Muscle Acetylcholine Receptor Research Articles

Estimating the pKa values of basic and acidic side chains in ion channels using electrophysiological recordings: A robust approach to an elusive problem

As a step toward gaining a better understanding of the physicochemical bases of pK(a)-value shifts in ion channels, we have previously proposed a method for estimating the proton affinities of systematically engineered ionizable side chains from the kinetic analysis of single-channel current recordings. We reported that the open-channel current flowing through mutants of the (cation-selective) muscle nicotinic acetylcholine receptor (AChR) engineered to bear single basic residues in the transmembrane portion of the pore domain fluctuates between two levels of conductance. Our observations were consistent with the idea that these fluctuations track directly the alternate protonation-deprotonation of basic side chains: protonation of the introduced basic group would attenuate the single-channel conductance, whereas its deprotonation would restore the wild-type-like level. Thus, analysis of the kinetics of these transitions was interpreted to yield the pK(a) values of the substituted side chains. However, other mechanisms can be postulated that would also be consistent with some of our findings but according to which the kinetic analysis of the fluctuations would not yield true pK(a)s. Such mechanisms include the pH-dependent interconversion between two conformations of the channel that, while both ion permeable, would support different cation-conduction rates. In this article, we present experimental evidence for the notion that the fluctuations of the open-channel current observed for the muscle AChR result from the electrostatic interaction between fixed charges and the passing cations rather than from a change in conformation. Hence, we conclude that bona fide pK(a) values can be obtained from single-channel recordings.

A Novel Labeling Approach Identifies Three Stability Levels of Acetylcholine Receptors in the Mouse Neuromuscular Junction In Vivo

BackgroundThe turnover of acetylcholine receptors at the neuromuscular junction is regulated in an activity-dependent manner. Upon denervation and under various other pathological conditions, receptor half-life is decreased.Methodology/Principal FindingsWe demonstrate a novel approach to follow the kinetics of acetylcholine receptor lifetimes upon pulse labeling of mouse muscles with 125I-α-bungarotoxin in vivo. In contrast to previous assays where residual activity was measured ex vivo, in our setup the same animals are used throughout the whole measurement period, thereby permitting a dramatic reduction of animal numbers at increased data quality. We identified three stability levels of acetylcholine receptors depending on the presence or absence of innervation: one pool of receptors with a long half-life of ∼13 days, a second with an intermediate half-life of ∼8 days, and a third with a short half-life of ∼1 day. Data were highly reproducible from animal to animal and followed simple exponential terms. The principal outcomes of these measurements were reproduced by an optical pulse-labeling assay introduced recently.Conclusions/SignificanceA novel assay to determine kinetics of acetylcholine receptor turnover with small animal numbers is presented. Our data show that nerve activity acts on muscle acetylcholine receptor stability by at least two different means, one shifting receptor lifetime from short to intermediate and another, which further increases receptor stability to a long lifetime. We hypothesize on possible molecular mechanisms.

Structure–activity relationship of ibogaine analogs interacting with nicotinic acetylcholine receptors in different conformational states

The interaction of ibogaine analogs with nicotinic acetylcholine receptors (AChRs) in different conformational states was studied by functional and structural approaches. The results established that ibogaine analogs: (a) inhibit (±)-epibatidine-induced Ca 2+ influx in human embryonic muscle AChRs with the following potency sequence (IC 50 in μM): (±)-18-methylaminocoronaridine (5.9 ± 0.3) ∼ (±)-18-methoxycoronaridine (18-MC) (6.8 ± 0.8) > (−)-ibogaine (17 ± 3) ∼ (+)-catharanthine (20 ± 1) > (±)-albifloranine (46 ± 13), (b) bind to the [ 3H]TCP binding site with higher affinity when the Torpedo AChR is in the desensitized state compared to that in the resting state. Similar results were obtained using [ 3H]18-MC. These and docking results suggest a steric interaction between TCP and ibogaine analogs for the same site, (c) enhance [ 3H]cytisine binding to resting but not to desensitized AChRs, with desensitizing potencies (apparent EC 50) that correlate very well with the p K i values in the desensitized state, and (d) there are good bilinear correlations between the ligand molecular volumes and their affinities in the desensitized and resting states, with an optimal volume of ∼345 Å 3 for the ibogaine site. These results indicate that the size of the binding sites for ibogaine analogs, located between the serine and nonpolar rings and shared with TCP, is an important structural feature for binding and for inducing desensitization.

