Mutations in different functional domains of the human muscle acetylcholine receptor alpha subunit in patients with the slow-channel congenital myasthenic syndrome

Congenital myasthenic syndromes are a group of rare genetic disorders that compromise neuromuscular transmission. A subset of these disorders, the slow-channel congenital myasthenic syndrome (SCCMS), is dominantly inherited and has been shown to involve mutations within the muscle acetylcholine receptor (AChR). We have identified three new SCCMS mutations and a further familial case of the alpha G153S mutation. Single channel recordings from wild-type and mutant human AChR expressed in Xenopus oocytes demonstrate that each mutation prolongs channel activation episodes. The novel mutations alpha V156M, alpha T254I and alpha S269I are in different functional domains of the AChR alpha subunit. Whereas alpha T254I is in the pore-lining region, like five of six previously reported SCCMS mutations, alpha S269I and alpha V156M are in extracellular domains. alpha S269I lies within the short extracellular sequence between M2 and M3, and identifies a new region of muscle AChR involved in ACh binding/channel gating. alpha V156M, although located close to alpha G153S which has been shown to increase ACh binding affinity, appears to alter channel function through a different molecular mechanism. Our results demonstrate heterogeneity in the SCCMS, indicate new regions of the AChR involved in ACh binding/channel gating and highlight the potential role of mutations outside the pore-lining regions in altering channel function in other ion channel disorders.     

Mode switching kinetics produced by a naturally occurring mutation in the cytoplasmic loop of the human acetylcholine receptor epsilon subunit

We describe the genetic and kinetic defects in a congenital myasthenic syndrome caused by heteroallelic mutations of the acetylcholine receptor (AChR) epsilon subunit gene. The mutations are an in-frame duplication of six residues in the long cytoplasmic loop (epsilon1254ins18) and a cysteine-loop null mutation (epsilonC128S). The epsilon1254 ins18 mutation causes mode switching in the kinetics of receptor activation in which three modes activate slowly and inactivate rapidly. The epsilon1245ins18-AChR at the endplate shows abnormally brief activation episodes during steady state agonist application and appears electrically silent during the synaptic response to acetylcholine. The phenotypic consequences are endplate AChR deficiency, simplification of the postsynaptic region, and compensatory expression of fetal AChR that restores electrical activity at the endplate and rescues the phenotype.     

Desensitization of mutant acetylcholine receptors in transgenic mice reduces the amplitude of neuromuscular synaptic currents

While the slow onset of desensitization of nicotinic acetylcholine receptors (AChRs), relative to the rate of acetylcholine removal, excludes this kinetic state from shaping synaptic responses in normal neuromuscular transmission, its role in neuromuscular disorders has not been examined. The slow-channel congenital myasthenic syndrome (SCCMS) is a disorder caused by point mutations in the AChR subunit-encoding genes leading to kinetically abnormal (slow) channels, reduced miniature endplate current amplitudes (MEPCs), and degeneration of the postsynaptic membrane. Because of this complicated picture of kinetic and structural change in the neuromuscular junction, it is difficult to assess the importance of the multiple factors that may be responsible for the reduced endplate current amplitudes, and ultimately the clinical syndrome. In order to address this we have used a transgenic mouse model for the SCCMS that has slow AChR ion channels and reduced endplate responsiveness in the absence of any of the degenerative changes. We found that the reduction in MEPC amplitudes in these mice could not be explained by either reduced AChR number or by reduced AChR channel conductance. Rather, we found that the mutant AChRs in situ manifested an activity-dependent reduction in sensitivity that caused diminished MEPC and endplate current amplitude with nerve stimulation. This observation demonstrates that the basis for the reduction in MEPC amplitudes in the SCCMS may be multifactorial. Moreover, these findings demonstrate that, under conditions that alter their rate of desensitization, the kinetic properties of nicotinic AChRs can control the strength of synaptic responses.     

Quinidine normalizes the open duration of slow-channel mutants of the acetylcholine receptor

Quinidine is a long-lived open-channel blocker of the wild-type endplate acetylcholine receptor (AChR). To test the hypothesis that quinidine can normalize the prolonged channel opening events of slow-channel mutants of human AChR, we expressed wild-type AChR and five well characterized slow-channel mutants of AChR in HEK 293 cells and monitored the effects of quinidine on acetylcholine-induced channel currents. Quinidine shortens the longest component of channel opening burst (tau3b) of both wild-type and mutant AChRs in a concentration-dependent manner, and 5 microM quinidine reduces tau3b of the mutant AChRs to that of wild-type AChRs in the absence of quinidine. Because this concentration of quinidine is attainable in clinical practice, the findings predict a therapeutic effect for quinidine in the slow-channel congenital myasthenic syndrome.     

