Surface Acetylcholine Receptors Research Articles

The MX-Helix of Muscle nAChR Subunits Regulates Receptor Assembly and Surface Trafficking.

Nicotinic acetylcholine receptors (AChRs) are pentameric channels that mediate fast transmission at the neuromuscular junction (NMJ) and defects in receptor expression underlie neuromuscular disorders such as myasthenia gravis and congenital myasthenic syndrome (CMS). Nicotinic receptor expression at the NMJ is tightly regulated and we previously identified novel Golgi-retention signals in the β and δ subunit cytoplasmic loops that regulate trafficking of the receptor to the cell surface. Here, we show that the Golgi retention motifs are localized in the MX-helix, a juxta-membrane alpha-helix present in the proximal cytoplasmic loop of receptor subunits, which was defined in recent crystal structures of cys-loop receptor family members. First, mutational analysis of CD4-MX-helix chimeric proteins showed that the Golgi retention signal was dependent on both the amphipathic nature of the MX-helix and on specific lysine residues (βK353 and δK351). Moreover, retention was associated with ubiquitination of the lysines, and βK353R and δK351R mutations reduced ubiquitination and increased surface expression of CD4-β and δ MX-helix chimeric proteins. Second, mutation of these lysines in intact β and δ subunits perturbed Golgi-based glycosylation and surface trafficking of the AChR. Notably, combined βK353R and δK351R mutations increased the amount of surface AChR with immature forms of glycosylation, consistent with decreased Golgi retention and processing. Third, we found that previously identified CMS mutations in the ε subunit MX-helix decreased receptor assembly and surface levels, as did an analogous mutation introduced into the β subunit MX-helix. Together, these findings indicate that the subunit MX-helix contributes to receptor assembly and is required for normal expression of the AChR and function of the NMJ. In addition, specific determinants in the β and δ subunit MX-helix contribute to quality control of AChR expression by intracellular retention and ubiquitination of unassembled subunits, and by facilitating the appropriate glycosylation of assembled surface AChR.

Suppression of acetylcholine receptor endocytosis in neuromuscular junction by carbonic anhydrase III in the pathogenesis of myasthenia gravis.

Abstract Myasthenia gravis is an autoimmune disorder of the neuromuscular junction manifestated as fatigable muscle weakness. The disorder typically caused by pathogenic autoantibodies against the postsynaptic acetylcholine receptor in the endplate of skeletal muscle. Our previous studies have identified carbonic anhydrase III as a specific protein insufficient in skeletal muscle from myasthenia gravis patient. In this study, we determined to investigate the underlying mechanism how carbonic anhydrase III insufficiency contributes to myasthenia gravis. Based on experimental autoimmune myasthenia gravis animal model and myocyte C2C12 cells, we find that inhibition of carbonic anhydrase III promotes acetylcholine receptor, which is through lipid raft-mediated pathway, leading to accelerated degradation of surface acetylcholine receptor. While activation of carbonic anhydrase III negatively regulates acetylcholine receptor degradation, through suppressing the endocytosis of surface acetylcholine receptor, thus contributes to attenuation of myasthenia gravis. Further study demonstrates that carbonic anhydrase III regulates endocytosis of AChR through actin polymerization and Src phosphorylation. Collectively, our study illustrates that carbonic anhydrase III in skeletal muscle cells is critical for acetylcholine receptor homeostasis in neuromuscular junction, while carbonic anhydrase III deficiency induces accelerated degradation of acetylcholine receptor, leading to myasthenia gravis. This novel mechanism highlights the essential role of carbonic anhydrase III in regualtion acetylcholine receptor endocytosis, providing a potential therapeutic approaches for myasthenia gravis.

