title: Reversal of agonist selectivity by mutations of conserved amino acids in the binding site of nicotinic acetylcholine receptors.
abstract: Homomeric alpha7 and heteromeric alpha4beta2 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 alpha7 receptors for which the alpha7-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 alpha4 receptors co-expressed with beta2 resulted in alpha4beta2 receptors for which ACh potency was relatively unchanged, although the efficacy of the alpha7-selective agonist 4OH-GTS-21 was increased greatly relative to that of ACh. We also investigated the significance of a conserved lysine (145 in alpha7), proposed to form a stable salt bridge with Asp-197 in the resting state of the receptor. Mutations of this residue in both alpha7 and alpha4 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 alpha4Y190F-binding site, large hydrophobic benzylidene anabaseines may close the C loop and initiate channel gating more effectively than the polar agonist ACh.
authors.affiliation: Department of Pharmacology and Therapeutics, University of Florida College of Medicine, Gainesville, Florida 32610, USA.
authors.forename: Nicole A
authors.initials: NA
authors.lastname: Horenstein
authors.forename: Thomas J
authors.initials: TJ
authors.lastname: McCormack
authors.forename: Clare
authors.initials: C
authors.lastname: Stokes
authors.forename: Ke
authors.initials: K
authors.lastname: Ren
authors.forename: Roger L
authors.initials: RL
authors.lastname: Papke
mesh_terms: D000691:Anabasine / Q000031:analogs & derivatives* / Q000494:pharmacology; D000818:Animals; D001597:Benzylidene Compounds / Q000494:pharmacology*; D006801:Humans; D015640:Ion Channel Gating / Q000187:drug effects / Q000235:genetics; D020125:Mutation, Missense; D018722:Nicotinic Agonists / Q000494:pharmacology*; D011485:Protein Binding; D017433:Protein Structure, Secondary / Q000235:genetics; D017434:Protein Structure, Tertiary / Q000235:genetics; D011978:Receptors, Nicotinic / Q000737:chemistry / Q000235:genetics / Q000378:metabolism*; D014982:Xenopus laevis; D064569:alpha7 Nicotinic Acetylcholine Receptor
chemical_list: C116666:3-(4-hydroxy-2-methoxybenzylidene)anabaseine; D001597:Benzylidene Compounds; C579167:Chrna7 protein, human; D018722:Nicotinic Agonists; D011978:Receptors, Nicotinic; D064569:alpha7 Nicotinic Acetylcholine Receptor; C403531:nicotinic receptor alpha4beta2; D000691:Anabasine