chamber. For recording, oocytes were kept under continuous
gravity-driven perfusion. Signals were amplified using a Geneclamp 500 amplifier (Axon Instruments, Foster City, CA). Voltage-command,
sampling, acquisition, and analysis were performed using a Digidata
1200 and the pClamp program (version 6.01, Axon Instruments). All
experiments were performed at room temperature (20-25°C).
For macroscopic whole-cell recordings, voltage clamp of oocytes was
performed using 3 M KCl-filled electrodes of 1-2 M
resistance. Junction potentials (typically <3-5 mV) were cancelled,
and contaminating endogenous
Ca2+-activated
Cl currents were suppressed by injecting
BAPTA (in mM: BAPTA 100, HEPES 10, CsOH 10, pH 7.2) into
oocytes by means of a third microelectrode and a pneumatic injector.
The final intra-oocyte BAPTA concentration was estimated to be 2-5
mM. Current recording and analysis were performed using
pClamp software.
The Ca2+/Na+
permeability ratio for each mutant used to generate transgenic mice was
measured by determining the change in reversal potential between
Ca2+- and
Na+- containing solutions. True
ACh-induced currents were obtained by digital subtraction of the
currents recorded during voltage ramps of  80 to +50 mV, both before
and after ACh perfusion (ACh = 0.1-40 µM). Reversal
potentials (Erev) were measured in
solutions of the following composition (in mM):
NaCl 100, HEPES 10, pH 7.2 with NaOH, or Ca50
(Ca2+Cl2 50, mannitol 50, HEPES 10, pH 7.2 with CaOH. Permeability ratios
(PCa/PNa)
were calculated as described (Mayer and Westbrook, 1987; Bertrand et
al., 1993). Computations were done by the constant field theory using
Goldman-Hodgkin-Katz equations for Na+,
K+, and Ca2+
permeabilities, yielding relative permeabilities. The activity coefficient for Ca2+ was 0.33, and
calculations were performed using the Solver tool from Excel
(Microsoft, Redmond, WA).
Single-channel recordings. Single-channel recordings were
performed on both cell-attached and outside-out patches at room temperature (22-24°C) using the same extracellular and intrapipette solutions (100 mM KCl, 2 mM
MgCl2, 10 mM HEPES, 10 mM EGTA, pH adjusted to 7.4 with KOH). In this
solution, the oocyte resting membrane potential was close to 0 mV. ACh
(2 µM) was included either in the bath solution
(outside-out recordings) or in the pipette solution (cell-attached
recordings). Pipettes made from KG33 borosilicate glass (Garner Glass,
Claremont, CA), treated with Sylgard, and fire polished had a
resistance of 10-15 M. Data were recorded with a Geneclamp500
amplifier (Axon Instruments; 100 G-CV5 headstage), using Fetchex
version 6.02 (Axon Instruments) in event-driven mode and digitized by a
Labmaster interface. Single-channel open time and conductance were
determined at different pipette voltages from 150 to +150 mV.
Single-channel open times were measured from the time constant of
exponential decays fitted to duration distributions of channel opening.
Single-channel conductances were determined by fitting Gaussians to
amplitude histograms.
AChR mutations and transgene constructs. We established
previously that overexpression of a mutant AChR subunit in mouse muscle gives rise to mice with prolonged endplate currents (Gomez et al.,
1996a, 1997). To develop a panel of transgenic mice with endplate
responses that are prolonged over a wide range, we constructed transgenes from a series of distinct AChR subunit mutations.
Site-directed mutagenesis of mouse , , and subunit cDNAs
(Lapolla et al., 1984; Boulter et al., 1985; Gardner, 1990) was
performed using mutagenic oligonucleotide primers and single-stranded
plasmid generated from the vector, pAlter, as per manufacturer's
instructions (Promega). The mutations were confirmed by
dideoxynucleotide sequence determination. The subunit mutation,
L269F, has been found in two families with moderately severe SCCMS
(Gomez and Gammack, 1995; Engel et al., 1996). Overexpression of this
mutation gave rise to several lines of mice that were found to develop
progressive weakness and degeneration of the postsynaptic membrane, as
is seen in the SCCMS (Gomez et al., 1997, 1998). The subunit
mutation, S262T, which is not known to be a spontaneously occurring
mutation, was generated during structure-function studies of the M2
domain (Charnet et al., 1992). The kinetic properties of S262T-AchRs and L269F-AChRs in vitro and in vivo have been
described previously (Engel et al., 1996; Gomez et al., 1996a, 1997).
The subunit mutations, L251T and C418W, were generated for
this study but have been used by others in structure-function studies
of the M2 (L251T) and M4 (C418W) domains (Filatov and White,
1995; Tamamizu et al., 1999). The properties of the AChRs expressing each of these mutations are described in Table 1.
The structure of the transgenes used to generate mice expressing each
of these mutations was identical to those described previously (Gomez
et al., 1996a, 1997). Each construct consisted of a minigene containing
3.3 kb of the promoter region from the mouse muscle creatine
phosphokinase gene (Johnson et al., 1989) joined to the 5' end of the
mutant AChR subunit cDNA, which in turn is joined to the 5' end of the
3' UTR of neo and the SV 40 small-t intron from the construct pRSVNEO
(Gorman et al., 1982).
Generation and characterization of slow-channel transgenic
mice. Transgenic mice were generated by pronuclear injection
followed by implantation into pseudopregnant females as described
previously (Gomez et al., 1996a, 1997). Transgenic mice were derived on
the FVB background (Taketo et al., 1991) with the exception of mice expressing S262T, which were made on the C57BL/6 × DBA/2 F2
background (Gomez et al., 1996a). For each transgene, two to four
founder lines, identified by analysis of tail DNA, were characterized initially. The expression levels of the transgenes were determined by
RNA blotting using skeletal muscle total RNA separated by gel electrophoresis (Gomez et al., 1997). Blots were hybridized with labeled AChR subunit cDNA probes and, for standardization of levels among lines of mice expressing different mutations, to a universal transgene-specific probe consisting of a 1000 bp 3' fragment from the
common tail sequence.
Characterization of neuromuscular synaptic currents. MEPCs
were recorded using the two-electrode voltage-clamp method as described previously (Maselli et al., 1989, 1991; Gomez et al., 1997). All microelectrode recordings were performed in the most posterior aspect
of the left hemidiaphragm muscle. Muscle fibers were viewed using an
upright microscope (Leitz Laborlux 12, Leitz Ltd., Heerbrugg, Switzerland) equipped with Hoffman interference contrast optics (magnification 250×). The preparations were bathed continuously in
Tyrode's solution with the following ionic composition (in mM): NaCl 140, KCl 2.6, MgCl2 0.4, CaCl2 2.5, NaH2PO4 0.4, pH 7.4),
bubbled with a 95% O2, 5%
CO2 gas mixture at 26°C. Voltage and current
electrodes were connected to an Axoclamp 2A amplifier (Axon
Instruments), and the signals were sampled at 10 kHz by a 12-bit
analog-to-digital converter (Data Translation 2818, Marlboro, MA).
