Mouse Acetylcholine Receptor Research Articles

Alterations in cardiac muscarinic acetylcholine receptors in mice with autoimmune myocarditis and association with circulating muscarinic receptor-related autoantibodies

Hearts from mice hyperimmunized with cardiac tissue were studied to evaluate the expression and biological activity of muscarinic cholinergic receptors and immunoglobulin G deposits along the immunization period. Mice were sacrificed at 10 day intervals from the first injection up to day 100. Simultaneously, the activity of autoantibodies against muscarinic receptors on normal hearts was also examined in sera. Hearts with autoimmune myocarditis showed a muscarinic receptor-related dysfunction, with an impaired response to exogenous muscarinic agonists and a significant reduction in muscarinic binding sites, both effects being maximum at 40-50 days post-immunization. In addition, serum or immunoglobulin G from mice with myocarditis were able to interact with muscarinic acetylcholine receptors displaying a partial agonist effect. Autoimmune sera and immunoglobulin G reduced heart contractility while inhibited 3H-QNB binding to cardiac acetylcholine receptors in a concentration dependent manner showing the highest effects at days 40-50 and decreased progressively thereafter. The development of muscarinic receptor-related cardiac dysfunction may be associated with the presence of circulating antibodies having muscarinic receptor activity. These studies are of relevance to clinical conditions such as Chagas' disease, where immunological processes involving the cholinergic system are considered to cause cardiomyopathy.

Mouse muscle epsilon- and gamma-containing acetylcholine receptors expressed in Xenopus laevis oocytes do not differ in their degradation half-lives.

Mouse acetylcholine receptors (AChRs) consisting of either the embryonic form (2 alpha, 1 beta, 1 delta and 1 gamma) or the adult form (2 alpha, 1 beta, 1 delta and 1 epsilon) were expressed in Xenopus laevis oocytes. As expected, the single channel conductance was approximately 52 pS and the exponential decay time constants were 2.2 and 8.2 ms for the gamma-AChR and respectively 65 pS and 0.6 and 2.8 ms for the epsilon-AChR. No difference was seen in the degradation rate between the gamma- and epsilon-containing AChRs, both having a half-life of about 5 days.

Differential interactions of gentamicin with mouse junctional and extrajunctional ACh receptors expressed in Xenopus oocytes

The nicotinic acetylcholine receptors (AChRs) from Torpedo electric organ and mouse muscles when expressed in Xenopus oocytes desensitize with different time courses. Initially, the role of cAMP-dependent phosphorylation on the γ subunits in the different desensitization rates was investigated by expressing normal and mutant AChRs in the oocytes cultured in the presence of gentamicin. Mutant Torpedo AChRs lacking the potential cAMP-dependent phosphorylation sites in the γ subunit appear to desensitize like normal Torpedo AChRs. Similarly, mutant mouse extrajunctional AChRs containing a newly created phosphorylation site in the γ subunit appeared to desensitize like normal mouse AChRs, which lack the potential cAMP-dependent phosphorylation site in the γ subunit. These results suggest that different rates of desensitization between the Torpedo and muscle extrajunctional AChRs are not attributable to differential cAMP-dependent phosphorylation of these AChRs. Subsequently, to determine whether gentamicin used in culturing oocytes differentially interacts with muscle junctional and extrajunctional AChRs, we analyzed rates of current decay following different gentamicin treatments. Both chronic and acute treatment with gentamicin profoundly accelerated the decay of whole-cell currents mediated by both types of AChR. The effect of prolonged gentamicin treatment on junctional AChRs was long lasting when compared to treatment on extrajunctional AChRs. Although the two types of AChR still desensitize differently in the absence of gentamicin, these results suggest that the characteristic desensitization of junctional and extrajunctional AChRs observed previously is largely due to differential interactions of gentamicin with the two types of AChR.

Cell type- and differentiation-dependent expression from the mouse acetylcholine receptor epsilon-subunit promoter.

