Postsynaptic Membranes at
the Neuromuscular Junction:
Molecular Organization
Secondary article
Article Contents
. Introduction
. Folds in the Postsynaptic Membrane of the
Neuromuscular Junction
Jean Cartaud, Institut Jacques Monod, Paris, France
Ekaterini Kordeli, Institut Jacques Monod, Paris, France
Annie Cartaud, Institut Jacques Monod, Paris, France
. Nicotinic Acetylcholine Receptors are Tightly Packed at
the Top of the Junctional Folds
Synaptic transmission at the neuromuscular junction requires the precise spatial
registration and a high degree of local molecular differentiation of both presynaptic and
postsynaptic membrane domains. At these two sites, the accumulation of various proteins,
particularly ion channels, is responsible for the regulated secretion of the neurotransmitter
by the nerve terminal and for the subsequent depolarization of the postsynaptic
membrane.
. The Synaptic Basal Lamina Contains Signals for
Neuromuscular Junction Specialization
Introduction
. Sodium Channels Accumulate at the Base of the
Junctional Folds
. Clustering of Nicotinic Receptors by the Protein
Rapsyn
. Organization of the Muscle Postsynaptic Membrane
by a Complex of Proteins that Binds the Basal Lamina
. Initiation of Assembly of the Postsynaptic Membrane
by Agrin Secreted by the Nerve
. Termination of the Action of Acetylcholine by
Acetylcholinesterase Located in the Synaptic Basal
Lamina
. Summary
Central to reliable synaptic transmission is the accumulation of two types of ion channels in the postsynaptic
membrane of the vertebrate neuromuscular junction, a
local differentiation of the sarcolemma. Directly across the
active zones of the nerve ending, the crests of the
postsynaptic membrane folds exhibit extraordinarily high
concentrations of ligand-gated Na 1 /K 1 channels, the
nicotinic acetylcholine receptors (AChR). Voltage-sensitive Na 1 channels accumulate in regions immediately
adjacent to the AChRs in the depths of the folds of the
postsynaptic membrane at a concentration at least tenfold
that found in the extrasynaptic sarcolemma. The two
classes of channels are essential for acetylcholine-mediated
membrane depolarization and initiation of the action
potential, respectively. Several peripheral proteins, such as
the 43 kDa protein rapsyn, dystrophin/utrophin, ankyrin
and syntrophins, colocalize with either AChRs or Na 1
channels and are believed to participate in their clustering
at synaptic sites. The postsynaptic differentiation also
encompasses the basal lamina, which locally contains high
concentrations of the enzyme acetylcholinesterase
(AChE), as well as several signalling molecules, such as
agrin, laminin and ARIA (AChR-inducing activity), all
involved in the differentiation of the synaptic structures.
The high degree of specialization of the postsynaptic
apparatus of the neuromuscular junction is the result of a
complex process involving not only membrane components but also the subsarcolemmal cytoskeleton and the
synaptic basal lamina.
Folds in the Postsynaptic Membrane of
the Neuromuscular Junction
Early electron-microscopic studies of the neuromuscular
junction demonstrated that the sarcolemma just beneath
the nerve terminal displays a shallow depression further
invaginated into deep and regular folds, the postjunctional
folds (Figure 1a). The crests of these folds are characterized
by electron-dense thickening corresponding to accumulation of AChR and are underlined by a dense submembraneous cytoskeleton. The mouths of the postjunctional
folds are precisely aligned with the presynaptic active
zones. This organization ensures that acetylcholine encounters a high concentration of AChRs within microseconds of release by the nerve terminal, thereby
facilitating membrane depolarization. The membrane in
the trough of the folds appears less electron-dense. The
Na 1 channels as well as several other molecules such as the
Na 1 /K 1 ATPase and the neural cell adhesion molecule
(N-CAM) are localized in this region. Interestingly, the
cytoskeletal elements associated with the crests or the
troughs of postsynaptic folds appear to be specific for each
domain (see below). The postsynaptic membrane thus has
the appearance of a mosaic structure in which islets of
densely packed AChRs, localized in register with the
presynaptic active zones, are embedded in a patch of Na 1
channels. This type of organization favours efficient
neuromuscular function.
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Postsynaptic Membranes at the Neuromuscular Junction: Molecular Organization
Figure 2 (a) Electron micrograph showing the densely packed AChR
rosettes in purified AChR-rich membrane from Torpedo electric tissue
(negative staining; magnification 300 000). Inset: Detail of an AChR
molecule exhibiting a pentameric structure; magnification 600 000. (b)
Electron micrograph of a freeze-etched AChR-rich postsynaptic membrane
fragment showing the geometrical arrangement of the AChRs at the
surface of the membrane (magnification 200 000).