Diacylglycerol levels modulate the cellular distribution of the nicotinic acetylcholine receptor

Diacylglycerol (DAG), a second messenger involved in different cell signaling cascades, activates protein kinase C (PKC) and D (PKD), among other kinases. The present work analyzes the effects resulting from the alteration of DAG levels on neuronal and muscle nicotinic acetylcholine receptor (AChR) distribution. We employ CHO-K1/A5 cells, expressing adult muscle-type AChR in a stable manner, and hippocampal neurons, which endogenously express various subtypes of neuronal AChR. CHO-K1/A5 cells treated with dioctanoylglycerol (DOG) for different periods showed augmented AChR cell surface levels at short incubation times (30min–4h) whereas at longer times (18h) the AChR was shifted to intracellular compartments. Similarly, in cultured hippocampal neurons surface AChR levels increased as a result of DOG incubation for 4h. Inhibition of endogenous DAG catabolism produced changes in AChR distribution similar to those induced by DOG treatment. Specific enzyme inhibitors and Western blot assays revealed that DAGs exert their effect on AChR distribution through the modulation of the activity of classical PKC (cPKC), novel PKC (nPKC) and PKD activity.

Detection of Acetylcholine Receptor Modulating Antibodies by Flow Cytometry

To determine the clinical utility and performance characteristics of a laboratory-adapted flow cytometric method for the detection of acetylcholine receptor (AChR) modulating antibodies in myasthenia gravis (MG). Serum samples from 120 healthy donors and 100 patients with suspected MG were assessed for the ability to reduce surface AChR concentrations (antigenic modulation) in RD (TE671) or DB40 human muscle cell lines by flow cytometry. Reference ranges were established by receiver operating characteristic curve analysis, and results were then compared with those of the current radioimmunoassay (RIA). Flow cytometric results from the RD cell line had an interpretive threshold of 46% modulation or greater and correlated best (98% sensitivity, 99% specificity) with those of the current RIA. The new flow cytometric method using the RD cell platform provided higher quality clinical results, a more robust and efficient assay format, a significant cost savings, and less environmental burden.

Regulation of muscle acetylcholine receptor turnover by β subunit tyrosine phosphorylation

At the neuromuscular junction (NMJ), the postsynaptic localization of muscle acetylcholine receptor (AChR) is regulated by neural signals and occurs via several processes including metabolic stabilization of the receptor. However, the molecular mechanisms that influence receptor stability remain poorly defined. Here, we show that neural agrin and the tyrosine phosphatase inhibitor, pervanadate slow the degradation of surface receptor in cultured muscle cells. Their action is mediated by tyrosine phosphorylation of the AChR β subunit, as agrin and pervandate had no effect on receptor half-life in AChR-β(3F/3F) muscle cells, which have targeted mutations of the β subunit cytoplasmic tyrosines. Moreover, in wild type AChR-β(3Y) muscle cells, we found a linear relationship between average receptor half-life and the percentage of AChR with phosphorylated β subunit, with half-lives of 12.7 and 23 h for nonphosphorylated and phosphorylated receptor, respectively. Surprisingly, pervanadate increased receptor half-life in AChR-β(3Y) myotubes in the absence of clustering, and agrin failed to increase receptor half-life in AChR-β(3F/3F) myotubes even in the presence of clustering. The metabolic stabilization of the AChR was mediated specifically by phosphorylation of βY390 as mutation of this residue abolished β subunit phosphorylation but did not affect δ subunit phosphorylation. Receptor stabilization also led to higher receptor levels, as agrin increased surface AChR by 30% in AChR-β(3Y) but not AChR-β(3F/3F) myotubes. Together, these findings identify an unexpected role for agrin-induced phosphorylation of β(Y390) in downregulating AChR turnover. This likely stabilizes AChR at developing synapses, and contributes to the extended half-life of AChR at adult NMJs.

Myasthenic syndrome AChRα C-loop mutant disrupts initiation of channel gating

Congenital myasthenic syndromes (CMSs) are neuromuscular disorders that can be caused by defects in ace-tylcholine receptor (AChR) function. Disease-associated point mutants can reveal the unsuspected functional significance of mutated residues. We identified two pathogenic mutations in the extracellular domain of the AChR α subunit (AChRα) in a patient with myasthenic symptoms since birth: a V188M mutation in the C-loop and a heteroallelic G74C mutation in the main immunogenic region. The G74C mutation markedly reduced surface AChR expression in cultured cells, whereas the V188M mutant was expressed robustly but had severely impaired kinetics. Single-channel patch-clamp analysis indicated that V188M markedly decreased the apparent AChR channel opening rate and gating efficiency. Mutant cycle analysis of energetic coupling among conserved residues within or dispersed around the AChRα C-loop revealed that V188 is functionally linked to Y190 in the C-loop and to D200 in β-strand 10, which connects to the M1 transmembrane domain. Furthermore, V188M weakens inter-residue coupling of K145 in β-strand 7 with Y190 and with D200. Cumulatively, these results indicate that V188 of AChRα is part of an interdependent tetrad that contributes to rearrangement of the C-loop during the initial coupling of agonist binding to channel gating.