The nicotinic acetylcholine receptor (AChR) in adult skeletal muscle is composed of alpha-, beta-, epsilon-, and delta-subunits and is localized at the neuromuscular junction; in contrast, the more diffusely distributed fetal form is composed of alpha-, beta-, gamma-, and delta-subunits. To define sequences necessary for the transcriptional regulation of the mouse epsilon-subunit gene, we sequenced and analyzed 1036 bp upstream of the transcription start site. Using deletion analysis of the 5'-flanking region linked to the bacterial chloramphenicol acetyltransferase (CAT) gene and transfection of the resulting constructs into established cell lines, we demonstrate that a 151 bp fragment exhibits cell type- and differentiation-specific promoter activity. This activity was independent of a myogenic factor putative binding site (E-box). However, transactivation experiments with recombinant myoD, myogenin, or MRF4 showed that the E-box was functional and that MRF4 preferentially transactivates the epsilon-promoter. Thus, like other AChR promoters, the proximal region of the epsilon-promoter contains information for cell type-specific and developmental regulation of CAT and can be transactivated by myogenic factors in cultured cell lines. Unlike the other AChR promoters characterized to date, epsilon-promoter function can be partially independent of myogenic factors of the helix-loop-helix class.

Pathogenic autoimmunity to affinity‐purified mouse acetylcholine receptor induced without adjuvant in BALB/c mice

Myasthenia gravis (MG) and experimental autoimmune myasthenia gravis (EAMG) are antibody-mediated disorders in which anti-acetylcholine receptor (anti-AChR) antibodies cause loss of muscle AChR and subsequent weakness. Many species are susceptible to induction of EAMG with purified xenogeneic AChR in adjuvant, but injection of Torpedo AChR without adjuvants can also induce evidence of EAMG. To see whether pathogenic autoimmunity could be induced in mice by isolated mouse AChR we injected BALB/c mice with several doses (1 pmole; about 0.1 microgram) of affinity-purified AChR (from the BC3H1 cell line but thought to be identical with denervated mouse muscle) intraperitoneally, without adjuvant, over a period of 10-22 weeks. Some of the mice became ill and died. High levels of serum anti-mouse AChR, directed mainly towards the main immunogenic region, were found and, in the survivors, correlated with loss of muscle AChR. Thus BALB/c mice can mount an autoimmune response to minute amounts of mouse AChR, without the use of adjuvants, and this response is very similar to that found in MG. This novel finding has implications regarding the etiology of the human disease.

The ligand binding domain of the nicotinic acetylcholine receptor: Immunological analysis

The interaction of the acetylcholine receptor (AChR) binding site domain with specific antibodies and with α-bungarotoxin (α-BTX) has been compared. The cloned and expressed ligand binding domain of the mouse AChR α-subunit binds α-BTX, whereas the mongoose-expressed domain is not recognized by α-BTX. On the other hand, both the mouse and mongoose domains bind to the site-specific monoclonal antibody 5.5. These results demonstrate that the structural requirements for binding of α-BTX and mcAb 5.5, both of which interact with the AChR binding site, are distinct from each other.

Desensitization of junctional and extrajunctional nicotinic ACh receptors expressed in Xenopus oocytes

Desensitization of nicotinic acetylcholine receptors (AChRs) was studied using the Xenopus oocyte expression system. Mouse and cat extrajunctional AChRs expressed in oocytes desensitized more slowly than Torpedo AChRs. Substitution of the mouse γ subunit into the Torpedo AChR reduced the rate of desensitization, making it similar to the mouse AChR. Likewise, substitution of the Torpedo γ subunit into the mouse extrajunctional AChR increased the rate of desensitization, making it similar to the Torpedo AChR. Furthermore, mouse junctional AChRs in which the γ subunit was replaced by the ϵ subunit desensitized more rapidly than either mouse or cat extrajunctional AChRs, and resembled Torpedo AChRs. Thus, in addition to other properties, junctional and extrajunctional AChRs differ with respect to desensitization, suggesting a possible role of desensitization in normal development of neuromuscular junction.

Use of Torpedo-mouse hybrid acetylcholine receptors reveals immunodominance of the alpha subunit in myasthenia gravis antisera.

