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NEUROPHARMACOLOGY

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

Department of Pharmacology and Therapeutics, University of Florida College of Medicine, Gainesville, Florida (D.K.W., C.S., R.L.P.); and Department of Chemistry, University of Florida, Gainesville, Florida (N.A.H.)

We have shown previously that a highly conserved Tyr in the nicotinic acetylcholine receptor (nAChR) ligand-binding domain (LBD) (7 Tyr188 or 4 Tyr195) differentially regulates the activity of acetylcholine (ACh) and the 7-selective agonist 3-(4-hydroxy,2-methoxybenzylidene)anabaseine (4OH-GTS-21) in 4β2 and 7 nAChR. In this study, we mutated two highly conserved LBD Trp residues in human 7 and 4β2 and expressed the receptors in Xenopus laevis oocytes. 7 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 4β2 receptors that showed increased responses to 4OH-GTS-21. Mutation of 7 Trp55 to Val resulted in receptors for which the partial agonist 4OH-GTS-21 became equally efficacious as ACh, whereas 4β2 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 7 Trp55 and β2 Trp57, mutations of 7 Trp149 or 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 7 Trp149 (4 Trp154) may be considered pillars upon which basic receptor function depends, whereas 7 Trp55 (β2 Trp57) and 7 Tyr188 (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.

Address correspondence to: Roger L. Papke, Department of Pharmacology and Therapeutics, University of Florida, P.O. Box 100267, Gainesville, FL 32610-0267. E-mail: rlpapke{at}ufl.edu