Intracellular distribution of functional M1‐muscarinic acetylcholine receptors in N1E‐115 neuroblastoma cells

Signaling by muscarinic agonists is thought to result from the activation of cell surface acetylcholine receptors (mAChRs) that transmit extracellular signals to intracellular systems. In N1E-115 neuroblastoma cells, we detected both plasma membrane and intracellular M(1) -mAChRs using both biochemical and pharmacological methods. In intact cells, both plasma membrane and intracellular M(1) -mAChRs were detected by the hydrophobic ligand probe, 1-quinuclidinyl-[phenyl-4-(3) H]-benzilate ([(3) H]-QNB) whereas the hydrophilic probe, 1-[N-methyl-(3) H] scopolamine ([(3) H]-NMS), detected only cell surface receptors. These probes detected comparable numbers of receptors in isolated membrane preparations. Immunohistochemical studies with M(1) -mAChR antibody also detected both cell-surface and intracellular M(1) -mAChRs. Carbachol-stimulated phosphatidylinositol hydrolysis and Ca(2+) mobilization were completely inhibited by a cell-impermeable M(1) antagonist, muscarinic toxin -7 and the G(q/11) inhibitor YM-254890. However, carbachol-stimulated extracellular-regulated kinase 1/2 activation was unaffected by muscarinic toxin-7, but was blocked by the cell-permeable antagonist, pirenzepine. extracellular regulated kinase 1/2 phosphorylation was resistant to blockade of G(q/11) (YM-254890) and protein kinase C (bisindolylmaleimide I). Our data suggest that the geographically distinct M(1) -mAChRs (cell surface versus intracellular) can signal via unique signaling pathways that are differentially sensitive to cell-impermeable versus cell-permeable antagonists. Our data are of potential physiological relevance to signaling that affects both cognitive and neurodegenerative processes.

Cholesterol Modulates the Rate and Mechanism of Acetylcholine Receptor Internalization

Stability of the nicotinic acetylcholine receptor (AChR) at the cell surface is key to the correct functioning of the cholinergic synapse. Cholesterol (Chol) is necessary for homeostasis of AChR levels at the plasmalemma and for ion translocation. Here we characterize the endocytic pathway followed by muscle-type AChR in Chol-depleted cells (Chol(-)). Under such conditions, the AChR is internalized by a ligand-, clathrin-, and dynamin-independent mechanism. Expression of a dominant negative form of the small GTPase Rac1, Rac1N17, abolishes receptor endocytosis. Unlike the endocytic pathway in control CHO cells (1), accelerated AChR internalization proceeds even upon disruption of the actin cytoskeleton. Under Chol(-) conditions, AChR internalization is furthermore found to require the activity of Arf6 and its effectors Rac1 and phospholipase D. The Arf6-dependent mechanism may constitute the default endocytic pathway followed by the AChR in the absence of external ligands, membrane Chol levels acting as a key homeostatic regulator of cell surface receptor levels.

Time Lapse in Vivo Visualization of Developmental Stabilization of Synaptic Receptors at Neuromuscular Junctions

The lifetime of nicotinic acetylcholine receptors (AChRs) in neuromuscular junctions (NMJs) is increased from <1 day to >1 week during early postnatal development. However, the exact timing of AChR stabilization is not known, and its correlation to the concurrent embryonic to adult AChR channel conversion, NMJ remodeling, and neuromuscular diseases is unclear. Using a novel time lapse in vivo imaging technology we show that replacement of the entire receptor population of an individual NMJ occurs end plate-specifically within hours. This makes it possible to follow directly in live animals changing stabilities of end plate receptors. In three different, genetically modified mouse models we demonstrate that the metabolic half-life values of synaptic AChRs increase from a few hours to several days after postnatal day 6. Developmental stabilization is independent of receptor subtype and apparently regulated by an intrinsic muscle-specific maturation program. Myosin Va, an F-actin-dependent motor protein, is also accumulated synaptically during postnatal development and thus could mediate the stabilization of end plate AChR.

