Abstract
Investigation of the mechanisms by which the subunits of ligand-gated ion channels fold and associate to form oligomers has been hampered by the lack of an in vitro system in which these reactions occur. We have established conditions in a rabbit reticulocyte translation system supplemented with canine pancreatic microsomes under which the alpha and delta subunits of the nicotinic acetylcholine receptor (AChR) fold and assemble to form a heterodimer with a cholinergic binding site comparable with that found in the intact AChR. Folding of the alpha subunit was followed by its ability to bind alpha-bungarotoxin. Folding efficiency was highly sensitive to changes in the redox potential of the translation medium and was favored by an oxidizing environment. Acquisition of the toxin binding conformation required N-linked core glycosylation but not oligosaccharide trimming, suggesting that oligosaccharide-dependent interaction of chaperones with the alpha subunit is not essential for correct subunit folding. The conformationally mature alpha subunit specifically associated with the delta subunit but not the beta subunit to form a heterodimer with a high affinity ligand-binding site. These data demonstrate, for the first time, correct folding and assembly of the AChR subunits in an in vitro system.
Highlights
The muscle acetylcholine receptor (AChR)1 is the best understood member of a family of ligand-gated ion channels that mediate fast synaptic transmission in the nervous system
Each of the subunits of the muscle AChR is synthesized as a separate polypeptide chain [8] that is translocated into the ER, where the signal sequence is cleaved [8], the core glycosyl residues are added [8, 9], and the N-terminal domain is folded [10, 11]
Folding of the ␣ Subunit in an in Vitro Translation System—In devising an in vitro translation system that would allow ␣ subunit folding, we focused initially on finding a redox potential that is compatible with both translation and disulfide bond formation
Summary
The muscle acetylcholine receptor (AChR) is the best understood member of a family of ligand-gated ion channels that mediate fast synaptic transmission in the nervous system. Studies of other glycoprotein complexes processed in the ER have demonstrated a critical role for molecular chaperones and folding enzymes in conformational maturation, oligomeric assembly, and retention of misfolded and unassembled monomers inside the ER lumen [22, 23] These factors facilitate protein folding and oligomerization by preventing irreversible aggregation and by catalyzing disulfide bond formation and prolyl isomerization [23,24,25,26]. Molecules such as BiP, Grp, and calnexin associate transiently with polypeptides that are in the process of folding [23, 27,28,29] and may play a role in subunit assembly [27, 30, 31]. The kinetics of the observed association and dissociation of the subunits with BiP
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