The Role of the Cystine Loop in Acetylcholine Receptor Assembly

• Published in: The Journal of biological chemistry Vol. 272; no. 33; pp. 20945 - 20953
• Main Authors: Green, William N., Wanamaker, Christian P.
• Format: Journal Article
• Language: English
• Published: United States Elsevier Inc 15.08.1997; American Society for Biochemistry and Molecular Biology
• Subjects: Amino Acid Sequence; Animals; Binding Sites; Bungarotoxins - metabolism; Cystine; Disulfides; Dithiothreitol - pharmacology; Methionine - metabolism; Molecular Sequence Data; Protein Conformation; Receptors, Cholinergic - chemistry; Torpedo
• ISSN: 0021-9258; 1083-351X
• DOI: 10.1074/jbc.272.33.20945

Nicotinic acetylcholine receptors (AChRs) are composed of α, β, γ, and δ subunits, assembled into α2βγδ pentamers. A highly conserved feature of ionotropic neurotransmitter receptors, such as AChRs, is a 15-amino acid cystine “loop.” We find that an intact cystine loop is necessary for complete AChR assembly. By preventing formation of the loop with 5 mm dithiothreitol, AChR subunits assemble into αβγ trimers, but the subsequent steps in assembly are blocked. When α subunit loop cysteines are mutated to serines, assembly is blocked at the same step as with dithiothreitol. In contrast, when β subunit loop cysteines are mutated to serines, assembly is blocked at a later step, i.e. after assembly of αβγδ tetramers and before the addition of the second α subunit. After formation of the cystine loop, the α subunit undergoes a conformational change, which buries the loop. This conformational change is concurrent with the step in assembly blocked by removal of the disulfide bond of the cystine loop, i.e. after assembly of αβγ trimers and before the addition of the δ subunit. The data indicate that the α subunit conformational change involving the cystine loop is key to a series of folding events that allow the addition of unassembled subunits.