Slow-channel transgenic mice: a model of postsynaptic organellar degeneration at the neuromuscular junction

The slow-channel congenital myasthenic syndrome (SCCMS) is a dominantly inherited disorder of neuromuscular transmission characterized by delayed closure of the skeletal muscle acetylcholine receptor (AChR) ion channel and degeneration of the neuromuscular junction. The identification of a series of AChR subunit mutations in the SCCMS supports the hypothesis that the altered kinetics of the endplate currents in this disease are attributable to inherited abnormalities of the AChR. To investigate the role of these mutant AChR subunits in the development of the synaptic degeneration seen in the SCCMS, we have studied the properties of the AChR mutation, epsilonL269F, found in a family with SCCMS, using both in vitro and in vivo expression systems. The mutation causes a sixfold increase in the open time of AChRs expressed in vitro, similar to the phenotype of other reported mutants. Transgenic mice expressing this mutant develop a syndrome that is highly reminiscent of the SCCMS. Mice have fatigability of limb muscles, electrophysiological evidence of slow AChR ion channels, and defective neuromuscular transmission. Pathologically, the motor endplates show focal accumulation of calcium and striking ultrastructural changes, including enlargement and degeneration of the subsynaptic mitochondria and nuclei. These findings clearly demonstrate the role of this mutation in the spectrum of abnormalities associated with the SCCMS and point to the subsynaptic organelles as principal targets in this disease. These transgenic mice provide a useful model for the study of excitotoxic synaptic degeneration.     

T helper cell recognition of muscle acetylcholine receptor in myasthenia gravis. Epitopes on the gamma and delta subunits

We tested the response of CD4+ cells and/or total lymphocytes from the blood of 22 myasthenic patients and 10 healthy controls to overlapping synthetic peptides, 20 residues long, to screen the sequence of the gamma and delta subunits of human muscle acetylcholine receptor (AChR). The gamma subunit is part of the AChR expressed in embryonic muscle and is substituted in the AChRs of most adult muscles by an epsilon subunit. The delta subunit is present in both embryonic and adult AChRs. Adult extrinsic ocular muscles, which are preferentially and sometimes uniquely affected by myasthenic symptoms, and thymus, which has a still obscure but important role in the pathogenesis of myasthenia gravis, express the embryonic gamma subunit. Anti-AChR CD4+ responses were more easily detected after CD8+ depletion. All responders recognized epitopes on both the gamma and delta subunits and had severe symptoms. In four patients the CD4+ cell response was tested twice, when the symptoms were severe and during a period of remission. Consistently, the response was only detectable, or larger, when the patients were severely affected.     

Neuronal nicotinic ACh receptor antibody in subacute autonomic neuropathy and cancer-related syndromes

BACKGROUND: Autoantibodies specific for the acetylcholine receptor (AChR) of skeletal muscle (containing the alpha1 subunit) impair neuromuscular transmission in myasthenia gravis (MG). AChRs mediating fast synaptic transmission through autonomic ganglia are structurally similar to muscle AChR, but contain the alpha3 subunit. We propose that ganglionic AChR autoimmunity may cause dysautonomia. OBJECTIVE: To test serum of patients with autonomic neuropathy for autoantibodies of neuronal ganglionic AChR specificity. METHODS: We developed an immunoprecipitation radioassay by complexing epibatidine (125I-labeled high affinity agonist) to a Triton X-100-solubilized AChR antigen from peripheral neuroblastoma membranes. Monoclonal rat immunoglobulins (IgG) specific for muscle or neuronal AChRs validated the assay's specificity. We tested serum from 52 healthy subjects, 12 patients with subacute autonomic neuropathy, and 248 patients with other neurologic disorders. RESULTS: Twelve patients had antibodies that bound unequivocally to ganglionic AChR. Five had subacute autonomic neuropathy, and three (of six tested) had Isaacs' syndrome; four of these eight had a carcinoma (lung, bladder, rectum, thyroid). The remaining four seropositive patients (two Lambert-Eaton syndrome, one dementia, one sensory neuronopathy) all had Ca2+ channel antibodies and three had small cell lung carcinoma. No healthy subject had ganglionic AChR antibodies, nor did 62 patients with MG and muscle AChR antibodies. CONCLUSION: Neuronal AChR antibodies are a novel serologic marker of neurologic autoimmunity. The pathogenicity of neuronal AChR autoantibodies in autonomic neuropathy, Isaacs' syndrome, or other neurologic disorders remains to be shown, as has been demonstrated for muscle AChR antibodies in MG. An autoimmune and potentially paraneoplastic etiology is implicated in seropositive patients.     

