mutations in congenital myasthenic syndromes reveal an e subunit

Mutations in congenital myasthenic syndromesreveal an e subunit C-terminal cysteine, C470,crucial for maturation and surface expressionof adult AChR

John Ealing1, Richard Webster1, Sharon Brownlow1, Amr Abdelgany1, Hans Oosterhuis2,{,

Francesco Muntoni3, David J. Vaux4, Angela Vincent1 and David Beeson1,*

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; Revised and Accepted September 25, 2002

Many congenital myasthenic syndromes (CMS) are associated with mutations in the genes encoding theacetylcholine receptor (AChR), an oligomeric protein with the structure a2bde. AChR deficiency is frequentlydue to homozygous or heteroallelic mutations in the AChR e subunit, most of which cause truncation of thepolypeptide chain and loss of surface expression of AChR. Here we identified mutations e1369delG andeY458X, located in the 18 amino acid e subunit C-terminus that lies extracellular to the M4 transmembranedomain. We then incorporated green fluorescent protein (GFP) into the intracellular loop between M3 and M4of mutant or wild-type e subunits and expressed the AChRs in RD or HEK 293 cells. AChR containing wild-type GFP-tagged e subunits were incorporated into the surface membrane, whereas the GFP-tagged AChRmutant e subunits co-localized with an endoplasmic reticulum (ER) marker and were not expressed on the cellsurface. In addition, mutant AChRs did not reach the cell surface, as measured by labelling of intact cells with125I-a-bungarotoxin and precipitation with an e-subunit-specific antiserum. Mutagenesis studies showed thatcysteine 470, located four amino acids from the C-terminus, is essential for a/e assembly and surfaceexpression of adult AChR. Replacement of cysteine 470 by serine does not restore a/e assembly or surfaceexpression. Our results provide the first use of GFP-tagged AChR as a tool for investigation of CMS anddemonstrate a previously undetermined role for a disulphide-bonded cystine in the e subunit C-terminus,which plays a crucial role in expression of the adult AChR.

INTRODUCTION

Muscle acetylcholine receptors (AChR) mediate synaptictransmission at the neuromuscular junction. They are membersof the ‘cystine loop’ ligand-gated ion channel superfamily thatincludes neuronal AChR, glycine receptors, GABAA receptorsand the 5HT3 receptor. These receptors have a pentamericstructure usually comprising several different subunits. Theprocess by which the different subunits are correctly assembledinto the mature functional protein is incompletely understood.For the AChR, it may take up to 2 h to complete (1) andrequires sequential subunit folding, post-translational modi-fication, subunit–subunit interactions and interaction withmolecular chaperones (2).

The transmembrane topology for each subunit consists of alarge N-terminal extracellular domain that contains the ligandbinding sites, glycosylation sites and the disulphide-linkedcystine loop, three closely linked transmembrane domains(M1–3), a large cytoplasmic domain followed by a fourthtransmembrane domain (M4) and a short stretch of extra-cellular C-terminal amino acids. Studies of the N-terminaldomains show that the AChR subunits contain specificrecognition signals for the initial steps of AChR assembly(3–7). In addition, conformational changes dependent uponformation of the cystine loop are required for oligomerization (8).Similarly, expression of chimeric subunits have identifiedregions in the cytoplasmic loop between M3 and M4 crucial forexpression of AChR on the cell surface (9–11). By contrast, the

*To whom correspondence should be addressed: Tel: þ44 1865222311; Fax: þ44 1865222402; Email: [email protected]{Deceased May 2002.

# 2002 Oxford University Press Human Molecular Genetics, 2002, Vol. 11, No. 24 3087–3096

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role of the C-terminal extracellular domain has not beenstudied in such detail.

Mutations in the AChR e subunit gene are the most commoncause of congenital myasthenic syndromes characterized bydeficiency of AChR at the endplate (12–20). The mutationsare found along the length of the e subunit gene, including thepromoter region (21–23) and result in frameshifts or nonsensecodons that truncate the subunit polypeptide chain, or loss ofresidues essential for AChR assembly or function. Expressionof the fetal (g) AChR subunit persists in human adult muscle(24), albeit at a low level and is thought to be able to substitutefor the dysfunctional e subunits (13).

We identified novel mutations close to the e subunitC-terminus in patients with congenital myasthenic syndromesand used a combination of e/GFP chimeric subunits, surfacelabelling of transfected cells and mutagenesis to investigatetheir effects on AChR expression. The results show thatcysteine residue 470 (C470), unique to the e subunit andlocated only four amino acids from the C-terminus, is essentialfor surface expression of adult AChR.

