Human Molecular Genetics, 2002, Vol. 11, No. 24 3087-3096
© 2002 Oxford University Press
Mutations in congenital myasthenic syndromes reveal an 
subunit C-terminal cysteine, C470, crucial for maturation and surface expression of adult AChR
1Neurosciences Group, Weatherall Institute of Molecular Medicine, The John Radcliffe, Oxford OX3 9DS, UK, 2The Department of Neurology, Academic Hospital, Groningen, The Netherlands, 3Department of Paediatrics, Imperial College, London, UK and 4Sir William Dunn School of Pathology, University of Oxford, UK
Received August 5, 2002; Accepted September 25, 2002
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ABSTRACT |
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 TOP ABSTRACT INTRODUCTIONRESULTSDISCUSSIONMATERIALS AND METHODSREFERENCES |
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Many congenital myasthenic syndromes (CMS) are associated with mutations in the genes encoding the acetylcholine receptor (AChR), an oligomeric protein with the structure
2ß
. AChR deficiency is frequently due to homozygous or heteroallelic mutations in the AChR subunit, most of which cause truncation of the polypeptide chain and loss of surface expression of AChR. Here we identified mutations 1369delG and Y458X, located in the 18 amino acid subunit C-terminus that lies extracellular to the M4 transmembrane domain. We then incorporated green fluorescent protein (GFP) into the intracellular loop between M3 and M4 of mutant or wild-type subunits and expressed the AChRs in RD or HEK 293 cells. AChR containing wild-type GFP-tagged subunits were incorporated into the surface membrane, whereas the GFP-tagged AChR mutant subunits co-localized with an endoplasmic reticulum (ER) marker and were not expressed on the cell surface. In addition, mutant AChRs did not reach the cell surface, as measured by labelling of intact cells with 125I--bungarotoxin and precipitation with an -subunit-specific antiserum. Mutagenesis studies showed that cysteine 470, located four amino acids from the C-terminus, is essential for / assembly and surface expression of adult AChR. Replacement of cysteine 470 by serine does not restore / assembly or surface expression. Our results provide the first use of GFP-tagged AChR as a tool for investigation of CMS and demonstrate a previously undetermined role for a disulphide-bonded cystine in the subunit C-terminus, which plays a crucial role in expression of the adult AChR. |
INTRODUCTION |
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TOPABSTRACTINTRODUCTIONRESULTSDISCUSSIONMATERIALS AND METHODSREFERENCES |
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Muscle acetylcholine receptors (AChR) mediate synaptic transmission at the neuromuscular junction. They are members of the ‘cystine loop’ ligand-gated ion channel superfamily that includes neuronal AChR, glycine receptors, GABAA receptors and the 5HT3 receptor. These receptors have a pentameric structure usually comprising several different subunits. The process by which the different subunits are correctly assembled into the mature functional protein is incompletely understood. For the AChR, it may take up to 2 h to complete (1) and requires sequential subunit folding, post-translational modification, subunit–subunit interactions and interaction with molecular chaperones (2).
The transmembrane topology for each subunit consists of a large N-terminal extracellular domain that contains the ligand binding sites, glycosylation sites and the disulphide-linked cystine loop, three closely linked transmembrane domains (M1–3), a large cytoplasmic domain followed by a fourth transmembrane domain (M4) and a short stretch of extracellular C-terminal amino acids. Studies of the N-terminal domains show that the AChR subunits contain specific recognition signals for the initial steps of AChR assembly (3–7). In addition, conformational changes dependent upon formation of the cystine loop are required for oligomerization (8). Similarly, expression of chimeric subunits have identified regions in the cytoplasmic loop between M3 and M4 crucial for expression of AChR on the cell surface (9–11). By contrast, the role of the C-terminal extracellular domain has not been studied in such detail.
Mutations in the AChR subunit gene are the most common cause of congenital myasthenic syndromes characterized by deficiency of AChR at the endplate (12–20). The mutations are found along the length of the subunit gene, including the promoter region (21–23) and result in frameshifts or nonsense codons that truncate the subunit polypeptide chain, or loss of residues essential for AChR assembly or function. Expression of the fetal (
) AChR subunit persists in human adult muscle (24), albeit at a low level and is thought to be able to substitute for the dysfunctional subunits (13).
We identified novel mutations close to the subunit C-terminus in patients with congenital myasthenic syndromes and used a combination of /GFP chimeric subunits, surface labelling of transfected cells and mutagenesis to investigate their effects on AChR expression. The results show that cysteine residue 470 (C470), unique to the subunit and located only four amino acids from the C-terminus, is essential for surface expression of adult AChR.
