Neuregulin/ErbB regulate neuromuscular junction development by phosphorylation of α-dystrobrevin

Neuregulin (NRG)/ErbB signaling is involved in numerous developmental processes in the nervous system, including synapse formation and function in the central nervous system. Although intensively investigated, its role at the neuromuscular synapse has remained elusive. Here, we demonstrate that loss of neuromuscular NRG/ErbB signaling destabilized anchoring of acetylcholine receptors (AChRs) in the postsynaptic muscle membrane and that this effect was caused by dephosphorylation of α-dystrobrevin1, a component of the postsynaptic scaffold. Specifically, in mice in which NRG signaling to muscle was genetically or pharmacologically abolished, postsynaptic AChRs moved rapidly from the synaptic to the perisynaptic membrane, and the subsynaptic scaffold that anchors the AChRs was impaired. These defects combined compromised synaptic transmission. We further show that blockade of NRG/ErbB signaling abolished tyrosine phosphorylation of α-dystrobrevin1, which reduced the stability of receptors in agrin-induced AChR clusters in cultured myotubes. Our data indicate that NRG/ErbB signaling maintains high efficacy of synaptic transmission by stabilizing the postsynaptic apparatus via phosphorylation of α-dystrobrevin1.     

Nicotine decreases agrin signaling and acetylcholine receptor clustering in C2C12 myotube culture

The clustering of acetylcholine receptors (AChRs) in skeletal muscle fibers is a critical event in neuromuscular synaptogenesis. AChRs in concert with other molecules form postsynaptic scaffolds in response to agrin released from motor neurons as motor neurons near skeletal muscle fibers in development. Agrin drives an intracellular signaling pathway that precedes AChR clustering and includes the tyrosine phosphorylation of AChRs. In C2C12 myotube culture, agrin application stimulates the agrin signaling pathway and AChR clustering. Previous studies have determined that the frequency of spontaneous AChR clustering is decreased and AChRs are partially inactivated when bound by the acetylcholine agonist nicotine. We hypothesized that nicotine interferes with AChR clustering and consequent postsynaptic scaffold formation. In the present study, C2C12 myoblasts were cultured with growth medium to stimulate proliferation and then differentiation medium to stimulate fusion into myotubes. They were bathed in a physiologically relevant concentration of nicotine and then subject to agrin treatment after myotube formation. Our results demonstrate that nicotine decreases agrin-induced tyrosine phosphorylation of AChRs and decreases the frequency of spontaneous as well as agrin-induced AChR clustering. We conclude that nicotine interferes with postsynaptic scaffold formation by preventing the tyrosine phosphorylation of AChRs, an agrin signaling event that precedes AChR clustering.     

Nitric oxide synthase activity is required for postsynaptic differentiation of the embryonic neuromuscular junction

Agrin, a synapse-organizing protein externalized by motor axons at the neuromuscular junction (NMJ), initiates a signaling cascade in muscle cells leading to aggregation of postsynaptic proteins, including acetylcholine receptors (AChRs). We examined whether nitric oxide synthase (NOS) activity is required for agrin-induced aggregation of postsynaptic AChRs at the embryonic NMJ in vivo and in cultured muscle cells. Inhibition of NOS reduced AChR aggregation at embryonic Xenopus NMJs by 50-90%, whereas overexpression of NOS increased AChR aggregate area 2- to 3-fold at these synapses. NOS inhibitors completely blocked agrin-induced AChR aggregation in cultured embryonic muscle cells. Application of NO donors to muscle cells induced AChR clustering in the absence of agrin. Our results indicate that NOS activity is necessary for postsynaptic differentiation of embryonic NMJs and that NOS is a likely participant in the agrin-MuSK signaling pathway of skeletal muscle cells.     