Figure 1 Electron micrographs of the postsynaptic apparatus of the
mouse neuromuscular junction. The postsynaptic membrane just beneath
the nerve ending (NE) is folded up into numerous folds. Note the electrondense appearance of the membrane at the top of the folds where AChRs are
concentrated (arrows in (b)). Magnifications: (a) 20 000, (b) 38 000.
Inset: The differential distributions of AChRs and Na 1 channels,
respectively, at the top and at the base of the folds are shown in this double
fluorescence picture obtained after labelling with fluorescein-conjugated
a-bungarotoxin (green fluorescence), a specific ligand of the AChR, and
with an antibody directed against Na 1 channel (red fluorescence).
Magnification 1600.
Nicotinic Acetylcholine Receptors are
Tightly Packed at the Top of the
Junctional Folds
Autoradiographic studies at the electron-microscopic level
of the mammalian neuromuscular junctions or of the eel
electromotor synapse have revealed an exceptional accumulation of acetylcholine binding sites (10 000 to 20 000
sites/mm2) within the postsynaptic membrane. The high
concentration of AChR at the synapse contrasts sharply
with the receptor density in the sarcolemma just a few
micrometres away from the synapse, where very low levels
were detected. Direct electron-microscopic visualization of
AChRs by freeze fracture/etching or negative staining
(Cartaud et al., 1976) has further disclosed that pentameric
receptors are organized in an almost crystalline array
within the plane of the juxtaneural postsynaptic membrane
(Figure 2). This supramolecular organization of the AChRs
is dependent upon interactions with the underlying
cytoskeleton. This was evidenced by experiments showing
that the stripping of peripheral components of postsynap2
tic membrane fragments leads to the loss of the organization and immobilization of the AChRs in the plane of the
membrane.
Sodium Channels Accumulate at the
Base of the Junctional Folds
Voltage-sensitive Na 1 channels mediate the initiation and
propagation of the action potential in excitable cells. Na 1
channels are a multigenic family of ion channels expressed
in the nervous system and in muscle fibres. Two Na 1
channel genes, SkM1 and SkM2, are specifically expressed
in skeletal muscle. Sodium channels have been localized in
the troughs of the postjunctional folds by immunocytochemistry and electron-microscopic autoradiography
(Figure 1, inset). An average site density of 2000 channels/
mm2, corresponding to a 10-fold enrichment as compared
to extrajunctional sarcolemma, has been deduced by
electrophysiological methods. Several peripheral proteins
such as syntrophin, dystrophin, ankyrin and spectrin
coextensively localize with Na 1 channels in the troughs
of the folds, suggesting that the cortical skeleton takes part
in synaptic localization. An interaction between Na 1
channels and ankyrin, which links ion channels and
spectrin, has been demonstrated in vitro. In addition,
association of Na 1 channels with the dystrophin complex
occurs via syntrophin (Colledge and Froehner, 1998).
Syntrophin and ankyrin associate with dystrophin and
spectrin, respectively, and, therefore, possibly provide a
specific link between the ion channels and the actin cortical
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Postsynaptic Membranes at the Neuromuscular Junction: Molecular Organization
skeleton. Clustering of Na 1 channels at the postsynaptic
membrane of the rat begins later than that of AChRs,
approximately one week after birth, and gradually
increases over the first 5 to 6 weeks. This observation
suggests that the clustering mechanisms for AChR and
Na 1 channels differ.
can also failed in rapsyn-deficient mice. These observations
demonstrate that rapsyn is necessary for the differentiation
of the postsynaptic apparatus in vivo. Rapsyn transcripts
are also found in ciliary ganglion neurons, suggesting that
rapsyn-like molecules may be involved in the clustering of
AChRs in neuronal synapses.
Clustering of Nicotinic Receptors by the The Synaptic Basal Lamina Contains
Protein Rapsyn
Signals for Neuromuscular Junction
Insight into the molecular mechanisms by which AChRs Specialization
are anchored at synaptic sites has been gained from studies
conducted on electric tissue from marine rays, i.e. Torpedo
sp. The electrocytes are flattened modified muscle cells that
are innervated by cholinergic synapses very similar to the
neuromuscular junctions. These electromotor synapses
cover a large part of the ventral surface of the cells and
serve as an excellent model system for biochemical studies
of the AChR and other synaptic molecules. Postsynaptic
membranes purified from Torpedo electrocytes contain, in
addition to the four AChR subunits (a, b, g and d), several
peripheral polypeptides, and in particular a 43 kDa
polypeptide named rapsyn (receptor-associated protein
at the synapse). Rapsyn and AChR extensively codistribute in the innervated membrane of the electrocyte and at
the crest of the folds of the mammalian neuromuscular
junction. Rapsyn is present at a 1:1 stoichiometry with
AChR and consequently may interact directly with it.