RESULTS

Clinical features

Six kinships were studied, five from Holland (25). Clinicalfeatures were previously described in detail (25,26) but, inbrief, the patients were of non-consanguineous parentage andhad symptoms of ptosis, external opthalmoplegia and variablegeneralized muscle weakness with onset in infancy. Antibodiesto the AChR were absent, but the patients showed apositive response to anticholinesterase treatments. Thesefeatures are typical of AChR deficiency syndrome (12,13,19).In accordance, analysis of a muscle biopsy from patient 3showed reduced endplate 125I-a-BuTx binding to around 10%of control values (26).

Mutational studies

SSCP, direct sequencing and restriction endonuclease digestionshowed that each of the five Dutch patients harbouredmutations in the AChR e subunit genes (Fig. 1A and B;Table 1). One patient is homozygous for a single nucleotidedeletion in exon 12, e1369delG and three are heteroallelic fore1369delG, also harbouring eY15H, e509insA and eR311Q.The fifth patient is homozygous for eR311Q. In addition,screening of patients from the UK with suspected AChRdeficiency syndrome revealed two brothers (family 6) with asecond mutation extracellular to M4: a T!A transversion atnucleotide 1374 which introduces a nonsense codon at aminoacid 458, Y458X (Fig. 1C). None of these mutations wereobserved in analysis of 120 control samples. As reported forother AChR deficiency mutations (12–16,19), expression inHEK 293 cells of AChR containing each of these mutationsresulted in loss of adult AChR surface expression (data fore1369delG and eY458X shown below). e1369delG haspreviously been identified in a CMS patient (18) and themutation eR311W has been reported to shorten AChRactivations as well as reduce AChR surface expression (13).

Analysis of single channel recordings of AChR containingeR311Q expressed in HEK 293 showed this mutation does notaffect AChR kinetics (data not shown). The M4 transmembranedomain is the last region on the e subunit for which there is adefined functional role. We therefore concentrated our studieson the disease mechanisms of e1369delG and eY458X, whichlie distal to M4 in the short C-terminal tail (Fig. 1D).

Expression of GFP-tagged human AChR

To visualize the fate of the mutant e1369delG subunitexpressed in HEK 293 or muscle cell lines, we incorporatedan enhanced green fluorescent protein (GFP) tag at the SfiIrestriction site located between the e subunit M3 and M4transmembrane domains. We first showed that wild-type AChRand AChR e-GFP expressed in HEK 293 cells gave similarlevels of 125I-a-BuTx surface binding and that they could beimmunoprecipitated by anti-e (Fig. 2B). Single channelanalysis showed that the AChR e-GFP is not altered in ionchannel characteristics (Fig. 2C and D; Table 2): the longestcomponent of the burst durations (t3) was 3.92� 0.2 and4.23� 0.3 for wild-type AChR and AChRe-GFP, respectivelyand the slope conductance was 60.2 pS, which is very close tothe figure of 62.2 pS we reported for human adult-type AChRunder similar conditions (27), and the figure of 60 pS for othermammalian adult-type AChR (28,29). None of the parametersmeasured for AChR and AChRe-GFP showed a significantdifference using Student’s t-test (t1, P¼ 0.94, t2, P¼ 0.55,t3, P¼ 0.53 and A1, P¼ 0.97, A2, P¼ 0.22, A3, P¼ 0.13).

Localization of GFP-tagged mutant AChR

The GFP-tag was also inserted into cDNAs containing thee1369delG and eY458X mutations. Expression of theseGFP-tagged e subunits in the RD muscle cell line (ECACCno. 85111502) resulted in two different patterns of fluorescencewhen viewed by confocal laser scanning microscopy. The wild-type GFP-tagged e subunit was present both within intracel-lular compartments and on the cell surface, indicated by thelabelled cellular processes (Fig. 3, upper left panel). Bycontrast, the mutant GFP-tagged e subunit was only presentwithin the cell (Fig. 3, lower left panel). The vector pCFP–ERcontains a mutated form of GFP that exhibits blue fluorescenceand contains a 50 calreticulin signal peptide and KDELtetrapeptide that localizes the expressed CFP to the endoplasmicreticulum (30) (Fig. 3, central panels). Overlay of the pCFP–ER

Table 1. Mutations affecting the C-terminus ofthe AChR e subunit in the five Dutch patients

Patient Mutations

1 (1) e1369delG, e1369delG2 (8) e1369delG, eY15H3 (5) e1369delG, eR311Q4 (4) e1369delG, e509insA5 (7) eR311Q, eR311Q

Clinical features of patients 1–5 were describedby Oosterhuis (25); patient number assigned in(25) is given in brackets.

