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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) (α7 Tyr188 or α4 Tyr195) differentially regulates the activity of acetylcholine (ACh) and the α7-selective agonist 3-(4-hydroxy,2-methoxybenzylidene)anabaseine (4OH-GTS-21) in α4β2 and α7 nAChR. In this study, we mutated two highly conserved LBD Trp residues in human α7 and α4β2 and expressed the receptors in Xenopus laevis oocytes. α7 Receptors with Trp55 mutated to Gly or Tyr became less responsive to 4OH-GTS-21, whereas mutation of the homologous Trp57 in β2 to Gly, Tyr, Phe, or Ala resulted in α4β2 receptors that showed increased responses to 4OH-GTS-21. Mutation of α7 Trp55 to Val resulted in receptors for which the partial agonist 4OH-GTS-21 became equally efficacious as ACh, whereas α4β2 receptors with the homologous mutation remained nonresponsive to 4OH-GTS-21. In contrast to the striking alterations in agonist activity profiles that were observed with mutations of α7 Trp55 and β2 Trp57, mutations of α7 Trp149 or α4 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 α7 Trp149 (α4 Trp154) may be considered pillars upon which basic receptor function depends, whereas α7 Trp55 (β2 Trp57) and α7 Tyr188 (α4 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.