Mutations in different functional domains of the human muscle acetylcholine receptor [alpha] subunit in patients with the slow-channel congenital myasthenic syndrome
Mutations in different functional domains of the human muscle acetylcholine receptor [alpha] subunit in patients with the slow-channel congenital myasthenic syndrome Rebecca Croxen1,+, Claire Newland1,+, David Beeson1,*, Hans Oosterhuis2, Guy Chauplannaz3, Angela Vincent1 and John Newsom-Davis1
1Neurosciences Group, Institute of Molecular Medicine, John Radcliffe Hospital, Oxford OX3 9DU, UK, 2Department of Neurology, Academic Hospital, Groningen, The Netherlands and 3Hôpital Neurologique et Université Claude Bernard, 69003 Lyon, France
Received February 24, 1997;Accepted February 28, 1997
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.
Neuromuscular transmission is impaired in myasthenic disorders of both autoimmune and genetic origin. The acquired autoimmune conditions, including myasthenia gravis and Lambert-Eaton myasthenic syndrome, are caused by antibodies to specific neuronal and muscle ion channels. The rarer inherited conditions, in which autoimmune features are absent, are collectively called congenital myasthenic syndromes (CMS). They can be associated with abnormalities affecting acetylcholine (ACh) release, acetylcholinesterase activity, AChR function and/or AChR number (1 ). Progress in determining the genetic defects has been greatest in disorders involving functional abnormalities of the AChR, in particular the slow-channel congenital myasthenic syndrome (SCCMS). This syndrome shows autosomal dominant inheritance although some cases appear to be sporadic. Muscle weakness may be apparent in infancy or may not develop until adulthood; cervical, scapular and finger extensor muscles are often particularly affected. Ultrastructural studies frequently show a focal endplate myopathy, particularly in affected muscles, and electromyography usually reveals a repetitive muscle response to a single nerve stimulus. Microelectrode studies on biopsied intercostal muscle show a characteristic prolonged decay of the miniature endplate potentials/currents. Although these data are consistent with prolonged AChR channel openings, patients nevertheless vary in their clinical, pathological and electrophysiological features.
Recent reports have identified six single-nucleotide mutations in AChR genes that underlie SCCMS (2 -6 ), five of which result in amino acid substitutions in regions of the AChR that contribute to the channel pore. Here we report three novel heterozygous mutations within the AChR [alpha]-subunit genes of SCCMS patients. These mutations result in substitutions in diverse regions of the AChR, including the ACh-binding pocket, the channel-lining domain and the extracellular M2-M3 loop. We also identify a further SCCMS case of a previously identified mutation (4 ). In expression studies of the human AChR in Xenopus oocytes, all four mutations give rise to prolonged channel activations, emphasizing the heterogeneity underlying SCCMS. Further study of the mechanisms that cause prolonged activations and their pathological consequences are likely to have implications for other excitotoxic neurodegenerative disorders.
Patient 1, a 34-year-old man, became aware of lower limb weakness at the age of 14. He later developed facial, neck and upper limb weakness and slight difficulty with chewing and swallowing. Eye movements were normal. He showed wasting of forearm and hand muscles. Antibodies to AChR were absent. Single nerve stimuli did not elicit repetitive compound muscle action potentials on electromyography. Miniature endplate potential amplitudes recorded from biopsied intercostal muscle were normal, but the rise time and decay phase were prolonged. Acetylcholinesterase staining at the endplate showed no obvious abnormality. Non-paternity was identified using highly polymorphic DNA fingerprinting markers (data not shown).
Patient 2 (a 60-year-old woman) and patient 3 (a 41-year-old woman) previously have been reported in detail as case 2 and case 1 respectively by Chauplannaz and Bady (7 ). Symptoms were first apparent at the age of 16 for patient 2 and in infancy for patient 3. Both patients had generalised weakness (sparing extraocular muscles), and also prominent wasting and weakness of finger extensor muscles. Patient 2 exhibited marked weakness and atrophy of cervical muscles and later developed respiratory problems due to diaphragmatic weakness. Deterioration occurred during pregnancy in patient 2 and in the months after pregnancy in patient 3. In both patients, antibodies to AChR were absent, and single nerve stimuli elicited repetitive compound muscle action potentials. Similar symptoms were reported in first degree relatives from both families, but DNA for analysis was only available from the affected mother of patient 3.
