Inter-residue coupling contributes to high-affinity subtype-selective binding of α-bungarotoxin to nicotinic receptors

The crystal structure of a pentameric alpha 7 ligand-binding domain chimaera with bound alpha-btx (alpha-bungarotoxin) showed that of the five conserved aromatic residues in alpha 7, only Tyr(184) in loop C of the ligand-binding site was required for high-affinity binding. To determine whether the contribution of Tyr(184) depends on local residues, we generated mutations in an alpha 7/5HT(3A) (5-hydroxytryptamine type 3A) receptor chimaera, individually and in pairs, and measured I-125-labelled alpha-btx binding. The results show that mutations of individual residues near Tyr(184) do not affect alpha-btx affinity, but pairwise mutations decrease affinity in an energetically coupled manner. Kinetic measurements show that the affinity decreases arise through increases in the alpha-btx dissociation rate with little change in the association rate. Replacing loop C in alpha 7 with loop C from the alpha-btx-insensitive alpha 2 or alpha 3 subunits abolishes high-affinity alpha-btx binding, but preserves acetylcholine-elicited single channel currents. However, in both the alpha 2 and alpha 3 construct, mutating either residue that flanks Tyr(184) to its alpha 7 counterpart restores high-affinity alpha-btx binding. Analogously, in alpha 7, mutating both residues that flank Tyr(184) to the alpha 2 or alpha 3 counterparts abolishes high-affinity a-btx binding. Thus interaction between Tyr(184) and local residues contributes to high-affinity subtype-selective alpha-btx binding.