Axonal filopodial asymmetry induced by synaptic target

During vertebrate neuromuscular junction (NMJ) assembly, motor axons and their muscle targets exchange short-range signals that regulate the subsequent steps of presynaptic and postsynaptic specialization. We report here that this interaction is in part mediated by axonal filopodia extended preferentially by cultured Xenopus spinal neurons toward their muscle targets. Immunoblotting and labeling experiments showed that basic fibroblast growth factor (bFGF) was expressed by muscle and associated with the cell surface, and treatment of cultured spinal neurons with recombinant bFGF nearly doubled the normal density of filopodia in neurites. This effect of bFGF was abolished by SU5402, a selective inhibitor of FGF-receptor 1 (FGFR1), and forced expression of wild-type or dominant-negative FGFR1 in neurons enhanced or suppressed the assembly of filopodia, respectively. Significantly, in nerve-muscle cocultures, knocking down bFGF in muscle decreased both the asymmetric extension of filopodia by axons toward muscle and the assembly of NMJs. In addition, neurons expressing dominant-negative FGFR1 less effectively triggered the aggregation of muscle acetylcholine receptors at innervation sites than did control neurons. These results suggest that bFGF activation of neuronal FGFR1 generates filopodial processes in neurons that promote nerve-muscle interaction and facilitate NMJ establishment.

Abelson Family Tyrosine Kinases Regulate the Function of Nicotinic Acetylcholine Receptors and Nicotinic Synapses on Autonomic Neurons

Abelson family kinases (AFKs; Abl1, Abl2) are non-receptor tyrosine kinases (NRTKs) implicated in cancer, but they also have important physiological roles that include regulating synaptic structure and function. Recent studies using Abl-deficient mice and the antileukemia drug STI571 [imatinib mesylate (Gleevec); Novartis], which potently and selectively blocks Abl kinase activity, implicate AFKs in regulating presynaptic neurotransmitter release in hippocampus and postsynaptic clustering of nicotinic acetylcholine receptors (nAChRs) in muscle. Here, we tested whether AFKs are relevant for regulating nAChRs and nAChR-mediated synapses on autonomic neurons. AFK immunoreactivity was detected in ciliary ganglion (CG) lysates and neurons, and STI571 application blocked endogenous Abl tyrosine kinase activity. With similar potency, STI571 specifically reduced whole-cell current responses generated by both nicotinic receptor subtypes present on CG neurons (α3*- and α7-nAChRs) and lowered the frequency and amplitude of α3*-nAChR-mediated excitatory postsynaptic currents. Quantal analysis indicated that the synaptic perturbations were postsynaptic in origin, and confocal imaging experiments revealed they were unaccompanied by changes in nAChR clustering or alignment with presynaptic terminals. The results indicate that in autonomic neurons, Abl kinase activity normally supports postsynaptic nAChR function to sustain nAChR-mediated neurotransmission. Such consequences contrast with the influence of Abl kinase activity on presynaptic function and synaptic structure in hippocampus and muscle, respectively, demonstrating a cell-specific mechanism of action. Finally, because STI571 potently inhibits Abl kinase activity, the autonomic dysfunction side effects associated with its use as a chemotherapeutic agent may result from perturbed α3*- and/or α7-nAChR function.

Effects of static magnetic fields on nicotinic cholinergic receptor function

Ligand-gated ion channel kinetics were studied in mammalian transfected cells encoding adult mouse muscle acetylcholine (ACh) receptors. We measured macroscopic and single-channel currents using the outside-out and cell-attached patch-clamp configurations. Cultured cells were exposed to moderate intensity inhomogeneous static magnetic fields up to 180 mT and measurements were performed for temperatures ranging from 5 to 50 °C. We found no significant changes in ACh-elicited macroscopic or single-channel currents. We observed the expected dependence in current decay constants with temperature, but negligible magnetic field influence on the channel's kinetics.

