The role of the cystine loop in acetylcholine receptor assembly.
Nicotinic acetylcholine receptors (AChRs) are composed of alpha, beta, gamma, and delta subunits, assembled into alpha2betagammadelta 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 alphabetagamma trimers, but the subsequent steps in assembly are blocked. When alpha subunit loop cysteines are mutated to serines, assembly is blocked at the same step as with dithiothreitol. In contrast, when beta subunit loop cysteines are mutated to serines, assembly is blocked at a later step, i.e. after assembly of alphabetagammadelta tetramers and before the addition of the second alpha subunit. After formation of the cystine loop, the alpha 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 alphabetagamma trimers and before the addition of the delta subunit. The data indicate that the alpha subunit conformational change involving the cystine loop is key to a series of folding events that allow the addition of unassembled subunits.
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Department of Pharmacological and Physiological Sciences, University of Chicago, Chicago, Illinois 60637, USA.
Wanamaker
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D000595:Amino Acid Sequence; D000818:Animals; D001665:Binding Sites; D002038:Bungarotoxins / Q000378:metabolism; D003553:Cystine; D004220:Disulfides; D004229:Dithiothreitol / Q000494:pharmacology; D008715:Methionine / Q000378:metabolism; D008969:Molecular Sequence Data; D011487:Protein Conformation; D011950:Receptors, Cholinergic / Q000737:chemistry*; D014101:Torpedo
D002038:Bungarotoxins; D004220:Disulfides; D011950:Receptors, Cholinergic; D003553:Cystine; D008715:Methionine; D004229:Dithiothreitol