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

ABSTRACT: The crystal structure of a pentameric ?7 ligand-binding domain chimaera with bound ?-btx (?-bungarotoxin) showed that of the five conserved aromatic residues in ?7, only Tyr¹?? in loop C of the ligand-binding site was required for high-affinity binding. To determine whether the contribution of Tyr¹?? depends on local residues, we generated mutations in an ?7/5HT(3A) (5-hydroxytryptamine type 3A) receptor chimaera, individually and in pairs, and measured ¹²?I-labelled ?-btx binding. The results show that mutations of individual residues near Tyr¹?? do not affect ?-btx affinity, but pairwise mutations decrease affinity in an energetically coupled manner. Kinetic measurements show that the affinity decreases arise through increases in the ?-btx dissociation rate with little change in the association rate. Replacing loop C in ?7 with loop C from the ?-btx-insensitive ?2 or ?3 subunits abolishes high-affinity ?-btx binding, but preserves acetylcholine-elicited single channel currents. However, in both the ?2 and ?3 construct, mutating either residue that flanks Tyr¹?? to its ?7 counterpart restores high-affinity ?-btx binding. Analogously, in ?7, mutating both residues that flank Tyr¹?? to the ?2 or ?3 counterparts abolishes high-affinity ?-btx binding. Thus interaction between Tyr¹?? and local residues contributes to high-affinity subtype-selective ?-btx binding.

SUBMITTER: Sine SM 

PROVIDER: S-EPMC3912756 | biostudies-literature | 2013 Sep

REPOSITORIES: biostudies-literature