Proteasome Inhibition with Bortezomib Depletes Plasma Cells and Autoantibodies in Experimental Autoimmune Myasthenia Gravis

Bortezomib, an inhibitor of proteasomes, has been reported to reduce autoantibody titers and to improve clinical condition in mice suffering from lupus-like disease. Bortezomib depletes both short- and long-lived plasma cells; the latter normally survive the standard immunosuppressant treatments targeting T and B cells. These findings encouraged us to test whether bortezomib is effective for alleviating the symptoms in the experimental autoimmune myasthenia gravis (EAMG) model for myasthenia gravis, a disease that is characterized by autoantibodies against the acetylcholine receptor (AChR) of skeletal muscle. Lewis rats were immunized with saline (control, n = 36) or Torpedo AChR (EAMG, n = 54) in CFA in the first week of an experimental period of 8 wk. After immunization, rats received twice a week s.c. injections of bortezomib (0.2 mg/kg in saline) or saline injections. Bortezomib induced apoptosis in bone marrow cells and reduced the amount of plasma cells in the bone marrow by up to 81%. In the EAMG animals, bortezomib efficiently reduced the rise of anti-AChR autoantibody titers, prevented ultrastructural damage of the postsynaptic membrane, improved neuromuscular transmission, and decreased myasthenic symptoms. This study thus underscores the potential of the therapeutic use of proteasome inhibitors to target plasma cells in Ab-mediated autoimmune diseases.

On the Mechanism Underlying the Irreversible Desensitization of Human Ganglionic Nicotinic Receptors

alpha3-containing nicotinic acetylcholine receptors (AChRs) are expressed in autonomic ganglia and are thought to mediate fast synaptic transmission. These receptors have largely eluded rigorous kinetic characterization due to a combination of poor expression in heterologous systems and the total loss of receptor responsiveness during the course of excised-patch electrophysiology experiments. Recently, it has been demonstrated that a cysteine-to-alanine mutation at the −4’ position of the cytosolic M1-M2 linker can prevent this rundown in whole-cell patches of rodent superior cervical ganglion neurons (Campanucci et al., 2010). This work also suggested that this rundown, which plays a pivotal role in diabetic neuropathy, is due to oxidation of these cysteine residues. Here, we heterologously expressed human alpha3-subunit-containing AChRs in HEK-293 cells and found that the −4’ cysteine mutation cannot prevent the irreversible rundown of currents observed in outside-out patches of membrane. Furthermore, we found that the irreversible loss of receptor responsiveness does not occur in the whole-cell or cell-attached configurations, implying that patch excision leads to this phenomenon. We also investigated the effects of the reciprocal −4’ cysteine mutation in all five subunits of the human muscle AChRs, which do not natively have cysteines at this position. We found that introduction of the cysteines does not lead to an irreversible rundown, although it does slow down the rate of recovery from desensitization. Taken together, our results thus far suggest that the mechanism underlying the rundown of currents mediated by alpha3-containing AChRs in outside-out patches is fundamentally different from that mediated by cysteine oxidation in whole-cell recordings.

Assigning Functional States to Structural Models of Nicotinic-Receptor Type ion Channels

Aromatic-aromatic interactions are a prominent feature of the crystal structure of ELIC a bacterial member of the nicotinic-receptor superfamily of ion channels where five pore-facing phenylalanines come together to form a structure akin to a narrow iris that occludes the transmembrane pore. To identify the functional state of the channel that this structure represents, we engineered phenylalanines at various pore-facing positions of the muscle acetylcholine receptor, including the position that aligns with the native phenylalanine 246 of ELIC, and assessed the consequences of such mutations using electrophysiological and toxin-binding assays. From our experiments, we conclude that the interaction among the side chains of pore-facing phenylalanines leads to the formation of a non-conductive conformation that is unresponsive to the application of acetylcholine and is highly stable even in the absence of ligand. Moreover, electrophysiological recordings from a GLIC channel (another bacterial member of the superfamily) engineered to have a ring of phenylalanines at the corresponding pore-facing position suggest that this novel refractory state is distinct from the well-known desensitized state. It seems reasonable to propose, then, that it is in this peculiar non-conductive conformation that the ELIC channel was crystallized. It seems also reasonable to propose that, in the absence of rings of pore-facing aromatic side chains, such stable conformation may never be attained by the acetylcholine receptor. Incidentally, we also noticed that the response of the proton-gated, wild-type GLIC channel to a fast change in pH from 7.4 to 4.5 (on the extracellular side) is only transient, with the evoked current fading completely in a matter of seconds. This raises the possibility that the crystal structures of GLIC obtained at pH 4.0 and pH 4.6 correspond to the (well-known) desensitized state.