Phosphoinositide 3-kinase acts through RAC and Cdc42 during agrin-induced acetylcholine receptor clustering

The formation of the neuromuscular junction (NMJ) is regulated by the nerve-derived heparan sulfate proteoglycan agrin and the muscle-specific kinase MuSK. Agrin induces a signal transduction pathway via MuSK, which promotes the reorganization of the postsynaptic muscle membrane. Activation of MuSK leads to the phosphorylation and redistribution of acetylcholine receptors (AChRs) and other postsynaptic proteins to synaptic sites. The accumulation of high densities of AChRs at postsynaptic regions represents a hallmark of NMJ formation and is required for proper NMJ function. Here we show that phosphoinositide 3-kinase (PI3-K) represents a component of the agrin/MuSK signaling pathway. Muscle cells treated with specific PI3-K inhibitors are unable to form full-size AChR clusters in response to agrin and AChR phosphorylation is reduced. Moreover, agrin-induced activation of Rac and Cdc42 is impaired in the presence of PI3-K inhibitors. PI3-K is localized to the postsynaptic muscle membrane consistent with a role during agrin/MuSK signaling. These results put PI3-K downstream of MuSK as regulator of AChR phosphorylation and clustering. Its role during agrin-stimulated Rac and Cdc42 activation suggests a critical function during cytoskeletal reorganizations, which lead to the redistribution of actin-anchored AChRs.     

Neural agrin increases postsynaptic ACh receptor packing by elevating rapsyn protein at the mouse neuromuscular synapse

Fluorescence resonance energy transfer (FRET) experiments at neuromuscular junctions in the mouse tibialis anterior muscle show that postsynaptic acetylcholine receptors (AChRs) become more tightly packed during the first month of postnatal development. Here, we report that the packing of AChRs into postsynaptic aggregates was reduced in 4-week postnatal mice that had reduced amounts of the AChR-associated protein, rapsyn, in the postsynaptic membrane (rapsyn(+/-) mice). We hypothesize that nerve-derived agrin increases postsynaptic expression and targeting of rapsyn, which then drives the developmental increase in AChR packing. Neural agrin treatment elevated the expression of rapsyn in C2 myotubes by a mechanism that involved slowing of rapsyn protein degradation. Similarly, exposure of synapses in postnatal muscle to exogenous agrin increased rapsyn protein levels and elevated the intensity of anti-rapsyn immunofluorescence, relative to AChR, in the postsynaptic membrane. This increase in the rapsyn-to-AChR immunofluorescence ratio was associated with tighter postsynaptic AChR packing and slowed AChR turnover. Acute blockade of synaptic AChRs with alpha-bungarotoxin lowered the rapsyn-to-AChR immunofluorescence ratio, suggesting that AChR signaling also helps regulate the assembly of extra rapsyn in the postsynaptic membrane. The results suggest that at the postnatal neuromuscular synapse agrin signaling elevates the expression and targeting of rapsyn to the postsynaptic membrane, thereby packing more AChRs into stable, functionally-important AChR aggregates.     

Lithium inhibits a late step in agrin-induced AChR aggregation

Agrin activates an intracellular signaling pathway to induce the formation of postsynaptic specializations on muscle fibers. In myotubes in culture, this pathway has been shown to include autophosphorylation of the muscle-specific kinase MuSK, activation of Src-family kinases, tyrosine phosphorylation of the acetylcholine receptor (AChR) beta subunit, a decrease in receptor detergent extractability, and the accumulation of AChRs into high-density aggregates. Here we report that treating chick myotubes with lithium prevented any detectable agrin-induced change in AChR distribution without affecting the number of AChRs or the agrin-induced change in AChR tyrosine phosphorylation and detergent extractability. Lithium treatment also increased the rate at which AChR aggregates disappeared when agrin was removed. The effects of lithium developed slowly over the course of approximately 12 h. Thus, sensitivity to lithium identifies a late step in the agrin signaling pathway, after agrin-induced MuSK and AChR phosphorylation, that is necessary for the recruitment of AChRs into visible aggregates.     