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Figure 1. (A) Direct DNA sequencing of PCR amplicons of exon 12 of the AChR e subunit gene showing deletion of a G nucleotide at position 1369 and (B)restriction endonuclease digestion of PCR amplicons of e subunit exons from affected individuals and family members confirming the presence of homozygous orheteroallelic mutations. Affected individuals (shaded symbols) carry two mutant alleles, whereas members carrying one (half-shaded) are unaffected. (C)Confirmation of mutation Y458X by direct DNA sequencing and restriction digests of exon 12 amplicons. (D) Schematic representation of the AChR e subunitshowing the positions of the two C-terminal mutations.

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Figure 2. Functional properties of human AChR containing an e-GFP-tag. (A) Schematic representation of the e subunit showing the position of the GFP-tag. (B)Expression of AChR and AChRe–GFP in HEK 293 cells. Surface AChR is detected by 125I-a-BuTx binding, and surface expression of AChRe-GFP confirmed byimmunoprecipitation with an e subunit-specific antiserum. Results are normalized to abde and represent the mean�SD of three experiments. (C) Single-channel record-ings showing typical activations and burst length distributions for wild-type and AChRe–GFP expressed in HEK293 cells. (Left) Example traces and (right) burst-durationhistograms fitted by the sum of three exponentials. (D) Slope conductance for AChRe–GFP (right) obtained by a plot of the mean single channel current amplitude versusthe patch potential (left). None of the characteristics of AChRe–GFP were significantly different from those of wild-type AChR (Table 2).



signal (pseudo-coloured red) with AChRe1369delG–GFP signals(pseudo-coloured green) demonstrates exact co-localization ofthe two signals with the green and red combining to give ayellow signal (Fig. 3, lower right panel), suggesting that themutant GFP-containing e subunit is retained within the ER. Bycontrast, with the wild-type e-GFP, fluorescence is located in theER (forming a yellow signal of co-localization with the ERmarker) and in other intracellular compartments and on the cellsurface (Fig. 4, upper right panel). The wild-type e-GFP surfacelabel is seen clearly in the processes extending between the twocells, visible in the upper right panel of Figure 3. Similar resultswere obtained with Y458X.

Deletion mutation of e subunit C-terminal region

The e subunit M4 domain is predicted to span amino acids436–455 and is followed by 18 amino acid residues (456–473)at the C-terminus extracellular to M4 (Fig. 4A). e1369delG

causes a frameshift at amino acid 457, just distal to the M4transmembrane domain and is predicted to generate 27missense amino acids followed by a nonsense codon (notshown). To investigate the effects of e1369delG and Y458X one subunit and AChR expression, we created a series of deletionmutants and transfected the cDNAs into HEK 293 cells.e1369delG and Y458X led to reduced surface AChR expres-sion as measured by 125I-a-BuTx labelling (Fig. 4B) andsimilar lack of expression was found with all mutants thatlacked the terminal four amino acids, CIQP. By contrast, morethan 60% of cell-surface expression was restored with e subunitlacking only the last three amino acids (I471X).Immunoprecipitation with an e subunit-specific antiserumconfirmed that only the e subunits containing C470 wereincorporated into surface adult AChR (Fig. 4B). Transfectionof e-omitted abd cDNAs generates around 40% of wild-type125I-a-BuTx surface binding and was used as a control for thespecificity of this antiserum.

Figure 3. Typical images of RD cells co-transfected with cDNAs encoding abd AChR subunits, GFP-tagged e (top panels) or e-mutant e1369delG (bottom panels)and a marker for the endoplasmic reticulum, pCFP–ER. Transfected cells were cultured for 36 h prior to imaging with a BioRad Radiance 2000 multiphoton micro-scope. GFP-tagged e subunits (left panels), pCFP–ER (central panels) are presented as grey scale. An overlay of the pCFP–ER (pseudo-coloured red) and GFP-tagged e subunits (pseudo-coloured green) are shown in the right panels.