Patient 4 (a 28-year-old female) was previously reported in detail as case 1 by Oosterhuis et al. (8 ). She developed arm weakness in the eighth month of her first pregnancy at the age of 23. She showed generalized myasthenic weakness and mild hand muscle wasting. Antibodies to AChR were absent and she reacted adversely to anticholinesterase drugs. Electromyography showed a repetitive muscle response to a single nerve stimulus and in vitro microelectrode studies showed prolonged decay time of miniature endplate potentials. Other family members were asymptomatic.
SSCP analysis. PCR products were amplified from cDNA derived from the patient's muscle biopsy, from their cultured muscle cells or from genomic DNA, and covered selected regions of the muscle AChR [alpha]-, [beta]-, [delta]- and [epsilon]-subunit coding sequence, including the M2 transmembrane domains. Amplified products were screened for mutations by single-strand conformation polymorphism (SSCP) analysis. For each patient a unique abnormal conformer was identified (Fig. 1 ) which was not evident in control individuals or when screening 60 individuals with other congenital myasthenic syndromes. These patient-specific aberrant conformers reside within exon 5 (patients 1 and 3) and exon 7 (patients 2 and 4) of the AChR [alpha]-subunit gene. The aberrant conformer, which was seen in analysis of exon 5 of the [alpha]-subunit gene from patient 3, was also detected in her symptomatic mother (Fig. 1 ).
To ascertain if the three novel mutations [alpha]V156M, [alpha]T254I and [alpha]S269I have an effect on channel function, we constructed expression vectors containing the respective mutations in the human [alpha]-subunit cDNA. Previous studies of the functional effects of SCCMS mutations have used mouse AChR (2 ,4 -6 ). In addition, we also constructed a cDNA expression vector containing the [alpha]G153S mutation. When this substitution was incorporated into mouse AChR expressed in HEK293 cells, it generated channels with prolonged activation episodes (4 ). Wild-type and mutant human [alpha]-subunit cRNAs were injected separately into Xenopus ooctyes in combination with cRNAs encoding the human AChR [beta], [delta] and [epsilon] subunits. Whole cell current recordings indicated that each of the mutated [alpha] subunits is assembled efficiently into functional AChRs (data not shown).
Single channel recordings from oocytes revealed that all four mutations prolonged the duration of bursts (Fig. 4 and Table 1 ). Wild-type and mutant AChRs exhibited three burst length components. The two briefest burst components ([tau]1 and [tau]2) of the mutant channels were similar to wild-type (1% significance level, t-test), whereas the longest burst components ([tau]3) were markedly prolonged (P <0.01). The degree by which [tau]3 was prolonged depended on the mutation: [tau]3 was increased ~7-fold by [alpha]T254I and [alpha]S269I, 6-fold by [alpha]G153S, but only 3-fold by [alpha]V156M. Although not analysed in detail, there appear to be differences in the structure of bursts for each mutation. For example, [alpha]G153S bursts contain a large number of openings that are of similar duration to wild-type (consistent with ref. 4 ), whereas [alpha]T254I bursts are composed of one or two very long openings. This indicates that although all four mutations prolong the duration of bursts they may do so through different mechanisms. All four mutations also resulted in a slight (6-9%) increase in the single channel conductance (P <0.05), measured as the current amplitude at -100 mV (Table 1 ).