Lithium inhibits a late step in agrin‐induced AChR aggregation

Agrin activates an intracellular signaling pathway to induce the formation of postsynaptic specializations on muscle fibers. In myotubes in culture, this pathway has been shown to include autophosphorylation of the muscle‐specific kinase MuSK, activation of Src‐family kinases, tyrosine phosphorylation of the acetylcholine receptor (AChR) β subunit, a decrease in receptor detergent extractability, and the accumulation of AChRs into high‐density aggregates. Here we report that treating chick myotubes with lithium prevented any detectable agrin‐induced change in AChR distribution without affecting the number of AChRs or the agrin‐induced change in AChR tyrosine phosphorylation and detergent extractability. Lithium treatment also increased the rate at which AChR aggregates disappeared when agrin was removed. The effects of lithium developed slowly over the course of approximately 12 h. Thus, sensitivity to lithium identifies a late step in the agrin signaling pathway, after agrin‐induced MuSK and AChR phosphorylation, that is necessary for the recruitment of AChRs into visible aggregates. © 2002 Wiley Periodicals, Inc. J Neurobiol 54: 346–357, 2003     

L-type calcium channels mediate acetylcholine receptor aggregation on cultured muscle

Agrin activation of muscle specific kinase (MuSK) initiates postsynaptic development on skeletal muscle that includes the aggregation of acetylcholine receptors (AChRs; Glass et al. [1996]: Cell 85: 513-523; Gautam et al. [1996]: Cell 85: 525-535). Although the agrin/MuSK signaling pathway remains largely unknown, changes in intracellular calcium levels are required for agrin-induced AChR aggregation (Megeath and Fallon [1998]: J Neurosci 18: 672-678). Here, we show that L-type calcium channels (L-CaChs) are required for full agrin-induced aggregation of AChRs and sufficient to induce agrin-independent AChR aggregation. Blockade of L-CaChs in muscle cultures inhibited agrin-induced AChR aggregation but not tyrosine phosphorylation of MuSK or AChR beta subunits. Activation of L-CaChs in the absence of agrin induced AChR aggregation but not tyrosine phosphorylation of MuSK or AChR beta subunits. Agrin responsiveness was significantly reduced in primary muscle cultures from the muscular dysgenesis mouse, a natural mutant, which does not express the L-CaCh. Our results establish a novel role for L-CaChs as important sources of the intracellular calcium necessary for the aggregation of AChRs.     

Affinity labelling of neuronal acetylcholine receptors localizes acetylcholine-binding sites to their beta-subunits

Neuronal nicotinic acetylcholine receptors (AChRs) from brains of chickens and rats consist of two types of subunits, alpha and beta, of which alpha shares some antigenic determinants with alpha-subunits from AChRs of electric organ and muscle [(1986) Biochemistry 25, 2082-2093; (1986) J. Neurosci. G, 3061-3069; (1986) Proc. Natl. Acad. Sci. USA, in press]. Here we demonstrate that after reduction with dithiothreitol (DTT) the AChRs can be specifically labelled with the acetylcholine-binding site directed reagent 4-(N-maleimido)benzyltri [3H]methylammonium iodide. Labelling of the beta-subunits of neuronal nicotinic AChRs indicates that the acetylcholine-binding site, and amino acids which may be homologous to Cys 192-193 of the alpha-subunits of AChRs from electric organ and muscle, are located on the beta-subunit of neuronal AChRs. These results suggest that although neuronal nicotinic AChRs have some structural homologies to AChRs from muscle and electric organs, the AChRs from these sources are quite distant relatives in an extended gene family.     