A single immunoglobulin-domain protein required for clustering acetylcholine receptors inC. elegans

At Caenorhabditis elegans neuromuscular junctions (NMJs), synaptic clustering of the levamisole-sensitive acetylcholine receptors (L-AChRs) relies on an extracellular scaffold assembled in the synaptic cleft. It involves the secreted protein LEV-9 and the ectodomain of the transmembrane protein LEV-10, which are both expressed by muscle cells. L-AChRs, LEV-9 and LEV-10 are part of a physical complex, which localizes at NMJs, yet none of its components localizes independently at synapses. In a screen for mutants partially resistant to the cholinergic agonist levamisole, we identified oig-4, which encodes a small protein containing a single immunoglobulin domain. The OIG-4 protein is secreted by muscle cells and physically interacts with the L-AChR/LEV-9/LEV-10 complex. Removal of OIG-4 destabilizes the complex and causes a loss of L-AChR clusters at the synapse. Interestingly, OIG-4 partially localizes at NMJs independently of LEV-9 and LEV-10, thus providing a potential link between the L-AChR-associated scaffold and local synaptic cues. These results add a novel paradigm for the immunoglobulin super-family as OIG-4 is a secreted protein required for clustering ionotropic receptors independently of synapse formation.

Characterization of peripheral blood acetylcholine receptor-binding B cells in experimental myasthenia gravis

In myasthenia gravis (MG), the neuromuscular transmission is impaired by antibodies (Abs) specific for muscle acetylcholine receptor (AChR). Anti-AChR Abs can be detected in the serum of MG patients, although their levels do not correlate with disease severity. In this study, we developed a flow cytometric assay for the detection of peripheral blood AChR-specific B cells to characterize B cell phenotypes associated with experimental autoimmune myasthenia gravis (EAMG). Alexa-conjugated AChR was used as a probe for AChR-specific B cells (B220+Ig+). Mice with EAMG had significantly elevated frequencies of AChR-specific IgG2+ and IgM+ B cells. While the frequencies of IgG2+ B cells and plasma anti-AChR IgG2 levels significantly correlated with the clinical grades of EAMG, the frequencies of IgM+ B cells and plasma anti-AChR IgM levels did not. These results indicate that the frequency of AChR-specific and IgG1+ (mouse IgG2 equivalent) peripheral blood B cells and anti-AChR IgG1 levels could be potential biomarkers for MG disease severity.

Sorting receptor Rer1 controls surface expression of muscle acetylcholine receptors by ER retention of unassembled α-subunits

The nicotinic acetylcholine receptor of skeletal muscle is composed of five subunits that are assembled in a stepwise manner. Quality control mechanisms ensure that only fully assembled receptors reach the cell surface. Here, we show that Rer1, a putative Golgi-ER retrieval receptor, is involved in the biogenesis of acetylcholine receptors. Rer1 is expressed in the early secretory pathway in the myoblast line C2C12 and in mouse skeletal muscle, and up-regulated during myogenesis. Upon down-regulation of Rer1 in C2C12 cells, unassembled acetylcholine receptor α-subunits escape from the ER and are transported to the plasma membrane and lysosomes, where they are degraded. As a result, the amount of fully assembled receptor at the cell surface is reduced. In vivo Rer1 knockdown and genetic inactivation of one Rer1 allele lead to significantly smaller neuromuscular junctions in mice. Our data show that Rer1 is a functionally important unique factor that controls surface expression of muscle acetylcholine receptors by localizing unassembled α-subunits to the early secretory pathway.

Proteomic analysis of chronic restraint stress-induced Gan (肝)-stagnancy syndrome in rats

To analyze the proteomic characteristics of Gan (肝)-stagnancy syndrome (GSS) by seeking the differential protein in blood and tissues of GSS model rats. GSS model rats were established by chronic restraint stress, keeping rats in restrain chamber for 6 h every day for 21 successive days. Their blood and liver samples were collected at the end of experiment for differential protein detection with methods of isoelectrofocusing and polyacrylamide SDS-PAGE, silver staining, and scanning. The gel images were analyzed with Imagemaster 2D Elite software, and the excavated differential protein spots were identified with matrix assistant laser resolving TOF mass spectrometry, Western blot, ELISA, and RT-PCR, respectively. A method for isolating the protein in blood serum and tissues by two-dimensional gel electrophoresis was established and optimized. Six serum proteins and three liver proteins that differentially expressed were identified. The down-regulated differential proteins in serum of GSS model rats were serum albumin precursor, beta 1 globin, antibody against muscle acetylcholine receptor, Ig lambda-2 C region, and transthyretin (TTR), and those in liver tissue were aryl sulfotransferase, enoyl-CoA hydratase, and TTR. TTR down-regulation was found in both serum and liver. Preliminary biological information analysis showed that these differential proteins involved in immune, neuroendocrine, nutrition, and substance metabolism. Proteomic analysis of differential proteins showed that TTR, aryl sulfotransferase, and enoyl-CoA hydratase expressions are downregulated in the GSS model rats, suggesting that the susceptibility of cancer could be enhanced by chronic stress.