Sequence and Structural Features of RNA Aptamer Against Myasthenic Autoantibodies

Myasthenia gravis (MG) is mainly caused by autoantibodies to postsynaptic nicotinic acetylcholine receptors (AChRs). Previously, we isolated an RNA aptamer with 2'-amino pyrimidines that inhibited both a rat monoclonal antibody, which recognizes the main immunogenic region on the AChR, and MG patient autoantibodies from down-modulating AChRs on human cells. In this study, secondary configuration and binding motif of the aptamer were characterized, and moreover, various mutant aptamer forms were generated to figure out sequence and structure requirements of the aptamer. Then, we found that intrinsic structure formation and sequence composition of the selected RNA aptamer specific to the antibody are required for the aptamer activity to inhibit the myasthenic autoantibody-mediated destruction of cell surface AChRs. Noticeably, we identified 47-mer minimized aptamer version, which can efficiently protect cells from the effects of the autoantibodies and could be optimally applicable for MG therapy.

Regulation of acetylcholine receptor clustering by ADF/cofilin-directed vesicular trafficking

SummaryPostsynaptic receptor localization is crucial for synapse development and function, but the underlying cytoskeletal mechanisms remain elusive. Using Xenopus neuromuscular junctions as a model, we here report that actin depolymerizing factor (ADF)/cofilin regulates actin-dependent vesicular trafficking of acetylcholine receptors (AChRs) to the postsynaptic membrane. We found that active ADF/cofilin was concentrated in small puncta adjacent to AChR clusters and spatiotemporally correlated with the formation and maintenance of surface AChR clusters. Importantly, increased actin dynamics, vesicular markers, and intracellular AChRs were all enriched at the sites of ADF/cofilin localization. Furthermore, a substantial amount of new AChRs was detected at these ADF/cofilin-enriched sites. Manipulation of either ADF/cofilin activity through its serine-3 phosphorylation or ADF/cofilin localization via 14-3-3 proteins markedly attenuated AChR insertion and clustering. These results suggest that spatiotemporally restricted ADF/cofilin-mediated actin dynamics regulate AChR trafficking during the development of neuromuscular synapses.

HSP90β Regulates Rapsyn Turnover and Subsequent AChR Cluster Formation and Maintenance

Rapsyn, an acetylcholine receptor (AChR)-interacting protein, is essential for synapse formation at the neuromuscular junction (NMJ). Like many synaptic proteins, rapsyn turns over rapidly at synapses. However, little is known about molecular mechanisms that govern rapsyn stability. Using a differential mass-spectrometry approach, we identified heat-shock protein 90beta (HSP90beta) as a component in surface AChR clusters. The HSP90beta-AChR interaction required rapsyn and was stimulated by agrin. Inhibition of HSP90beta activity or expression, or disruption of its interaction with rapsyn attenuated agrin-induced formation of AChR clusters in vitro and impaired the development and maintenance of the NMJ in vivo. Finally, we showed that HSP90beta was necessary for rapsyn stabilization and regulated its proteasome-dependent degradation. Together, these results indicate a role of HSP90beta in NMJ development by regulating rapsyn turnover and subsequent AChR cluster formation and maintenance.

Nicotinic acetylcholine receptor is internalized via a Rac-dependent, dynamin-independent endocytic pathway.

Endocytosis of the nicotinic acetylcholine receptor (AChR) is a proposed major mechanism of neuromodulation at neuromuscular junctions and in the pathology of synapses in the central nervous system. We show that binding of the competitive antagonist α-bungarotoxin (αBTX) or antibody-mediated cross-linking induces the internalization of cell surface AChR to late endosomes when expressed heterologously in Chinese hamster ovary cells or endogenously in C2C12 myocytes. Internalization occurs via sequestration of AChR–αBTX complexes in narrow, tubular, surface-connected compartments, which are indicated by differential surface accessibility of fluorescently tagged αBTX–AChR complexes to small and large molecules and real-time total internal reflection fluorescence imaging. Internalization occurs in the absence of clathrin, caveolin, or dynamin but requires actin polymerization. αBTX binding triggers c-Src phosphorylation and subsequently activates the Rho guanosine triphosphatase Rac1. Consequently, inhibition of c-Src kinase activity, Rac1 activity, or actin polymerization inhibits internalization via this unusual endocytic mechanism. This pathway may regulate AChR levels at ligand-gated synapses and in pathological conditions such as the autoimmune disease myasthenia gravis.