We report three new mutations, [alpha]V156M, [alpha]T254I and [alpha]S269I, within the AChR [alpha]-subunit gene from SCCMS patients, and a further familial case of the [alpha]G153S mutation (4 ). Of the six previously reported SCCMS mutations, five occur in pore-forming regions of the [alpha], [beta] and [epsilon] subunits (2 ,3 ,5 ,6 ). The mutations we detected lie in diverse regions of the [alpha] subunit, yet cause a similar change in AChR function, namely an increase in the duration of activations. Functional characterization not only confirms the pathogenicity of the mutations but also provides insights into the structure and function of the AChR.
It has been proposed that the [alpha] subunit plays a major role in ACh binding and signal transduction (11 ,13 ). In adult mammalian AChR, the two [alpha] subunits show non-equivalence both at the ACh-binding sites (14 ) and within the pore-lining region (15 ). Thus, within a patient who is heterozygous for an [alpha]-subunit mutation, four adult AChR subtypes may be expressed, each of which could be functionally distinct. The heterogeneity of both the mutations and channel subtypes is likely to contribute to the variation in severity of disease phenotype.
The mutations we identified probably prolong receptor activations through different mechanisms. [alpha]G153S and [alpha]V156M are situated close to [alpha]W149 which has been shown to contribute to the ACh-binding pocket (16 ). It has been demonstrated by single channel analysis of mouse AChR that [alpha]G153S causes prolonged receptor activations primarily by reducing the rate of dissociation of ACh from the AChR, thereby increasing the number of reopenings during ACh occupancy (4 , see also Fig. 4 ). Qualitative comparison of [alpha]V156M and [alpha]G153S single channel currents indicates different mechanisms of actions (Fig. 4 ). The apparently longer duration of individual openings within a burst suggests that the [alpha]V156M mutation may stabilize the open state, although further analysis is required to investigate the exact mechanism of action. These results are consistent with single channel analysis of mutations at [alpha]Y190 and [alpha]D200 which, although within or close to the ACh-binding site as identified by affinity labelling studies (9 ,10 ), show effects on gating as well as binding (14 ,17 ).
[alpha]T254I is situated three residues C-terminal to the central leucine of the pore-lining M2 domain. Mutations that underlie SCCMS have also been identified within the M2 domain of the [beta] and [epsilon] subunits, and in the outer third of the [alpha]-subunit M1 domain which also contributes to the pore (Fig. 5 ) (2 ,3 ,5 ,6 ). These mutations stabilize the open state of the AChR, resulting in longer individual openings. We obtained qualitatively similar single channel recordings from AChR containing the [alpha]T254I mutation, suggesting that [alpha]T254I also stabilizes the open state. Based on the substituted-cysteine accessibility method, [alpha]T254I and two of the other SCCMS mutations, [epsilon]T264P and [epsilon]L269F, are not exposed to the channel lumen, whereas [beta]L262M, [beta]V266M and [alpha]N217K are exposed (18 ). Thus the same functional effect can result from mutations at different positions within the pore-lining domains, including residues that either do, or do not, face the channel lumen.
[alpha]S269I, located midway along the extracellular loop between M2 and M3, is in a region not previously identified in muscle AChR as being involved in ACh binding or channel gating. We have yet to determine the mechanism by which this mutation prolongs receptor activations, but [alpha]S269I could have a distant effect on the ACh-binding sites, which current models suggest are 25-30 Å above the membrane surface (11 ,19 ,20 ), or may directly affect the transduction of conformational changes involved in channel gating. Indeed, residues located in the extracellular M2-M3 loop of homomeric glycine and neuronal acetylcholine receptors have been reported to affect the coupling between agonist binding and channel gating. These include the glycine receptor [alpha]1 subunit mutation associated with hereditary hyperekplexia (21 -25 ) and a neuronal [alpha]7 subunit mutation at a homologous position to [alpha]S269I (26 ).