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.

Bridging the Gap between Structural Models of Nicotinic Receptor Superfamily Ion Channels and Their Corresponding Functional States

Aromatic–aromatic interactions are a prominent feature of the crystal structure of ELIC [Protein Data Bank (PDB) code 2VL0], a bacterial member of the nicotinic receptor superfamily of ion channels where five pore-facing phenylalanines come together to form a structure akin to a narrow iris that occludes the transmembrane pore. To identify the functional state of the channel that this structure represents, we engineered phenylalanines at various pore-facing positions of the muscle acetylcholine (ACh) receptor (one position at a time), including the position that aligns with the native phenylalanine 246 of ELIC, and assessed the consequences of such mutations using electrophysiological and toxin-binding assays. From our experiments, we conclude that the interaction among the side chains of pore-facing phenylalanines, rather than the accumulation of their independent effects, leads to the formation of a nonconductive conformation that is unresponsive to the application of ACh and is highly stable even in the absence of ligand. Moreover, electrophysiological recordings from a GLIC channel (another bacterial member of the superfamily) engineered to have a ring of phenylalanines at the corresponding pore-facing position suggest that this novel refractory state is distinct from the well-known desensitized state. It seems reasonable to propose then that it is in this peculiar nonconductive conformation that the ELIC channel was crystallized. It seems also reasonable to propose that, in the absence of rings of pore-facing aromatic side chains, such stable conformation may never be attained by the ACh receptor. Incidentally, we also noticed that the response of the proton-gated wild-type GLIC channel to a fast change in pH from pH 7.4 to pH 4.5 (on the extracellular side) is only transient, with the evoked current fading completely in a matter of seconds. This raises the possibility that the crystal structures of GLIC obtained at pH 4.0 (PDB code 3EHZ) and pH 4.6 (PDB code 3EAM) correspond to the to the (well-known) desensitized state.

Mechanism of verapamil action on wild‐type and slow‐channel mutant human muscle acetylcholine receptor

Verapamil, a Ca(2+) channel blocker widely used in clinical practice, also affects the properties of frog and mouse muscle acetylcholine receptor (AChR). Here, we examine the mechanism of action of verapamil on human wild-type and slow-channel mutant muscle AChRs harboring in any subunit a valine-to-alanine mutation of 13' residue of the pore-lining M2 transmembrane segment. Verapamil, after a pre-treatment of 0.5-10 s, accelerated the decay of whole-cell or macroscopic outside-out currents within milliseconds of ACh application even at clinically attainable doses. Recordings of unitary events in the cell-attached and outside-out configurations showed that verapamil does not alter single-channel conductance, but reduces channel open probability, by prolonging the dwell time into the closed state for wild-type and all mutant AChR. The duration of channel openings decreased only for the epsilonV265A-AChR, by shortening the longest exponential component of the open-time distribution. These results provide a rationale for the therapeutic use of verapamil in the slow-channel syndrome and emphasize the major role played by epsilon subunit in controlling the functional properties of human muscle AChR, as revealed by the peculiar alterations imparted by mutations in this subunit.

In vivo adsorption of autoantibodies in myasthenia gravis using Nanodisc-incorporated acetylcholine receptor

Autoantibodies directed against the skeletal muscle acetylcholine receptor (AChR) play a critical role in the pathogenesis of the autoimmune disease, myasthenia gravis (MG). The pathogenic importance of anti-AChR antibodies is substantiated clinically by the often dramatic clinical improvement that follows removal of circulating antibodies utilizing extracorporeal plasma exchange (PE). Unfortunately, the effects of PE are non-specific as immunoglobulins (IgG) and other plasma proteins are removed in addition to anti-AChR IgG. In this study, we have successfully incorporated the AChR protein purified from Torpedo californicus into a Nanodisc (ND) membrane scaffold protein/phospholipid structure. We go on to demonstrate the effectiveness of this ND–AChR complex, administered intravenously, in the in vivo down-modulation of anti-AChR antibodies and subsequent amelioration of clinical disease in the experimental murine model of MG. These results provide proof-of-principle for the in vivo antigen-specific reduction of pathogenic anti-AChR antibodies utilizing ND–AChR particles. Further development of this strategy may provide an effective, antigen-specific, and readily accessible acute therapy for exacerbating MG or myasthenic crisis.