Cell culture-based analysis of postsynaptic membrane assembly in muscle cells

We report a method for studying postsynaptic membrane assembly utilizing the replating of aneural cultures of differentiated skeletal muscle cells onto laminin-coated surfaces. A significant limitation to the current cell culturebased approaches has been their inability to recapitulate the multistage surface acetylcholine receptor (AChR) redistribution events that produce complex AChR clusters found at the intact neuromuscular junction (NMJ). By taking advantage of the ability of substrate laminin to induce advanced maturation of AChR aggregates on the surface of myotubes, we have developed a secondary-plating method that allows more precise analysis of the signaling events connecting substrate laminin stimulation to complex AChR cluster formation. We validate the utility of this method for biochemical and microscopy studies by demonstrating the roles of RhoGTPases in substrate laminin-induced complex cluster assembly.

β-Catenin Regulates Acetylcholine Receptor Clustering in Muscle Cells through Interaction with Rapsyn

Agrin is believed to be a factor used by motoneurons to direct acetylcholine receptor (AChR) clustering at the neuromuscular junction. However, exactly how agrin mediates this effect remains unclear. Here we demonstrate that the beta-catenin interacts with rapsyn, a molecule key for AChR clustering. Agrin stimulation increases the association of beta-catenin with surface AChRs. Suppression of beta-catenin expression inhibited agrin-induced AChR clustering, suggesting a necessary role of beta-catenin in this event. The beta-catenin action did not appear to require the function of T-cell factors (TCFs), suggesting a mechanism independent of TCF-mediated transcription. In contrast, prevention of beta-catenin from interacting with alpha-catenin attenuated agrin-induced AChR clustering. These results suggest that beta-catenin may serve as a link between AChRs and alpha-catenin-associated cytoskeleton, revealing a novel function of beta-catenin in synaptogenesis.

Effect of sera from AChR-antibody negative myasthenia gravis patients on AChR and MuSK in cell cultures

A proportion of patients with myasthenia gravis (MG) do not have antibodies to the acetylcholine receptor (AChR). Some of these patients have antibodies to muscle specific kinase (MuSK), whereas others have neither antibody (seronegative MG, SNMG). Both MuSK antibody positive MG (MuSK-MG) and SNMG are antibody-mediated diseases but how they cause neuromuscular junction failure is not clear. One possibility is that they reduce the clustering and expression of AChRs. We looked at the effects of MuSK-MG and SNMG sera/IgG on surface AChR distribution and expression, and AChR subunit and MuSK mRNA by quantitative RT-PCR, in TE671 and C2C12 myotubes. In TE671 cells MuSK-MG sera reduced AChR expression by about 20%, but had no effect on AChR subunit or MuSK mRNA expression. In C2C12 myotubes, MuSK-MG sera caused a reduction in the number of agrin-induced clusters, but the clusters became larger and there was no significant effect on total surface AChR numbers or AChR subunit or MuSK mRNA. By contrast, SNMG sera not only reduced AChR numbers by about 20% in TE671 cells, but modestly upregulated AChR γ subunit expression in TE671 cells and both AChR γ subunit and MuSK expression in C2C12 myotubes. Thus, although these results have, disappointingly, demonstrated little effect of MuSK antibodies on AChR expression, they do imply that SNMG antibodies act on AChR-associated pathways.

Agrin and laminin induce acetylcholine receptor clustering by convergent, Rho GTPase-dependent signaling pathways

During neuromuscular junction formation, extracellular matrix-mediated signals cause muscle surface acetylcholine receptors (AChRs) to aggregate at synaptic sites. Two extracellular matrix proteins, agrin and laminin, have each been shown to initiate signaling pathways that culminate in AChR clustering in cultured muscle cells. Here we present evidence that laminin-induced AChR clustering is mediated by the activation of the Rho GTPases Cdc42, Rac and Rho. Clustering in response to laminin is blocked by the dominant negative mutants Cdc42N17, RacN17 and RhoN19, as well as by the Rho inhibitor C3 transferase. Moreover, laminin-induced AChR clustering is impaired by the Rho kinase inhibitor Y-27632. Agrin-induced AChR clustering has previously been shown to require activation of Cdc42, Rac and Rho. Therefore, although agrin and laminin use distinct transmembrane receptors to initiate AChR clustering, their signaling pathways converge at the level of Rho GTPase activation.