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RESULTS |
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TOPABSTRACTINTRODUCTIONRESULTSDISCUSSIONMATERIALS AND METHODSREFERENCES |
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Clinical features
Six kinships were studied, five from Holland (25). Clinical features were previously described in detail (25,26) but, in brief, the patients were of non-consanguineous parentage and had symptoms of ptosis, external opthalmoplegia and variable generalized muscle weakness with onset in infancy. Antibodies to the AChR were absent, but the patients showed a positive response to anticholinesterase treatments. These features are typical of AChR deficiency syndrome (12,13,19). In accordance, analysis of a muscle biopsy from patient 3 showed reduced endplate 125I-
-BuTx binding to around 10% of control values (26).
Mutational studies
SSCP, direct sequencing and restriction endonuclease digestion showed that each of the five Dutch patients harboured mutations in the AChR subunit genes (Fig. 1A and B; Table 1). One patient is homozygous for a single nucleotide deletion in exon 12, 1369delG and three are heteroallelic for 1369delG, also harbouring Y15H, 509insA and R311Q. The fifth patient is homozygous for R311Q. In addition, screening of patients from the UK with suspected AChR deficiency syndrome revealed two brothers (family 6) with a second mutation extracellular to M4: a T
A transversion at nucleotide 1374 which introduces a nonsense codon at amino acid 458, Y458X (Fig. 1C). None of these mutations were observed in analysis of 120 control samples. As reported for other AChR deficiency mutations (12–16,19), expression in HEK 293 cells of AChR containing each of these mutations resulted in loss of adult AChR surface expression (data for 1369delG and Y458X shown below). 1369delG has previously been identified in a CMS patient (18) and the mutation R311W has been reported to shorten AChR activations as well as reduce AChR surface expression (13). Analysis of single channel recordings of AChR containing R311Q expressed in HEK 293 showed this mutation does not affect AChR kinetics (data not shown). The M4 transmembrane domain is the last region on the subunit for which there is a defined functional role. We therefore concentrated our studies on the disease mechanisms of 1369delG and Y458X, which lie distal to M4 in the short C-terminal tail (Fig. 1D).
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Expression of GFP-tagged human AChR
To visualize the fate of the mutant
1369delG subunit expressed in HEK 293 or muscle cell lines, we incorporated an enhanced green fluorescent protein (GFP) tag at the SfiI restriction site located between the subunit M3 and M4 transmembrane domains. We first showed that wild-type AChR and AChR -GFP expressed in HEK 293 cells gave similar levels of 125I--BuTx surface binding and that they could be immunoprecipitated by anti- (Fig. 2B). Single channel analysis showed that the AChR -GFP is not altered in ion channel characteristics (Fig. 2C and D; Table 2): the longest component of the burst durations (
3) was 3.92±0.2 and 4.23±0.3 for wild-type AChR and AChR-GFP, respectively and the slope conductance was 60.2 pS, which is very close to the figure of 62.2 pS we reported for human adult-type AChR under similar conditions (27), and the figure of 60 pS for other mammalian adult-type AChR (28,29). None of the parameters measured for AChR and AChR-GFP showed a significant difference using Student's t-test (1, P=0.94, 2, P=0.55, 3, P=0.53 and A1, P=0.97, A2, P=0.22, A3, P=0.13).
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Localization of GFP-tagged mutant AChR
The GFP-tag was also inserted into cDNAs containing the
1369delG and Y458X mutations. Expression of these GFP-tagged subunits in the RD muscle cell line (ECACC no. 85111502) resulted in two different patterns of fluorescence when viewed by confocal laser scanning microscopy. The wild-type GFP-tagged subunit was present both within intracellular compartments and on the cell surface, indicated by the labelled cellular processes (Fig. 3, upper left panel). By contrast, the mutant GFP-tagged subunit was only present within the cell (Fig. 3, lower left panel). The vector pCFP–ER contains a mutated form of GFP that exhibits blue fluorescence and contains a 5' calreticulin signal peptide and KDEL tetrapeptide that localizes the expressed CFP to the endoplasmic reticulum (30) (Fig. 3, central panels). Overlay of the pCFP–ER signal (pseudo-coloured red) with AChR1369delG–GFP signals (pseudo-coloured green) demonstrates exact co-localization of the two signals with the green and red combining to give a yellow signal (Fig. 3, lower right panel), suggesting that the mutant GFP-containing subunit is retained within the ER. By contrast, with the wild-type -GFP, fluorescence is located in the ER (forming a yellow signal of co-localization with the ER marker) and in other intracellular compartments and on the cell surface (Fig. 4, upper right panel). The wild-type -GFP surface label is seen clearly in the processes extending between the two cells, visible in the upper right panel of Figure 3. Similar results were obtained with Y458X.