The nicotinic acetylcholine receptor (AChR), a pentameric complex of alpha 2 beta gamma delta subunits, is the autoantigen in the human autoimmune disease myasthenia gravis (MG). Anti-AChR antibodies are found in approximately 90% of MG patients and using indirect methods (competitive binding to solubilized AChR), peptides, or synthetic peptides, the majority of these antibodies have been shown to bind to the AChR alpha subunit. In order to determine directly the AChR subunit specificities of MG antibodies, we employed as antigens a novel set of hybrid AChR composed of species cross-reacting and non-cross-reacting subunits stably expressed in fibroblasts. Sequence similarities of homologous subunits among species can vary widely, with mammalian subunits having 87%-96% identity and Torpedo-mammalian subunits having 54%-80% identity. These findings are reflected in antigenic specificities, with human anti-AChR antisera frequently recognizing mouse AChR but rarely recognizing Torpedo. By establishing separate cell lines stably expressing all-Torpedo, all-mouse, and different combinations of Torpedo and mouse subunits, we were able to provide the first direct evidence of a predominant anti-alpha subunit specificity in MG antisera. Functional hybrid AChR stably expressed in an intact cell membrane provide us with a system that best mimics the in vivo environment of the MG antibody in a binding assay. Such a system allows us to investigate a perplexing observation in the field: a poor correlation between the patient's clinical status and antibody titer. Those antibodies which can interfere with AChR function, such as ones with the ability to cross-link AChR and induce their accelerated internalization and degradation (antigenic modulation) might represent a subpopulation of MG antibodies important in disease induction or maintenance. In this report, we demonstrate that wild-type and hybrid AChR expressed in fibroblasts can be antigenically modulated by intermolecular cross-linking antibodies as AChR are in native muscle cells. Because we can monitor dynamic interactions between AChR and MG antibodies, this system may allow us to define crucial pathogenic epitopes in MG by expressing hybrid, chimeric, and mutant AChR.

Subunit folding and alpha delta heterodimer formation in the assembly of the nicotinic acetylcholine receptor. Comparison of the mouse and human alpha subunits

We have used the mouse alpha (alpha M) and human alpha (alpha H) subunits to investigate the molecular mechanisms of assembly of the mammalian acetylcholine receptor (AChR) transiently expressed in COS cells. COS cells expressing hybrid receptors incorporating alpha H along with other mouse subunits exhibited a 2-fold higher level of surface alpha-bungarotoxin (BuTx) binding than cells expressing the wild-type mouse AChR. When expressed either alone or with the delta subunit in COS cells, alpha H acquired the BuTx binding conformation (alpha Tx) more efficiently than did alpha M. By oligonucleotide-directed mutagenesis we showed that 2 residues in the amino-terminal domain were responsible for the differences between alpha M and alpha H. Alpha MST, the modified mouse alpha subunit, both folded more efficiently to form alpha Tx and was more effective in forming a stable alpha delta heterodimer than was alpha M. The kinetics of alpha Tx and alpha delta heterodimer formation revealed that the delta subunit increased the conversion of immature forms of the alpha subunit into the BuTx binding form and therefore provides evidence for interaction between the delta subunit and the immature form of the alpha subunit. These results provide evidence of the importance of the amino-terminal domains of the AChR subunits in the assembly process.

Cooperation between the products of different nuclei in hybrid myotubes produces localized acetylcholine receptor clusters.

Cultured myotubes form clusters of acetylcholine receptors (AChRs) spontaneously and at sites of nerve-muscle contact. To investigate the cellular mechanisms by which spontaneous clusters are formed, we have made hybrid myotubes between a mouse muscle cell variant, S27, that does not cluster AChRs, and one that does not make AChRs. We have also made hybrid myotubes using S27 and quail muscle cells. In both cases, clusters of AChRs were found near the non-S27 nuclei; in the case of the interspecific hybrids, mouse AChRs were associated with extracellular matrix components contributed by the quail nuclei. Our results suggest that AChRs made by one nucleus can be clustered by localized extracellular matrix produced by a different nucleus and provide an example of nuclear cooperation between the products of different nuclei within multinucleated muscle fibers.