Exponential fits to the MEPC decays were performed using a nonlinear
least square fitting routine. Because endplate currents in transgenic
animals might reflect the activity of mixed populations of mutant and
wild-type AChRs, leading to complex decay phases, both exponential
decay time constants and the areas under the MEPCs were compared for
each transgenic line. Grouped mean and SE were determined by the method
of weighted least squares (Weisberg, 1985). The mean area under the
MEPC is an estimate of the total amount of charge passing during
receptor activation. Areas for each MEPC were obtained using a graphics
program (Photoshop, Adobe, San Jose, CA). An average of 13 MEPCs were
used to estimate the mean MEPC area for each transgene line.
Detection and quantitation of Ca2+-overloaded
endplates. Mice were killed by anesthetic overdose either at rest
or after an exercise protocol (see below). The number of
Ca2+-overloaded neuromuscular junctions
throughout the muscles of each transgenic line was estimated using a
histological stain for motor endplates and accumulated
Ca2+ on adjacent sections.
Ca2+ accumulation (ionized
Ca2+) at endplates was detected using the
glyoxal bis 2 hydroxyanil (GBHA) method (Kashiwa, 1970; Evans, 1974;
Bodensteiner and Engel, 1978). Motor endplates were localized using the
stain for endplate-specific enzyme acetylcholinesterase (AChE) (Namba
et al., 1967). Serial 8 µM frozen sections were taken
from seven muscle groups, the muscular diaphragm, gastrocnemius,
tibialis anterior, biceps, triceps, and the flexors and extensors of
the forepaw. To facilitate counting and allow simultaneous labeling and
examination of sections from each of these muscle groups, adjacent
serial sections from each muscle group for each animal were arranged
together on glass microscope slides. To prevent experimenter bias, the
identities for each mouse were encoded. To estimate the number of motor
endplates in these muscle sections, the first and third of three serial slides were stained for AChE. The brown-stained endplate regions in the
seven sections of each first and third slide were counted by direct
visualization, and the average number of endplates for the two slides
was taken as the number of endplates present on the second slide. An
average of 640 endplates was counted for each mouse. Slides with great
differences in the endplate number were recounted and checked for loss
of sections. Cholinesterase-reactive regions in muscle spindles, which
were easily distinguished from normal muscle fibers, were not counted.
To estimate the fraction of endplates that have become overloaded with
Ca2+, the second slide of each series of
three was stained using the GBHA procedure, and the number of
red-black-stained, Ca2+-overloaded
endplates was expressed as the ratio of the total number of
cholinesterase-labeled endplates that were stained with GBHA.
Electron microscopy. For ultrastructural studies, mice were
perfused transcardially with 2% glutaraldehyde. Forelimb flexor muscles were then removed and fixed overnight in fresh glutaraldehyde and rinsed in 0.1 M phosphate buffer. The muscle
bellies were teased into 1 mm bundles, trimmed to lengths of 3 mm,
osmicated, and embedded in Epon. Regions containing endplates were
localized in toluidine blue-stained thick sections, and thin sections
were obtained and viewed as described previously (Gomez et al., 1984, 1997).
Exercise and measurement of strength. Muscle strength was
assessed using a wire-hang test (Gomez et al., 1997), in which the ability of mice to grip and remain suspended from a wire was expressed as the mean of five trials. Briefly, mice were allowed to grip a taut
wire suspended at a height of 35 cm by their front claws and were
observed for periods of up to 60 sec. The possible outcomes consisted
of escape or persistence on the wire for the entire period (score of
100%) or falling off before 1 min was reached (scored as the fraction
of a minute). Five sets of three repetitions were conducted. Mice were
tested with encoded identities and using littermate controls.
To increase neuromuscular synaptic activity, mice were subjected to a
30 min period of exercise that consisted of gently pulling the tail
while the mouse gripped the bars of the cage top with forelimb and
hindlimb claws. This led to a uniform routine of gentle exercise for
limb muscle.
Quantitation of AChRs. The number of AChRs per endplate was
estimated by determining the number of bungarotoxin (-BT)
binding sites per cholinesterase-stained endplate region in teased
muscle fibers segments using 125I--BT
(Pestronk et al., 1985). Mice aged 16-21 months were killed by
anesthetic overdose. The brachioradialis of the right forelimb was
excised close to the origin. The excised muscle was (1) incubated at
37°C for 4 hr in Ham's F-12 culture medium containing 0.15 µg/ml
125I--BT (specific activity 150-300
Ci/mmol), (2) rinsed 12 times at 5 min intervals with HBSS containing
0.5% BSA (fraction V), buffered to pH 7.2 with HEPES, (3) incubated at
4°C overnight, (4) washed five times at 5 min intervals, (5) pinned
at resting length for fixation with 2.5% glutaraldehyde for 30 min,
and (6) washed three times at 15 min intervals with 0.1 M sodium acetate. The labeled, washed, and fixed
muscle was gently teased to spread apart the muscle fibers and was
stained for AChE to localize endplate regions (Namba et al., 1967).
Sections of identical size with and without endplate regions were
trimmed from each muscle belly. The number of endplates in the small
bundles and the radioactivity were counted, and the specific
125I--BT-binding to the endplates was
determined by subtracting the background activity of the adjacent,
endplate-free bundles.
Statistics. All statistical comparisons for
single measurements were made using the Student's t test.
Because strength testing and MEPC recordings were made on different
animals in unpaired mouse groups for each transgene, a Matlab (The
Mathworks, Natick, MA) routine was used to estimate a normally
distributed set of paired variables to make possible a regression
analysis. For this purpose, a weighted average of the normalized SDs
(SD/mean) was made for the measurements for each transgenic mouse line.
A normally distributed set of data points about the mean for each
x-y coordinate pair was calculated. It was
assumed that the points were uniformly distributed in direction and
normally distributed in distance from the mean.
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RESULTS |
In vitro characterization of mouse of AChR
subunit mutations
To generate lines of transgenic mice with differing synaptic
current properties, we chose AChR subunit mutations predicted to affect
ion channel kinetics over a wide range. The mean single-channel open
time and conductance recorded from expression studies in Xenopus oocytes for the four mutations used in this study
are compared with those of wild-type AChRs in Table
1 and Figure 1. The duration of the channel openings
ranges from 0.8 msec in wild type to 150 msec in AChRs expressing the
L251T mutation. The single-channel conductance, determined from the
current-voltage relationship with K+ as
the predominant charge-carrying ion, varies little for the mutant AChRs
(72-83 pS) (Table 1, Fig. 1B). The
Ca2+/Na+
relative permeabilities of the mutant AChRs are more variable than are
the monovalent cation conductances, as shown on Figure 1C.
