Extrajunctional Acetylcholine Receptors Research Articles

Early Changes in Skeletal Muscle of Young C22 Mice, A Model of Charcot-Marie-Tooth 1A.

Background:The C22 mouse is a Charcot-Marie-Tooth 1A transgenic model with minimal axonal loss.Objective:To analyse early skeletal muscle changes resulting from this dysmyelinating neuropathy.Methods:Histology of tibialis anterior muscles of C22 mice and wild type litter mate controls for morphometric analysis and (immuno-)histochemistry for known denervation markers and candidate proteins identified by representational difference analysis (RDA) based on mRNA from the same muscles; quantitative PCR and Western blotting for confirmation of RDA findings.Results:At age 10 days, morphometry was not different between groups, while at 21 days, C22 showed significantly more small diameter fibres, indicating the onset of atrophy at an age when weakness becomes detectable. Neither (immuno-)histochemistry nor RDA detected extrajunctional expression of acetylcholine receptors by age 10 and 21 days, respectively. RDA identified some mRNA up-regulated in C22 muscles, among them at 10 days, prior to detectable weakness or atrophy, integral membrane protein 2a (Itm2a), eukaryotic initiation factor 2, subunit 2 (Eif2s2) and cytoplasmic phosphatidylinositol transfer protein 1 (Pitpnc1). However, qPCR failed to measure significant differences. In contrast, Itm2a and Eif2s2 mRNA were significantly down-regulated comparing 21 versus 10 days of age in both groups, C22 and controls. Western blotting confirmed significant down-regulation of ITM2A protein in C22 only.Conclusion:Denervation-like changes in this model develop slowly with onset of atrophy and weakness at about three weeks of age, before detection of extrajunctional acetylcholine receptors. Altered Itm2a expression seems to begin early as an increase, but becomes distinct as a decrease later.

Activity-dependent vs. neurotrophic modulation of acetylcholine receptor expression: Evidence from rat soleus and extensor digitorum longus muscles confirms the exclusive role of activity.

Evoked electrical muscle activity suppresses the transcription of mRNAs for acetylcholine receptors in extrajunctional myonuclei. Muscle denervation or disuse releases such inhibition and extrajunctional receptors appear. However, in soleus muscles paralysed with nerve-applied tetrodotoxin, a restricted perijunctional region has been described where myonuclei remain inhibited, a finding attributed to nerve-derived trophic factor(s). Here, we reinvestigate extrajunctional acetylcholine receptor expression in soleus and extensor digitorum longus muscles up to 90days after denervation or up to 20days of disuse, to clarify the role of trophic factors, if any. The perijunctional region of soleus muscles strongly expressed acetylcholine receptors during the first 2-3weeks of denervation. After 2-3months, this expression had disappeared. No perijunctional expression was seen after paralysis by tetrodotoxin or botulinum toxin A. In contrast, the extensor digitorum longus never displayed suppressed perijunctional acetylcholine receptor expression after any treatment, suggesting that it is an intrinsic property of soleus muscles. Soleus denervation only transiently removed the suppression, and its presence in long-term denervated soleus muscles contradicts any contribution from nerve-derived trophic factor(s). In conclusion, our results confirm that evoked electrical activity is the physiological factor controlling the expression of acetylcholine receptors in the entire extrajunctional membrane of skeletal muscles.