The postsynaptic submembrane machinery at the neuromuscular junction: Requirement for rapsyn and the utrophin/dystrophin-associated complex

Neuromuscular synapse formation is brought about by a complex bi-directional exchange of information between the innervating motor neuron and its target skeletal muscle fiber. Agrin, a heparin sulfate proteoglycan, is released from the motor nerve terminal to activate its muscle-specific kinase (MuSK) receptor that leads to a second messenger cascade requiring rapsyn to ultimately bring about AChR clustering in the muscle membrane. Rapsyn performs many functions in skeletal muscle. First, rapsyn and AChRs co-target to the postsynatic apparatus. Second, rapsyn may self associate to stabilize and promote AChR clustering. Third, rapsyn is essential for AChR cluster formation. Fourth, rapsyn is required to transduce the agrin-evoked MuSK phosphorylation signal to AChRs. Finally, rapsyn links AChRs to the utrophin-associated complex, which appears to be required for AChR stabilization as well as maturation of the neuromuscular junction. Proteins within the utrophin-associated complex such as α-dystrobrevin and α-syntrophin are also important for signaling events that affect neuromuscular synapse stability and function. Here we review our current understanding of the role of the postsynaptic-submembrane machinery involving rapsyn and the utrophin-associated complex at the neuromuscular synapse. In addition we briefly review how these studies of the neuromuscular junction relate to GABAergic and glycinergic synapses in the CNS.     

Structure and transmembrane nature of the acetylcholine receptor in amphibian skeletal muscle as revealed by cross-reacting monoclonal antibodies

A collection of 126 monoclonal antibodies (mAbs) made against acetylcholine receptors (AChRs) from the electric organs of Torpedo californica or Electrophorus electricus was tested for cross-reactivity with AChRs in cryostat sections of skeletal muscle from Rana pipiens and Xenopus laevis by indirect immunofluorescence. 49 mAbs (39%) cross-reacted with AChRs from Rana, and 25 mAbs (20%) cross-reacted with AChRs from Xenopus. mAbs specific for each of the four subunits of electric organ AChR (alpha, beta, gamma, delta) cross-reacted with AChRs from each amphibian species. mAbs cross-reacting with Xenopus AChRs were, with one exception, a subset of the mAbs cross-reacting with Rana AChRs. The major difference detected between the two species was in binding by mAbs specific for the main immunogenic region (MIR) of the alpha-subunit. Whereas 22 of 33 anti-MIR mAbs tested cross-reacted with Rana AChRs, only one of these mAbs cross-reacted with Xenopus AChRs. Some (32) of the cross-reacting mAbs were tested for binding to AChRs in intact muscle. 21 of these mAbs bound to AChRs only when membranes were made permeable with saponin. Electron microscopy using immunoperoxidase or colloidal gold techniques revealed that these mAbs recognize cytoplasmic determinants and that mAbs that do not require saponin in order to bind AChRs in intact muscle recognize extracellular determinants. These results suggest that AChRs in skeletal muscle of Rana and Xenopus are composed of subunits corresponding to the alpha-, beta-, gamma-, and delta-subunits of AChRs from fish electric organs. The subunit specificity of mAbs whose binding was examined by electron microscopy suggests that parts of each subunit (alpha, beta, gamma, delta) are exposed on the cytoplasmic surface and that, as in AChRs from fish electric organs and mammalian muscle, the MIR on alpha-subunits of Rana AChRs is exposed on the extracellular surface.     

A role for acetylcholine receptors in their own aggregation on muscle cells

Both neurotrophic factors and activity regulate synaptogenesis. At neuromuscular synapses, the neural factor agrin released from motor neuron terminals stimulates postsynaptic specialization by way of the muscle specific kinase MuSK. In addition, activity through acetylcholine receptors (AChRs) has been implicated in the stabilization of pre- and postsynaptic contacts on muscle at various stages of development. We show here that activation of AChRs with specific concentrations of nicotine is sufficient to induce AChR aggregation and that this induction requires the function of L-type calcium channels (L-CaChs). Furthermore, AChR function is required for agrin induced AChR aggregation in C2 muscle cells. The same concentrations of nicotine did not induce observable tyrosine phosphorylation on either MuSK or the AChR beta subunit, suggesting significant differences between the mechanisms of agrin and activity induced aggregation. The AChR/L-CaCh pathway provides a mechanism by which neuromuscular signal transmission can act in concert with the agrin-MuSK signaling cascade to regulate NMJ formation.     