Transformation of a non-enzymatic toxin into a toxoid by genetic engineering

Curaremimetic toxins are typical non-enzymatic toxins that bind to their target [the nicotinic acetylcholine receptor (AChR)] through multiple residues. Nevertheless, we show that the concomitant substitutions of only three of the ten functionally important residues of such a toxin sufficed to cause an affinity decrease of the toxin for AChR that is higher than four orders of magnitude. Despite these triple mutations, the overall conformation of the mutated protein remains similar to that of a related recombinant toxin, as judged from both circular dichroism analysis and investigation of antigenicity, using monoclonal and polyclonal antibodies. Furthermore, we show that the detoxified toxin is capable of eliciting antibodies that neutralize the binding of a wild-type toxin to AChR. Therefore, transformation of a non-enzymatic toxin into a toxoid can be achieved, like in the case of enzymatic toxins, by introducing a small number of mutations at positions identified to be critical for expression of toxicity.     

Mutation in the M1 domain of the acetylcholine receptor alpha subunit decreases the rate of agonist dissociation

We describe the kinetic consequences of the mutation N217K in the M1 domain of the acetylcholine receptor (AChR) alpha subunit that causes a slow channel congenital myasthenic syndrome (SCCMS). We previously showed that receptors containing alpha N217K expressed in 293 HEK cells open in prolonged activation episodes strikingly similar to those observed at the SCCMS end plates. Here we use single channel kinetic analysis to show that the prolonged activation episodes result primarily from slowing of the rate of acetylcholine (ACh) dissociation from the binding site. Rate constants for channel opening and closing are also slowed but to much smaller extents. The rate constants derived from kinetic analysis also describe the concentration dependence of receptor activation, revealing a 20-fold shift in the EC50 to lower agonist concentrations for alpha N217K. The apparent affinity of ACh binding, measured by competition against the rate of 125I-alpha-bungarotoxin binding, is also enhanced 20-fold by alpha N217K. Both the slowing of ACh dissociation and enhanced apparent affinity are specific to the lysine substitution, as the glutamine and glutamate substitutions have no effect. Substituting lysine for the equivalent asparagine in the beta, epsilon, or delta subunits does not affect the kinetics of receptor activation or apparent agonist affinity. The results show that a mutation in the amino-terminal portion of the M1 domain produces a localized perturbation that stabilizes agonist bound to the resting state of the AChR.     

Molecular genetics of ion channel diseases

Many physiological processes depend upon the proper functioning of plasma membrane ion channels. This is most apparent in absorptive and secretory epithelia, and in electrically excitable tissues such as nerve and muscle. Disturbances in the operation of ion channels in these settings can alter normal physiology and cause disease. This review illustrates the use of molecular genetics in identifying hereditary diseases caused by mutations in genes which encode various skeletal muscle ion channels. Recent advances in the discovery of genetic mutations in the skeletal muscle voltage-gated sodium channel in certain forms of periodic paralysis, mutations in the skeletal muscle chloride channel gene in myotonia congenita, and defects in two distinct calcium channels that underlie disorders of excitation-contraction coupling (murine muscular dysgenesis, malignant hyperthermia susceptibility) will be presented. In each case, prior knowledge of abnormal ion channel function prompted the search for mutations in candidate genes. This work is beginning to shed new light on the relationship between ion channel structure and function by studies of naturally occurring channel mutations.     

Multiple virus genes involved in the nematode transmission of pea early browning virus

Gorlin syndrome is an autosomal dominant disorder characterized by multiple basal cell carcinomas, medulloblastomas, ovarian fibromas, and a variety of developmental defects. All affected individuals share certain key features, but there is significant phenotypic variability within and among kindreds with respect to malformations. The gene (NBCCS) maps to chromosome 9q22, and allelic loss at this location is common in tumors from Gorlin syndrome patients. Two recessive cancer-predisposition syndromes, xeroderma pigmentosum group A (XPAC) and Fanconi anemia group C (FACC), map to the NBCCS region; and unusual, dominant mutations in these genes have been proposed as the cause of Gorlin syndrome. This study presents cytogenetic and molecular characterization of germ-line deletions in one patient with a chromosome 9q22 deletion and in a second patient with a deletion of 9q22-q3l. Both have typical features of Gorlin syndrome plus additional findings, including mental retardation, conductive hearing loss, and failure to thrive. That Gorlin syndrome can be caused by null mutations (deletions) rather than by activating mutations has several implications. First, in conjunction with previous analyses of allelic loss in tumors, this study provides evidence that associated neoplasms arise with homozygous inactivation of the gene. In addition, dominant mutations of the XPAC and FACC1 genes can be ruled out as the cause of Gorlin syndrome, since the two patients described have null mutations. Finally, phenotypic features that show variable expression must be influenced by genetic background, epigenetic effects, somatic mutations, or environmental factors, since these two patients with identical alterations (deletions) of the Gorlin syndrome gene have somewhat different manifestations of Gorlin syndrome.     