Succinylcholine-induced Hyperkalemia and Beyond

The mechanism of succinylcholine-induced hyperkalemia was studied in three lesions affecting canine gastrocnemius muscle. Dogs were treated for 1 month before study: 10 with normal activity, 5 with unilateral sciatic nerve section, active on 3 legs, 5 with unilateral cast immobilization of a hind limb and pelvis, active on 3 legs, and 7 inactive with T6 section of the spinal cord. Succinylcholine responses were determined during thiopental―ha lothane (mean expired halothane 1.0 ± 0.2%) endotracheal anesthesia with arterial carbon dioxide tension of 38—42 mmHg, arterial oxygen tension of 100—120 mmHg, and muscle and body temperatures maintained at 37° ± 0.2°C. The investigators isolated and collected the venous drainage of gastrocnemius muscle and measured its total blood flow. Muscle potassium release and oxygen consumption were calculated as blood flow x (arterial content — venous content). Succinylcholine-induced gastrocnemius potassium release was greatest after both sciatic and cord section; oxygen consumption was increased in parallel. Disuse atrophy of one leg slightly increased both values but was insufficient to produce systemic hyperkalemia. Reuptake of potassium followed succinylcholine-induced release. Given before succinylcholine, modest doses of gallamine slightly modified the release of potassium, and total paralysis by gallamine blocked it.

“Necklace” fibers as a late clue to the interpretation of the forgotten “trilaminar” fibers

In a recent article by Bevilacqua et al. [1] on ‘‘necklace fibers, a new histological marker of late-onset MTM1related centronuclear myopathy’’, impressive light micrographs of such fibers are presented which closely resemble those previously described as ‘‘trilaminar’’ fibers [2–4], as far as the different techniques applied at the earlier and present times allow identification. Both the clinical and the structural changes appear rather similar within a wide spectrum of individual variations. Ringel et al. [2] in 1978 reported on a sporadic myopathy with pronounced rigidity, lack of spontaneous movements, and increased CK values. Electromyography appeared to be normal. The muscle biopsy at the age of 7 weeks showed numerous fibers with three concentric zones leading to the designation as trilaminar fibers. The innermost zone showed accumulations of mitochondria, glycogen, and electron dense material with only few remnants of myofibrils. The intermediate zone consisted of myofibrils showing Z-band streaming. The outer zone resembled a sarcoplasmic mass. Histochemistry revealed extrajunctional acetylcholine receptor (AChR) between the intermediate and outer zone. Increased muscle tone was attributed to a disturbed neural influence, while increased CK activity was interpreted as being more characteristic for a primary muscle lesion. The female case of Schroder and Schonberger published in 1981 [3, 4] was clearly a familial one, deceasing at the age of 18 months from bronchopneumonia. The older brother was born with bilateral club feet dying at the age of 3 months due to aspiration pneumonia. A younger brother showed delayed motor milestones with tremor since birth. He died at the age of 9 months. One sister and the parents appeared to be unaffected. Therefore, autosomal recessive inheritance was assumed. Muscle biopsy showed at the light microscopic level in semithin sections many muscle fibers with an outer light zone, an intermediate darker ring, and a relatively light center (Fig. 1a–d). At the electron microscopic level (Fig. 2a, b), there were glycogen granules, mitochondria, components of the T-system and microtubules in the outer zone. The intermediate ring showed an increased density of myofibrillar structures, while the center was rather disorganized. The nuclei were located in the peripheral, not in the central zone similar to those in the recent biopsies. The trilaminar fibers were smaller than the normal ones but not severely atrophic. To me, there appears to be no essential difference between ‘‘necklace’’ and ‘‘trilaminar’’ fibers, although the published electron micrographs of necklace fibers show less impressive or representative changes than the light micrographs and the electron micrographs previously published of the trilaminar fibers. The anti-desmin and antiaB-crystalline labelling of the intermediate ring in necklace fibers corresponds quite closely to the electron dense intermediate zone of trilaminar fibers, which I would have liked to call ‘‘cockade’’ (in French ‘‘cocarde’’, in German: Kokarden-) fibers if there would not have been the national emblematic implication. ‘‘Trilaminar’’ or ‘‘necklace’’ fibers are obviously very rare because in 40 years (between 1965 and 2005) looking at more than 12,000 muscle biopsies there was not a single other one with similar changes, including myotonic dystrophy. Nevertheless, it is highly appreciated that the changes have now been attributed to a distinct molecular genetically defined myopathy, which is due to mutations in the phosphoinositide phosphatase myotubularin 1 protein (MTM1) gene [1]. J. M. Schroder (&) Department of Neuropathology, Aachen University Hospital, RWTH Aachen, Pauwelsstr. 30, 52074 Aachen, Germany e-mail: jmschroder@ukaachen.de