Table 2. Burst durations of wild-type and GFP-tagged AChRs

n t1 (SEM) (ms) A1 (%) t2 (ms) A2 (%) t3 (ms) A3 (%)

AChR 3 0.075 (0.01) 35.8 (1.4) 1.245 (0.17) 23.9 (7.0) 3.923 (0.20) 40.9 (7.3)AChRe–GFP 5 0.074 (0.01) 35.9 (2.0) 1.128 (0.14) 41.2 (8.6) 4.226 (0.32) 22.8 (6.7)

Burst length time constants (tn) and their relative area (An) are represented as mean (SEM); n¼ number of patches analysed.



The C-terminal cysteine is not present in any of the other humanAChR subunits, but is conserved within the e subunit betweenspecies (Fig. 5A). We therefore mutated this residue to eitheralanine (eC470A) or serine (eC470S; Fig. 5B). Both mutationsseverely reduced surface expression of adult AChR, to levelssimilar to those obtained with the truncation mutant C470X.

To investigate the effect of the e1369delG mutation and the Cterminal variants on AChR a/e subunit assembly, which takesplace in the ER, we determined intracellular 125I-a-BuTx bindingto ae complexes expressed in HEK 293 cells (Fig. 5C).Expression of a subunit alone gave �20% of the toxin bindingobserved for wild-type ae complexes. When an alanine or serinewas substituted for eC470, 125I-a-BuTx binding to ae complexesincreased above that for a alone, although the binding was stillmuch reduced compared with wild-type ae complexes. Thissuggests that in HEK 293 cells the mutant e subunits do notinteract efficiently with the a subunits to create the 125I-a-BuTxbinding site (or that they modify 125I-a-BuTx binding).

DISCUSSION

In investigating the pathogenic effects of C-terminal mutationsin the e subunit of the human AChR, we found a previously

unrecognized role for a C-terminal cysteine in AChRmaturation and surface expression. We first labelled humanAChR by incorporating a GFP-tag without affecting itsfunctional properties and used expression studies of GFP–AChR to study the subcellular localization of the mutantAChR. The GFP-tagged mutant AChR e subunit, expressed ina muscle cell line, was retained within the ER and a cysteineresidue 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 wassubstituted for C470, this residue is likely to form a disulphidebond crucial for AChR folding/assembly.

To investigate the fate of mutant AChRs, we tagged theAChRs by incorporating GFP into the cytoplasmic domain ofthe mutant and wild-type e subunits. In contrast to resultsobtained from other members of the cystine loop ion channelfamily, such as neuronal a7 AChR subunits, GABAA andglycine receptors for which N- or C-terminal GFP–fusionconstructs can generate expressed receptors (31,32), previousattempts to fuse a GFP-tag to the C-terminus of subunits ofmuscle AChR resulted in loss of AChR expression (33). Thismay reflect our proposed role for the C-terminal region in AChRassembly. We found that, as reported for mouse AChR (33),

Figure 4. Expression of AChR containing mutant e subunits in HEK 293 cells. (A) Illustration of the deletion mutations constructed within the 18 amino acid tailsequence of the e subunit C-terminal to M4. (B) Total 125I-a-BuTx binding to surface of HEK 293 cells transfected with cDNAs encoding wild-type abde, wild-type abd and abd plus e subunit C-terminal deletion mutants. (C) Surface 125I-a-BuTx binding precipitated with an AChR e subunit-specific antiserum (anti-e).Results are normalized for 125I-a-BuTx binding to abde and represent the mean� SD of four experiments. Control cells were transfected with pcDNA3.1.



insertion of the GFP tag in the cytoplasmic domain between M3and M4 (avoiding the amphipathic region, MA) can generatetagged human AChR with functional properties that are notsignificantly different from wild-type. Moreover, the presence ofthe GFP-tag does not appear to interfere with rapsyn-inducedclustering of the AChR (33) (D. Beeson, unpublished data).Thus GFP-tagged AChR should provide a powerful tool for thestudy of the localization, assembly and trafficking of the humanAChR and for the investigation of disease mechanisms.

The GFP-tagged e1369delG subunit was retained in the ERas shown by co-localization with pCFP–ER. A strict require-ment for correctly folded and assembled AChR is requiredbefore 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 sequentialprocess that may take several hours (1). Unassembled AChRare retained within the ER where they accumulate or aredegraded. Most studies of assembly have identified regions andspecific residues within the extracellular N-terminal domainthat govern the initial subunit interactions (2). More recently,studies on the AChR g subunit resulted in the proposal that theN-terminal domain mediates the initial subunit associations,whereas signals in its C-terminal half are required forsubsequent subunit interactions (10). In addition, studies of a3 amino acid deletion in the b subunit have identified a regionin the cytoplasmic loop between M3 and M4 where thesecondary structure is crucial for interaction between the b andd subunits (11).

