Junctional Acetylcholine Receptors Research Articles

A CHRNE frameshift mutation causes congenital myasthenic syndrome in young Jack Russell Terriers

Congenital myasthenic syndromes (CMSs) are a group of rare genetic disorders of the neuromuscular junction resulting in structural or functional causes of fatigable weakness that usually begins early in life. Mutations in pre-synaptic, synaptic and post-synaptic proteins have been demonstrated in human cases, with more than half involving aberrations in nicotinic acetylcholine receptor (AChR) subunits. CMS was first recognized in dogs in 1974 as an autosomal recessive trait in Jack Russell Terriers (JRTs). A deficiency of junctional AChRs was demonstrated. Here we characterize a CMS in 2 contemporary cases of JRT littermates with classic clinical and electromyographic findings, and immunochemical confirmation of an approximately 90% reduction in AChR protein content. Loci encoding the 5 AChR subunits were evaluated using microsatellite markers, and CHRNB1 and CHRNE were identified as candidate genes. Sequences of the splice sites and exons of both genes revealed a single base insertion in exon 7 of CHRNE that predicts a frameshift mutation and a premature stop codon. We further demonstrated this pathogenic mutation in CHRNE in archival tissues from unrelated JRTs studied 34 years ago.

The neuromuscular activity of Bothriopsis bilineata smaragdina (forest viper) venom and its toxin Bbil-TX (Asp49 phospholipase A2) on isolated mouse nerve-muscle preparations.

The presynaptic action of Bothriopsis bilineata smaragdina (forest viper) venom and Bbil-TX, an Asp49 PLA2 from this venom, was examined in detail in mouse phrenic nerve-muscle (PND) preparations invitro and in a neuroblastoma cell line (SK-N-SH) in order to gain a better insight into the mechanism of action of the venom and associated Asp49 PLA2. In low Ca(2+) solution, venom (3μg/ml) caused a quadriphasic response in PND twitch height whilst at 10μg/ml the venom additionally induced an abrupt and marked initial contracture followed by neuromuscular facilitation, rhythmic oscillations of nerve-evoked twitches, alterations in baseline and progressive blockade. The venom slowed the relaxation phase of muscle twitches. In low Ca(2+), Bbil-TX [210nM (3μg/ml)] caused a progressive increase in PND twitch amplitude but no change in the decay time constant. Venom (10μg/ml) and Bbil-TX (210nM) caused minor changes in the compound action potential (CAP) amplitude recorded from sciatic nerve preparations, with no significant effect on rise time and latency; tetrodotoxin (3.1nM) blocked the CAP at the end of the experiments. In mouse triangularis sterni nerve-muscle (TSn-m) preparations, venom (10μg/ml) and Bbil-TX (210nM) significantly reduced the perineural waveform associated with the outward K(+) current while the amplitude of the inward Na(+) current was not significantly affected. Bbil-TX (210nM) caused a progressive increase in the quantal content of TSn-m preparations maintained in low Ca(2+) solution. Venom (3μg/ml) and toxin (210nM) increased the calcium fluorescence in SK-N-SH neuroblastoma cells loaded with Fluo3 AM and maintained in low or normal Ca(2+) solution. In normal Ca(2+), the increase in fluorescence amplitude was accompanied by irregular and frequent calcium transients. In TSn-m preparations loaded with Fluo4 AM, venom (10μg/ml) caused an immediate increase in intracellular Ca(2+) followed by oscillations in fluorescence and muscle contracture; Bbil-TX did not change the calcium fluorescence in TSn-m preparations. Immunohistochemical analysis of toxin-treated PND preparations revealed labeling of junctional ACh receptors but a loss of the presynaptic proteins synaptophysin and SNAP25. Together, these data confirm the presynaptic action of Bbil-TX and show that it involves modulation of K(+) channel activity and presynaptic protein expression.