Kinetic behavior of cloned mouse acetylcholine receptors. A semi-autonomous, stepwise model of gating

This model assumes that any conformational changes consequent to binding one ligand are kinetically undetectable (on the time scale of the instrumentation) and/or irrelevant to the gating process. We have analysed AChR activation kinetics according to an alternative scheme that explicitly incorporates agonist-induced conformational changes. In this stepwise model, each subunit undergoes an isomerization after binding an agonist, with ionic conduction occurring only after both subunits have activated: -100 mV). Kinetic analysis followed the Horn and Lange (1983) full maximum likelihood method, with a first-order correction for missed events (Roux and Sauve, 1985). After selecting a subset of bursts that was apparently homogeneous, the rates of either activation scheme were iteratively adjusted by a Simplex algorithm until the -log likelihood was a minimum. The significance of the log likelihood ratios (LLR) was examined both by resampling and simulation methods (Horn, 1987). At 1,000 ~M, the predominant component of gaps with bursts has a time constant of 64 ~s, indicating an effective opening rate at this concentration of 15,600 S-I. The results of the likelihood fit to a patch exposed to 10 ~M ACh are shown below (all rates are S-I or S-l ~M-l). The association and dissociation rates of the stepwise scheme were assumed to be equal and independent, so that each model had the same number of independent parameters (six).

Multiple binding sites for myogenic regulatory factors are required for expression of the acetylcholine receptor gamma-subunit gene.

The nicotinic acetylcholine receptor (AChR) is a critical component of the mechanism responsible for signal transduction between nerve and muscle. Induction of AChR gene expression occurs during myogenesis when myoblasts exit the cell cycle and fuse to form myotubes. Induction of muscle-specific genes during differentiation is believed to be mediated through the activities of members of the MyoD family of myogenic regulatory factors. At least two of the factors, MyoD and myogenin, function as transcriptional activators. Here we present evidence that MyoD is capable of activating transcription of the mouse AChR gamma subunit gene. We have identified seven potential MyoD-binding sites (E-boxes) within the 5'-flanking DNA of the gamma subunit gene. We demonstrate that two small regions of the 5'-flanking DNA, one containing a pair of E-boxes and the other containing a single E-box, are required for gamma subunit expression in mouse muscle cells. DNase I footprinting indicates that the E-box proximal to the transcriptional initiation site is capable of interacting directly with MyoD. Furthermore, site-directed mutational analysis indicates that the single E-box is necessary for transactivation by MyoD and expression of the gamma subunit promoter region in muscle cells.

Stable expression of the mouse nicotinic acetylcholine receptor in mouse fibroblasts. Comparison of receptors in native and transfected cells

A stable cell line expressing mouse acetylcholine receptors (AChRs), named AM4, was established by cotransfecting into NIH 3T3 fibroblasts, alpha-, beta-, gamma-, and delta-subunit cDNAs plus the neor gene by calcium phosphate precipitation. Surface AChRs on AM4 cells contain all four subunits, sediment as a single approximately 9 S peak on sucrose gradients, and have the same ratio of alpha- to beta-subunits as surface AChRs from mouse BC3H-1 cells. The surface AChRs exhibit pharmacological properties identical to those obtained for BC3H-1 cells, including the association and dissociation rates of alpha-bungarotoxin, a low affinity and cooperative instantaneous dose-response curve, cooperative steady state agonist binding and desensitization, cooperative enhancement of agonist binding affinity by local anesthetics, and distinct affinities for curariform antagonists. Patch clamp measurements on AM4 cells reveal AChR single channel properties identical to those obtained from BC3H-1 cells, including a single class of channels with a conductance of 56 pS, short and long duration openings at low and high agonist concentrations, brief and intermediate closed duration components at low agonist concentrations, and six distinct closed duration components at high agonist concentrations. The biochemical, pharmacological, and single channel measurements indicate at least 95% of the surface AChRs on AM4 cells are alpha 2 beta gamma delta pentamers.

Chronic murine experimental myasthenia gravis: Strength testing and serology

CB6 ( Balb c × C57B1 6 F1) and C57B1 6 (B6) mice were hyperimmunized with Torpedo acetylcholine receptor (AChR) for 7 months. Control groups were hyperimmunized with bovine serum albumin. Antibody titers against Torpedo AChR rose quickly, reaching plateau levels by 3–4 months, while antibody to mouse AChR lagged by a few months, reaching plateau levels in 5 months. After the last immunization the mice maintained a state of stable autoimmunity for 9 months with high levels of antibodies against Torpedo and mouse AChR. Fatigability was measured on a programmable treadmill and remained present through the 9 months after the last immunization. CB6 mice had less weakness than the B6 mice, but the latter strain when immunized with BSA had more “false-positive” weakness. Titers of antibodies did not correlate with the degree of weakness measured on the treadmill. Despite the weakness and the high titers of anti-AChR antibodies, sera from myasthenic mice, in contrast to sera from myasthenic humans, were not able to block bungarotoxin binding to native AChR on the surface of BC3H1 cells.