HGF induction of postsynaptic specializations at the neuromuscular junction

A critical event in the formation of vertebrate neuromuscular junctions (NMJs) is the postsynaptic clustering of acetylcholine receptors (AChRs) in muscle. AChR clustering is triggered by the activation of MuSK, a muscle-specific tyrosine kinase that is part of the functional receptor for agrin, a nerve-derived heparan sulfate proteoglycan (HSPG). At the NMJ, heparan sulfate (HS)-binding growth factors and their receptors are also localized but their involvement in postsynaptic signaling is poorly understood. In this study we found that hepatocyte growth factor (HGF), an HS-binding growth factor, surrounded muscle fibers and was localized at NMJs in rat muscle sections. In cultured Xenopus muscle cells, HGF was enriched at spontaneously occurring AChR clusters (hot spots), where HSPGs were also concentrated, and, following stimulation of muscle cells by agrin or cocultured neurons, HGF associated with newly formed AChR clusters. HGF presented locally to cultured muscle cells by latex beads induced new AChR clusters and dispersed AChR hot spots, and HGF beads also clustered phosphotyrosine, activated c-Met, and proteins of dystrophin complex; clustering of AChRs and associated proteins by HGF beads required actin polymerization. Lastly, although bath-applied HGF alone did not induce new AChR clusters, addition of HGF potentiated agrin-dependent AChR clustering in muscle. Our findings suggest that HGF promotes AChR clustering and synaptogenic signaling in muscle during NMJ development.     

Nitric oxide and cyclic GMP regulate early events in agrin signaling in skeletal muscle cells

Agrin released from motor nerve terminals directs differentiation of the vertebrate neuromuscular junction (NMJ). Activity of nitric oxide synthase (NOS), guanylate cyclase (GC), and cyclic GMP-dependent protein kinase (PKG) contributes to agrin signaling in embryonic frog and chick muscle cells. Stimulation of the NO/cyclic GMP (cGMP) pathway in embryos potentiates agrin's ability to aggregate acetylcholine receptors (AChRs) at NMJs. Here we investigated the timing and mechanism of NO and cGMP action. Agrin increased NO levels in mouse C2C12 myotubes. NO donors potentiated agrin-induced AChR aggregation during the first 20 min of agrin treatment, but overnight treatment with NO donors inhibited agrin activity. Adenoviruses encoding siRNAs against each of three NOS isoforms reduced agrin activity, indicating that these isoforms all contribute to agrin signaling. Inhibitors of NOS, GC, or PKG reduced agrin-induced AChR aggregation in mouse muscle cells by ∼ 50%. However, increased activation of the GTPase Rac1, an early step in agrin signaling, was dependent on NOS activity and was mimicked by NO donors and a cGMP analog. Our results indicate that stimulation of the NO/cGMP pathway is important during the first few minutes of agrin signaling and is required for agrin-induced Rac1 activation, a key step leading to reorganization of the actin cytoskeleton and subsequent aggregation of AChRs on the surface of skeletal muscle cells.     

Molecular regulation of postsynaptic differentiation at the neuromuscular junction

The neuromuscular junction (NMJ) is a synapse that develops between a motor neuron and a muscle fiber. A defining feature of NMJ development in vertebrates is the re-distribution of muscle acetylcholine (ACh) receptors (AChRs) following innervation, which generates high-density AChR clusters at the postsynaptic membrane and disperses aneural AChR clusters formed in muscle before innervation. This process in vivo requires MuSK, a muscle-specific receptor tyrosine kinase that triggers AChR re-distribution when activated; rapsyn, a muscle protein that binds and clusters AChRs; agrin, a nerve-secreted heparan-sulfate proteoglycan that activates MuSK; and ACh, a neurotransmitter that stimulates muscle and also disperses aneural AChR clusters. Moreover, in cultured muscle cells, several additional muscle- and nerve-derived molecules induce, mediate or participate in AChR clustering and dispersal. In this review we discuss how regulation of AChR re-distribution by multiple factors ensures aggregation of AChRs exclusively at NMJs.     

