ALLOSTERIC TRANSITIONS OF THE ACETYLCHOLINE-RECEPTOR PROBED AT THE AMINO-ACID LEVEL WITH A PHOTOLABILE CHOLINERGIC LIGAND

Structural changes occurring upon desensitization of the Torpedo marmorata acetylcholine receptor were monitored with tritiated p-(N,N-dimethyl)aminobenzenediazonium fluoroborate, a reversible competitive antagonist in the dark, which may serve as a photoaffinity probe of the area of the receptor molecule with which cholinergic ligands interact. Addition of meproadifen, an allosteric effector that stabilizes the high-affinity desensitized state of the receptor upon binding to a site topographically distinct from the cholinergic ligand-binding domains, caused a major increase in labeling of the alpha-subunit, a smaller increase in the delta-subunit, and decreased labeling in the gamma-subunit, thus revealing changes in the alpha and non-alpha subunits' contribution to cholinergic ligand binding. Also, in agreement with the tighter binding of cholinergic ligands to the desensitized receptor, differential labeling of three peptide loops of the alpha-subunit was detected: while Tyr-190, Cys-192, and Cys-193 were labeled in a roughly identical manner in both resting and desensitized conformations, the labeling of Tyr-93 and Trp-149 increased up to 6-fold in the desensitized state. Tyr-93 and Trp-149 belong to separate regions containing strictly conserved "canonical" amino acids, common to all nicotinic, gamma-aminobutyrate, and glycine receptor subunits. These regions are thus likely to play a critical role in the regulation of ligand-gated ion channels.