Single-channel properties of mouse— Torpedo acetylcholine receptor hybrids expressed in Xenopus oocytes

This report analyzes the contribution of individual nicotinic acetylcholine receptor (AChR) subunits to the single-channel properties of the AChR ion channel. By in vitro synthesis of mRNA from cDNa clones encoding each AChR subunit (α, β, γ, and δ) from mouse BC3H-1 cells and Torpedo electric organ and microinjection of appropriate mRNA combinations into Xenopus oocytes, we studied the single-channel properties of both ‘homologous’ (all subunits from the same species) and ‘hybrid’ (subunits from both species) AChRs as they were expressed in the oocyte membrane. AChR expression was determined by surface binding of 125I-labeled α-bungarotoxin to intact oocytes, and those with binding sites of 1 fmol/cell or more were chosen for patch-clamp studies. Our results indicate the following: (1) Species difference in single-channel conductance can be explained largely by the charge distribution flanking the M2 transmembrane domain. (2) The α and δ subunits from mouse AChR independently lengthen the channel open time, in some cases by 10-fold; the β subunit from mouse shortens the channel open time; the mouse γ subunit lengthens open time less dramatically. (3) Voltage sensitivity, as measured by the ratio of channel open times at −60 mV and +60 mV, is influenced by the β and δ subunits, in agreement with our previous study by two-electrode voltage-clamp recording. We conclude that single-channel properties of the AChR are governed by multiple elements located on different AChR subunits.

Efficiency of acetylcholine receptor subunit assembly and its regulation by cAMP.

Assembly of nicotinic acetylcholine receptor (AChR) subunits was investigated using mouse fibroblast cell lines stably expressing either Torpedo (All-11) or mouse (AM-4) alpha, beta, gamma, and delta AChR subunits. Both cell lines produce fully functional cell surface AChRs. We find that two independent treatments, lower temperature and increased intracellular cAMP can increase AChR expression by increasing the efficiency of subunit assembly. Previously, we showed that the rate of degradation of individual subunits was decreased as the temperature was lowered and that Torpedo AChR expression was acutely temperature sensitive, requiring temperatures lower than 37 degrees C. We find that Torpedo AChR assembly efficiency increases 56-fold as the temperature is decreased from 37 to 20 degrees C. To determine how much of this is a temperature effect on degradation, mouse AChR assembly efficiencies were determined and found to be only approximately fourfold more efficient at 20 than at 37 degrees C. With reduced temperatures, we can achieve assembly efficiencies of Torpedo AChR in fibroblasts of 20-35%. Mouse AChR in muscle cells is also approximately 30% and we obtain approximately 30% assembly efficiency of mouse AChR in fibroblasts (with reduced temperatures, this value approaches 100%). Forskolin, an agent which increases intracellular cAMP levels, increased subunit assembly efficiencies twofold with a corresponding increase in cell surface AChR. Pulse-chase experiments and immunofluorescence microscopy indicate that oligomer assembly occurs in the ER and that AChR oligomers remain in the ER until released to the cell surface. Once released, AChRs move rapidly through the Golgi membrane to the plasma membrane. Forskolin does not alter the intracellular distribution of AChR. Our results indicate that cell surface expression of AChR can be regulated at the level of subunit assembly and suggest a mechanism for the cAMP-induced increase in AChR expression.

Immunological heterogeneity of autoreactive T lymphocytes against the nicotinic acetylcholine receptor in myasthenic patients

The response of human T lymphocytes against the nicotinic acetylcholine receptor (AChR) was studied in five patients with myasthenia gravis (MG) and in six healthy donors using either native Torpedo AChR or recombinant protein derived from the mammalian AChR alpha subunit (X4, residues 6-216 of mouse AChR alpha subunit). The present study demonstrates that (a) AChR-specific T helper cell lines can be generated from MG patients [either from peripheral blood lymphocytes (PBL) or from thymocytes] as well as from PBL of normal controls, (b) lymphocytes from MG patients, but not from controls, recognize the mammalian AChR but not the Torpedo receptor, (c) in humans, the HLA-DR2-associated T cell epitope is probably located in the region of residues 162-216 of the AChR alpha subunit and (d) there is a considerable heterogeneity of autoreactive T cell responses: (i) T cell lines from different HLA-type donors have distinct epitope profiles; (ii) the epitope specificity of the PBL-derived T cell line is different from that of the thymocyte-derived line; (iii) the epitope specificities of patient-derived T cell lines are different from those generated from normal controls who share the same HLA phenotype.