Calcium ions contribute ~6% of the total inward current through endplate AChR (27 ). It has been proposed that the pathological consequences of prolonged AChR channel activations result from excess Ca2+ entry at the neuromuscular junction, leading to inhibition of mitochondrial respiration and activation of degenerative enzymes (28 ). The excitotoxic effects of excess Ca2+ may result in the endplate myopathy that frequently is seen in affected muscles from SCCMS patients, and lead to impaired synaptic transmission because of the loss of AChR associated with the destruction of junctional folds (2 ,5 ,6 ,8 ,28 ). The slight increase in single channel conductance we observed for all four mutations may also contribute to Ca2+ overload. Another cause of muscle dysfunction may be that, at physiological rates of stimulation, the prolonged endplate potentials summate, leading to persistent depolarization of the endplate and consequent failure of neuromuscular transmission due to inactivation of perijunctional sodium channels. In addition, the increased propensity for desensitization reported for certain mutations (4 ,6 ) may lead to fewer AChRs available for repetitive signal transmission. However, questions remain as to why there is selective muscle weakness and variability in clinical symptoms. It is of interest that pregnancy was associated with deterioration in each of our three female patients.
Mutations in SCCMS patients are single amino acid changes that cause a pathologic gain of function of the AChR. They are located in various domains of the AChR and exert a similar pathogenic functional effect through different molecular mechanisms. Characterization of muscle AChR kinetic abnormalities, including the recently characterized low-affinity fast-channel syndrome (29 ), are providing insights into the AChR structure-function relationships not revealed from in vitro mutagenesis studies. Other conditions have been linked to mutations in the M2 region of neuronal nicotinic AChRs, including autosomal dominant nocturnal frontal lobe epilepsy (30 ), and the degeneration of a small subset of neurons that results from the deg-3 mutation of Caenorhabditis elegans (31 ). Similarities between these disorders and the SCCMS suggests that elucidation of muscle AChR abnormalities may be a useful model on which to base screening and characterization of mutations in related ligand-gated ion channels of the central nervous system.
mRNA and DNA samples. RNA was isolated from cultured muscle cells or biopsied muscle using RNAzolT B (Cinna/Biotecx) and following the instructions provided by the manufacturer. First-strand cDNA was prepared from 10 [mu]g of mRNA by using oligo(dT)12-18 primer and a mixture of SuperScriptT II RNase H- and M-MLV reverse transcriptases (BRL). Genomic DNA was isolated from peripheral blood using the NucleonT II DNA extraction kit (Scotlab). Approval for the use of human muscle tissues was received from the Central Oxford Research Ethics Committee. Polymerase chain reaction and SSCP analysis. PCR amplifications for SSCP analysis were performed on genomic DNA and cDNA for regions of the AChR [alpha]-, [beta]-, [delta]- and [epsilon]-subunit genes.Primers were derived from published sequences (32 -35 ). The PCR amplifications for SSCP which gave rise to unique conformers for the SCCMS patients were performed using the following primer pairs: 5'-CAG ATG GTG ACT TTG CTA TTG TC-3' and 5'-CTG AAA CCA CCC TTA TCA TAT GTG-3' to amplify a 230 bp fragment spanning exon 5 of the [alpha] subunit; 5'-GAA GAT GAC TCT GAG CAT CTC T-3' and 5'-GAT GAT GGA GGC AAT GAC GAA C-3' to amplify an 148 bp fragment of exon 7 of the [alpha] subunit. A typical PCR reaction for SSCP analysis included reaction buffer [60 mM Tris-HCl, 15 mM (NH4)2SO4, 1.5-2.0 mM MgCl2, pH 8.5(Invitrogen)], 1.25 [mu]M each primer, 200 [mu]M dCTP, dGTP and dTTP, 0.075 [mu]l of [35S]dATP[alpha]S (>1000 Ci/mmol; Amersham), 50 [mu]M dATP, with ~50 ng of genomic DNA and 0.25 U of Taq DNA polymerase (Perkin-Elmer) in a 5 [mu]l reaction. A typical cycling protocol comprised (i) denaturation at 94oC for 3 min; (ii) 33 cycles of 94oC for 1 min, 60oC for 40 s and 72oC for 30 s. Samples were mixed with 5 [mu]l of gel loading buffer (95% formamide, 20 mM EDTA, 0.05% bromophenol blue, 0.05% Xylene Cyanol FF), denatured and loaded on a 8.3% polyacrylamide gel (100:1 acrylamide:bis) containing 5% glycerol. Gels were run at 6 W for ~19 h at 4oC, and exposed to autoradiography for 17 h or longer.
