Skeletal Muscle Acetylcholine Receptor Research Articles

Acetylcholinesterase activity of skeletal muscle in a non-immunogenic model for myasthenia gravis in rats.

Myasthenia gravis is caused by an autoimmune attack to acetylcholine receptors of skeletal muscle. Acetylcholine release from motor nerve terminals is upregulated in patients with myasthenia gravis and also in rat "myasthenic" models, dependent on the reduction of the number of acetylcholine receptors. This study addresses the question as to whether at "myasthenic" endplates there are changes in the activity of acetylcholinesterase. To this end we studied acetylcholinesterase activity in junctional and extrajunctional regions of dilator naris, extensor digitorum longus, and hemidiaphragm muscles from rats with alpha-bungarotoxin-induced myasthenia gravis. In all studied muscles from "myasthenic" rats there was no significant change of junctional acetylcholinesterase activity. In contrast, in dilator naris and extensor digitorum longus muscles, there was a 60% and 30% increase of extrajunctional acetylcholinesterase activity. There was no significant change in the extrajunctional activity in hemidiaphragm muscles. Velocity sedimentation analysis revealed that the increase in extrajunctional activity in extensor digitorum longus muscles could be attributed to an increase of the activity of the G4 form of acetylcholinesterase. Treatment of rats with 6.4 microgh(-1) neostigmine bromide for 29 days had no influence on junctional and extrajunctional acetylcholinesterase activity of extensor digitorum longus muscles from rats with alpha-bungarotoxin-induced myasthenia gravis.

Myoid cell differentiation of rat thymus-derived progenitor cell line established by coculture with HTLV-I-producing human T cells

P042 Thymus-derived myoid precursor cell line (ST1), which differentiates to myoid cells in the growth arrest condition, was established by the co-cultivating F344 rat thymic cells with human T-lymphotropic virus type-I (HTLV-I)-producing human lymphoid cells. No integration of HTLV-I was detected in ST1 cells by Southern blot hybridization. In the serum starvation condition as the trigger of differentiation, ST1 cells began to fuse creating multinuclear giant cells, and formed a myotube-like structure. Finally, these cells started to contract spontaneously. Under the serum starvation condition, ST1B cells sub-cloned from ST1 cells became to express muscle specific proteins including MyoD1, α-sarcomeric actin, skeletal muscle myosin, myoglobin, desmin and acetylcholine receptor. From the ultrastructural investigation, it was observed that ST1B cells created the aggregates of thick and thin filaments with Z-band-like composition, then forming sarcomeric structures. Transplantation of ST1B cells into nude mice produced small tumor nodules showing clear differentiation to skeletal muscle cells. This cell line did not have colony forming activities in soft agar. These results demonstrate that ST1B cells retain some physiologically normal phenotypes of thymic myoid cells. ST1B cells were also found to specifically adhere to freshly isolated thymocytes in vitro, suggesting some specific cross talking with thymocytes. This system would be a good system to investigate the functional role of thymic myoid cells in terms of differentiation, selection and maturation of thymocytes as well as pathogenesis of HTLV-I associated polymyositis (PM).

Disorders of neuromuscular junction ion channels

Ion channel defects produce a clinically diverse set of disorders that range from cystic fibrosis and some forms of migraine to renal tubular defects and episodic ataxias. This review discusses diseases related to impaired function of the skeletal muscle acetylcholine receptor and calcium channels of the motor nerve terminal. Myasthenia gravis is an autoimmune disease caused by antibodies directed toward the skeletal muscle acetylcholine receptor that compromise neuromuscular transmission. Congenital myasthenias are genetic disorders, a subset of which are caused by mutations of the acetylcholine receptor. Lambert-Eaton myasthenic syndrome is an immune disorder characterized by impaired synaptic vesicle release likely related to a defect of calcium influx. The disorders will illustrate new insights into synaptic transmission and ion channel structure that are relevant for all ion channel disorders.

Expression of acetylcholine receptor mRNAs in atrophying and nonatrophying skeletal muscles of old rats.