Two adjacent MyoD1-binding sites regulate expression of the acetylcholine receptor α-subunit gene

Several genes encoding putative myogenic regulatory factors have been isolated on the basis of their ability to convert nonmuscle cells into myoblasts. Four of these genes code for nuclear proteins that belong to a larger family characterized by a conserved helix-loop-helix motif required for DNA-binding and dimerization. At least one protein, MyoD1, can function as a transcription factor and activate muscle-specific genes during differentiation. But the promoter of the delta-subunit gene of the mouse acetylcholine receptor (AChR) was recently reported to be functional in the absence of MyoD1 binding sites and it has been suggested that the genes coding for the AChR could be regulated independently of MyoD1 protein. Here, we identify two functional MyoD1-binding sites in the muscle-specific enhancer of the chicken AChR alpha-subunit gene that are essential for full activity in transfected myotubes.

Autoimmune T lymphocytes in myasthenia gravis. Determination of target epitopes using T lines and recombinant products of the mouse nicotinic acetylcholine receptor gene.

Oligoclonal and cloned T lines from peripheral blood or thymuses of patients with myasthenia gravis (MG) were selected for reactivity against nicotinic acetylcholine receptors (AChR) from Torpedo california, or against a recombinant fusion peptide, X4, representing the extracellular portion of the mouse AChR alpha-chain. All cell lines expressed the CD4 membrane phenotype, and their antigen reactivity was blocked by antibodies against monomorphic HLA DR/DP determinants. Using a panel of fusion proteins of different, overlapping mouse AChR alpha-chain sequences, a major T cell epitope was localized between amino acid positions 85 and 142. This determinant was distinct from the humoral main immunogenic region, which has been identified on the sequence 61-76. The response pattern of uncloned T lines from three patients with different HLA haplotypes suggests, however, that in any one MG patient T lymphocytes may recognize more than one autoantigenic epitope on the AChR alpha-chain, and that the T lymphocyte response profiles vary among individual patients.

Stepwise activation of the mouse acetylcholine receptor delta- and gamma-subunit genes in clonal cell lines.

We used the DNase I-hypersensitive sites around the mouse acetylcholine receptor delta-subunit gene as a guide toward the cloning and sequencing of delta and gamma transcriptional regulatory regions and as a means to assess chromatin structural activation of the delta- and gamma-subunit genes during myogenesis. Genomic cloning of hypersensitive sites downstream of the delta-subunit gene revealed the presence of the gamma-subunit gene approximately 5 kilobases away; the hypersensitive sites mapped to the 5' end of the gamma-subunit gene. Sequence comparison of restriction fragments containing hypersensitive sites in analogous locations at the 5' ends of the delta- and gamma-subunit genes uncovered little overall homology between the two genomic fragments; however, an 11- of 13-base-pair match between the two sequences was found. Homologs to this sequence were also found to be present in the upstream regions of the chicken alpha- and mouse beta-subunit genes. By RNase protection and primer extension analyses, the delta-subunit gene transcription start site was mapped to 56 base pairs upstream of the initiator ATG codon. Clonal cell lines with various potentials to differentiate to the skeletal muscle phenotype were examined for their hypersensitive site pattern within the delta-gamma locus. Only remote hypersensitive sites flanking the locus appear in pluripotential mesodermal cells. A cell line of determined but inducible myoblasts expressed only one more intergenic site, while in permissively differentiating myoblasts hypersensitive sites were already present at the 5' ends of the delta and gamma genes prior to differentiation. Terminal differentiation resulted in an identical pattern of hypersensitive sites in all muscle cell lines examined so far, with an intergenic site near the gamma-subunit gene being the only site specific to the differentiated muscle phenotype.