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Deletion mutation of
subunit C-terminal regionThe
subunit M4 domain is predicted to span amino acids 436–455 and is followed by 18 amino acid residues (456–473) at the C-terminus extracellular to M4 (Fig. 4A). 1369delG causes a frameshift at amino acid 457, just distal to the M4 transmembrane domain and is predicted to generate 27 missense amino acids followed by a nonsense codon (not shown). To investigate the effects of 1369delG and Y458X on subunit and AChR expression, we created a series of deletion mutants and transfected the cDNAs into HEK 293 cells. 1369delG and Y458X led to reduced surface AChR expression as measured by 125I--BuTx labelling (Fig. 4B) and similar lack of expression was found with all mutants that lacked the terminal four amino acids, CIQP. By contrast, more than 60% of cell-surface expression was restored with subunit lacking only the last three amino acids (I471X). Immunoprecipitation with an subunit-specific antiserum confirmed that only the subunits containing C470 were incorporated into surface adult AChR (Fig. 4B). Transfection of -omitted ß cDNAs generates around 40% of wild-type 125I--BuTx surface binding and was used as a control for the specificity of this antiserum.
The C-terminal cysteine is not present in any of the other human AChR subunits, but is conserved within the subunit between species (Fig. 5A). We therefore mutated this residue to either alanine (C470A) or serine (C470S; Fig. 5B). Both mutations severely reduced surface expression of adult AChR, to levels similar to those obtained with the truncation mutant C470X.
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To investigate the effect of the
1369delG mutation and the C terminal variants on AChR / subunit assembly, which takes place in the ER, we determined intracellular 125I--BuTx binding to complexes expressed in HEK 293 cells (Fig. 5C). Expression of subunit alone gave 
20% of the toxin binding observed for wild-type complexes. When an alanine or serine was substituted for C470, 125I--BuTx binding to complexes increased above that for alone, although the binding was still much reduced compared with wild-type complexes. This suggests that in HEK 293 cells the mutant subunits do not interact efficiently with the subunits to create the 125I--BuTx binding site (or that they modify 125I--BuTx binding). |
DISCUSSION |
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TOPABSTRACTINTRODUCTIONRESULTSDISCUSSIONMATERIALS AND METHODSREFERENCES |
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In investigating the pathogenic effects of C-terminal mutations in the
subunit of the human AChR, we found a previously unrecognized role for a C-terminal cysteine in AChR maturation and surface expression. We first labelled human AChR by incorporating a GFP-tag without affecting its functional properties and used expression studies of GFP–AChR to study the subcellular localization of the mutant AChR. The GFP-tagged mutant AChR subunit, expressed in a muscle cell line, was retained within the ER and a cysteine residue at position 470, just four residues from the C-terminus, was found to be crucial for surface expression of adult AChR. Since surface AChR expression was lost even when serine was substituted for C470, this residue is likely to form a disulphide bond crucial for AChR folding/assembly.
To investigate the fate of mutant AChRs, we tagged the AChRs by incorporating GFP into the cytoplasmic domain of the mutant and wild-type subunits. In contrast to results obtained from other members of the cystine loop ion channel family, such as neuronal 7 AChR subunits, GABAA and glycine receptors for which N- or C-terminal GFP–fusion constructs can generate expressed receptors (31,32), previous attempts to fuse a GFP-tag to the C-terminus of subunits of muscle AChR resulted in loss of AChR expression (33). This may reflect our proposed role for the C-terminal region in AChR assembly. We found that, as reported for mouse AChR (33), insertion of the GFP tag in the cytoplasmic domain between M3 and M4 (avoiding the amphipathic region, MA) can generate tagged human AChR with functional properties that are not significantly different from wild-type. Moreover, the presence of the GFP-tag does not appear to interfere with rapsyn-induced clustering of the AChR (33) (D. Beeson, unpublished data). Thus GFP-tagged AChR should provide a powerful tool for the study of the localization, assembly and trafficking of the human AChR and for the investigation of disease mechanisms.