Early detection of denervated muscle fibers in hindlimb muscles after sciatic nerve transection in wild type mice and in the G93A mouse model of amyotrophic lateral sclerosis

The cell adhesion molecule N-CAM is localized to the adult neuromuscular junction but is also expressed in the extrajunctional membrane of denervated muscles concurrent with extrajunctional acetylcholine receptors. Here we used N-CAM immunohistochemistry to determine whether we could detect early denervation in hindlimb muscles of the G93A transgenic mouse model of amyotrophic lateral sclerosis (ALS). In denervated wild type mouse muscles, N-CAM immunoreactivity on the sarcolemma of all fiber types and within the sarcoplasm of only type IIA fibers was detected at day 2: ∼30% of the muscle fibers in cross-section were fully circumscribed by N-CAM immunoreactivity and ∼25% of fibers were incompletely circumscribed. The proportion of the latter fibers remained constant over the next 8 days as the proportions of the former fibers increased exponentially. Thereafter, fully circumscribed muscle fibers increased to a maximum by 30 days with a concomitant fall in the incompletely circumscribed fibers. Hence, early muscle denervation was detected by the incomplete circumscription of fiber membranes by N-CAM immunoreactivity with full circumscription and intracellular localization indicating more long-term denervation. In the G93A transgenic mouse, rapid denervation of fast-twitch muscles was readily detected by a corresponding proportion of muscle fibers in cross-section with positive N-CAM immunoreactivity. The proportions of incompletely and completely circumscribed muscle fibers corresponded well with the rate of decline in intact motor units and reduced muscle contractile forces. Progressively more fully circumscribed muscle fibers became evident with age. We conclude that the N-CAM immunoreactivity on muscle fiber membranes in muscle cross-sections provides a sensitive means of detecting early muscle fiber denervation.

Prolongation of Evoked and Spontaneous Synaptic Currents at the Neuromuscular Junction after Activity Blockade Is Caused by the Upregulation of Fetal Acetylcholine Receptors

It has been shown previously in a number of systems that after an extended block of activity, synaptic strength is increased. We found that an extended block of synaptic activity at the mouse neuromuscular junction, using a tetrodotoxin cuff in vivo, increased synaptic strength by prolonging the evoked endplate current (EPC) decay. Prolongation of EPC decay was accompanied by only modest prolongation of spontaneous miniature EPC (MEPC) decay. Prolongation of EPC decay was reversed when quantal content was lowered by reducing extracellular calcium. These findings suggested that the cause of EPC prolongation was presynaptic in origin. However, when we acutely inhibited fetal-type acetylcholine receptors (AChRs) using a novel peptide toxin (alphaA-conotoxin OIVA[K15N]), prolongation of both EPC and MEPC decay were reversed. We also blocked synaptic activity in a mutant strain of mice in which persistent muscle activity prevents upregulation of fetal-type AChRs. In these mice, there was no prolongation of EPC decay. We conclude that upregulation of fetal-type AChRs after blocking synaptic activity causes modest prolongation of MEPC decay that is accompanied by much greater prolongation of EPC decay. This might occur if acetylcholine escapes from endplates and binds to extrajunctional fetal-type AChRs only during large, evoked EPCs. Our study is the first to demonstrate a functional role for upregulation of extrajunctional AChRs.

