The effects of inhibiting oligosaccharide trimming by 1-deoxynojirimycin on the nicotinic acetylcholine receptor.
The nicotinic acetylcholine receptor has a subunit stoichiometry of alpha 2 beta gamma delta; all 5 subunits contain N-linked oligosaccharides. We investigated what role trimming of the oligosaccharides played in the post-translational processing of the subunits and assembly of the receptor by examining the receptor synthesized in the presence of an inhibitor of oligosaccharide trimming, 1-deoxynojirimycin. BC3H-1 cells express one-third fewer receptors when grown in the presence of 1-deoxynojirimycin. The receptor subunits that are expressed have decreased mobility by sodium dodecyl sulfate-polyacrylamide gel electrophoresis, indicating an inhibition of oligosaccharide trimming. In control cells, 40% of the translated alpha subunit acquires the capacity to bind alpha-bungarotoxin with a half-time of 40 min before assembly with the other subunits; the rest is rapidly degraded. In 1-deoxynojirimycin-treated cells approximately the same amount of alpha subunit is translated as in control cells, but that alpha subunit is degraded more rapidly, and only 25% acquires the capacity to bind alpha-bungarotoxin. From these results, we conclude that oligosaccharide processing either may aid in protecting the alpha subunit primary translation product from degradation or may be required for the conformational change or other post-translational modification(s) necessary for formation of the alpha-bungarotoxin binding form of the alpha subunit, which is then protected from proteolytic degradation. The cell surface receptor that is expressed in the presence of 1-deoxynojirimycin, however, is not altered in its affinity for cholinergic ligands. Thus, we conclude that differential N-linked oligosaccharide trimming of the 2 alpha subunits does not appear to play a part in the differences in affinities of the 2 alpha subunits for cholinergic ligands.
D017485:1-Deoxynojirimycin; D000818:Animals; D002038:Bungarotoxins / Q000378:metabolism; D002462:Cell Membrane / Q000378:metabolism; D002478:Cells, Cultured; D005944:Glucosamine / Q000031:analogs & derivatives / Q000494:pharmacology; D007700:Kinetics; D046911:Macromolecular Substances; D051379:Mice; D009844:Oligosaccharides / Q000378:metabolism*; D011499:Protein Processing, Post-Translational / Q000187:drug effects*; D011978:Receptors, Nicotinic / Q000187:drug effects / Q000235:genetics* / Q000378:metabolism
D002038:Bungarotoxins; D046911:Macromolecular Substances; D009844:Oligosaccharides; D011978:Receptors, Nicotinic; D017485:1-Deoxynojirimycin; D005944:Glucosamine