Structure-Activity Relationship of Quaternary Acetylcholinesterase Inhibitors – Outlook for Early Myasthenia Gravis Treatment

Myasthenia gravis is a rare autoimmune neuromuscular junction disorder mainly caused by antibodies being targeted against the muscle acetylcholine receptors (AChRs). The loss of AChRs leads to a defect in neuromuscular transmission resulting in muscle weakness and fatigue. Although once an often fatal illness, Myasthenia gravis can now be well managed with relatively safe and effective treatments. However, the severe myasthenic cases associated with thymus tumors remain often fatal exception in the management of the disease. The early treatment includes the use of acetylcholinesterase inhibitors (AChEI) which enhance neuromuscular transmission. To ensure a peripheral effect, charged molecules are used, particularly quaternary ammonium salts. The structure of AChEIs has been continuously modified to obtain the optimal ratio between AChE inhibition and potential side-effects. This review summarizes progress in the use of quaternary compounds as AChE inhibitors in vitro with respect to their structure and inhibitory ability. Namely, carbamic acid esters, piperidinium and pyridinium salts, bisquaternary pyridinium salts and heterogeneous quaternary inhibitors are all discussed. Among data found in the literature, many compounds have shown promising inhibition of AChE when compared to commercial standards (pyridostigmine, neostigmine). Besides a promising inhibitory ability, selectivity for AChE versus butyrylcholinesterase (BChE) for the most potent compounds (sub-nanomolar IC(50)) was also identified.

Acetylcholine negatively regulates development of the neuromuscular junction through distinct cellular mechanisms

Emerging evidence suggests that the neurotransmitter acetylcholine (ACh) negatively regulates the development of the neuromuscular junction, but it is not clear if ACh exerts its effects exclusively through muscle ACh receptors (AChRs). Here, we used genetic methods to remove AChRs selectively from muscle. Similar to the effects of blocking ACh biosynthesis, eliminating postsynaptic AChRs increased motor axon branching and expanded innervation territory, suggesting that ACh negatively regulates synaptic growth through postsynaptic AChRs. However, in contrast to the effects of blocking ACh biosynthesis, eliminating postsynaptic AChRs in agrin-deficient mice failed to restore deficits in pre- and postsynaptic differentiation, suggesting that ACh negatively regulates synaptic differentiation through nonpostsynaptic receptors. Consistent with this idea, the ACh agonist carbachol inhibited presynaptic specialization of motorneurons in vitro. Together, these data suggest that ACh negatively regulates axon growth and presynaptic specialization at the neuromuscular junction through distinct cellular mechanisms.

Clinical implication of peripheral CD4 +CD25 + regulatory T cells and Th17 cells in myasthenia gravis patients

Myasthenia gravis (MG) is an autoimmune disorder generally mediated by antibodies against the acetylcholine receptors (AChR) of the skeletal muscles. CD4 T cells help B cells to produce antibodies through their production of cytokines or chemokines. In this study, we evaluated the frequency of regulatory (Treg) and IL-17 producing CD4 T-cell subsets (Th17) in peripheral blood mononuclear cells (PBMCs) of patients with MG. The transcription factor, forkhead transcription factor (Foxp3), is essential for T-cell regulatory function, and the orphan nuclear receptor, RORγT, is important in Th17 cell differentiation. In MG patients, Foxp3 mRNA expression in PBMCs was lower than those in healthy subjects ( p = 0.007), while there was no significant difference of RORγT mRNA expression between MG patients and healthy subjects. Glucocorticoid-induced tumour-necrosis-factor receptor-related protein (GITR) is expressed predominantly on CD4 +CD25 + Treg cells. We found that the number of GITR +CD4 +CD25 + T cells in peripheral lymphocytes in MG patients was lower than that in healthy subjects ( P < 0.01). In addition, there was a significant positive correlation between the change of the frequency of GITR +CD4 +CD25 + T cells and the changing rate in quantitative myasthenia gravis scores (%) ( p = 0.03). Furthermore, there was a significant negative correlation between the change of the percentage of GITR +CD4 +CD25 + T cells (% lymphocytes) and the changing rate of daily PSL doses (%) ( P = 0.002). The relative RORγT levels in PBMCs negatively correlated with the Th1/Th2 ratio in CD4 + cells in MG patients ( p = 0.014). In conclusion, our findings suggest that Th17 cells affect the production of autoantibodies through their influence on the Th1- and Th2-cytokine balance in PBMCs of MG patients. On the other hand, Treg cells are suggested to be involved in the clinical condition or severity of MG disease.