We examined the age-related association in skeletal muscle between atrophy and expression of mRNAs encoding both the gamma-subunit of the nicotinic acetylcholine receptor (AChR), and myogenin, a transcription factor that upregulates expression of the gamma-subunit promoter. Gastrocnemius and biceps brachii muscles were collected from young (2-mo-old), adult (18-mo-old), and old (31-mo-old) Fischer 344/Brown Norway F1 generation cross male rats. In the gastrocnemius muscles of old vs. young and adult rats, lower muscle mass was accompanied by significantly elevated AChR gamma-subunit and myogenin mRNA levels. In contrast, the biceps brachii muscle exhibited neither atrophy nor as drastic a change in AChR gamma-subunit and myogenin mRNA levels with age. Expression of the AChR epsilon-subunit mRNA did not change with age in either gastrocnemius or biceps brachii muscles. Thus changes in skeletal muscle AChR gamma-subunit and myogenin mRNA levels may be more related to atrophy than to chronological age in old rats.

An acetylcholine receptor alpha subunit promoter confers intrathymic expression in transgenic mice. Implications for tolerance of a transgenic self-antigen and for autoreactivity in myasthenia gravis.

Myasthenia gravis (MG) is an autoimmune disease targeting the skeletal muscle acetylcholine receptor (AChR). Although the autoantigen is present in the thymus, it is not tolerated in MG patients. In addition, the nature of the cell bearing the autoantigen is controversial. To approach these questions, we used two lineages of transgenic mice in which the beta-galactosidase (beta-gal) gene is under the control of a 842-bp (Tg1) or a 3300-bp promoter fragment (Tg2) of the chick muscle alpha subunit AChR gene. In addition to expression in muscle cells, thymic expression was observed in both mouse lines (mainly in myoid cells in Tg1 and myoid cells and epithelial cells in Tg2). After challenge with beta-gal, Tg1 mice produced Th2-dependent anti-beta-gal antibodies, while Tg2 mice were almost unresponsive. By contrast, in a proliferation assay both Tg lines were unresponsive to beta-gal. Cells from Tg1 mice produce Th2-dependent cytokine whereas cells from Tg2 mice were nonproducing in response to beta-gal. These data indicate that the level of expression in Tg1 mice could be sufficient to induce tolerance of Th1 cells but not of Th2 cells, while both populations are tolerated in Tg2 mice. These findings are compatible with the hypothesis that AChR expression is not sufficiently abundant in MG thymus to induce a full tolerance.

Development of an assay for modulating anti-acetylcholine receptor autoantibodies using human rhabdomyosarcoma cell line

Three types of autoantibodies against the acetylcholine receptors (AChR) of skeletal muscle are detectable in patients with myasthenia gravis including binding, blocking, and modulating anti-AChR antibodies. Modulating autoantibodies correlate best with the severity of the disease, but are also technically most difficult to measure because the assay generally requires fresh human muscle cells. We have developed an assay for the modulation of anti-AChR antibodies using a rhabdomyosarcoma (RD) cell line expressing AChR on the cell surface. By decreasing the FetalClone III serum from 10% to 0.5% in Eagles Minimal Essential Medium (EMEM) we were able to increase the number of AChR on RD cells to meet the need of sensitivity of the assay. The extent of modulation was determined as the percent of AChR internalized in the presence or absence of modulating autoantibodies. Less than 6% modulation was found with the normal serum (n = 42). The CVs of both the intra- and day-to-day precision were less than 20%. When clinical samples (n = 105) were assayed in our laboratory and also at Nichols Institute, a correlation coefficient of 0.816 was obtained. The selection of RD cell line, the success of increasing the expression of the AChR on RD cells and the use of 125I alpha-bungarotoxin of high specific activity allowed the establishment of an assay which can be used in routine clinical laboratory for the measurement of modulating anti-AChR autoantibodies for the management of patients with myasthenia gravis.