Chemical crosslink experiments with bifunctional reagents
indeed showed that rapsyn is in close proximity to the
AChR b subunit. These data are consistent with a
structural role for rapsyn in AChR anchoring to the
postsynaptic membrane skeleton. Moreover, molecular
biology and genetic approaches revealed that rapsyn
functions in AChR clustering. Cotransfection experiments
in heterologous systems demonstrate that rapsyn is
necessary for AChR clustering. Rapsyn comprises several
structural domains: an amino-terminal myristoylation site,
eight tandem tetratricopeptide repeats (TRPs) and a
carboxy-terminal zinc ring finger domain. Transfection
experiments with chimaeric polypeptides containing critical structural domains of rapsyn have shown that the
myristoylated amino-terminal 15 amino acids are sufficient
for membrane targeting. The first two TRP repeats mediate
self-association of rapsyn, whereas a coiled-coil domain
within TRP 8 mediates interaction with AChRs (Ramarao
and Cohen, 1998). Recently, rapsyn-deficient (‘knockout’)
mice have been obtained by homologous recombination.
These animals are paralysed, have difficulty in breathing
and die soon after birth. Differentiation of their postsynaptic apparatus is absent, despite a normal level of
expression of AChRs (Gautam et al., 1995). Moreover,
clustering of other synaptic components, such as the
synaptic ARIA receptors (ErbBs) utrophin and dystrogly-
Along with the differentiation of the sarcolemma, the basal
lamina under the nerve terminal is biochemically and
functionally specialized. The idea that the synaptic basal
lamina contains an activity that induces presynaptic and
postsynaptic differentiation originated from developmental and regeneration studies. The synaptic basal lamina
mimics the motor neuron in inducing the clustering of
AChRs and associated proteins, suggesting that it contains
the motor neuron regulatory signal(s). These studies led
McMahan and colleagues to purify an AChR-aggregating
factor from extracellular matrix of Torpedo electric tissue.
This molecule, named agrin, is a multidomain 400 kDa
heparan sulfate proteoglycan. Sequences in the N-terminal
region are responsible for association with laminin 2 and
laminin 4, the two laminin isoforms present in the muscle
synaptic basal lamina. The C-terminal agrin fragment
containing four EGF-like and three laminin-like globular
(G) domains is sufficient to induce AChR clustering in
cultured myotubes. Several agrin isoforms endowed with
very different AChR-aggregating efficiencies result from
alternative splicing at two sites in the C-terminus: agrin
isoforms containing inserts of 4 and 8–19 amino acids
appear to be the most active and, interestingly, are
expressed in motor neurons during synaptogenesis (Ruegg
and Bixby, 1998). Agrin has also been shown to be involved
in the aggregation of several components that are normally
aggregated in the postsynaptic region such as Na 1
channel, rapsyn, acetylcholinesterase, heparan sulfate
proteoglycan and dystrophin/utrophin. ‘Knockout’ mice
lacking agrin have abrogated presynaptic specializations,
have no postsynaptic apparatus and die perinatally. These
studies argue in favour of an essential role of agrin in
orchestrating the synaptic differentiation at the vertebrate
neuromuscular junction.
Several other components of the synaptic basal lamina
such as synaptic laminins, basic FGF, etc. are capable of
inducing in vitro AChR aggregation and are thus
potentially involved in synaptic differentiation. In particular, laminin seems to increase the aggregating activity of
agrin. A surprising observation is that charged latex beads
can trigger the formation of a postsynaptic apparatus at
sites of contact with myotubes. Such experiments in vitro
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Postsynaptic Membranes at the Neuromuscular Junction: Molecular Organization
are difficult to interpret. ‘Knockout’ experiments seem to
be more reliable tools for the identification of the
physiologically active factors engaged in synaptic differentiation.
In addition to factors involved in the clustering of
AChRs, the synaptic basal lamina accommodates several
nerve-derived trophic factors such as ARIA, a member of
the neuregulin/heregulin family of ligands, shown to
induce AChR gene expression in subsynaptic nuclei. This
transcriptional upregulation concerns several other synaptic proteins including Na 1 channel, AChE and utrophin.
The synaptic basal lamina therefore appears to be a
reservoir of nerve-derived (and also muscle-derived)
signalling molecules involved in various aspects of synaptic
specialization.
