Epibatidine binds with unique site and state selectivity to muscle nicotinic acetylcholine receptors

Ligand binding sites in fetal (α₂βγδ) and adult (α₂βδε) muscle acetylcholine receptors are formed by αδ, αγ, or αε subunit pairs. Each type of binding site shows unique ligand selectivity due to different contributions by the δ, γ, or ε subunits. The present study compares epibatidine and carbamylcholine binding in terms of their site and state selectivities for muscle receptors expressed in human embryonic kidney 293 cells. Measurements of binding to αγ, αδ, and αε intracellular complexes reveal opposite site selectivities between epibatidine and carbamylcholine; for epibatidine the rank order of affinities is αε > αγ > αδ, whereas for carbamylcholine the rank order is αδ αε ≃ > αγ. Because the relative affinities of intracellular complexes resemble those of receptors in the closed activable state, the results suggest that epibatidine binds with unique site selectivity in activating the muscle receptor. Measurements of binding at equilibrium show that both enantiomers of epibatidine bind to adult and fetal receptors with shallow but monophasic binding curves. However, when receptors are fully desensitized, epibatidine binds in a biphasic manner, with dissociation constants of the two components differing by more than 170-fold. Studies of subunit-omitted receptors (α₂βδ₂, α₂βγ₂, and α₂βε₂) reveal that in the desensitized state, the αδ interface forms the low affinity epibatidine site, whereas the αγ and αε interfaces form high affinity sites. In contrast to epibatidine, carbamylcholine shows little site selectivity for desensitized fetal or adult receptors. Thus epibatidine is a potentially valuable probe of acetylcholine receptor binding site structure and of elements that confer state-dependent selectivities of the binding the sites.