The effects of T cell subpopulations and recombinant interleukin (IL)-2 on peripheral B cell function in patients with myasthenia gravis

Myasthenia gravis is an autoimmune disease mediated by antibodies to acetylcholine receptors (AChR) of skeletal muscle. The production of anti-AChR antibodies has been shown to be T cell dependent. To elucidate the mechanism(s) of anti-AChR antibody production in myasthenic patients, we studied the effects of regulatory T cells and/or IL-2 on the differentiation of AChR-primed B cells, with use of AChR stimulation for the induction of anti-AChR antibodies in vitro. Our data suggest that CD8+ T cells possess some complicated functions. CD8+ T cells could not only provide help for B cells to secrete anti-AChR antibody, but also possibly inhibit response of CD4+ T cells or kill B cells, then repress anti-AChR antibody production in MG patients. There might be some defect either in the number or function of CD8+ T cell in MG patients. Exogenous IL-2 could completely restore the suppression activity of CD8+ T cells in anti-AChR antibody production in vitro.

Slow-Channel Transgenic Mice: A Model of Postsynaptic Organellar Degeneration at the Neuromuscular Junction

The slow-channel congenital myasthenic syndrome (SCCMS) is a dominantly inherited disorder of neuromuscular transmission characterized by delayed closure of the skeletal muscle acetylcholine receptor (AChR) ion channel and degeneration of the neuromuscular junction. The identification of a series of AChR subunit mutations in the SCCMS supports the hypothesis that the altered kinetics of the endplate currents in this disease are attributable to inherited abnormalities of the AChR. To investigate the role of these mutant AChR subunits in the development of the synaptic degeneration seen in the SCCMS, we have studied the properties of the AChR mutation, epsilonL269F, found in a family with SCCMS, using both in vitro and in vivo expression systems. The mutation causes a sixfold increase in the open time of AChRs expressed in vitro, similar to the phenotype of other reported mutants. Transgenic mice expressing this mutant develop a syndrome that is highly reminiscent of the SCCMS. Mice have fatigability of limb muscles, electrophysiological evidence of slow AChR ion channels, and defective neuromuscular transmission. Pathologically, the motor endplates show focal accumulation of calcium and striking ultrastructural changes, including enlargement and degeneration of the subsynaptic mitochondria and nuclei. These findings clearly demonstrate the role of this mutation in the spectrum of abnormalities associated with the SCCMS and point to the subsynaptic organelles as principal targets in this disease. These transgenic mice provide a useful model for the study of excitotoxic synaptic degeneration.

Cytokines and the pathogenesis of myasthenia gravis.

Myasthenia gravis (MG) and its animal model experimental autoimmune myasthenia gravis (EAMG) are caused by autoantibodies against nicotinic acetylcholine receptor (AChR) in skeletal muscle. The production of anti-AChR antibodies is mediated by cytokines produced by CD4+ and CD8+ T helper (Th) cells. Emerging investigations of the roles of cytokines in MG and EAMG have revealed that the Th2 cell related cytokine interleukin 4 (IL-4), an efficient growth promoter for B-cell proliferation and differentiation, is important for anti-AChR antibody production. IL-6 and IL-10 have similar effects. The Th1 cytokine IFN-gamma is important in inducing B-cell maturation and in helping anti-AChR antibody production and, thereby, for induction of clinical signs and symptoms. Results from studies of time kinetics of cytokines imply that IFN-gamma is more agile at the onset of EAMG, probably being one of the initiating factors in the induction of the disease, and IL-4 may be mainly responsible for disease progression and persistance. Even though other Th1 cytokines like IL-2, tumor necrosis factor alpha (TNF-alpha), and TNF-beta as well as the cytolytic compound perforin do not directly play a role in T-cell-mediated help for anti-AChR antibody production, they are actually involved in the development of both EAMG and MG, probably by acting in concert with other cytokines within the cytokine network. In contrast, transforming growth factor beta (TGF-beta) exerts immunosuppressive effects which include the down-regulation of both Th1 and Th2 cytokines in MG as well as EAMG. Suppressive effects are also exerted by interferon alpha (IFN-alpha). Based on elucidation of the role of cytokines in EAMG and MG, treatments that up-modulate TGF-beta or IFN-alpha and/or suppress cytokines that help B-cell proliferation could be useful to improve the clinical outcome.