Figure 5. (A) Alignment of the C-terminal extracellular domains for the human muscle a, b, g, d and e subunits and of the e subunits from human, calf, rat andmouse. (B) Surface expression of AChR containing mutations of eC470 in HEK 293 cells. Surface 125I-a-BuTx binding was precipitated with an AChR e subunit-specific antiserum (anti-e). Results are normalized for 125I-a-BuTx binding to abde and represent the mean�SD of four experiments. (C) Triton X-100 extracts ofHEK 293 cells transfected with ae, a and ae-mutant cDNAs precipitated with an AChR a subunit-specific antiserum (anti-a). Results are normalized for binding toae and represent the mean�SD for three experiments.



Our results focussed on a previously unidentified role of theC-terminus as crucial for the assembly of the adult AChR andfor its exit from the ER. Analysis of previous reports providesadditional support for the crucial role of this region of the esubunit. Transfection of HEK 293 cells with a chimera inwhich the C-terminal region of e was replaced by g, abde459g,reduced surface a-BuTx binding (10); and a chimera composedof the extracellular e subunit N-terminus, the b subunitmembrane-spanning regions (M1–M4) and the e subunitextracellular C-terminal domains was reported to substitutefor the e subunit in supporting surface AChR expression (4).By contrast, although a broadly equivalent chimera composedof the C-terminal domain of the g subunit and thetransmembranous regions of the d subunit prevented surfaceexpression (10), it was concluded that no general C-terminalmotif supports maturation of heterodimers prior to theirassembly into abg trimers.

Our results highlight the critical role of a single residue,eC470, in the C-terminus and show that it is likely to form adisulphide bond and play a role additional to that of theN-terminal extracellular cystine loop structure that is commonto all members of this ion channel superfamily. Mutation of theN-terminal cystine loops on both a and b subunits are thoughtto block conformational changes necessary for AChR assembly(8). Naturally occurring mutations in the human AChRN-terminal cystine loop, such as eC128S (35) or bC128R(D. Beeson, unpublished data), are null mutations and mutationat eC128S reduces a-BuTx binding to aeC128S complexes toless than that of the a subunits alone (35), probably because ofinstability and rapid degradation of the dimers (36). Bycontrast, the eC470S did not have such a ‘dominant negative’effect on 125I-a-BuTx binding (Fig. 5C) and the robustfluorescent signal observed for the e-GFP mutants indicatesthat rapid degradation in the ER is unlikely. These observationssuggest that loss of eC470 only partially reduces the efficiencyof the steps involved in the initial ae association and may alsoaffect subsequent steps in pentamer assembly, transfer from theER or incorporation into the plasma membrane.

The residue with which the C-terminal eC470 interacts is notknown. The e subunit has an extracellular cysteine at position190 that is conserved across species and could potentially bondto eC470. In Torpedo electric organ, the penultimate aminoacid of the d subunit is a cysteine residue (C500) thought tomediate dimerization of the AChR through forming adisulphide bond with another d subunit (37), althoughexpression of Torpedo AChR subunits in mammalian cellsdoes not result in this AChR dimer formation (6). Indeed thereis, as yet, no evidence that mammalian adult AChR exists asdimers. Alternatively, C470 may interact with a non-AChRsubunit chaperone protein. Native AChRs have been shown tointeract with ER chaperones such as BiP and Calnexin (38–40)and, more recently, the product of the ric-3 gene in C. eleganshas been localized to the ER and shown to be essential for thefinal 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 deficiencysyndromes are located in the e subunit gene. We show here thateven mutations in the extreme C-terminal domain can causeAChR deficiency, if they result in loss of eC470, and we beginto provide a greater understanding of the mechanisms through

which these mutations result in AChR deficiency. Many AChRe subunit mutations are ‘private’ and there are few clearexamples of founder effects. The exception until now ise1267delG, a mutation that is common in patients in south-eastern Europe of gypsy ethnic origin (17), but four out of thefive AChR deficiency patients from Holland, analysed here,harboured e1369delG, suggesting a common founder for thismutation.

