Mutations in congenital myasthenic syndromes reveal an epsilon subunit C-terminal cysteine, C470, crucial for maturation and surface expression of adult AChR.

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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 alpha(2)betadelta epsilon. AChR deficiency is frequently due to homozygous or heteroallelic mutations in the AChR epsilon subunit, most of which cause truncation of the polypeptide chain and loss of surface expression of AChR. Here we identified mutations epsilon 1369delG and epsilon Y458X, located in the 18 amino acid epsilon 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 epsilon subunits and expressed the AChRs in RD or HEK 293 cells. AChR containing wild-type GFP-tagged epsilon subunits were incorporated into the surface membrane, whereas the GFP-tagged AChR mutant epsilon 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 (125)I-alpha-bungarotoxin and precipitation with an epsilon-subunit-specific antiserum. Mutagenesis studies showed that cysteine 470, located four amino acids from the C-terminus, is essential for alpha/epsilon assembly and surface expression of adult AChR. Replacement of cysteine 470 by serine does not restore alpha/epsilon 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 epsilon subunit C-terminus, which plays a crucial role in expression of the adult AChR.