ARIA: a neuromuscular junction neuregulin.

Motor neurons influence the expression and the distribution of acetylcholine receptors in skeletal muscle. Molecules that mediate this carefully choreographed interaction have recently been identified. One of them, ARIA, is a polypeptide purified from chicken brain on the basis of its ability to stimulate the synthesis of muscle acetylcholine receptors. The predicted amino acid sequence suggests that ARIA is synthesized as a transmembrane precursor protein and that it is a member of a family of ligands that activate receptor tyrosine kinases related to the epidermal growth factor receptor. Certain features of the ligand family (the neuregulins) and their receptors (erbBs) are reviewed. Evidence that ARIA plays an important role at developing and mature neuromuscularjunctions is discussed.

Identification of a Determinant of Acetylcholine Receptor Gating Kinetics in the Extracellular Portion of the γ Subunit

A large body of structure-function studies has identified many of the functional motifs underlying ion permeation through acetylcholine receptor (AChR) channels. The structural basis of channel gating kinetics is, however, incompletely understood. We have previously identified a novel shorter form of the AChR gamma subunit, which lacks the 52 amino acids within the extracellular amino-terminal half, encoded by exon 5. To define the contribution of the missing domain to AChR channel function, we have transiently coexpressed the mouse short gamma subunit [gamma(s)] with alpha, beta and delta subunits in human cells and recorded single-channel currents from the resulting AChRs. Our findings show that replacement of the gamma by the gamma(s) subunit confers a long duration characteristic to AChR channel openings without altering unitary conductance sizes or receptor affinity for the transmitter. We also show that alpha beta gamma(s) delta AChR channels exhibit a peculiar voltage sensitivity characterized by a short opening duration when the membrane potential is hyperpolarized. Together, these findings indicate that the domain in the extracellular amino-terminal half of the gamma subunit that encompasses a conserved disulphide loop and a critical tyrosine residue implicated in receptor oligomerization and insertion at the cell surface is a functional motif that also modulates AChR channel gating kinetics. The results also provide a molecular explanation of the functional diversity exhibited by skeletal muscle AChRs during development.

Membrane tethering enables an extracellular domain of the acetylcholine receptor alpha subunit to form a heterodimeric ligand-binding site.

The first step of assembly of the nicotinic acetylcholine receptor (AChR) of adult skeletal muscle is the specific association of the alpha subunit with either delta or epsilon subunits to form a heterodimer with a ligand-binding site. Previous experiments have suggested that het erodimer formation in the ER arises from interaction between the luminal, NH2-terminal domains of the subunits. When expressed in COS cells with the delta subunit, however, the truncated NH2-terminal domain of the subunit folded correctly but did not form a heterodimer. Association with the delta subunit occurred only when the NH2-terminal domain was retained in the ER and was tethered to the membrane by its own M1 transmembrane domain, by the transmembrane domain of another protein, or by a glycolipid link. In each case, the ligand-binding sites of the resulting heterodimers were indistinguishable from that formed when the full-length alpha subunit was used. Attachment to the membrane may promote interaction by concentrating or orienting the subunit; alternatively, a membrane-bound factor may facilitate subunit association.

Maturation of the Acetylcholine Receptor in Skeletal Muscle: Regulation of the AChR γ-to-ϵ Switch

