Abstract
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.
Highlights
acetylcholine receptors (AChRs) assembly is a slow process that takes ;2 h to complete [2], assembly intermediates have been difficult to isolate
The Conformational Change Occurs before Formation of the BuTx Binding Site and the Addition of the d Subunit—The only AChR subunit complexes that appear to be recognized by the cystine loop mAb are abg trimers
The strong conservation of the cystine loop among the different ionotropic neurotransmitter receptors and throughout evolution suggests that this structure plays a vital role with respect to the receptors
Summary
(Received for publication, November 6, 1996, and in revised form, April 24, 1997). William N. Mutated a or b subunits lacking the cystine loop disulfide bond assembled with other wild type subunits [14, 15] and produced functional receptors [15] Based on these data, it was suggested that formation of the cystine loop is not required for subunit assembly but instead plays an important role in the rate that subunits are degraded and in the efficiency of transport of AChRs to the cell surface. It was suggested that formation of the cystine loop is not required for subunit assembly but instead plays an important role in the rate that subunits are degraded and in the efficiency of transport of AChRs to the cell surface Contrary to this viewpoint, we demonstrate in this paper that an intact cystine loop is essential for proper AChR subunit assembly.
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