MATERIALS AND METHODS

Mutational analysis

Approval for the use of human tissues was received from theCentral Oxford Research Ethics Committee. DNA was isolatedfrom peripheral blood using the NucleonTM II DNA extractionkit (Nucleon Biosciences). RNA was prepared from muscletissue using RNAzol B (AMS Biotechnology). Exons withinthe AChR a, b, d and e subunit genes were screened formutations using single-strand conformation polymorphismanalysis (SSCP) as described previously (42). Amplicons fromexons showing abnormal conformers were subject to directautomated sequencing (Oxford University, Department ofBiochemistry DNA Sequencing Facility) and changes in theDNA sequence confirmed by restriction endonuclease diges-tion. Oligonucleotide 50-GTATGGCCTCTGTGTCGATGTCC-ATCTTG-30 was used to create an XcmI restriction site which islost in patients harbouring the mutation e509insA.

Expression constructs

cDNAs encoding the AChR a, b, d and e subunits (43) weresubcloned into pcDNA3.1 (Invitrogen Ltd). Naturally occurringmutations Y458X and e1369delG were introduced into the esubunit cDNA sequence using the GeneEditor1 in vitromutagenesis kit (Promega Corp.). Artificial mutations wereintroduced into the e subunit cDNA using 30 reverse PCRoligonucleotide primers that contained both the mutant DNAsequence and a 30 NotI restriction site. Amplicons containingthe mutant cDNA sequences were generated using therespective 30 mutant reverse primers in combination with theforward primer 50-GCCACGCTCATTGTCATGAATTGC-30,located in the sequence that encodes the M3 transmembranedomain. SfiI/NotI restriction sites were used for ligation of themutant cDNA sequence into wild-type e subunit cDNA withinthe pcDNA3.1 vector. Mutant cDNAs were sequenced toconfirm the presence of the mutation and absence of additionalDNA changes. GFP-tagged AChR e subunits were generatedusing pEGFP-N1 (BD Biosciences). PCR was used to amplifythe GFP tag, which was then ligated into the e subunit cDNA atthe SfiI restriction site located between transmembrane domainsM3 and M4. DNA sequencing confirmed the presence andcorrect reading frame of the inserted GFP-tag sequence.

Confocal laser scanning microscopy

Cells from the rhabdomyosarcoma cell line RD were grown insix-well tissue culture plates containing 25 mm diameterglass cover slips and transfected using polyethyleneimine with



GFP-tagged e subunit cDNAs. Transfection of cells withpECFP-ER (BD Biosciences), which contains a 50 calreticulinsignal peptide and a 30 KDEL tetrapeptide ER-retentionsequence was used for localization of the ER (30). Two dayspost-transfection, cells were mounted in a live cell chambermaintained at 37�C and viewed using 457 and 488 nm laserlines on a Bio-Rad Radiance 2000 MP microscope. Data wascollected with Lasersharp 2000 software, analysed withConfocal Assistant and presented using Adobe Photoshop 5.Single GFP and CFP images are presented as grey-scale; forthe merged images, GFP is shown in green but the CFP channelis shown in red to facilitate comparison.

Patch-clamp recordings and analysis

Recordings were performed in the cell-attached patch configu-ration using standard methods previously described (44).Single-channel currents were amplified with an Axopatch 200Bamplifier (Axon Instruments Inc.), sampled to hard disk at90 kHz and filtered for analysis to a final cumulative fc of4.37 kHz; resolution was set at 45 ms. Recordings of burstactivity (100 nM ACh in pipette) were made with pipettepotential set at þ80 mV. Single channel conductance (100 mM

ACh in pipette) was assessed at different patch potentials.Channel transitions were detected by 50% amplitude thresholdcrossings (pClamp6). Bursts were defined (44) and histogramsof burst duration were fitted to the sum of three exponentials bymaximum log likelihood.

Expression studies

Wild-type and mutant AChR e subunit cDNAs, in combinationwith wild-type a, b and d subunit cDNAs were transfected intoHEK 293 cells grown on six-well tissue culture plates usingpolyethyleneimine. Surface AChR expression was determined2 days post-transfection by incubating cells in 10 nM

125I-a-bungarotoxin (125I-a-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. Surface125I-a-BuTx-AChR containing the e subunit was then deter-mined by immunoprecipitation with an e subunit-specificantiserum (27). 125I-a-BuTx bound to a and ae variants wasdetermined on Triton X-100 extracts of transfected cells, usingan a subunit-specific antiserum (27).

ACKNOWLEDGEMENTS

J.E. was supported by a Wellcome Trust Clinical TrainingFellowship. Work on congenital myasthenic syndromes isfunded by the Muscular Dystrophy Campaign/MyastheniaGravis Association and the Medical Research Council.

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mutations in congenital myasthenic syndromes reveal an e subunit

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