Organization of the Muscle
Postsynaptic Membrane by a Complex
of Proteins that Binds the Basal Lamina
Several spectrin-like molecules, including an isoform of bspectrin, dystrophin and utrophin (previously called
dystrophin-related protein), have been localized to the
postsynaptic membrane of the neuromuscular junction.
Dystrophin, the product of a gene that is defective in
Duchenne and Becker muscular dystrophies, is a major
peripheral cytoskeletal polypeptide located at the cytoplasmic face of the sarcolemma and accumulates in the
troughs of the junctional folds. In contrast, utrophin, the
product of a gene closely related to dystrophin, is located
exclusively at the crests of postsynaptic folds in normal
muscle. At the muscle cell surface, dystrophin interacts
with extracellular laminin indirectly via dystroglycan, a
transmembrane dystrophin-associated glycoprotein. At
the cytoplasmic face of the sarcolemma, dystrophin
directly binds to the underlying actin cytoskeleton. It is
likely that dystroglycan, which also associates with
utrophin, has a role in anchoring AChRs to the underlying
cytoskeleton. The fact that dystroglycan and AChRs are
both directly linked to rapsyn argues in favour of such a
hypothesis. A dramatic disorganization of many neuromuscular junctions has been observed in chimaeric mice
lacking dystroglycan in striated muscles, suggesting that
the dystroglycan subcomplex has important synaptic
function (Côté et al., 1999). a-Dystroglycan, the extracellularly exposed dystroglycan moiety, is also able to bind
agrin and, as such, has been proposed to participate in
agrin signalling. For instance, a-dystroglycan could
participate indirectly in AChR clustering by increasing
the local concentration of active agrin presented to the
signal-transducing receptor (see next paragraph). Most
probably, dystroglycan mediates the formation of AChR
macroclusters rather than the initial clustering events.
4
Accumulation of dystroglycan at the postsynaptic membrane may also help to stabilize the neuromuscular
junction via multiple interactions with the basal lamina.
Initiation of Assembly of the
Postsynaptic Membrane by Agrin
Secreted by the Nerve
MuSK (muscle-specific kinase), a transmembrane receptor
tyrosine kinase highly expressed in skeletal muscle and
Torpedo electric tissue, appears to be a critical element in
the agrin signalling pathway. As muscle matures, MuSK
becomes strictly localized at the neuromuscular junction, a
strategic position for its recognition and mediation of
nerve-derived signals. ‘Knockout’ mice lacking MuSK are
paralysed because they lack neuromuscular junctions (preand postsynaptic aspects), and die at birth, owing to their
inability to breathe (DeChiara et al., 1996). Interestingly,
the phenotype of these animals closely resembles that of
mice lacking agrin. These striking similarities suggested
that agrin acts via the MuSK receptor. Indeed, it was
shown that agrin fails to induce AChR clustering in
myotubes from MuSK-deficient mice, and otherwise that
agrin induces rapid and specific tyrosine phosphorylation
of MuSK. Nevertheless, agrin does not bind directly to the
MuSK ectodomain. An additional myotube-specific activity or component – the myotube-associated specific
Figure 3 Electron micrographs of negatively stained AChE molecules. The
asymmetric form A12 (inset) aggregates at low ionic strength and in the
presence of polyanions into discrete assemblies containing up to six
molecules organized head to tail at both ends of a bundle of collagenic
subunits. Similar aggregates are likely to occur in situ in the basal lamina
(see Figure 4). Arrows point to the collagenic tail in the isolated A12
molecule in the inset and in one aggregate. Magnification 300 000.
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Postsynaptic Membranes at the Neuromuscular Junction: Molecular Organization
component, or MASC – is thus necessary for agrinmediated signalling. Any of the agrin-binding factors
present at the muscle surface such as dystroglycan,
integrins, etc. could, in principle, be the coreceptor. The
events in agrin signalling downstream of MuSK are still
poorly understood. Rapsyn seems to have an important
function there. Data suggest a cascade model in which
agrin first clusters MuSK, then MuSK clusters an
unknown transmembrane linker protein (rapsyn-associated transmembrane linker or RATL), the linker protein
ECM
PM
P
(a)
ECM
PM
(b)
α
AChR
MuSK/
MASC
RATL
β
Dystroglycan
P
NaCh
AChE
Agrin
Rapsyn
Ankyrin
Syntrophin
Spectrin
Actin
Utrophin/
Dystrophin
Laminin
Figure 4 Model of the molecular specialization in the postsynaptic membrane and basal lamina at the neuromuscular junction. Distinct molecules are
segregated in the crests (a) and the troughs (b) of the postjunctional folds. The exclusive localization of MuSK/MASC complex and RATL at the crests of
the folds is speculative. The representation of the extracellular matrix is oversimplified to highlight the possible association of AChE with laminin or agrin.
