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
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.
* 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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