Rearrangement of nicotinic receptor alpha subunits during formation of theligand binding sites

Muscle nicotinic acetylcholine receptors (AChRs) are pentamers that contain two a subunits a beta, gamma (or epsilon), and delta subunit. In this pape r, we have characterized subunit processing and folding events leading to f ormation of the two AChR ligand binding sites. a subunit residues, 187-199, which are part of overlapping ACh and alpha -bungarotoxin (Bgt) binding si tes on AChRs, were assayed using a monoclonal antibody (mAb) specific for t hese residues. We found that this region was inaccessible to the mAb early during AChR assembly but became accessible as the first of two Bgt binding sites formed later during assembly, indicating that the region changes conf ormation as the Bgt binding site appears. Without previous reduction, 20% o f the alpha subunits could be alkylated by bromoacetylcholine bromide as th e first ACh binding site formed, which further indicated that the disulfide bond between cysteines 192 and 193 does not form until the first ACh bindi ng site appears soon after Bgt binding site formation. When alpha subunits were mutated to add a glycosylation site at residue 187, the number of Bgt binding sites increased threefold, AChRs assembled more efficiently, and 2. 5-fold more AChRs reached the cell surface. Our results indicate that bindi ng site formation involves a rate-limiting rearrangement of the a subunit t hat exposes the 187-199 region to the endoplasmic reticulum lumen and deter mines when cysteines 192 and 193 disulfide bond.