Schwann Cells Express Active Agrin and Enhance Aggregation of Acetylcholine Receptors on Muscle Fibers

To explore novel roles of glial cells in synaptic function and formation, we examined the expression of agrin in frog Schwann cells and tested their role in the aggregation of acetylcholine receptors (AChRs). Using reverse transcription-PCR, we found that Schwann cells along nerve fibers in tadpoles expressed only the inactive agrin isoform B0 but began to also express active agrin isoforms B11 and B19 at approximately metamorphosis. During nerve regeneration in the adult, the expression of these active agrin isoforms in Schwann cells was upregulated, including the appearance of the most potent isoform, B8. This upregulation was induced by regenerating axons but not by nerve injury per se. In muscle cultures, the presence of adult Schwann cells enhanced the number and the total area of AChR aggregates 2.2- and 4.5-fold, respectively, and this enhancement was eliminated by heparin treatment. Furthermore, adult Schwann cells in culture expressed active agrin isoforms and produced agrin protein. Using a novel technique to selectively ablate perisynaptic Schwann cells (PSCs) at the neuromuscular junction, we found that PSCs also expressed active agrin isoforms B11 and B19, and these active isoforms were upregulated, including the appearance of B8, during reinnervation. Observation in vivo showed that extrajunctional AChR aggregates were associated with PSC sprouts after nerve injury and subsequent reinnervation. These results suggest that, contrary to the prevailing view that only neurons express active agrin, glial cells also express active agrin and play a role in the aggregation of AChRs both in vitro and in vivo.

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.

INSIGHTS INTO SUCCINYLCHOLINE-INDUCED HYPOTENSION AND MALIGNANT HYPERTHERMIA IN SUSCEPTIBLE SWINE

S383 INTRODUCTION: Administration of succinylcholine (SCH), a depolarizing muscle relaxant, to swine susceptible to malignant hyperthermia (MH) causes immediate, severe arterial hypotension, and fulminant episodes of MH. [1] However, it was previously reported that these responses could not be prevented by pretreatment with clinical doses of pancuronium. [1] The aims of our study were to gain new insights into the genotype-phenotype association underlying these SCH responses. METHODS: The in vivo effects of SCH were contrasted between swine with the MH mutation (n=10) and normal animals (n=6). Parameters studied included: ECG, heart rate, invasive blood pressure (BP), pulmonary artery (PA), central venous, left and right ventricular pressures (CVP, LVP, RVP), cardiac output (CO), endtidal CO2, axilla, esophageal, PA and liver temperatures and arterial blood gases. After a control period (PaCO2 40 +/- 2 mmHg, temperature 38 +/- 0.5 [inverted exclamation mark]C), SCH, 2 mg/kg was given i.v. and all parameters were measured for 15 min. To potentially alter the phenotypic responses various pretreatments were given to MH swine: 1) ranitidine and diphenhydramine (n=1), 2) dantrolene (n=2), 3) atropine (n=1), and 4) supra-clinical doses of vecuronium (n=3). In addition, responses of femoral artery vascular rings and whole isolated hearts isolated from both genotypes were studied in vitro. The femoral arteries were prepared without removing the endothelium and at 37 [inverted exclamation mark]C, the effects of SCH, 0.06 mM, on isometric tension was recorded. The hearts were removed using a standard cardioplegic procedure and then function was re-initiated in a four chamber working mode. Subsequently, the effects of SCH on left and right ventricular pressures, and contractility were analyzed. Statistics were performed using repeated measures ANOVA and Bonferroni multiple comparison post test. RESULTS: In vivo, SCH caused significant, early onset and severe decreases in BP, CO, LVP, and LV contractility in MH swine but not in normals: subsequently episodes of MH occurred in susceptible animals. Pretreatment with either antihistamines or atropine did not prevent either of these responses. Therapeutic doses of dantrolene prevented MH, but not the SCH-induced hypotension. In contrast, supra-clinical doses of vecuronium prevented both the hypotension and MH. In vitro, SCH was found to have no differential effects on either responses in the vascular rings or isolated hearts between the genotypes. DISCUSSION: Severe hypotension occurs before muscle hypermetabolism in swine with the MH genotype. Associated with this hypotensive reaction are changes in cardiac performance. However, in vitro neither in the functional hearts nor vascular rings did SCH induce differential response between the genotypes. Thus, the cardiovascular responses are likely of secondar origin relative to primary defects within the skeletal muscles. Our observations that only supra-clinical pretreatment doses of vecuronium prevented the systemic hypotension and MH episodes may suggest that SCH effects on both junctional and extrajunctional acetylcholine (ACH) receptors ultimately causes all phenotypic responses (cardiovascular and skeletal muscular). It was previously reported that extrajunctional ACH are upregulated in swine with this MH mutation. [2] In conclusion, SCH-induced hypotension in swine with the MH mutation is due to activation of nicotinic receptors: complete blockage of these receptors will also prevent the initiation of MH.