Abnormal Spreading and Subunit Expression of Junctional Acetylcholine Receptors of Paraspinal Muscles in Scoliosis Associated With Syringomyelia

A comparative study was performed among 2 groups of patients: Group A with scoliosis and syringomyelia and Group B with idiopathic scoliosis. To investigate the denervation of paraspinal muscles and analyze its association with scoliosis in patients with syringomyelia. The mechanism by which scoliosis develops secondary to syringomyelia remains unclear. Some authors hypothesize that scoliosis may be caused by an alteration in the innervation of the trunk musculature. Few studies, however, have been reported to testify the presence of denervation of the paraspinal muscles in scoliotic patients with syringomyelia. Forty-one children were enrolled in the study and were divided into 2 groups. Group A consisted of 25 patients with scoliosis associated with syringomyelia. Group B included 16 adolescents with idiopathic scoliosis. Bilateral biopsy of paraspinal muscles was performed during scheduled spinal surgery. Distribution of acetylcholine receptors (AChRs) and acetylcholinesterase was investigated by immunofluorescence staining. RT-PCR was performed to determine the AChRs subunit mRNA expression. Immunostaining showed that 56% patients in Group A were scored positive for the loss of localization of AChRs to neuromuscular junction, while all Group B patients were negative. gamma-AChR subunit expression was detected in 65% patients in Group A while absent in all samples in Group B. There was no statistical significance of both the positive rate of abnormal spreading and that of gamma subunit expression of AChRs between the convex and concave side in Group A. The denervation of paraspinal muscles is present in some patients with scoliosis associated with syringomyelia. The size of the syrinx and the degree of cerebellar tonsillar descent might seem to have no relation to the denervation of paraspinal muscles.

P58. Abnormal Spread of Junctional Acetylcholine Receptor of Paravertebral Muscles in Scoliosis Associated With Syringomyelia

P58. Abnormal Spread of Junctional Acetylcholine Receptor of Paravertebral Muscles in Scoliosis Associated With Syringomyelia

Structural determinants of the reliability of synaptic transmission at the vertebrate neuromuscular junction.

The reliability of neuromuscular transmission depends on the size and molecular organization of the neuromuscular junction. Comparative studies show that the quantal release per unit area is similar at neuromuscular junctions in a number of species in spite of wide variation in synaptic area. They also show an inverse relationship between the size of the nerve terminal and the extent of postsynaptic folding. Evidence is presented supporting the view that the folds, and the voltage-gated sodium channels present in them, effectively amplify synaptic currents. How are the size and molecular organization of the neuromuscular junction determined? Studies with botulinum toxin, including our new work on humans, reveal striking "adaptive plasticity" of the nerve terminal. However, the links between synaptic size and effective transmission remain unclear. On the postsynaptic side, we have shown that mRNA encoding sodium channels is concentrated at the adult junction. During development, mRNA accumulates just before the protein it encodes. Throughout development the sodium channels are associated with ankyrinG and both proteins are initially excluded from the junctional acetylcholine receptor cluster, possibly accounting for the formation of the boundary between the domains occupied by the two key postsynaptic ion channels. These findings have important clinical implications. Reduced transmitter release may result from small nerve terminals as much as from defective release. Abnormal folding is likely to reduce the reliability of transmission. A better understanding of how the structural features that influence the reliability of the neuromuscular transmission are controlled should be of general interest to neuroscientists and of use to clinicians.

Looking back on the discovery of alpha-bungarotoxin.

This review is a personal narration by a retiring pharmacologist from Taiwan who looks back at his discovery of alpha-bungarotoxin from the historical perspective of Taiwan during the last 50 years, with accounts of his experiences and his efforts to overcome hardship. How the alpha-toxin was isolated and characterized as an irreversible specific nicotinic acetylcholine (ACh) receptor antagonist, and how it subsequently became a useful experimental probe are presented here. The dilemma of differentiating the actions of tubocurarine and alpha-bungarotoxin is analyzed. The author also outlines findings based on work done in his laboratory using alpha-bungarotoxin as a tool on particular aspects of synaptic transmission. These include presynaptic receptor for positive feedback of transmitter release, explosive release of ACh, up- and downregulation of ACh receptors after chronic drug treatment, autodesensitization of junctional ACh receptors, differences in action between natural transmitter and exogenous agonists and that between junctional and extrajunctional ACh receptors. Some experimental pitfalls, in which biomedical scientists are frequently trapped, are raised. Finally, some anecdotes are appended from which the reader may further understand scientific life in the 20th century, including its joys and regrets.