The apparent increase in Ca2+ permeability
for L251T over wild type, i.e., 0.75 versus 0.61, for example, may
relate to the addition of a polar residue in the ion permeation pathway
(Imoto et al., 1991). On the other hand, the critical effect on
Ca2+ permeability of the threonine at
position 6' in the M2 domain in the S262T mutation suggests a
previously unexpected effect of pore size on divalent cation
conductance. Permeability to Ba2+ was
similarly reduced for S262T (data not shown). Thus, this panel of
four AChR mutations with open times that vary >100-fold over that of
wild type had a fourfold difference in relative
Ca2+/Na+
permeability and displayed minimal changes in conductance, allowing us
to generate a panel of transgenic mice with widely differing synaptic
currents.
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Figure 1.
In vitro kinetic and permeation
properties of a panel of mutant mouse AChRs used in transgenic mice.
A, Single-channel recordings (left) and
open duration histograms (right) for mouse wild-type and
AChR mutants expressed in Xenopus oocytes. Recordings
were performed at 100 mV and 2.0 µM ACh. Marquardt
least squares fitting in pClamp 6 allowed resolution of two open
components for each mutant (Table 1). B, Single-channel
conductance obtained from oocyte patch-clamp recordings. There is no
significant difference between the conductance for sodium among
any of the mutants. C, Permeability of wild-type and
mutant AChRs to calcium relative to sodium. The relative calcium
permeability of L251T (n = 17) AChRs is ~33%
greater than wild-type AChRs (n = 19;
p < 0.005). The relative calcium permeability of
S262T AChRs (n = 20) is ~67% less than wild
type (p < 0.001).
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A panel of mutant AChR subunit transgenes perturbs synaptic
currents over a wide range
Using the mutant AChR subunit transgene constructs, we established
at least three founder lines for each transgene (Gomez et al., 1996a,
1997). The levels of expression of the mutant AChR subunit transgenes
in skeletal muscle varied over a wide range, but at least two founder
lines for each transgene had high levels of transgene expression. We
selected lines with matched levels of high expression by hybridizing
RNA blots with a common sequence probe present in all AChR subunit
transgenes (Fig. 2A).
To compare the levels of expression of the transgenes with those of
endogenous AChR subunits, we hybridized RNA blots with a probe
generated from subunit cDNA (Fig. 2B). As noted
previously, the levels of transgene expression greatly exceed those of
endogenous AChR subunits (Merlie and Sanes, 1985; Gomez et al., 1996a).
Although this degree of overexpression should lead, in theory, to
complete substitution of the wild-type with mutant subunits
(Bhattacharyya et al., 1997; Gomez et al., 1997), it is difficult to
estimate the effect of the localized, subsynaptic expression of
endogenous AChR subunits (Merlie and Sanes, 1985; Brenner et al.,
1990).
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Figure 2.
Analysis of transgene mRNA expression. Expression
of the mutant AChR mRNA in the panel of transgenic mice was comparable
between several lines and well above that of endogenous mouse AChR
subunit genes. A, Matched expression of AChR subunit
transgenes. Top panel, Ten micrograms of total muscle
RNA per mouse were hybridized with common sequence probe for all
transgenes. Exposure was 4 d. Bottom panel, Same
blot rehybridized with MCK cDNA to ensure uniform loading. Exposure was
4 hr. Lane 1, S262T, line 40; lane 2,
C418W, line 42; lane 3, L251T, line 39;
lane 4, L251T, line 36; lane 5,
L269F, line 14; lane 6, L269F, line 5; lane
7, control muscle. B, Overexpression of mutant
subunits. Top panel, Total muscle RNA probed with
mouse subunit cDNA. Exposure was 5 d. Endogenous subunit
mRNA is undetectable at this explosure time. Lane 1,
C418W, line 42; lane 2, L251T, line 39;
lane 3, L251T, line 36; lane 4,
control muscle. Bottom panel, Same blot reprobed with
MCK cDNA. Exposure was 4 hr. Size markers are in
kilobases.
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The duration of the MEPCs in mice expressing each mutant was
significantly increased over control mice for each transgenic line,
presumably reflecting the effects of the mutant AChRs on channel open
time recorded in vitro (Table 1). The
 s and 2 time
constants increased successively in transgenic lines S262T, C418W, L251T, and L269F. Figure
3 compares the typical traces from
control and transgenic mice expressing each mutation. In the C418W,
L251T, and L269F transgenic mice, the decay phases could be
resolved well into two exponents (1 and
2), with 2 proportions of ~43, 17, and 45%, respectively. Based on the time constants of decay of wild-type MEPCs and single-channel data (Gomez et
al., 1996a, 1997; Bhattacharyya et al., 1997), the
1 may not necessarily reflect the open
duration of a population of wild-type AChRs (Table 1). Nevertheless, in
several cases the 2 values, which varied
nearly 20-fold over wild-type s, are close to
the long opening component of the corresponding mutant AChRs expressed
in oocytes. The slow component, 2, of the MEPC decay phase in L251T mice, although increased to 12.4 msec, is smaller than the 2 component of other lines of
slow-channel mice and does not reflect the presence of the very long
opening events (150 msec) resolved in oocyte expression studies. This
may arise from the effect of coexpression of wild-type subunits in
muscle or a stabilizing effect of the mutant AChRs when associated with the subsynaptic apparatus compared with the oocyte membrane. Similar disparities have been seen between expression studies of recombinant AChRs bearing SCCMS mutations and single-channel studies using dissociated muscle fibers bearing the same mutation (Engel et al.,
1996; Gomez et al., 1997). The value for MEPC area, which has the units
of picocoulombs, is an estimate of amount of charge entering during
synaptic activity for each transgenic line (Table 1). The mean MEPC
area was significantly greater for C418W and L269F mice than for
wild type (p < 0.01). The effect of increases in MEPC duration on MEPC area in L251T mice was perhaps offset by
the reduced MEPC amplitudes and low proportion of slow MEPC decay
phase.
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Figure 3.
Miniature endplate current traces from individual
diaphragm muscle fibers of wild type (A),
S262T (B), C418W (C),
L251T (D), and L269F
(E). The prolonged and prominent bi-exponential
decay phases correlate with the open times of the mutant channels
expressed in the mice (Table 1). A, Average rise,
0.42 ± 0.47 msec; s, 1.34 msec;
amplitude (Amp), 4.15 ± 1.16 nA. B, Average
rise, 0.32 ± 0.09 msec; s = 2.34 msec; Amp,
2.69 ± 0.47 nA. C, Average rise, 0.25;
1 = 1.52, 2 = 9.89 msec; Amp,
3.48 nA. D, Average rise, 0.18 ± 0.14 msec;
1 = 0.72 msec, 2 = 8.37 msec;
Amp, 3.31 ± 0.65 nA. E, Average rise, 0.37 ± 0.33 msec; 1 = 0.17 msec, 2 = 15.87 msec; Amp, 2.58 nA. Calibration: 1 nA, 15 msec.
