Sine_2013_Biochem.J_454_311

The crystal structure of a pentameric alpha7 ligand-binding domain chimaera with bound alpha-btx (alpha-bungarotoxin) showed that of the five conserved aromatic residues in alpha7, only Tyr184 in loop C of the ligand-binding site was required for high-affinity binding. To determine whether the contribution of Tyr184 depends on local residues, we generated mutations in an alpha7/5HT3A (5-hydroxytryptamine type 3A) receptor chimaera, individually and in pairs, and measured 125I-labelled alpha-btx binding. The results show that mutations of individual residues near Tyr184 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 alpha7 with loop C from the alpha-btx-insensitive alpha2 or alpha3 subunits abolishes high-affinity alpha-btx binding, but preserves acetylcholine-elicited single channel currents. However, in both the alpha2 and alpha3 construct, mutating either residue that flanks Tyr184 to its alpha7 counterpart restores high-affinity alpha-btx binding. Analogously, in alpha7, mutating both residues that flank Tyr184 to the alpha2 or alpha3 counterparts abolishes high-affinity alpha-btx binding. Thus interaction between Tyr184 and local residues contributes to high-affinity subtype-selective alpha-btx binding.