Sequence analysis. PCR-amplified fragments of genomic DNA and cDNA were purified from agarose gels using the QIAGEN gel extraction kit. Direct cycle-sequencing was performed using the fmolTDNA sequencing kit (Promega). Subcloned PCR-amplified cDNA fragments were sequenced using the T7 SequenaseT version 2.0 DNA sequencing kit (USB).
Expression of wild-type and mutant human AChR in Xenopus oocytes. cDNAs encoding the human muscle AChR [alpha] and [delta] subunits (32 ) were ligated into a form of the vector pGEMHE (36 ) modified both to include a 3' poly(A) tract and for transcription to be initiated from the SP6 promoter. cDNAs encoding the human muscle AChR [beta] and [epsilon] subunits (32 ) were ligated into vector pGEM3Z (Promega) modified to include an ~100 nucleotide poly(A) tract. cDNA fragments containing the respective mutations were obtained by RT-PCR on RNA isolated from the patient muscle biopsies or cultured muscle cells. The primer sets were 5'-CAA TGG GTG GAT TAC AAC CTA AA-3' and 5'-CTG AGT CTG TGG GCA GGT AGA-3' for patients 1 and 3, and 5'-GAA AGC GAC CAG CCA GAC CTG A-3' and 5'-TAA GCT CAG CTC ATT TTC TGC-3' for patients 2 and 4. PCR products were subcloned into pGEM5Z (Promega); mutant cDNAs were identified and confirmed by sequencing and ligated in-phase into the [alpha]-subunit expression vector at the BglII-XmaI sites (patients 1 and 3) and XmaI-SalI sites (patients 2 and 4) within the [alpha]-subunit coding sequence. cRNAs encoding each subunit were synthesized using the mMESSAGE mMACHINE[middot] SP6 in vitro transcription kit (Ambion) and the translational activity of the resultant cRNAs tested using rabbit reticulocyte lysates (Promega). Reaction mixtures were diluted between 1- and 100-fold before injection into Xenopus oocytes as described (15 ).
Patch-clamp recording and analysis. Single channel recordings were made from outside-out patches held at -100 mV and at 19-22oC with 0 or 50-200 nM ACh. The pipette solution contained (in mM) 80 KF, 20 KCl, 10 HEPES, 10 EGTA (pH 7.4) and the bath solution was composed of (in mM) 115 NaCl, 2.5 KCl, 1.8 CaCl2, 10 HEPES (pH 7.2). Currents were recorded at 50 kHz with an Axopatch-1D patch-clamp amplifier (Axon Instruments) and stored on video tape. For analysis, recordings were further filtered at 9 kHz (-3 dB, Bessel filter), digitized at 90 kHz on to hard disk and analysed with the program pClamp 6 (Axon Instruments). Channel opening and closing transitions were detected by crossing a threshold set at 50% of the open amplitude. Current amplitude was estimated from all-point amplitude histograms. Most channel events were ~60 pS in amplitude; occasionally observed smaller amplitude channel events were excluded from analysis. Bursts were defined as a group of openings separated by closed intervals shorter than a certain critical duration. The critical closed time was determined for each patch from the closed time distribution to give an equal proportion of misclassified short intervals within bursts and long intervals between bursts (37 ). Histograms of shut times and burst durations were fitted to the sum of exponentials by maximum likelihood (events shorter than 50 [mu]s were excluded from the fit).
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*To whom correspondence should be addressed. Tel: +44 1865 222326; Fax: +44 1865 222402; Email: neurosciences@imm.ox.ac.uk
+These authors contributed equally to this work
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