Phenotype variation and newcomers in ion channel disorders

Ion channels are part of a large family of macromolecules whose functions include the control and maintenance of electrical potential across cell membranes, secretion and signal transduction. Close inspection of the physiological processes involved in channel function and the secondary structure of various ion channels has served as a basis for subdividing ion channels into a number of superfamilies. The voltage-gated ion channels are one of these superfamilies. Recent work has shown that mutations in various ion channel genes are responsible for a number of neuromuscular and neurological disorders. Correlation of the various mutations with the clinical phenotype is providing us with insight into the pathophysiology of these channel proteins. Interestingly, different mutations within the same gene may cause quite distinct clinical disorders, while mutations in different channel genes may result in very similar phenotypes (genetic heterogeneity). Examples of phenotypic variation and genetic heterogeneity are presented in the context of the periodic paralytic disorders of skeletal muscle, episodic ataxia, migraine, long QT syndrome and paroxysmal dyskinesia. Some of these disorders are known to be caused by mutations in ion channel genes, while in the episodic movement disorders, ion channel genes are considered excellent candidate genes.     

Myasthenia gravis: pathogenesis and therapeutic approach on the basis of molecular structure and immunogenicity of acetylcholine receptor

Recent research for myasthenia gravis has been advanced by the availability of primary sequence of acetylcholine receptor (AChR) precursor and AChR transmembrane topography. (1) Synthetic peptides of AChR alpha-subunit defined myasthenogenic sites recognized by blocking or modulating antibody; their capability of inducing an animal model suggests that synthetic antigens contain not only B-cell sites but also T-cell sites which interact with restricted MHC class II molecules. (2) In view of the conformation-dependent B-cell site expected at beta-turn and the MHC-restricted T-cell site expected at amphipathic alpha-helix, conformationally modified peptides were synthesized by linking them; some were in combination with artificially sequenced peptide to favor an enhanced beta-turn. (3) Experiments by the use of these synthetic peptides demonstrated that such antigens were enhanced in myasthenogenicity. However, the immunogenic conformation for the induction of animal model did not necessarily assume that for the detection of antibody in native AChR-immunized rats. (4) Peptides synthesized to assume specifically for binding with antibody were used as a tool for immunoadsorption via plasma perfusion in myasthenic patients, resulting in clinical improvement.     

Immunoadsorption therapy for myasthenia gravis

The results of a multicentre trial were analysed to evaluate the efficacy of immunoadsorption therapy for severe generalised myasthenia gravis. Twenty patients with myasthenia gravis who were concurrently receiving high dose prednisolone and azathioprine therapy were treated with an affinity-type adsorbent, using tryptophan-linked polyvinyl alcohol gel (IM-TR), according to a standardised treatment protocol. The 20 patients received five adsorption treatments within a period of 10 days. In 11, pronounced improvement of myasthenic weakness was seen and long-term remission was maintained. The treatment was especially effective in patients with thymic hyperplasia. Circulating acetylcholine receptor (AChR) antibodies were reduced by about 60% by treating one plasma volume. There was no difference in the rate of removal of the AChR antibodies between patients with thymic hyperplasia and patients with thymoma. No serious complications occurred during 100 procedures. It was concluded that the immunoadsorption therapy with IM-TR is useful in controlling symptoms in patients with severe myasthenia gravis who are otherwise unresponsive.     