The GFP-tagged 1369delG subunit was retained in the ER as shown by co-localization with pCFP–ER. A strict requirement for correctly folded and assembled AChR is required before AChR can exit the ER and passage to the Golgi (34). Slow post-translational folding and processing are integrally involved in oligomerization, which is thought to be a sequential process that may take several hours (1). Unassembled AChR are retained within the ER where they accumulate or are degraded. Most studies of assembly have identified regions and specific residues within the extracellular N-terminal domain that govern the initial subunit interactions (2). More recently, studies on the AChR subunit resulted in the proposal that the N-terminal domain mediates the initial subunit associations, whereas signals in its C-terminal half are required for subsequent subunit interactions (10). In addition, studies of a 3 amino acid deletion in the ß subunit have identified a region in the cytoplasmic loop between M3 and M4 where the secondary structure is crucial for interaction between the ß and subunits (11).
Our results focussed on a previously unidentified role of the C-terminus as crucial for the assembly of the adult AChR and for its exit from the ER. Analysis of previous reports provides additional support for the crucial role of this region of the subunit. Transfection of HEK 293 cells with a chimera in which the C-terminal region of was replaced by , ß459, reduced surface -BuTx binding (10); and a chimera composed of the extracellular subunit N-terminus, the ß subunit membrane-spanning regions (M1–M4) and the subunit extracellular C-terminal domains was reported to substitute for the subunit in supporting surface AChR expression (4). By contrast, although a broadly equivalent chimera composed of the C-terminal domain of the subunit and the transmembranous regions of the subunit prevented surface expression (10), it was concluded that no general C-terminal motif supports maturation of heterodimers prior to their assembly into ß trimers.
Our results highlight the critical role of a single residue, C470, in the C-terminus and show that it is likely to form a disulphide bond and play a role additional to that of the N-terminal extracellular cystine loop structure that is common to all members of this ion channel superfamily. Mutation of the N-terminal cystine loops on both and ß subunits are thought to block conformational changes necessary for AChR assembly (8). Naturally occurring mutations in the human AChR N-terminal cystine loop, such as C128S (35) or ßC128R (D. Beeson, unpublished data), are null mutations and mutation at C128S reduces -BuTx binding to C128S complexes to less than that of the subunits alone (35), probably because of instability and rapid degradation of the dimers (36). By contrast, the C470S did not have such a ‘dominant negative’ effect on 125I--BuTx binding (Fig. 5C) and the robust fluorescent signal observed for the -GFP mutants indicates that rapid degradation in the ER is unlikely. These observations suggest that loss of C470 only partially reduces the efficiency of the steps involved in the initial association and may also affect subsequent steps in pentamer assembly, transfer from the ER or incorporation into the plasma membrane.
The residue with which the C-terminal C470 interacts is not known. The subunit has an extracellular cysteine at position 190 that is conserved across species and could potentially bond to C470. In Torpedo electric organ, the penultimate amino acid of the subunit is a cysteine residue (C500) thought to mediate dimerization of the AChR through forming a disulphide bond with another subunit (37), although expression of Torpedo AChR subunits in mammalian cells does not result in this AChR dimer formation (6). Indeed there is, as yet, no evidence that mammalian adult AChR exists as dimers. Alternatively, C470 may interact with a non-AChR subunit chaperone protein. Native AChRs have been shown to interact with ER chaperones such as BiP and Calnexin (38–40) and, more recently, the product of the ric-3 gene in C. elegans has been localized to the ER and shown to be essential for the final assembly and transport of AChR to the cell surface (41). This will need to be tested in future studies.
The majority of mutations underlying AChR deficiency syndromes are located in the subunit gene. We show here that even mutations in the extreme C-terminal domain can cause AChR deficiency, if they result in loss of C470, and we begin to provide a greater understanding of the mechanisms through which these mutations result in AChR deficiency. Many AChR subunit mutations are ‘private’ and there are few clear examples of founder effects. The exception until now is 1267delG, a mutation that is common in patients in south-eastern Europe of gypsy ethnic origin (17), but four out of the five AChR deficiency patients from Holland, analysed here, harboured 1369delG, suggesting a common founder for this mutation.
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MATERIALS AND METHODS |
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TOPABSTRACTINTRODUCTIONRESULTSDISCUSSIONMATERIALS AND METHODSREFERENCES |
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Mutational analysis
Approval for the use of human tissues was received from the Central Oxford Research Ethics Committee. DNA was isolated from peripheral blood using the NucleonTM II DNA extraction kit (Nucleon Biosciences). RNA was prepared from muscle tissue using RNAzol B (AMS Biotechnology). Exons within the AChR
, ß, and subunit genes were screened for mutations using single-strand conformation polymorphism analysis (SSCP) as described previously (42). Amplicons from exons showing abnormal conformers were subject to direct automated sequencing (Oxford University, Department of Biochemistry DNA Sequencing Facility) and changes in the DNA sequence confirmed by restriction endonuclease digestion. Oligonucleotide 5'-GTATGGCCTCTGTGTCGATGTCCATCTTG-3' was used to create an XcmI restriction site which is lost in patients harbouring the mutation 509insA.