Neuromuscular transmission and its pharmacological blockade. Part 2: Pharmacology of neuromuscular blocking agents.

Clinically, neuromuscular blockade is induced with either depolarizing or non-depolarizing relaxants. Suxamethonium is the only depolarizing relaxant still in use. It is hydrolysed in the plasma by pseudo-cholinesterase (plasma cholinesterase). In some patients and in particular diseases the plasma cholinesterase activity is low and hence the effect of suxamethonium prolonged. Suxamethonium is characterized by side-effects such as myalgia, fasciculations and increase in intraocular, intracranial and intragastric pressure. More serious adverse reactions are masseter muscle spasm and potassium release, in patients with some neuromuscular diseases and increase in extrajunctional acetylcholine receptors. As non-depolarizing muscle relaxants benzylisoquinolines and steroidal compounds are mainly used. Each relaxant has its own pharmacological characteristics. The effect of most relaxants depends on liver and renal function because the pharmacokinetic behaviour is strongly dependent on these organs. Also, acid base balance disturbances, change in temperature, and neurological diseases have an effect on the profile of the relaxants. A number of drugs (anaesthetics, antibiotics, antiepileptics, etc.) have an effect on neuromuscular transmission, and thus interact with the relaxants. Some non-depolarizing relaxants cause histamine release and cardiovascular effects.

Anesthetic Considerations in Myelopathy Associated with Human T-Cell Lymphotropic Virus Type I (HTLV-I-Associated Myelopathy

Anesthetic Considerations in Myelopathy Associated with Human T-Cell Lymphotropic Virus Type I (HTLV-I-Associated Myelopathy

Neurotoxins in the study of neural regulation of membrane proteins in skeletal muscle

The discovery and purification of several neurotoxins, including α-bungarotoxin and tetrodotoxin has provided very high-affinity ligands which have proved to be central to the elucidation of the neural control of skeletal muscle membrane proteins and to the purification and characterization of the nicotinic acetylcholine receptor (AChR) and the Na + channel, respectively. This review describes the use of neurotoxins for quantification and localization of receptors and ion channels in normal and denervated skeletal muscles with particular emphasis on the appropriateness of the muscle preparation and ligand used in the studies. It is now clear that the nerve controls the synthesis and spatial distribution of AChRs and Na + channels by regulating gene expression in extrajunctional and subjunctional nuclei. The down-regulation of extrajunctional AChRs is primarily mediated by neuromuscular activity and the concentration of AChRs and Na + channels in specific membrane domains at the neuromuscular junction is controlled by a number of neurotrophic substances at the neuromuscular junction. These include agrin, ARIA, and CGRP.

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.

Phenytoin Induced Resistance to Vecuronium

Chronic phenytoin therapy causes resistance to some nondepolarising muscle relaxants. We have confirmed that this resistance is seen with vecuronium and suggest that at least a week of phenytoin therapy is required for a significant effect to develop. The mechanism of this resistance is not known. We have shown that an exaggerated rise in serum potassium after succinylcholine does not occur in patients with demonstrated resistance to vecuronium from chronic phenytoin therapy. This would suggest that significant extrajunctional acetylcholine receptor proliferation is an unlikely mechanism.