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Transgenic mouse strength varies with AChR mutation
The principal clinical features of impaired neuromuscular
transmission in the SCCMS are weakness and fatigability (Engel et al.,
1982; Gomez et al., 1996b). We demonstrated previously that L269F
mice have overt weakness (Gomez et al., 1997) and that S262F mice
are sensitive to low doses of curare (Gomez et al., 1996a). To compare
the effect of the different AChR mutations on muscle strength we
compared forelimb strength of mice from each transgenic line using a
standardized, timed-grip strength test (Gomez et al., 1997). Weakness
ranged from mild in the case of S262F mice to severe in the case of
the L269F line (Fig. 4A, left axis
label, dark bars). There appeared to be a progression of increasingly poor performance in mice between lines in the order
S262F, C418W, L251T, and L269F. Mice from three lines, C418W (p < 0.025; n = 5),
L251T (p < 0.015; n = 4),
and L269F (p<<0.001; n = 8), performed
significantly more poorly than control mice (n = 25).
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Figure 4.
Transgene-specific neuromuscular weakness
correlates with MEPC current kinetics in slow-channel transgenic mice.
A, Slow-channel mice exhibit a broad range of muscle
weakness (left axis, dark bars). Strength
testing was performed using a wire-hang paradigm (see Materials and
Methods). Results are expressed as percentage of perfect score. The
mean scores of C418W (p < 0.025;
n = 5), L251T (p < 0.015; n = 4), and L269F (p<<0.001;
n = 8), but not that of S262F
(p = 0.5; n = 4), were
significantly worse than age-matched control mice
(n = 25). Performance by L269F mice was also
significantly poorer than C418W mice (p < 0.02). Slow-channel mice have diminished miniature endplate current
(MEPC) amplitudes (right axis,
light bars). MEPC amplitudes (nanoamps) were recorded
during voltage-clamp analysis of excised diaphragms of control
(n = 5 mice) or S262F (n = 4), C418W (n = 7), L251T
(n = 6), or L269F (n = 8)
transgenic mice. The mean MEPC amplitude was significantly lower than
control mice in L269F transgenic mice (p < 0.025). B, L269F (n = 7)
transgenic mice have reduced endplate AChRs compared with control
(p < 0.025; n = 4), as
determined by 125I BT binding (see Materials and
Methods). C, Plot of strength performance versus the
slow decay time constant (2) of bi-exponential
MEPCs for each transgenic mouse line. For this plot the value for
s was used for wild type because no 2
value was resolved for wild-type mice. D, Plot of
strength performance versus the log of MEPC area as expressed in
picocoulombs. Mouse lines for C and D:
open circle, wild type; filled square,
S262; filled triangle, C418W; filled
diamond, L251T; filled circle,
L269F.
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The amplitude of the MEPC is a direct measure of synaptic strength and
is a critical component of the safety factor of neuromuscular transmission. The MEPC amplitudes were diminished in each line, in
parallel to the strength performance, although the reduction reached
statistical significance only in L269F mice
(p < 0.025; n = 8) (Fig.
4A, right axis label, light
bars). The fact that strength performance was inversely
proportional to MEPC amplitude for each transgenic line suggested that
muscle weakness in these lines was predominantly of neuromuscular origin.
The postsynaptic causes of weakness and reduced MEPC amplitude
include functional changes in AChR gating, reduced conductance of the
AChR ion channel, reduced number and density of functional AChRs, and
structural aspects of endplate degeneration (Engel et al., 1982;
Bhattacharyya et al., 1997; Gomez et al., 1997, 1998, 2002; Milone et
al., 1997). Figure 1B demonstrates that all the
mutant AChRs used in this in vivo study have conductances similar to monovalent cations. We estimated the density of endplate AChRs by measuring the number of -BT binding sites on muscle segments containing endplates. Endplate AChR numbers were significantly reduced to 50% of control (p < 0.025) in the
L269F transgenic mice, the line that had the greatest weakness and
reduction of MEPC amplitudes, but were normal in other lines (Fig.
4B). This suggests that reduction in AChR number
plays a prominent role in neuromuscular weakness in L269F mice.
Additional mechanisms must contribute to the weakness in the other
lines of slow-channel mice.
Changes in gating of AChRs bearing SCCMS mutations will predominantly
affect the MEPC decay phase, although mutations with slowed activation
rates that appear to reduce AChR responses (Gomez et al., 2002) have
rarely been identified. To visualize how estimates of muscle
strength for each transgenic line vary with line-specific differences
in MEPC kinetics, we plotted the strength scores as a function of
2 (Fig. 4C),
s (data not shown), and the mean MEPC area
(Fig. 4D) for each transgenic line.
2 and s best reflect the duration of the endplate current, whereas MEPC area is an estimate
of total charge entry during synaptic activity. The small number of
mean data points and large variance for each value preclude an accurate
prediction of a mathematical relationship. Nevertheless, it is clear
that the strength for the mouse lines varies inversely with the
duration of the MEPC and charge entry during synaptic activity.
Although these relationships are based on unpaired transgene-specific measurements, we used weighted averages of the normalized SDs for the
data from each mouse group and transgene to estimate a normal
distribution of data points for a regression analysis. Of the three
MEPC variables, s, 2,
and area, the highest correlation coefficients were obtained for
regressions between strength and MEPC area: r = 0.66 for a linear fit and r = 0.71 for an exponential fit.
As is evident from the plot (Fig. 4D), if such a
relationship exists between strength performance and MEPC area, the
L251T transgenic mice tend to perform somewhat more poorly than
predicted. When the data points from the L251T mice are removed, the
correlation coefficients increase to r = 0.73 and
r = 0.85 for linear and exponential fits, respectively.
This suggests that AChRs bearing L251T mutation may have a greater
adverse effect on strength than predicted for estimates of MEPC
duration, possibly because of the presence of additional properties.
Severe weakness is associated with active calcium overload
of endplates
The greater adverse effect of the L251T mutation on the
strength and viability of the NMJ in slow-channel transgenic mice could
be attributable to the fact that this mutation has additional properties not revealed in the voltage-clamp studies. Differences in
Ca2+ permeability between SCCMS mutants is
not readily apparent by comparing MEPCs or channel kinetics as they are
routinely recorded. Figure 1C demonstrates that there are
significant differences in relative
Ca2+/Na+
permeability between the same mutations as judged by reversal potential
studies in oocytes. To explore the effects of different transgene
mutations on Ca2+ entry into endplate
regions, we used the histochemical stain GBHA as an indicator of gross
Ca2+ overload. The GBHA stain is estimated
to detect free Ca2+ in tissues at
concentrations of 1-2 mM (Bodensteiner and
Engel, 1978). It does not detect the Ca2+
present in the sarcoplasmic reticulum or mitochondrial compartments, presumably because the Ca2+ is protein
bound (Schwartz et al., 1967; Kashiwa, 1970; Bodensteiner and Engel,
1978). We determined the proportion of endplates that were overloaded
with histochemically detectable Ca2+ for
each transgenic line (Evans, 1974; Kawabuchi, 1982; Gomez et al., 1997)
in a panel of seven muscle regions, using a histochemical stain for
cholinesterase to localize endplates on adjacent sections. In muscle
from normal mice or from mice bearing the S262T mutation, there were
no sites of staining with GBHA, either at endplate regions or elsewhere
(Fig.