Sciatin: a myotrophic protein increases the number of acetylcholine receptors and receptor clusters in cultured skeletal muscle

Factors present in neural extracts or in media conditioned by neurons have been shown by others to increase both the number of acetylcholine receptors (AChRs) and the number of receptor clusters in cultures of embryonic skeletal muscle. We have recently shown that the glycoprotein, sciatin, exerts trophic effects on developing muscle in vitro. In the present study, we investigated the effect of sciatin on AChRs in aneural cultures of chick skeletal muscle. Sciatin caused a significant increase in the number of AChRs/dish as measured by binding of ¹²⁵I-α-bungarotoxin (α-Btx) and in acetylcholinesterase (AChE) activity/dish in differentiating muscle cells. The increase in AChRs elicited by sciatin was due solely to increased receptor synthesis and incorporation. The rate of AChR synthesis in sciatin-treated cultures was as much as five times the control rate and was significantly reduced by cycloheximide (10 μM). AChR degradation was unaffected by the myotrophic protein. Although the number of AChRs/dish was increased by sciatin during myogenesis, AChR specific activity, expressed as picomoles ¹²⁵I-α-Btx bound/mg cell protein, was only transiently increased by the myotrophic protein. This contrasted with AChE specific activity in sciatin-treated cultures which remained elevated throughout differentiation. Autoradiographs of ¹²⁵I-α-Btx-labeled cultures showed that sciatin caused an increase in the number and size of AChR “hot spots” and maintained the integrity of these AChR clusters in aneural muscle cultures for up to 5 weeks. At this time control cultures had completely degenerated. The mechanism by which sciatin enhanced the synthesis of AChRs appeared to be distinct from that of tetrodotoxin (TTX), an agent which abolishes muscle activity. However, like theophylline, sciatin might evoke increased synthesis of AChRs via regulation of cyclic AMP since the myotrophic protein increased cAMP both in cells and in conditioned medium. The results of this study suggest that sciatin may be related to the diffusible factor(s) from motor neurons described by others which has trophic effects on AChRs. Furthermore, we suggest that this myotrophic protein may be responsible for the clustering of AChRs and maintenance of receptor clusters at neuromuscular junctions in developing avian muscle.     

Neuregulin inhibits acetylcholine receptor aggregation in myotubes

The high local concentration of acetylcholine receptors (AChRs) at the vertebrate neuromuscular junction results from their aggregation by the agrin/MuSK signaling pathway and their synthetic up-regulation by the neuregulin/ErbB pathway. Here, we show a novel role for the neuregulin/ErbB pathway, the inhibition of AChR aggregation on the muscle surface. Treatment of C2C12 myotubes with the neuregulin epidermal growth factor domain decreased the number of both spontaneous and agrin-induced AChR clusters, in part by increasing the rate of cluster disassembly. Upon cluster disassembly, AChRs were internalized into caveolae (as identified by caveolin-3). Time-lapse microscopy revealed that individual AChR clusters fragmented into puncta, and application of neuregulin accelerated the rate at which AChR clusters decreased in area without affecting the density of AChRs remaining in individual clusters (as measured by the fluorescence intensity/unit area). We propose that this novel action of neuregulin regulates synaptic competition at the developing neuromuscular junction.     