Morphological correlates of the differential responses of muscles to vecuronium

We have noted previously that duration of vecuronium block correlated with fibre size in six muscle groups in the goat. Electrophysiological considerations suggest that the important factor should be the number of acetylcholine receptors (AChR) relative to fibre size. However, this hypothesis could not be verified in the goat because the number of AChR was relatively constant in the different muscles despite differences in fibre size. Therefore, in this study, we have investigated the relationship between sensitivity to vecuronium, as reflected by the ED50 and duration of block, of six muscles in the cat and the number of AChR per unit fibre cross-sectional area (CSA). The ED50 and duration of action (time to 50% recovery of the first twitch after a dose of 15 micrograms kg-1) of vecuronium in the tibialis cranialis, soleus, rectus abdominis, masseter, diaphragm and thyroarytenoideus muscles were determined during train-of-four stimulation and EMG recording in seven cats anaesthetized with pentobarbital. CSA of the muscle fibres and number of junctional AChR in these muscles were measured by histological methods and 125I-alpha-bungarotoxin binding assay, respectively, and the number of AChR per unit fibre CSA calculated. The association between muscle response (ED50 and duration of block) and fibre CSA or number of AChR per unit fibre CSA was then tested by regression analyses. Duration of block varied between the six muscles (mean 8.9 (SEM 2.6) to 20.3 (3.1) min; P = 0.0001) but ED50 did not (7.5 (1.5) to 15.6 (2.5) micrograms kg-1; P = 0.185). Fibre CSA and number of AChR per unit fibre CSA also varied between these muscles (P = 0.0001). Duration to 50% TI recovery was prolonged in muscles with a low number of AChR relative to fibre CSA (r2 = 0.30; P = 0.0002) and the ED50 increased as the number of AChR per fibre CSA increased (r2 = 0.240; P = 0.0016). These results in the cat suggest that the number of junctional AChR relative to fibre CSA is a morphological predictor of the differential sensitivities of muscles to neuromuscular blocking agents.

Mechanism of neuromuscular blockade induced by phenthonium, a quaternary derivative of (-)-hyoscyamine, in skeletal muscles.

1. The mechanisms underlying the postjunctional blockade induced by phenthonium [N-(4-phenyl) phenacyl 1-hyoscyamine] were investigated in mammalian and amphibian muscles. This muscarinic antagonist was previously shown to enhance specifically the spontaneous acetylcholine (ACh) release at concentrations that blocked neuromuscular transmission. 2. In both rat diaphragm and frog sartorius muscles, phenthonium (Phen, 1-100 microM) depressed the muscle twitches elicited by nerve stimulation (IC50: 23 microM and 5 microM, respectively), and blocked the nerve-evoked muscle action potential. The neuromuscular blockade was not reversed after incubation with neostigmine. 3. Equal concentrations of Phen decreased the rate of rise and prolonged the falling phase of the directly elicited action potential in frog sartorius muscle fibres, indicating that the drug also affects the sodium and potassium conductance. 4. Phen (50 and 100 microM) protected the ACh receptor against alpha-bungarotoxin (BUTX) blockade in the mouse diaphragm allowing recording of endplate potentials and action potentials after 5 h wash with physiological salt solution. 5. Phen (10-100 microM) produced a concentration- and voltage-dependent decrease of the endplate current (e.p.c.), and induced nonlinearity of the current-voltage relationship. At high concentrations Phen also shortened the decay time constant of e.p.c (tau(e.p.c.)) and reduced its voltage sensitivity. 6. At the same range of concentrations, Phen also reduced the initial rate of [125I]-BUTX binding to junctional ACh receptors of the rat diaphragm (apparent dissociation constant = 24 microM), the relationship between the degree of inhibition and antagonist concentration being that expected for a competitive mechanism. 7. It is concluded that Phen affects the electrical excitability of the muscle fibre membrane, and blocks neuromuscular transmission through a mechanism that affects the agonist binding to its recognition site and ionic channel conductance of the nicotinic ACh receptor.

Acetylcholine Receptors in Innervated Muscles of DystrophicmdxMice Degrade as after Denervation

Acetylcholine receptors (AChRs) are present at the top of the postsynaptic membrane of the neuromuscular junction (NMJ) at very high density, possibly anchored to cytoskeletal elements. The present study investigated whether AChR degradation is affected in animals lacking dystrophin, a protein that is an integral part of the cytoskeletal complex and is missing in Duchenne muscular dystrophy. The animal model for Duchenne muscular dystrophy, the mutant mdx mouse, was used to determine whether disruption of the cytoskeleton, caused by the absence of dystrophin, affects AChR degradation. Of the two populations of junctional AChRs, Rs (expressed in innervated adult muscles) and Rr (expressed in embryonic or denervated muscles), only Rs are affected in mdx animals. In innervated mdx soleus, diaphragm, and sternomastoid muscles, the AChRs have an accelerated degradation rate (t1/2 of approximately 3-5 d), similar to that acquired by Rs in control muscles after denervation. The Rs in mdx NMJs do not accelerate further when the muscles are denervated. The absence of dystrophin does not affect the degradation rate of the Rr AChRs (t1/2 of 1 d), which are expressed after denervation in mdx as in control muscles. These results suggest that dystrophin or an intact cytoskeletal complex may be required for neuronal stabilization of Rs receptors at the adult neuromuscular junctions.