5A,B).
GBHA stain was present at some endplate regions in mice expressing the
C418W, L251T, and L269F mutant subunits, although in
very different proportions (Fig. 5A,B). Similar findings were
obtained in mice from at least two founder lines for each transgene,
indicating that the effect on Ca2+
overload was not specific to transgene integration site. The proportion
of Ca2+-overloaded endplates detected in
resting L251T mice (2.8%) was only slightly greater than that in
C418W mice (0.47%; p = 0.08). However, the
proportion of GBHA-stained endplates in resting L269F transgenic
mice was significantly greater than that of the other resting mice
(p  0.001).
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Figure 5.
Calcium accumulation at motor endplates
differs with transgenic expression of distinct AChR mutations.
A, Sections of forelimb flexor muscles from resting
S262T, C418W, L251T, and L269F mice demonstrate differences
in degree of GBHA staining for different transgenic lines. Sections in
the left panels were stained for cholinesterase to
localize endplates (cholinesterase is brown) (Namba et
al., 1967) and counterstained with hematoxylin and eosin. The
adjacent serial sections in the right panels were
stained for glyoxal bis 2 hydroxyanil (Evans, 1974)
(GBHA, dark red or black)
and methylene blue. There is no GBHA staining at control (data not
shown) or S262T endplates. Scale bar, 25 µm. B,
Calcium accumulation at motor endplates differs according to mutation
and increases with exercise. The proportions of motor endplates stained
histochemically with GBHA in C418W, L251T, and L269F mice at
rest or after exercise is displayed in vertical bars
labeled below. Dark bars indicate the lines that develop
endplate myopathy (Fig. 6). At rest, no GBHA stain was detected at
control or S262T endplates, whereas 0.47% were labeled in C418W
mice, 2.8% in L251T, and 19% in L269F mice. The proportion of
Ca2+-overloaded endplates detected in resting
L251T mice (n = 6) was slightly greater than
that in C418W mice (0.47%; p = 0.08;
n = 6). The proportion of GBHA-stained endplates in
resting L269F transgenic mice was significantly greater than that of
the other resting mice (p 0.001;
n = 5). After exercise (see Materials and Methods),
there was still no GBHA staining at motor endplates or elsewhere in
muscle fibers of wild-type or S262T transgenic mice, whereas marked
increases in GBHA staining were seen in the other three transgenic
lines after exercise. In exercised C418W transgenic mice
(n = 5), the proportion of endplates stained for
GBHA increased to 3.9%, an eightfold increase
(p < 0.01). In L251T transgenic mice
(n = 4) and L269F transgenic mice
(n = 4), the proportion of GBHA-stained endplates
increased to 26% (10-fold; p < 0.002) and 33%
(1.7-fold; p < 0.04), respectively. The
proportions of Ca2+-overloaded endplates after
exercise in L251T transgenic mice and L269F transgenic mice were
significantly greater than the Ca2+-overloaded
endplates in exercised C418W transgenic mice
(p < 0.01).
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To further explore the relationship between synaptic activity and
Ca2+ overload in slow-channel transgenic
mice, we investigated the effect of changes in neuromuscular activity
on endplate Ca2+ overload. We found that
exercise had a marked and selective effect on
Ca2+ overload of transgenic endplates
(Fig. 5B). Mice were given a 30 min standardized exercise
protocol consisting of forced gripping of the cage top with all four
paws. No GBHA staining was detected at motor endplates or elsewhere in
muscle fibers of exercised wild-type or S262T-transgenic mice. In
contrast, in mice bearing the C418W, L251T, and L269F
mutations, exercise had a potent effect on the proportion of endplates
overloaded with Ca2+. In C418W
transgenic mice, the proportion of endplates stained for GBHA increased
to 3.9%, an eightfold increase (p < 0.01). In
L251T and L269F transgenic mice, the proportion of GBHA-stained endplates increased to 26% (10-fold; p < 0.002) and
33% (1.7-fold; p < 0.04), respectively. Moreover, the
proportions of Ca2+-overloaded endplates
after exercise in L251T transgenic mice and L269F transgenic mice
were significantly greater than the Ca2+-overloaded endplates in exercised
C418W transgenic mice (p < 0.01). Thus, in
mice expressing mutant AChR subunits, Ca2+
accumulation at motor endplates increased with neuromuscular activity.
Structural correlates of impaired neuromuscular transmission
Classical SCCMS is associated with striking degenerative
changes of the postsynaptic membrane, referred to as endplate
myopathy (Engel et al., 1982). We previously reported similar
degenerative changes of the endplate region in L269F mice (Gomez et
al., 1997). Comparison of the NMJ at the ultrastructural level in the
four transgenic lines studied here showed that S262T and C418W
mice had essentially normal endplates (Fig.
6A), although a few
vacuoles were found in some C418W endplates. In both L251T (Fig.
6B) and L269F (Fig. 6C) transgenic
mice, however, numerous endplates had marked ultrastructural changes.
The most prominent abnormalities were collections of membrane-bound
structures that in some cases filled the junctional sarcoplasm and
appeared to arise from dilated sarcoplasmic reticulum. These
vacuole-like structures were often small and uniform-sized in L251T
mice, but in L269F mice and occasionally in L251T mice these
vacuoles were quite variable in size and filled with fluffy or granular
material (Fig. 6C). In both transgenic lines, several other
ultrastructural abnormalities typical of the endplate myopathy in the
SCCMS were present and included degenerating nuclei, dilated or
degenerating mitochondria, and membranous or granular debris. Although
difficult to quantitate, these abnormalities, especially the nuclear
degeneration, were qualitatively more abundant in L269F transgenic
mice. We found that 56% of endplates were abnormal in L251T mice,
compared with 66% of endplates in L269F mice. Degenerating nuclei
were seen in 25% of L251T mice and in 55% of L269F mice. The
pronounced nuclear degeneration in L269F mice most likely
contributes to the marked reduction in endplate AChRs in L269F
transgenic mice because AChR subunit mRNA transcription is restricted
to the subsynaptic nuclei (Merlie and Sanes, 1985; Brenner et al.,
1990). These results support earlier findings which demonstrated that
the severe ultrastructural changes in the NMJ seen in SCCMS can
underlie progressive changes in muscle strength (Gomez et al., 2002).
Moreover, the presence of severe endplate myopathy is consistent with
the intense endplate calcium overload and the greater degree of
weakness in L251T and L269F mice.
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Figure 6.
Endplate myopathy occurs in slow-channel
transgenic mice with severe calcium overload. A,
Neuromuscular junction from a 2-month-old C418W mouse showing
essentially normal ultrastructure of nerve terminus (white
arrowheads), postsynaptic folds (black arrow),
and junctional sarcoplasm (asterisk).
