Formation of a ligand-binding site for the acetylcholine receptor in vitro.
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.
Shtrom
S S
SS
Department of Biochemistry and Biophysics, University of California, San Francisco, California 94143, USA.
Hall
Z W
ZW
D000818:Animals; D001665:Binding Sites; D002038:Bungarotoxins / Q000378:metabolism; D002474:Cell-Free System; D004285:Dogs; D006031:Glycosylation; D007700:Kinetics; D008024:Ligands; D046911:Macromolecular Substances; D051379:Mice; D008970:Molecular Weight; D014176:Protein Biosynthesis; D017510:Protein Folding; D011817:Rabbits; D011978:Receptors, Nicotinic / Q000096:biosynthesis / Q000737:chemistry* / Q000378:metabolism*; D012156:Reticulocytes / Q000378:metabolism
D002038:Bungarotoxins; D008024:Ligands; D046911:Macromolecular Substances; D011978:Receptors, Nicotinic