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 hav e established conditions in a rabbit reticulocyte translation system s upplemented with canine pancreatic microsomes under which the alpha an d delta subunits of the nicotinic acetylcholine receptor (AChR) fold a nd assemble to form a heterodimer with a cholinergic binding site comp arable with that found in the intact AChR. Folding of the alpha subuni t was followed by its ability to bind alpha-bungarotoxin. Folding effi ciency was highly sensitive to changes in the redox potential of the t ranslation medium and was favored by an oxidizing environment. Acquisi tion of the toxin binding conformation required N-Linked core glycosyl ation but not oligosaccharide trimming, suggesting that oligosaccharid e-dependent interaction of chaperones with the alpha subunit is not es sential for correct subunit folding. The conformationally mature alpha subunit specifically associated with the delta subunit but not the be ta subunit to form a heterodimer with a high affinity ligand-binding s ite. These data demonstrate, for the first time, correct folding and a ssembly of the AChR subunits in an in vitro system.