title: Differentiating enantioselective actions of GABOB: a possible role for threonine 244 in the binding site of GABA(C) ρ(1) receptors.
abstract: Designing potent and subtype-selective ligands with therapeutic value requires knowledge about how endogenous ligands interact with their binding site. 4-Amino-3-hydroxybutanoic acid (GABOB) is an endogenous ligand found in the central nervous system in mammals. It is a metabolic product of GABA, the major inhibitory neurotransmitter. Homology modeling of the GABA(C) ρ(1) receptor revealed a potential H-bond interaction between the hydroxyl group of GABOB and threonine 244 (T244) located on loop C of the ligand binding site of the ρ(1) subunit. Using site-directed mutagenesis, we examined the effect of mutating T244 on the efficacy and pharmacology of GABOB and various ligands. It was found that mutating T244 to amino acids that lacked a hydroxyl group in their side chains produced GABA insensitive receptors. Only by mutating ρ(1)T244 to serine (ρ(1)T244S) produced a GABA responsive receptor, albeit 39-fold less sensitive to GABA than ρ(1)wild-type. We also observed changes in the activities of the GABA(C) receptor partial agonists, muscimol and imidazole-4-acetic acid (I4AA). At the concentrations we tested, the partial agonists antagonized GABA-induced currents at ρ(1)T244S mutant receptors (Muscimol: ρ(1)wild-type, EC(50) = 1.4 μM; ρ(1)T244S, IC(50) = 32.8 μM. I4AA: ρ(1)wild-type, EC(50) = 8.6 μM; ρ(1)T244S, IC(50) = 21.4 μM). This indicates that T244 is predominantly involved in channel gating. R-(-)-GABOB and S-(+)-GABOB are full agonists at ρ(1)wild-type receptors. In contrast, R-(-)-GABOB was a weak partial agonist at ρ(1)T244S (1 mM activates 26% of the current produced by GABA EC(50) versus ρ(1)wild-type, EC(50) = 19 μM; I(max) 100%), and S-(+)-GABOB was a competitive antagonist at ρ(1)T244S receptors (ρ(1)wild-type, EC(50) = 45 μM versus ρ(1)T244S, IC(50) = 417.4 μM, K(B) = 204 μM). This highlights that the interaction of GABOB with T244 is enantioselective. In contrast, the potencies of a range of antagonists tested, 3-aminopropyl(methyl)phosphinic acid (3-APMPA), 3-aminopropylphosphonic acid (3-APA), S- and R-(3-amino-2-hydroxypropyl)methylphosphinic acid (S-(-)-CGP44532 and R-(+)-CGP44533), were not altered. This suggests that T244 is not critical for antagonist binding. Receptor gating is dynamic, and this study highlights the role of loop C in agonist-evoked receptor activation, coupling agonist binding to channel gating.
authors.affiliation: Faculty of Pharmacy, The University of Sydney, Sydney, NSW 2006, Australia.
authors.forename: Izumi
authors.initials: I
authors.lastname: Yamamoto
authors.forename: Nathan
authors.initials: N
authors.lastname: Absalom
authors.forename: Jane E
authors.initials: JE
authors.lastname: Carland
authors.forename: Munikumar R
authors.initials: MR
authors.lastname: Doddareddy
authors.forename: Navnath
authors.initials: N
authors.lastname: Gavande
authors.forename: Graham A R
authors.initials: GA
authors.lastname: Johnston
authors.forename: Jane R
authors.initials: JR
authors.lastname: Hanrahan
authors.forename: Mary
authors.initials: M
authors.lastname: Chebib
mesh_terms: D000818:Animals; D001665:Binding Sites; D003627:Data Interpretation, Statistical; D004305:Dose-Response Relationship, Drug; D015195:Drug Design; D055724:Electrophysiological Phenomena; D018755:GABA Agonists / Q000737:chemistry / Q000494:pharmacology*; D018756:GABA Antagonists / Q000737:chemistry / Q000494:pharmacology; D006801:Humans; D008024:Ligands; D008195:Lymnaea; D008958:Models, Molecular; D016297:Mutagenesis, Site-Directed; D009865:Oocytes / Q000378:metabolism; D018408:Patch-Clamp Techniques; D018080:Receptors, GABA-B / Q000187:drug effects*; D013237:Stereoisomerism; D013912:Threonine / Q000737:chemistry* / Q000235:genetics; D014982:Xenopus laevis; D005680:gamma-Aminobutyric Acid / Q000031:analogs & derivatives* / Q000737:chemistry / Q000494:pharmacology
chemical_list: D018755:GABA Agonists; D018756:GABA Antagonists; D008024:Ligands; D018080:Receptors, GABA-B; C004480:4-amino-3-hydroxybutyric acid; D013912:Threonine; D005680:gamma-Aminobutyric Acid