Expression constructs
cDNAs encoding the AChR , ß, and subunits (43) were subcloned into pcDNA3.1 (Invitrogen Ltd). Naturally occurring mutations Y458X and 1369delG were introduced into the subunit cDNA sequence using the GeneEditor® in vitro mutagenesis kit (Promega Corp.). Artificial mutations were introduced into the subunit cDNA using 3' reverse PCR oligonucleotide primers that contained both the mutant DNA sequence and a 3' NotI restriction site. Amplicons containing the mutant cDNA sequences were generated using the respective 3' mutant reverse primers in combination with the forward primer 5'-GCCACGCTCATTGTCATGAATTGC-3', located in the sequence that encodes the M3 transmembrane domain. SfiI/NotI restriction sites were used for ligation of the mutant cDNA sequence into wild-type subunit cDNA within the pcDNA3.1 vector. Mutant cDNAs were sequenced to confirm the presence of the mutation and absence of additional DNA changes. GFP-tagged AChR subunits were generated using pEGFP-N1 (BD Biosciences). PCR was used to amplify the GFP tag, which was then ligated into the subunit cDNA at the SfiI restriction site located between transmembrane domains M3 and M4. DNA sequencing confirmed the presence and correct reading frame of the inserted GFP-tag sequence.
Confocal laser scanning microscopy
Cells from the rhabdomyosarcoma cell line RD were grown in six-well tissue culture plates containing 25 mm diameter glass cover slips and transfected using polyethyleneimine with GFP-tagged subunit cDNAs. Transfection of cells with pECFP-ER (BD Biosciences), which contains a 5' calreticulin signal peptide and a 3' KDEL tetrapeptide ER-retention sequence was used for localization of the ER (30). Two days post-transfection, cells were mounted in a live cell chamber maintained at 37°C and viewed using 457 and 488 nm laser lines on a Bio-Rad Radiance 2000 MP microscope. Data was collected with Lasersharp 2000 software, analysed with Confocal Assistant and presented using Adobe Photoshop 5. Single GFP and CFP images are presented as grey-scale; for the merged images, GFP is shown in green but the CFP channel is shown in red to facilitate comparison.
Patch-clamp recordings and analysis
Recordings were performed in the cell-attached patch configuration using standard methods previously described (44). Single-channel currents were amplified with an Axopatch 200B amplifier (Axon Instruments Inc.), sampled to hard disk at 90 kHz and filtered for analysis to a final cumulative fc of 4.37 kHz; resolution was set at 45 µs. Recordings of burst activity (100 nM ACh in pipette) were made with pipette potential set at +80 mV. Single channel conductance (100 µM ACh in pipette) was assessed at different patch potentials. Channel transitions were detected by 50% amplitude threshold crossings (pClamp6). Bursts were defined (44) and histograms of burst duration were fitted to the sum of three exponentials by maximum log likelihood.
Expression studies
Wild-type and mutant AChR subunit cDNAs, in combination with wild-type , ß and subunit cDNAs were transfected into HEK 293 cells grown on six-well tissue culture plates using polyethyleneimine. Surface AChR expression was determined 2 days post-transfection by incubating cells in 10 nM125I--bungarotoxin (125I--BuTx) with 1 mg/ml BSA for 30 min. Cells were washed four times with PBS and extracted in 1.25% Triton X-100, in 60 mM Tris–HCl (pH 7.4), 100 mM NaCl, 1 mM EDTA, 0.5 mM phenylmethysulphonyl fluoride. Surface 125I--BuTx-AChR containing the subunit was then determined by immunoprecipitation with an subunit-specific antiserum (27). 125I--BuTx bound to and variants was determined on Triton X-100 extracts of transfected cells, using an subunit-specific antiserum (27).
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ACKNOWLEDGEMENTS |
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J.E. was supported by a Wellcome Trust Clinical Training Fellowship. Work on congenital myasthenic syndromes is funded by the Muscular Dystrophy Campaign/Myasthenia Gravis Association and the Medical Research Council.
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FOOTNOTES |
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* To whom correspondence should be addressed: Tel: +44 1865222311; Fax: +44 1865222402; Email: dbeeson{at}hammer.imm.ox.ac.uk
Deceased May 2002. |
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