During the development of the mammalian neuromuscular junction, acetylcholine receptors (AChRs) become localized to the postsynaptic muscle membrane. As this process nears completion, the fetal form of the receptor, containing a γ subunit (composition α2βγδ) is gradually replaced by an ϵ subunit-containing adult form (α2βϵδ). To understand how this transition is controlled, we compared the expression and regulation of the AChR γ and ϵ subunits in developing, adult, and cultured muscles. Immunostaining with subunit-specific antibodies showed that replacement of γ subunit- by ϵ subunit-containing AChRs occurs largely during the first postnatal week in fast-twitch muscles, and occurs homogeneously throughout individual endplates. In the slow-twitch soleus, however, this transition is delayed, and in the multiply innervated slow fibers of extraocular muscle, γ subunit expression persists into adulthood. The transcriptional bases of the AChR subunit transition, and of these intermuscular variations, were demonstrated in mice bearing transgenes containing promoter elements from the AChR γ and ϵ subunit genes, each coupled to a nuclear-localized β-galactosidase (nlacZ) reporter. We show that transgene expression is stimulated by the nerve-derived inducer of AChR expression, ARIA, in myotubes cultured from γ–nlacZ as well as ϵ–nlacZ mice. However, the expression of γ–nlacZ, but not ϵ–nlacZ, is increased by treatment of myotubes with TTX, and the ARIA sensitivity of γ–nlacZ is dependent on the electrical state of the myotube. Thus, the promoters of the γ and ϵ subunit genes may integrate ARIA- and activity-dependent signals in different ways to generate their complementary patterns of expression.

Specific removal of antiacetylcholine receptor antibodies in patients with myasthenia gravis

In short-term therapy for myasthenia gravis caused by an antibody-mediated attack on the acetylcholine receptor (AChR) in skeletal muscle, a specific system for antibody removal, the use of tryptophan-bound immunoadsorbent and synthetic AChR peptide-bound immunoadsorbent, offers advantages over plasma exchange. These two types of immunoadsorption provided selective or semi-selective removal of pathogenic substances from the circulation without the use of plasma products, and minimized side-effects. A difficulty is that the former removed about 65% of the total IgG, and the latter removed only a fraction of the pathogenic antibodies. In neither case can a radical method of treatment for myasthenia gravis be expected. Hopefully, an adsorbent which has a well-balanced bioimmunological specific binding reaction and physicochemical adsorptive affinity will be developed in the future.

Specific removal of antiacetylcholine receptor antibodies in patients with myasthenia gravis

In short-term therapy for myasthenia gravis caused by an antibody-mediated attack on the acetylcholine receptor (AChR) in skeletal muscle, a specific system for antibody removal, the use of tryptophan-bound immunoadsorbent and synthetic AChR peptide-bound immunoadsorbent, offers advantages over plasma exchange. These two types of immunoadsorption provided selective or semi-selective removal of pathogenic substances from the circulation without the use of plasma products, and minimized side-effects. A difficulty is that the former removed about 65% of the total IgG, and the latter removed only a fraction of the pathogenic antibodies. In neither case can a radical method of treatment for myasthenia gravis be expected. Hopefully, an adsorbent which has a well-balanced bioimmunological specific binding reaction and physicochemical adsorptive affinity will be developed in the future.

ARIA can be released from extracellular matrix through cleavage of a heparin-binding domain.

ARIA, or acetylcholine receptor-inducing activity, is a polypeptide that stimulates the synthesis of acetylcholine receptors in skeletal muscle. Here we demonstrate that the ability of ARIA to induce phosphorylation of its receptor in muscle is blocked by highly charged glycosaminoglycans. ARIA constructs lacking the NH2-terminal portion, containing an immunoglobulin-like domain, are fully active and are not inhibited by glycosaminoglycans. Limited proteolysis of ARIA with subtilisin blocks the glycosaminoglycan interaction by degrading this NH2-terminal portion, but preserves the active, EGF-like domain. We also show that ARIA can be released from freshly dissociated cells from embryonic chick spinal cord and cerebellum by either heparin, high salt or limited proteolysis with subtilisin, suggesting that ARIA is bound to the extracellular matrix through charged interactions. We present a model of how ARIA may be stored in extracellular matrix at developing synapses and how its release may be mediated by local proteolysis.