Myasthenia gravis: recognition of a human autoantigen at the molecular level

The symptoms of myasthenia gravis are primarily or exclusively due to an autoimmune response against the muscle nicotinic acetylcholine receptor (AChR) and this has been the object of intensive investigations for almost 20 years. A detailed picture at the molecular level of the interaction of this autoantigen with the key elements involved in the autoimmune response, such as anti-AChR antibodies, the T-cell receptor and restricting major histocompatibility complex molecules, is now emerging for both human myasthenia gravis and its experimental model, experimental autoimmune myasthenia gravis. Here, Maria Pia Protti and colleagues focus on the molecular interactions occurring in human myasthenia gravis and summarize recent information on pathogenic mechanisms of the autoimmune response, and the structure of epitopes recognized by B cells and CD4+ T cells of myasthenic patients on the AChR molecule.     

Intrinsic dynamics and mechanosensory modulation of non-nutritive sucking in human infants

The rate constants of acetylcholine receptor channels (AChR) desensitization and recovery were estimated from the durations and frequencies of clusters of single-channel currents. Diliganded-open AChR desensitize much faster than either unliganded- or diliganded-closed AChR, which indicates that the desensitization rate constant depends on the status of the activation gate rather than the occupancy of the transmitter binding sites. The desensitization rate constant does not change with the nature of the agonist, the membrane potential, the species of permeant cation, channel block by ACh, the subunit composition (epsilon or gamma), or several mutations that are near the transmitter binding sites. The results are discussed in terms of cyclic models of AChR activation, desensitization, and recovery. In particular, a mechanism by which activation and desensitization are mediated by two distinct, but interrelated, gates in the ion permeation pathway is proposed.     

Paradigmatic obstacles to improving the health of populations--implications for health policy

Over the past few decades much effort has been expended elucidating the key domains of the nicotinic acetylcholine receptor (AChR) responsible for agonist binding, ion conduction, and gating. An emerging concept in the receptor field has been to consider the receptor entity as a signal transducer that suffers modulatory control by allosterically acting ligands. Of particular interest are the molecules that inhibit the agonist-evoked ion flux activity in a noncompetitive manner: the so-called noncompetitive inhibitors (NCIs). The actual knowledge on the action of NCIs was obtained by using several drugs from exogenous origin. However, several lines of investigation indicate that the receptor protein can be modulated by endogenous substances other than acetylcholine. In this regard, we outline the progress evidenced on the localization of binding sites for drugs of endogenous origin that have been found to directly interact with the AChR in a noncompetitive fashion. Among them we can quote lipids such as steroids and fatty acids, the neurotransmitter 5-hydroxytryptamine (5-HT) and related compounds, as well as the neuropeptide substance P. We present the available experimental evidence indicating the existence of both luminal (located into the ion channel) and nonluminal (located out of the ion channel) binding sites for endogenous NCIs. Particularly, the binding site for substance P is found in the delta M2 domain. In addition, the locus for 5-HT is putatively located in the ion channel close to the serine ring, whereas the binding site for two competitive antagonists of 5-HT receptors (e.g., methysergide and spiperone) is located closer to the external end of the ion channel. Instead, fatty acid and steroid molecules bind to nonluminal sites. More specifically, fatty acids may bind to the annular lipid domain of the AChR or/and to the high-affinity quinacrine site (a NCI from exogenous origin) which is located at a nonannular lipid domain. Additionally, steroids may bind to a site located on the extracellular hydrophilic domain of the AChR or/and at the lipid-protein interface, specifically, at the annular lipid domain and/or close to the nonannular quinacrine binding site.     

Mutation of RFXAP, a regulator of MHC class II genes, in primary MHC class II deficiency

BACKGROUND: Major-histocompatibility-complex (MHC) class II deficiency is an autosomal recessive primary immunodeficiency disease in which MHC class II molecules are absent. It is a genetically heterogeneous disease of gene regulation resulting from defects in several transactivating genes that regulate the expression of MHC class II genes. The mutations responsible for MHC class II deficiency are classified according to complementation group (a group in which the phenotype remains uncorrected in pairwise fusions of cells). There are three known complementation groups (A, B, and C). METHODS: To elucidate the genetic defect in patients with MHC class II deficiency that was not classified genetically, we performed direct complementation assays with the three genes known to regulate the expression of MHC class II genes, CIITA, RFX5, and RFXAP, and the relevant mutations were identified in each patient. RESULTS: Mutations in the RFXAP gene were found in three patients from unrelated families, and the resulting defect was classified as belonging to a novel complementation group (D). Transfection with the wild-type RFXAP gene restored the expression of MHC class II molecules in the patients' cells. CONCLUSIONS: Mutations in a novel MHC class II transactivating factor, RFXAP, can cause MHC class II deficiency. These mutations abolish the expression of MHC class II genes and lead to the same clinical picture of immunodeficiency as in patients with mutations in the other two MHC class II regulatory genes.