Anionic residue in the α-subunit of the nicotinic acetylcholine receptor contributing to subunit assembly and ligand binding

Abstract

To ascertain the anionic sites on the nicotinic receptor to which acetylcholine and other quaternary ammonium ligands bind, we have examined the role of an aspartyl residue (Asp-152) in the α-subunit. Prior photolytic labeling with agonist analogues of the neighboring residues Trp-149 and Tyr- 151 suggests that their side chains reside on the binding face (also termed the (+)- or counterclockwise face) of the α-subunit. Asp-152 presents an anionic charge in the vicinity of these aromatic residues. Modification of the aspartate to asparagine (D152N) creates a glycosylation signal (Asn-152- Gly-Ser), and we find, on the basis of altered electrophoretic migration, that glycosylation occurs at this position upon cotransfection of the mutant α-subunit with β-, γ-, and δ-subunits. Glycosylation results in a reduction in the capacity of the receptor to assemble; this reduction is manifest in the initial step of dimer formation between the αγ- and αδ- subunits. The α-subunit mutant receptor reaching the assembled pentamer exhibits an altered selectivity for certain ligands. Little reduction in α- bungarotoxin binding is observed, whereas affinities for agonists and competitive alkaloid antagonists are reduced substantially. Separation of the contributions of charge removal and glycosylation addition shows that both factors affect agonist affinity, with the charge influence being far more predominant. These findings raise the possibility that a component of the coulombic attraction stabilizing the binding of agonists comes from the aspartyl residue at position 152 in the α-subunit.