Number of junctional acetylcholine receptors: control by neural and muscular influences in the rat.

1. The number of acetylcholine receptors (AChRs) per neuromuscular junction in soleus muscles of adult rats was estimated from counts of 125I-alpha-bungarotoxin binding sites. The muscles were either denervated, denervated and electrically stimulated, paralysed by botulinum toxin (BoTX), or paralysed by tetrodotoxin (TTX). 2. After denervation, the number of junctional AChRs was normal after 18 days and then fell to 54 and 35% of normal after 33 and 57 days, respectively. 3. Direct high frequency muscle stimulation (100 Hz) maintained a normal number of junctional AChRs for at least 2 months when the stimulation started on the day of denervation. When the stimulation was started progressively later, the effect of the stimulation on AChR number disappeared within about a week. The disappearance was gradual and appeared to affect all the muscle fibres equally. 4. Stimulation at 100 Hz, starting on the day of denervation and stopping after 18 days, did not prevent the endplates from losing AChRs during the subsequent 15 days without stimulation. Thus 100 Hz stimulation and innervation are not equivalent in their effects on junctional AChR number. 5. Direct low frequency muscle stimulation from the day of denervation did not maintain a normal number of junctional AChRs, as the number of AChRs fell to 70 and 62% of normal after 33 days of stimulation at 20 and 10 Hz, respectively. 6. Endplates paralysed by BoTX or TTX for 33 days lost about as many junctional AChRs (54 and 55%) as endplates denervated for 33 days (46%). Direct stimulation at 100 Hz during the last 15 days of BoTX treatment reduced but did not prevent this AChR loss (36% loss at 33 days). 7. The results show that when motor nerve terminals in rat soleus muscles are removed by axotomy, they leave a 'trace' which, in conjunction with appropriate muscle stimulation, can maintain a normal number of AChRs in the postsynaptic region. In non-stimulated muscles the trace responsible for this maintenance disappears within about a week. In stimulated muscles it persists for at least 2 months. From indirect evidence it appears that the trace is a factor, or the postsynaptic effect of a factor, released by impulse activity in the nerve, and that its degradation after denervation is accelerated by the acute effects of nerve degeneration.

Tunicamycin alters channel gating characteristics of junctional and extrajunctional acetylcholine receptors expressed in Xenopus oocytes

The acute effects of tunicamycin on mouse junctional and extrajunctional acetylcholine receptors (AChRs) expressed in Xenopus oocytes, distinct from its ability to block N-glycosylation of the protein, was examined at the single channel level. Tunicamycin reduced the frequency of ACh-activated single channel openings and prolonged the mean channel open times of AChRs in a dose-dependent manner, without affecting inward conductances. Continuous application of ACh to both junctional and extrajunctional AChR channels very slowly decreased the number of channel opening events, and tunicamycin accelerated this process. These results suggest that tunicamycin alters channel gating kinetics and accelerates transition towards a desensitized state.

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.

Properties of acetylcholine receptors in adult rat skeletal muscle fibers in culture.

The distribution and biophysical properties of acetylcholine receptors were studied, using morphological and patch-clamp techniques, in adult rat skeletal muscle fibers dissociated by collagenase and maintained in culture. Up to ten hours after dissociation, there were no changes in either the distribution or the biophysical properties of junctional acetylcholine receptors. In long-term culture (5 to 14 days), a new type of acetylcholine receptor was inserted all over the muscle fibers; the channel properties were characterized by a longer open time and a smaller conductance, similar to what has been observed in in vivo denervated muscles. Using autoradiography, we found that during culture an impaired incorporation of new acetylcholine receptors in the former endplates caused a progressive decrease in the density of junctional acetylcholine receptors. This contrasts with muscle fibers denervated in vivo, where the density of receptors does not change after denervation.