Overexpression of Rapsyn in Rat Muscle Increases Acetylcholine Receptor Levels in Chronic Experimental Autoimmune Myasthenia Gravis

The primary autoantigen in myasthenia gravis, the acetylcholine receptor (AChR), is clustered and anchored in the postsynaptic membrane of the neuromuscular junction by rapsyn. Previously, we found that overexpression of rapsyn by cDNA transfection protects AChRs in rat muscles from antibody-mediated loss in passive transfer experimental autoimmune myasthenia gravis (EAMG). Here, we determined whether rapsyn overexpression can reduce or even reverse AChR loss in muscles that are already damaged by chronic EAMG, which mimics the human disease. Active immunization against purified AChR was performed in female Lewis rats. Rapsyn overexpression resulted in an increase in total muscle membrane AChR levels, with some AChR at neuromuscular junctions but much of it in extrasynaptic membrane regions. At the ultrastructural level, most endplates in rapsyn-treated chronic EAMG muscles showed increased damage to the postsynaptic membrane. Although rapsyn overexpression stabilized AChRs in intact or mildly damaged endplates, the rapsyn-induced increase of membrane AChR enhanced autoantibody binding and membrane damage in severe ongoing disease. Thus, these results show the complexity of synaptic stabilization of AChR during the autoantibody attack. They also indicate that the expression of receptor-associated proteins may determine the severity of autoimmune diseases caused by anti-receptor antibodies.

Agrin elicits membrane lipid condensation at sites of acetylcholine receptor clusters in C2C12 myotubes

The formation of the neuromuscular junction is characterized by the progressive accumulation of nicotinic acetylcholine receptors (AChRs) in the postsynaptic membrane facing the nerve terminal, induced predominantly through the agrin/muscle-specific kinase (MuSK) signaling cascade. However, the cellular mechanisms linking MuSK activation to AChR clustering are still poorly understood. Here, we investigate whether lipid rafts are involved in agrin-elicited AChR clustering in a mouse C2C12 cell line. We observed that in C2C12 myotubes, both AChR clustering and cluster stability were dependent on cholesterol, because depletion by methyl-beta-cyclodextrin inhibited cluster formation or dispersed established clusters. Importantly, AChR clusters resided in ordered membrane domains, a biophysical property of rafts, as probed by Laurdan two-photon fluorescence microscopy. We isolated detergent-resistant membranes (DRMs) by three different biochemical procedures, all of which generate membranes with similar cholesterol/GM1 ganglioside contents, and these were enriched in several postsynaptic components, notably AChR, syntrophin, and raft markers flotillin-2 and caveolin-3. Agrin did not recruit AChRs into DRMs, suggesting that they are present in rafts independently of agrin activation. Consequently, in C2C12 myotubes, agrin likely triggers AChR clustering or maintains clusters through the coalescence of lipid rafts. These data led us to propose a model in which lipid rafts play a pivotal role in the assembly of the postsynaptic membrane at the neuromuscular junction upon agrin signaling.

Autoimmune Channelopathies and Related Neurological Disorders

Ion channels are crucial elements in neuronal signaling and synaptic transmission, and defects in their function are known to underlie rare genetic disorders, including some forms of epilepsy. A second class of channelopathies, characterized by autoantibodies against ligand- and voltage-gated ion channels, cause a variety of defects in peripheral neuromuscular and ganglionic transmission. There is also emerging evidence for autoantibody-mediated mechanisms in subgroups of patients with central nervous system disorders, particularly those involving defects in cognition or sleep and often associated with epilepsy. In all autoimmune channelopathies, the relationship between autoantibody specificity and clinical phenotype is complex. But with this new information, autoimmune channelopathies are detected and treated with increasing success, and future research promises new insights into the mechanisms of dysfunction at neuronal synapses and the determinants of clinical phenotype.