Anti-agrin staining is absent at abandoned synaptic sites of frog neuromuscular junctions

The neuromuscular junction is a plastic structure and is constantly undergoing changes as the nerve terminals that innervate the muscle fiber extend and retract their processes. In vivo observations on developing mouse neuromuscular junctions revealed that prior to the retraction of a nerve terminal the acetylcholine receptors (AChRs) under that nerve terminal disperse. Agrin is a protein released by nerve terminals that binds to synaptic basal lamina and directs the aggregation of AChRs and acetylcholinesterase (AChE) in and on the surface of the myotube. Thus, if the AChRs under a nerve terminal disperse, then the cellular signaling mechanism by which agrin maintains the aggregation of those AChRs must have been disrupted. Two possibilities that could lead to the disruption of the agrin induced aggregation are that agrin is present at the synaptic basal lamina but is unable to direct the aggregation of AChRs, or that agrin has been removed from the synaptic basal lamina. Thus, if agrin were blocked, one would expect to see anti-agrin staining at abandoned synaptic sites; whereas if agrin were removed, anti-agrin staining would be absent at abandoned synaptic sites. We find that anti-agrin staining and α-bungarotoxin staining are absent at abandoned synaptic sites. Further, in vivo observations of retracting nerve terminals confirm that agrin is removed from the synaptic basal lamina within 7 days. Thus, while agrin will remain bound to synaptic basal lamina for months following denervation, it is removed within days following synaptic retraction. © 1996 John Wiley & Sons, Inc.     

Stabilization of Acetylcholine Receptors by Exogenous ATP and Its Reversal by cAMP and Calcium

Innervation of the neuromuscular junction (nmj) affects the stability of acetylcholine receptors (AChRs). A neural factor that could affect AChR stabilization was studied using cultured muscle cells since they express two distinct populations of AChRs similar to those seen at the nmjs of denervated muscle. These two AChR populations are (in a ratio of 9 to 1) a rapidly degrading population (Rr) with a degradation half-life of ~1 d and a slowly degrading population (Rs) that can alternate between an accelerated form (half-life ~3–5 d) and a stabilized form (half-life ~10 d), depending upon the state of innervation of the muscle.

Previous studies have shown that elevation of intracellular cAMP can stabilize the Rs, but not the Rr. We report here that in cultured rat muscle cells, exogenous ATP stabilized the degradation half-life of Rr and possibly also the Rs. Furthermore, pretreatment with ATP caused more stable AChRs to be inserted into the muscle membrane. Thus, in the presence of ATP, the degradation rates of the Rr and Rs overlap. This suggests that ATP released from the nerve may play an important role in the regulation of AChR degradation. Treatment with either the cAMP analogue dibutyryl-cAMP (dB-cAMP) or the calcium mobilizer ryanodine caused the ATP-stabilized Rr to accelerate back to a half-life of 1 d. Thus, at least three signaling systems (intracellular cAMP, Ca²⁺, and extracellular ATP) have the potential to interact with each other in the building of an adult neuromuscular junction.

     

Developmental increase in the amount of rapsyn per acetylcholine receptor promotes postsynaptic receptor packing and stability

Neuromuscular synaptic transmission depends upon tight packing of acetylcholine receptors (AChRs) into postsynaptic AChR aggregates, but not all postsynaptic AChRs are aggregated. Here we describe a new confocal Fluorescence Resonance Energy Transfer (FRET) assay for semi-quantitative comparison of the degree to which AChRs are aggregated at synapses. During the first month of postnatal life the mouse tibialis anterior muscle showed increases both in the number of postsynaptic AChRs and the efficiency with which AChR was aggregated (by FRET). There was a concurrent two-fold increase in immunofluorescent labeling for the AChR-associated cytoplasmic protein, rapsyn. When 1-month old muscle was denervated, postsynaptic rapsyn immunostaining was reduced, as was the efficiency of AChR aggregation. In vivo electroporation of rapsyn-EGFP into muscle fibers increased postsynaptic rapsyn levels. Those synapses with higher ratios of rapsyn-EGFP to AChR displayed a slower metabolic turnover of AChR. Conversely, the reduction of postsynaptic rapsyn after denervation was accompanied by an acceleration of AChR turnover. Thus, a developmental increase in the amount of rapsyn targeted to the postsynaptic membrane may drive enhanced postsynaptic AChRs aggregation and AChR stability within the postsynaptic membrane.