Effect of myasthenic IgG on degradation of junctional acetylcholine receptor

We investigated the effect of the IgG from patients with myasthenia gravis (MG) on the degradation of normal rat junctional acetylcholine receptor (AChR) labeled with 125I-alpha-bungarotoxin (BuTx) and calculated the degradation rate (DR). The DR for the IgG from these patients was significantly higher than that from healthy volunteers and patients with other autoimmune diseases. For MG, DR was significantly correlated with the severity of the disease but not with anti-AChR antibody titer. DR was accelerated by IgG from patients with generalized MG whose antibody titers were in the normal range and by IgG from patients with ocular MG. These results indicate that measurement of the DR of junctional AChR in normal rats is more closely correlated with the severity of the disease than is measurement of anti-AChR antibody and that the former is a sensitive and confirmatory method for evaluating MG.

Degradation of two AChR populations at rat neuromuscular junctions: regulation in vivo by electrical stimulation

The effect of electrical stimulation on the stability of junctional ACh receptors (AChR) on soleus muscles of Wistar rats was compared to that of denervation and reinnervation. Denervation causes the degradation rate of the slowly degrading AChRs (Rs) at the neuromuscular junction to accelerate and be replaced by rapidly degrading AChRs (Rr), while reinnervation restabilizes the accelerated Rs. Electrical stimulation initiated at the time of denervation prevented the acceleration of the Rs. It could not, however, reverse the effect of denervation if initiated after the AChRs became destabilized, nor could it slow the degradation rate of the Rr. We conclude that electrical stimulation of denervated muscle downregulates the expression of the Rr and prevents the destabilization of Rs.

Long‐Term Treatment with Glucocorticoids Increases Synthesis and Stability of Junctional Acetylcholine Receptors on Innervated Cultured Human Muscle

We have studied the effect of long-term treatment with hydrocortisone on the expression of acetylcholine receptors (AChRs) at the neuromuscular junctions of human muscle cultured in monolayer and innervated de novo by fetal rat spinal cord motoneurons. Hydrocortisone increased accumulation of junctional AChRs in a dose- and time-dependent fashion. This increase was due to both decreased degradation and increased synthesis of AChRs. Other glucocorticoids, dexamethasone and prednisolone, exerted similar effects. Our study demonstrates a novel action of glucocorticoids on human junctional AChRs.

Relationship of a dystrophin-associated glycoprotein to junctional acetylcholine receptor clusters in rat skeletal muscle

The relationship of a member of the transmembrane dystrophin-associated glycoprotein (DAG) complex to acetylcholine receptors (AChRs) was investigated using immunofluorescence techniques at rat neuromuscular junctions (NMJs) viewed en face. These results were compared with those from a similar previous study of dystrophin and an autosomal homologue (utrophin or dystrophin-related protein, DRP) (Bewick et al. NeuroReport 1992; 3; 857–860). The region of highest 43 K DAG (43DAG) labelling projected beyond the AChRs by ∼0.3 μm, as does that for dystrophin. By contrast DRP labelling precisely co-localizes with the AChRs. These results suggest that at the NMJ, the region of high 43DAG concentration encompasses the area of highest intensity labelling for both DRP and dystrophin.

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.

The influence of basic fibroblast growth factor on acetylcholine receptors in cultured muscle cells

Acetylcholine receptors (AChRs) in Xenopus muscle cells undergo changes in channel kinetics during development in culture and these changes are somehow related to innervation. Recently we showed that basic fibroblast growth factor (bFGF), when locally applied, can mimic the effect of nerve in inducing a postsynaptic-type development. In this study, we examined where bFGF can influence the developmental changes of AChRs. Patch clamp method was employed to record single AChR channel currents from cultured Xenopus myotomal muscle cells and the kinetics of low-conductance AChR channels were analyzed. In cultures treated with 1 μg/ml bFGF at an early stage (stage 23), the burst duration of low-conductance AChR channels showed a 1.5-fold decrease between the first and second day in culture, while it underwent a remarkable 3-fold decrease during the same period in the control. Histogram analyses showed that the low-conductance channels were composed of a fast and a slow component and that the decrease in burst duration was due to a shift in the population from the slow to the fast. bFGF treatment appeared to slow down this shift by retaining the slow channels for a longer period of time. This effect is probably not due to channel modulation as the burst duration of short channel in older cells (stage 40) was not affected by bFGF. These data suggest that bFGF may enhance the metabolic stability of intrinsically short-lived AChRs. This effect seems to parallel the stabilization of junctional AChRs at the innervated endplate. Thus, bFGF, or a related polypeptide growth factor, may mediate this and other innervation-induced changes in the postsynaptic membrane.

Degradation of junctional acetylcholine receptors: Role of muscle acitvity

Degradation of junctional acetylcholine receptors: Role of muscle acitvity