title: The activity of GAT107, an allosteric activator and positive modulator of α7 nicotinic acetylcholine receptors (nAChR), is regulated by aromatic amino acids that span the subunit interface.
abstract: GAT107, the (+)-enantiomer of racemic 4-(4-bromophenyl)-3a,4,5,9b-tetrahydro-3H-cyclopenta[c]quinoline-8-sulfonamide, is a strong positive allosteric modulator (PAM) of α7 nicotinic acetylcholine receptor (nAChR) activation by orthosteric agonists with intrinsic allosteric agonist activities. The direct activation produced by GAT107 in electrophysiological studies is observed only as long as GAT107 is freely diffusible in solution, although the potentiating activity primed by GAT107 can persist for over 30 min after drug washout. Direct activation is sensitive to α7 nAChR antagonist methyllycaconitine, although the primed potentiation is not. The data are consistent with GAT107 activity arising from two different sites. We show that the coupling between PAMs and the binding of orthosteric ligands requires tryptophan 55 (Trp-55), which is located at the subunit interface on the complementary surface of the orthosteric binding site. Mutations of Trp-55 increase the direct activation produced by GAT107 and reduce or prevent the synergy between allosteric and orthosteric binding sites, so that these mutants can also be directly activated by other PAMs such as PNU-120596 and TQS, which do not activate wild-type α7 in the absence of orthosteric agonists. We identify Tyr-93 as an essential element for orthosteric activation, because Y93C mutants are insensitive to orthosteric agonists but respond to GAT107. Our data show that both orthosteric and allosteric activation of α7 nAChR require cooperative activity at the interface between the subunits in the extracellular domain. These cooperative effects rely on key aromatic residues, and although mutations of Trp-55 reduce the restraints placed on the requirement for orthosteric agonists, Tyr-93 can conduct both orthosteric activation and desensitization among the subunits.
authors.affiliation: From the Department of Pharmacology and Therapeutics, University of Florida College of Medicine, Gainesville, Florida 32610.
authors.forename: Roger L
authors.initials: RL
authors.lastname: Papke
authors.forename: Nicole A
authors.initials: NA
authors.lastname: Horenstein
authors.forename: Abhijit R
authors.initials: AR
authors.lastname: Kulkarni
authors.forename: Clare
authors.initials: C
authors.lastname: Stokes
authors.forename: Lu W
authors.initials: LW
authors.lastname: Corrie
authors.forename: Cheol-Young
authors.initials: CY
authors.lastname: Maeng
authors.forename: Ganesh A
authors.initials: GA
authors.lastname: Thakur
mesh_terms: D000494:Allosteric Regulation / Q000187:drug effects; D019943:Amino Acid Substitution; D024322:Amino Acids, Aromatic / Q000737:chemistry / Q000235:genetics / Q000378:metabolism; D000818:Animals; D004789:Enzyme Activation / Q000187:drug effects; D020536:Enzyme Activators / Q000737:chemistry / Q000494:pharmacology*; D005260:Female; D006801:Humans; D020125:Mutation, Missense; D017434:Protein Structure, Tertiary; D011804:Quinolines / Q000737:chemistry / Q000494:pharmacology*; D013449:Sulfonamides / Q000737:chemistry / Q000494:pharmacology*; D014982:Xenopus laevis; D064569:alpha7 Nicotinic Acetylcholine Receptor / Q000037:antagonists & inhibitors* / Q000737:chemistry / Q000235:genetics / Q000378:metabolism*
chemical_list: C578201:4-(4-bromophenyl)-3a,4,5,9b-tetrahydro-3H-cyclopenta(c)quinoline-8-sulfonamide; D024322:Amino Acids, Aromatic; D020536:Enzyme Activators; D011804:Quinolines; D013449:Sulfonamides; D064569:alpha7 Nicotinic Acetylcholine Receptor
keywords: Allosteric Regulation; Electrophysiology; Mutagenesis Site-specific; Nicotinic Acetylcholine Receptors; Structural Biology; Voltage Clamp