ECM, extracellular matrix; PM, postsynaptic membrane.
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Postsynaptic Membranes at the Neuromuscular Junction: Molecular Organization
clusters rapsyn, which in turn clusters AChRs. Finally,
tyrosine protein phosphorylation is an important regulatory mechanism in AChR clustering. Inhibitors of tyrosine
kinases prevent both agrin-induced AChR clustering and
AChR phosphorylation. The kinase(s) that stimulates
AChR tyrosine phosphorylation is distinct from MuSK,
suggesting that MuSK activation might be required for
activation of still unidentified downstream kinases.
Termination of the Action of
Acetylcholine by Acetylcholinesterase
Located in the Synaptic Basal Lamina
Efficient inactivation of the neurotransmitter in the
synaptic cleft is necessary for repeated neurotransmission.
At the neuromuscular junction, this is achieved by rapid
hydrolysis of acetylcholine by the enzyme AChE. To
accomplish this, AChE molecules must be correctly
located and concentrated at synaptic sites within the
specialized synaptic basal lamina. In mammals the
catalytic domain of AChE is encoded by a single gene that
can give rise to a variety of transcripts by alternative
splicing in the C-terminal domain. The alternative Cterminal domains are not required for catalytic activity,
but are important for the cellular localization of the
enzyme. The various molecular forms generated by
alternative splicing of the catalytic subunit and by
combinations with structural subunits (e.g. the collagenic
or Q subunit), allow multiple modes of anchoring at the cell
surface. Most of the AChE at the neuromuscular junction
is anchored to the basal lamina via the collagenous subunit
or ‘tail’. This main synaptic form, called A12, is an
asymmetric collagen-tailed molecule, combining 3 structural and 12 catalytic subunits (Figure 3). The asymmetric
‘tailed’ forms of the enzyme associate in vitro to form
discrete supramolecular assemblies in the presence of
polyanionic components such as heparan sulfates. The
enzyme probably takes advantage of this property for its
attachment to the synaptic basal lamina via electrostatic
interactions with ‘receptor’ sites: the heparan sulfate
proteoglycans such as perlecan and agrin (Peng et al.,
1999). High levels of AChE at synaptic sites also result
from the spatial regulation of gene expression in the
multinucleated muscle fibre by nerve-derived factors,
similarly to AChR subunits, utrophin, etc. Finally, the
stoichiometry and spatial organization of AChE catalytic
sites relative to AChR sites at the neuromuscular junction
are possibly regulated by interactions between elements of
the postsynaptic membrane and of the basal lamina.
Dystroglycan and agrin are good candidates for such
interactions, because of their strategic location and their
respective partners.
6
Summary
The differentiation of the neuromuscular junction depends
on a complex interplay of inductive interactions between
the nerve and the muscle. According to the ‘agrin
hypothesis’, this nerve-derived factor orchestrates the
assembly of the postsynaptic membrane. Indeed, agrin is
capable of mustering a series of synaptic molecules such as
AChR, Na 1 channel, AChE, rapsyn, components of the
dystrophin/utrophin complex, etc. A receptor tyrosine
kinase, MuSK, serves as the signalling component of a
receptor complex that specifically responds to neural agrin
isoforms. At the cytoplasmic face of the postsynaptic
membrane, elements of the cortical skeleton relay agrin
signalling. Rapsyn, the AChR-associated peripheral protein, is recruited in the cascade of events downstream of
MuSK and induces AChR clustering. Dystroglycan, the
major agrin-binding site in the postsynaptic membrane
also participates, probably independently from MuSK, in
the consolidation of AChR macroclusters. Owing to its
capacity to bind laminin and agrin, dystroglycan is
probably responsible for the interaction between the
postsynaptic membrane and the synaptic basal lamina.
An attractive hypothesis is that dystroglycan–agrin interaction also regulates the organization of the asymmetric
AChE form in the synaptic basal lamina (Figure 4).
References
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Postsynaptic Membranes at the Neuromuscular Junction: Molecular Organization
Further Reading
Burden S (1998) The formation of the neuromuscular synapses. Genes
and Development 12: 133–148.
Duclert A and Changeux JP (1995) Acetylcholine receptor gene
expression at the developing neuromuscular junction. Physiological
Review 75: 339–368.
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Sanes JR and Lichtman JW (1999) Development of the vertebrate
neuromuscular junction. Annual Review of Neuroscience 22: 389–442.
ENCYCLOPEDIA OF LIFE SCIENCES / & 2001 Nature Publishing Group / www.els.net
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