B, Neuromuscular junction from 2-month-old L251T
mouse. The junctional sarcoplasm is filled with myriads of vacuole-like
structures ranging in size from 0.05 to 1 µm
(asterisk). Vacuoles are empty or filled with fluffy or
granular material. Profiles of nuclei are present at either side of the
accumulation of vacuoles. One nucleus appears severely degenerated
(black arrow). In B and C
the secondary synaptic folds and clefts are absent, and the nerve
terminals (white arrowheads) are small and barely
recognizable at the outer surface of the bulging postsynaptic regions.
C, Neuromuscular junction from a L269F mouse at 2 months. Vacuoles fill the junctional sarcoplasm and are present within the underlying sarcomeres
(asterisk). The subsynaptic mitochondria are abnormally
enlarged compared with those in the nerve terminus. Some show
accumulations of dark, dense granules consistent with calcium (data not
shown). Others contain multiple clear inclusions (black
arrowhead). Some mitochondria are pathologically dilated
(black arrow). One subsynaptic nucleus has normal
ultrastructure. Scale bars: A, 0.9 µm;
B, 4.1 µm; C, 1.5 µm.
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DISCUSSION |
The succinct termination of excitatory synaptic responses is
important for reproducible activation and recovery of excitatory synapses. In the SCCMS, progressive weakness and degeneration of the
postsynaptic portion of the neuromuscular junction is associated with
missense mutations in the AChR that lead to prolonged opening of the
AChR ion channel (Engel et al., 1982, 1996; Gomez et al., 1996b, 1997).
This suggests that proper termination of synaptic responses is
necessary to preserve the viability of the neuromuscular junction. The
presence of deposits of Ca2+ at the
neuromuscular junctions of these patients strongly suggests a role for
Ca2+ overload in the pathogenesis of
endplate degeneration (Engel et al., 1982).
The strength of neuromuscular transmission depends on several
structural, cellular, and neurophysiological aspects of the neuromuscular junction, including cleft width, activity of AChE, and
the density and channel properties of AChRs (Engel et al., 1982;
Oosterhuis et al., 1987; Gomez et al., 1996b, 1997, 2002). Some of
these properties are altered in the SCCMS and its animal model. In this
study we have used transgenic mice expressing different AChR subunit
mutations to determine the relationships between kinetic properties of
synaptic currents and the strength of neuromuscular transmission, focal
Ca2+ overload, and ultrastructural
indicators of neuromuscular disease. The results are summarized in
Table 2.
The limitations of this approach relate to issues of transgene
expression. Expression of the transgenes derived by pronuclear injection occurs in combination with, rather than in place of, that of
the endogenous subunits. The mutant phenotype relies on overexpression
of the mutant gene. Although the bulk expression of these transgenes is
considerably greater than that of endogenous genes, variations in
expression between muscle fibers and even between myonuclei may arise
because of position effects conferred by the sites of transgene
integration. Such variations may affect a proportion of mutant AChRs in
a given endplate and hence endplate kinetics. This is particularly the
case for the expression of subunit mutants, given the subunit
stoichiometry of 2. We believe the conclusions are valid
for several reasons. (1) To minimize the effects of regional
differences in expression, all comparisons between transgenes were made
on measurements averaged over multiple mice, muscle groups, and muscle
fibers. (2) There were no appreciable differences between lines bearing
distinct integrations of the same transgene, suggesting that position
effects could not explain the differences. (3) All transgenes were
expressed at levels at least 100-fold above endogenous subunits (Gomez
et al., 1996a). Moreover, there were no differences in
electrophysiological abnormalities in mice expressing the S262T
transgene over a 50-fold range (Gomez et al., 1996a), indicating that
the mutant phenotype was not sensitive to transgene expression level.
In the panel of slow-channel transgenic mice, neuromuscular
weakness and reduction in MEPC amplitudes correlated well over a broad
range of muscle strength. Milder weakness and reduced MEPC amplitudes
were seen in S262F and C418W mice, which had kinetic changes but
little or no Ca2+ overload. The mild
impairment of neuromuscular transmission could not be attributed to
structural changes or to AChR loss and is likely caused by additional
functional changes arising at least in part from the kinetic
disturbance (Milone et al., 1997; Gomez et al., 2002). Previous studies
have shown that delayed AChR activation or increased desensitization
may contribute significantly to reduced synaptic strength in some SCCMS
patients or slow-channel mice (Bhattacharyya et al., 1997; Gomez et
al., 2002). Functional changes are likely to include intrinsic
properties of mutant AChRs, effects of smaller increases in
Ca2+ entry on the rate of desensitization,
blockade, and compensatory changes in the neuromuscular transmission
(Ochoa et al., 1989; Siara et al., 1990; Nojima et al., 1994; Fenster
et al., 1999).
Greater weakness and the most severe endplate myopathy were
seen in the L251T and L269F transgenic mice. Intense overload of endplates with Ca2+, at rest or
with exercise, attributable to the prolonged MEPC duration or a higher
Ca2+ permeability overload, appear to be
most closely associated with the typical changes in the SCCMS. Weakness
was greatest in L269F mice, the line that displayed the greatest
perturbations in channel kinetics, active endplate
Ca2+ overload, endplate myopathy, and
endplate AChR loss. Thus, both channel gating activity and ion
permeation are critical to maintain the ionic environment of the
subsynaptic region and lead to active Ca2+
overload, degeneration of the endplate, and impaired neuromuscular transmission when disrupted.
The findings in this study demonstrate that
Ca2+ overload occurs as a direct result of
mutant AChR channel activity. Ca2+
overload in some slow-channel transgenic mice and patients is detectable using a relatively insensitive histochemical assay. The GBHA
stain, which is the most sensitive for soluble
Ca2+, has an estimated detection limit of
1-2 mM (Bodensteiner and Engel, 1978). Using this same
assay we also detected a severalfold increase of
Ca2+ overloaded endplates in transgenic
mice after brief exercise. These results show that for some mutants,
Ca2+ overload is governed by the
Ca2+ permeability, open duration, and even
frequency of opening of the AChR channel at the
neuromuscular junction. The muscle AChR ion channel is
highly permeable to Ca2+, which represents
~2-3% of ions that comprise the adult endplate current (Decker and
Dani, 1990). Nevertheless, Ca2+ does not
accumulate at normal endplates unless AChE is inhibited pharmacologically (Evans, 1974; Kawabuchi, 1982). Changes in the kinetics of the channel opening affect the quantity of
Ca2+ that enters the junctional sarcoplasm
during synaptic activity. The findings in this study suggest that there
are discrete thresholds for the duration of synaptic currents
beyond which Ca2+ overload and endplate
degeneration occur. The determinants of these thresholds are complex
and related to the size of the neuromuscular junction and muscle fiber
and Ca2+-buffering systems present in the
junctional sarcoplasm (Kohr and Mody, 1991; Gunter and Gunter, 1994;
Hartmann et al., 1994).