Differential Expression Patterns of Five Acetylcholine Receptor Subunit Genes in Rat Muscle During Development

The spatial and temporal expression patterns of five genes which encode the alpha-, beta-, gamma-, delta- and epsilon-subunits of the nicotinic acetylcholine receptor in skeletal muscle were followed during development in the rat by in situ hybridization analysis. Three major developmental phases, characterized by specific expression patterns, could be distinguished. (i) During myogenic differentiation alpha-, beta-, gamma- and delta-subunit genes are activated and transcripts are expressed in muscle precursor cells at embryonic day 12 (E12) and during subsequent cell fusion. (ii) Following innervation of myotubes at approximately E15-E17 the mRNA of the alpha-, beta-, gamma- and delta-subunit genes accumulate in synaptic and decrease in extrasynaptic fibre regions during early synaptogenesis. The mRNA of the epsilon-subunit gene becomes detectable first in subsynaptic nuclei 2-3 days after innervation has occurred. (iii) During postnatal development alpha-, beta- and delta- subunit transcript levels are reduced predominantly in extrasynaptic fibre segments and show significant differences in distribution depending on the muscle subtype whereas the gamma-subunit mRNA disappears completely within the first postnatal week in all muscles. In contrast, the epsilon-subunit gene is transcribed only in subsynaptic myonuclei throughout development and in the adult muscle.

A role for calcitonin gene-related peptide in the regulation of rat skeletal muscle G 4 acetylcholinesterase

Calcitonin gene-related peptide (CGRP) acts as an anterograde trophic agent which regulates skeletal muscle acetylcholine receptor function. We examined whether CGRP also influences other synaptic transmission-related molecules, i.e. acetylcholinesterase (AChE) forms. Results show that: (a) CGRP associated with rat anterior gracilis muscle endplates declines following obturator nerve transection; (b) exogenous CGRP treatment has a selective, innervation-like effect on the globular tetramer (G4) of AChE in gracilis motor endplates; and (c) this effect is reversed by the CGRP receptor antagonist hCGRP8-37. We conclude that exogenous CGRP, and/or a biologically active CGRP fragment(s), influences G4 AChE levels through specific CGRP-CGRP receptor interactions. This conclusion is consistent with the notion that motor nerve-derived CGRP participates in the trophic control of G4 AChE at the neuromuscular junction.

A leucine‐to‐phenylalanine substitution in the acetylcholine receptor ion channel in a family with the slow‐channel syndrome

The slow-channel syndrome is one of several congenital myasthenic syndromes that result from inherited abnormalities of the ion channel of the skeletal muscle acetylcholine receptor (AChR). The ion channel is formed by the second transmembrane domains (M2) of the four AChR subunits. We screened the genomic DNA of one family with the slow-channel syndrome for mutations in the coding sequences for the M2 domains of the four AChR subunits and report the identification of a missense mutation that causes a leucine-to-phenylalanine substitution at position 269 of the epsilon subunit in three affected members of a family with the slow-channel syndrome. We propose that this mutation may be responsible for the disease.

Two forms of acetylcholine receptor gamma subunit in mouse muscle.

Nicotinic acetylcholine receptors (nAcChoRs) of skeletal muscle are heterosubunit ligand-gated channels that mediate signal transmission from motor nerves to muscle. While cloning murine nAcChoR subunits, to gain an insight into the receptor diversity across species, we detected two forms of gamma subunits in the myogenic C2C12 cell line. Both forms are functional when expressed in Xenopus oocytes. One gamma subunit [long gamma (gamma 1)] was almost identical to that previously cloned in the murine BC3H-1 tumor cell line. The second form of gamma subunit [short gamma (gamma s)] lacked 156 bp (52 amino acids) in the extracellular N terminus, adjoining the hydrophobic segment M1, which corresponds to the fifth exon of the gamma-subunit gene. The two forms of gamma subunit coexist during myogenesis in vitro and in 17-day embryonic and denervated adult muscle fibers in vivo. However, the gamma s variant was the only form of gamma subunit in newborn muscle. In dissociated muscle fibers of newborn mice, AcCho-evoked channel openings were more prolonged when compared with C2C12 myotubes or denervated adult muscle fibers. The gamma s subunit may, thus, contribute to the structural and functional diversity of nAcChoRs in muscle cells.