Nicotinic Acetylcholine Receptor mRNAs in Myasthenic Thymuses: Association with Intrathymic Pathogenesis of Myasthenia Gravis

Reverse transcription and the polymerase chain reaction showed that the α-, β- and γ-subunit genes of nicotinic acetylcholine receptor (AChR) in human skeletal muscle were expressed in myasthenia gravis (MG) thymuses (3 thymomas and 7 hyperplastic thymuses). A study of the expression of these subunit mRNAs in a variety of human tissues showed that α-subunit mRNA was expressed in human thymus and cerebral cortex, and that all three subunits were expressed only in human child thymus. Our results suggest that a complete AChR similar to extrajunctional (embryonic) muscle AChR is expressed in MG thymuses and that intrathymic AChR might be the primary antigen which induces autoimmune responses to muscle AChR.

Phenytoin-induced resistance to vecuronium.

Chronic phenytoin therapy causes resistance to some nondepolarising muscle relaxants. We have confirmed that this resistance is seen with vecuronium and suggest that at least a week of phenytoin therapy is required for a significant effect to develop. The mechanism of this resistance is not known. We have shown that an exaggerated rise in serum potassium after succinylcholine does not occur in patients with demonstrated resistance to vecuronium from chronic phenytoin therapy. This would suggest that significant extrajunctional acetylcholine receptor proliferation is an unlikely mechanism.

Congruity of acetylcholine receptor, acetylcholinesterase, and Dolichos biflorus lectin binding glycoprotein in postsynaptic‐like sarcolemmal specializations in noninnervated regenerating rat muscles

Noninnervated regenerating muscles are able to form focal postsynaptic-like sarcolemmal specializations either in places of the former motor endplates ("junctional" specializations) or elsewhere along the muscle fibers (extrajunctional specializations). The triple labeling histochemical method was introduced to analyse the congruity of focalization in such specializations of 3 synaptic components: acetylcholinesterase (AChE), acetylcholine receptor (AChR), and a specific synaptic glycoprotein which binds Dolichos biflorus lectin (DBAR). Noninnervated regenerating soleus and extensor digitorum longus (EDL) muscles of the rat were examined and compared with denervated muscles of neonatal and adult rats. All junctional sarcolemmal specializations in noninnervated regenerating muscles accumulated AChE and AChR. Localization of the 2 components was identical within the limits of resolution of the method. DBAR could not be demonstrated in junctional specializations in 17-day-old regenerating muscles. It seems that an agrin-like inducing substance in the former junctional basal lamina invariably triggers the accumulation of both AChE and AChR in the underlying sarcolemma of the regenerating muscle fiber. However, accumulation of DBAR would probably require the presence of the motor nerve. In most of the extrajunctional sarcolemmal specializations in 8-day-old regenerating soleus and EDL muscles, both AChE and AChR accumulated. However, about 10 percent of AChE accumulations lacked AChR and about 35% of AChR accumulations lacked AChE. Even greater variability was observed in 17-day-old regenerating muscles. The presence of DBAR in the extrajunctional postsynaptic-like sarcolemmal specializations could not be demonstrated. Similar extrajunctional sarcolemmal specializations were observed in denervated postnatal rat muscles. About 70% contained both AChE and AChR, and 30% contained only AChR, but none contained DBAR. In denervated mature muscles, sparse extrajunctional AChR accumulations did not contain detectable amounts of AChE. The ability to form complex postsynaptic-like sarcolemmal specializations in the absence of nerve, which is probably inherent to noninnervated immature muscle fibers, may be reduced with muscle maturation. Variable accumulation of individual components in the postsynaptic-like specializations indicates that different triggering factors may be involved in their accumulation or, at least, the mechanisms of their accumulation can function relatively independently.

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.

ATP activates junctional and extrajunctional acetylcholine receptor channels in isolated adult rat muscle fibres

Single-channel recordings in the cell-attached configuration were made on freshly dissociated and cultured rat muscle fibres. We demonstrate that ATP (50–100 μM) elicits single channel currents in freshly isolated fibres. These ATP-activated channels have a slope conductance similar to that determined for ACh-activated channels but exhibit a shorter open time. We also show that ATP activates extrajunctional AChR-channels in the membrane of long-term cultured muscle fibres. Here the ATP activated channel has a slope conductance and open time characteristics similar to those of the ACh-activated channel.