On the basis of the ultrastructural data, we predict that
Ca2+ accumulates in the sarcoplasm, the
sarcoplasmic reticulum, and the mitochondria (Fig. 6) (Gomez et al.,
1997). The marked Ca2+ overload is
associated with severe ultrastructural changes at the neuromuscular
junction. The dilated vacuoles, localized contractures, and
degenerating mitochondria and nuclei, termed endplate myopathy, which
were seen in the L251T and L269F mice have been described previously in both mice and patients (Engel et al., 1982; Gomez et al.,
1996b, 1997; Milone et al., 1997). Excessive intracellular Ca2+ levels might activate a number of
harmful enzymatic or free radical degradative pathways. We have found
evidence that the Ca2+-activated protease,
calpain, contributes to impaired neuromuscular transmission in L269F
mice (J. Groshong, B. P. S. Vohra, M. J. Spencer, and C. M. Gomez, unpublished observations). Moreover, in mice and patients,
multiple components of the caspase cascade appear to elicit a process
of localized subcellular apoptosis (Vohra, R. Zayas, Groshong, R. A. Maselli, R. L. Wollmann, and Gomez, unpublished observations).
The findings reported here may have a number of important
clinical implications. First, the capacity of some
Ca2+ buffering systems, such as the
mitochondria, may diminish with age (Martinez et al., 1992; Satrustegui
et al., 1996), such that a given mutation might not lead to a phenotype
until advanced age. This fact could provide an explanation for late
onset or progression of some forms of the disease despite the lifelong presence of the mutation (Engel et al., 1982; Croxen et al., 1997). Second, deterioration of such buffering systems in excitatory pathways,
for example, in mitochondrial disorders (Brini et al., 1999; Wasniewska
et al., 2001), may be a route by which normal amounts of
Ca2+ entry may lead to neurodegeneration.
Third, because neuromuscular activity increases the extent of
Ca2+ overload, the value of exercise in
the long-term management of the SCCMS is uncertain. Finally, the
biochemical processes coupling Ca2+
overload to endplate degeneration are complex and may include activation of Ca2+-activated neutral
protease, calpain, Dnase, or damage to cells through overproduction of
free radicals (Choi, 1992). Collectively, these results suggest
that agents that either serve to block
Ca2+ entry through mutant ion channels or
that enhance Ca2+ buffering would be
additional neuroprotective strategies for this syndrome and those such
as stroke and epilepsy, which are associated with excitotoxic neuronal stress.
| |
FOOTNOTES |
Received March 15, 2002; revised May 6, 2002; accepted May 10, 2002.
This work was supported by National Institutes of Health Grants
RO1 NS33202 and RO1 NS36809 and the Muscular Dystrophy Association to
C.M.G., and by North Atlantic Treaty Organization Grant CRG 972059, Association Francaise contre les Myopathies, Association pour la
Recherche contre le Cancer, and Ligue Nationale contre le Cancer to
P.C. We thank Drs. John H. Anderson and Bruce Lynn for helpful discussions.
Correspondence should be addressed to Dr. Christopher M. Gomez, Box
295, Departments of Neuroscience and Neurology, 420 Delaware Street
Southeast, Minneapolis, MN 55455. E-mail:
gomez001{at}tc.umn.edu.
| |
REFERENCES |
-
Bertrand D,
Galzi JL,
Devillers TA,
Bertrand S,
Changeux JP
(1993)
Mutations at two distinct sites within the channel domain M2 alter calcium permeability of neuronal alpha 7 nicotinic receptor.
Proc Natl Acad Sci USA
90:6971-6975[Abstract/Free Full Text].
-
Bhattacharyya BJ,
Day J,
Gundeck JE,
Leonard S,
Wollmann RW,
Gomez C
(1997)
Desensitization of mutant acetylcholine receptors in transgenic mice reduces the amplitude of synaptic currents.
Synapse
27:367-377[Medline].
-
Bodensteiner JB,
Engel AG
(1978)
Intracellular calcium accumulation in Duchenne dystrophy and other myopathies: a study of 567,000 muscle fibers in 114 biopsies.
Neurology
28:439-446[Abstract/Free Full Text].
-
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 -subunit of a mouse acetylcholine receptor.
J Neurosci
5:2545-2552[Abstract].
-
Brenner HR,
Witzemann V,
Sakmann B
(1990)
Imprinting of acetylcholine receptor messenger RNA accumulation in mammalian neuromuscular synapses.
Nature
344:544-547[Medline].
-
Brini M,
Pinton P,
King MP,
Davidson M,
Schon EA,
Rizzuto R
(1999)
A calcium signaling defect in the pathogenesis of a mitochondrial DNA inherited oxidative phosphorylation deficiency.
Nat Med
5:951-954[Web of Science][Medline].
-
Charnet P,
Labarca C,
Lester HA
(1992)
Structure of the gamma-less nicotinic acetylcholine receptor: learning from omission.
Mol Pharmacol
41:708-717[Abstract].
-
Choi DW
(1992)
Excitotoxic cell death.
J Neurobiol
23:1261-1276[Web of Science][Medline].
-
Choi DW
(1994)
Calcium and excitotoxic neuronal injury.
Ann NY Acad Sci
747:162-171[Web of Science][Medline].
-
Croxen R,
Newland C,
Beeson D,
Oosterhuis H,
Chauplannaz G,
Vincent A,
Newsomdavis J
(1997)
Mutations in different functional domains of the human muscle acetylcholine receptor alpha subunit in patients with the slow-channel congenital myasthenic syndrome.
Hum Mol Genet
6:767-774[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].
-
Engel AG
(1994)
Myasthenic syndromes.
In: Myology (Engel AG,
Franzini-Armstrong C,
eds), pp 393-418. New York: McGraw-Hill.
-
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,
Sieb JP,
Sine SM
(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].
-
Fenster CP,
Beckman ML,
Parker JC,
Sheffield EB,
Whitworth TL,
Quick MW,
Lester RA
(1999)
Regulation of alpha4beta2 nicotinic receptor desensitization by calcium and protein kinase C.
Mol Pharmacol
55:432-443[Abstract/Free Full Text].
-
Filatov GN,
White MM
(1995)
The role of conserved leucines in the M2 domain of the acetylcholine receptor in channel gating.
Mol Pharmacol
48:379-384[Abstract].
-
Gardner PD
(1990)
Nucleotide sequence of the epsilon-subunit of the mouse muscle nicotinic acetylcholine receptor.
Nucleic Acids Res
18:6714[Free Full Text].
-
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/Free Full Text].
-
Gomez CM,
Wollmann RL,
Richman DP
(1984)
Induction of the morphologic changes of both acute and chronic experimental myasthenia by monoclonal antibody directed against acetylcholine receptor.
Acta Neuropathol
63:131-143[Medline].
-
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
(1996b)
A subunit mutation in the acetylcholine receptor channel gate causes severe slow-channel syndrome.
Ann Neurol
39:717-723.
