Differential Regulation of Receptor Activation and Agonist Selectivity by Highly Conserved Tryptophans in the Nicotinic Acetylcholine Receptor Binding Site

We have shown previously that a highly conserved Tyr in the nicotinic acetylcholine receptor (nAChR) ligand-binding domain (LBD) (alpha 7 Tyr188 or alpha 4 Tyr195) differentially regulates the activity of acetylcholine (ACh) and the alpha 7-selective agonist 3-(4-hydroxy, 2-methoxybenzylidene) anabaseine (4OH-GTS-21) in alpha 4 beta 2 and alpha 7 nAChR. In this study, we mutated two highly conserved LBD Trp residues in human alpha 7 and alpha 4 beta 2 and expressed the receptors in Xenopus laevis oocytes. alpha 7 Receptors with Trp55 mutated to Gly or Tyr became less responsive to 4OH-GTS-21, whereas mutation of the homologous Trp57 in beta 2 to Gly, Tyr, Phe, or Ala resulted in alpha 4 beta 2 receptors that showed increased responses to 4OH-GTS-21. Mutation of alpha 7 Trp55 to Val resulted in receptors for which the partial agonist 4OH-GTS-21 became equally efficacious as ACh, whereas alpha 4 beta 2 receptors with the homologous mutation remained non-responsive to 4OH-GTS-21. In contrast to the striking alterations in agonist activity profiles that were observed with mutations of alpha 7 Trp55 and beta 2 Trp57, mutations of alpha 7 Trp149 or alpha 4 Trp154 universally resulted in receptors with reduced function. Our data support the hypothesis that some conserved residues in the nAChR LBD differentially regulate receptor activation by subtype-selective agonists, whereas other equally well conserved residues play fundamental roles in receptor activation by any agonist. Residues like alpha 7 Trp149 (alpha 4 Trp154) may be considered pillars upon which basic receptor function depends, whereas alpha 7 Trp55 (beta 2 Trp57) and alpha 7 Tyr188 (alpha 4 Tyr195) may be fulcra upon which agonists may operate differentially in specific receptor subtypes, consistent with the hypothesis that ACh and 4OH-GTS-21 are able to activate nAChR in distinct ways.