Reversal of agonist selectivity by mutations of conserved amino acids in the binding site of nicotinic acetylcholine receptors

Homomeric alpha 7 and heteromeric alpha 4 beta 2 nicotinic acetylcholine receptors (nAChR) can be distinguished by their pharmacological properties, including agonist specificity. We introduced point mutations of conserved amino acids within the C loop, a region of the receptor critical for agonist binding, and we examined the expression of the mutant receptors in Xenopus oocytes. Mutation of either a conserved C loop tyrosine (188) to phenylalanine or a nearby conserved aspartate (197) to alanine resulted in alpha 7 receptors for which the alpha 7-selective agonist 3-(4-hydroxy,2-methoxybenzylidene)anabaseine (4OH-GTS-21) had roughly the same potency as for wild-type receptors, whereas the physiologic agonist acetylcholine (ACh) showed drastically reduced potency for these mutant receptors. Corresponding mutations in alpha 4 receptors co-expressed with 62 resulted in alpha 4 beta 2 receptors for which ACh potency was relatively unchanged, although the efficacy of the alpha 7-selective agonist 4OH-GTS-21 was increased greatly relative to that of ACh. We also investigated the significance of a conserved lysine (145 in alpha 7), proposed to form a stable salt bridge with Asp-197 in the resting state of the receptor. Mutations of this residue in both alpha 7 and alpha 4 resulted in receptors that were largely unresponsive to both ACh and 4OH-GTS-21. Our results suggest that initiation of gating depends both on specific interactions between residues in the C loop domain and, depending on receptor subtype, the physiochemical properties of the agonist, so that in the altered environment of the alpha 4Y190F-binding site, large hydrophobic benzylidene anabaseines may close the C loop and initiate channel gating more effectively than the polar agonist ACh.