-
Gomez CM,
Maselli R,
Gundeck JE,
Chao M,
Day JW,
Tamamizu S,
Lasalde JA,
McNamee M,
Wollmann RL
(1997)
Slow-channel transgenic mice: a model of postsynaptic organellar degeneration at the neuromuscular junction.
J Neurosci
17:4170-4179[Abstract/Free Full Text].
-
Gomez CM,
Maselli R,
Williams JM,
Bhattacharyya BB,
Wollmann RL,
Day JW
(1998)
Genetic manipulation of AChR responses in transgenic mice points to multiple causes of weakness in slow-channel syndrome.
Ann NY Acad Sci
841:167-180[Medline].
-
Gomez C,
Maselli R,
Vohra B,
Navedo M,
Stiles J,
Charnet P,
Schott K,
Rojas L,
Keesey J,
Verity A,
Wollmann R,
Lasalde-Dominicci J
(2002)
Novel delta subunit mutation in slow-channel syndrome causes severe weakness by novel mechanisms.
Ann Neurol
51:102-112[Web of Science][Medline].
-
Gorman CM,
Merlino GT,
Willingham MC,
Pastan I,
Howard BH
(1982)
The Rous sarcoma virus long terminal repeat is a strong promoter when introduced into a variety of eukaryotic cells by DNA-mediated transfection.
Proc Natl Acad Sci USA
79:6777-6781[Abstract/Free Full Text].
-
Gunter KK,
Gunter TE
(1994)
Transport of calcium by mitochondria.
J Bioenerg Biomembr
26:471-485[Web of Science][Medline].
-
Hartmann H,
Eckert A,
Muller WE
(1994)
Disturbances of the neuronal calcium homeostasis in the aging nervous system.
Life Sci
55:2011-2018[Web of Science][Medline].
-
Imoto K,
Konno T,
Nakai J,
Wang F,
Mishina M,
Numa S
(1991)
A ring of uncharged polar amino acids as a component of channel constriction in the nicotinic acetylcholine receptor.
FEBS Lett
289:193-200[Web of Science][Medline].
-
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
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].
-
Kohr G,
Mody I
(1991)
Endogenous intracellular calcium buffering and the activation/inactivation of HVA calcium currents in rat dentate gyrus granule cells.
J Gen Physiol
98:941-967[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].
-
Lerche H,
Jurkat-Rott K,
Lehmann-Horn F
(2001)
Ion channels and epilepsy.
Am J Med Genet
106:146-159[Web of Science][Medline].
-
Martinez SA,
Blanco P,
Satrustegui J
(1992)
Calcium binding to the cytosol and calcium extrusion mechanisms in intact synaptosomes and their alterations with aging.
J Biol Chem
267:4672-4679[Abstract/Free Full Text].
-
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].
-
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].
-
Mayer ML,
Westbrook GL
(1987)
The physiology of excitatory amino acids in the vertebrate central nervous system.
Prog Neurobiol
28:197-276[Web of Science][Medline].
-
Merlie JP,
Sanes JR
(1985)
Concentration of acetylcholine receptor mRNA in synaptic regions of adult muscle fibres.
Nature
317:66-68[Medline].
-
Milone M,
Wang HL,
Ohno K,
Fukudome T,
Pruitt JN,
Bren N,
Sine SM,
Engel AG
(1997)
Slow-channel myasthenic syndrome caused by enhanced activation, desensitization, and agonist binding affinity attributable to mutation in the m2 domain of the acetylcholine receptor subunit.
J Neurosci
17:5651-5665[Abstract/Free Full Text].
-
Muley S,
Gomez C
(2002)
Congenital myasthenic syndromes.
In: Mysathenia gravis and related disorders (Kaminiski H,
ed), pp 309-326. Totowa, NJ: Humana.
-
Namba T,
Nakamura T,
Grob D
(1967)
Staining for nerve fiber and cholinesterase activity in fresh frozen sections.
Am J Clin Pathol
47:75-79.
-
Nojima H,
Kimura I,
Kimura M
(1994)
The evidence of accelerative interaction between cAMP-dependent protein kinase and external calcium for the desensitization of nicotinic acetylcholine receptor channel in mouse skeletal muscle cells.
Neurosci Lett
167:113-116[Medline].
-
Ochoa EL,
Chattopadhyay A,
McNamee MG
(1989)
Desensitization of the nicotinic acetylcholine receptor: molecular mechanisms and effect of modulators.
Cell Mol Neurobiol
9:141-178[Web of Science][Medline].
-
Oosterhuis HJ,
Newsom-Davis J,
Wokke JH,
Molenaar PC,
Weerden TV,
Oen BS,
Jennekens FG,
Veldman H,
Vincent A,
Wray DW,
Prior C,
Murray NMF
(1987)
The slow channel syndrome. Two new cases.
Brain
110:1061-1079[Abstract/Free Full Text].
-
Pestronk A,
Drachman DB,
Self SG
(1985)
Measurement of junctional acetylcholine receptors in myasthenia gravis: clinical correlates.
Muscle Nerve
8:245-251[Web of Science][Medline].
-
Satrustegui J,
Villalba M,
Pereira R,
Bogonez E,
Martinez SA
(1996)
Cytosolic and mitochondrial calcium in synaptosomes during aging.
Life Sci
59:429-434[Medline].
-
Schwartz M,
Kashiwa HK,
Jacobson A,
Rehm WS
(1967)
Concentration and localization of calcium in frog gastric mucosa.
Am J Physiol
212:241-246[Free Full Text].
-
Siara J,
Ruppersberg JP,
Rudel R
(1990)
Human nicotinic acetylcholine receptor: the influence of second messengers on activation and desensitization.
Pflügers Arch
415:701-706[Medline].
-
Steinlein OK
(2001)
Genes and mutations in idiopathic epilepsy.
Am J Med Genet
106:139-145[Web of Science][Medline].
-
Taketo M,
Schroeder AC,
Mobraaten LE,
Gunning KB,
Hanten G,
Fox RR,
Roderick TH,
Stewart CL,
Lilly F,
Hansen CT
(1991)
FVB/N: an inbred mouse strain preferable for transgenic analyses.
Proc Natl Acad Sci USA
88:2065-2069[Abstract/Free Full Text].
-
Tamamizu S,
Lee Y,
Hung B,
McNamee MG,
Lasalde-Dominicci JA
(1999)
Alteration in ion channel function of mouse nicotinic acetylcholine receptor by mutations in the M4 transmembrane domain.
J Membr Biol
170:157-164[Web of Science][Medline].
-
Wasniewska M,
Karczmarewicz E,
Pronicki M,
Piekutowska-Abramczuk D,
Zablocki K,
Popowska E,
Pronicka E,
Duszynski J
(2001)
Abnormal calcium homeostasis in fibroblasts from patients with Leigh disease.
Biochem Biophys Res Commun
283:687-693[Web of Science][Medline].
-
Weisberg S
(1985)
In: Applied linear regression, Ed 2. New York: Wiley.
Copyright © 2002 Society for Neuroscience 0270-6474/02/22156447-11$05.00/0
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