Differentiating Enantioselective Actions of GABOB: A Possible Role for Threonine 244 in the Binding Site of GABA_C ρ₁ 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 sitedirected 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 wildtype, 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. KEYWORDS: T244, channel gating, enantioselective actions of GABOB, loop C, coupling agonist binding, channel gating 4-Amino-3-hydroxybutanoic acid (GABOB) is an endogenous molecule found within the CNS, possessing anticonvulsant properties.1 It is formed by two metabolic pathways: either by the metabolism of putrescine to γ-aminobutyric acid (GABA) and then hydroxylation of GABA at the third carbon (C3), 2,3 or by the hydroxylation of putrescine to 2-hydroxyputrescine and then oxidative N-dealkylation to GABOB.3 There are conflicting results concerning the concentration of GABOB in the CNS, with concentration ranging between <0.01 and 4.8 μmol/g for rat and bovine brain, respectively. H...

A molecular characterization of the agonist binding site of a nematode cys‐loop GABA receptor

“…Previous research on GABA A and GABA C receptors found that a conserved threonine at positions 202 and 244, respectively, in binding loop C plays a key role in the activation of the channel by GABA where a change to a serine causes a significant change in GABA sensitivity (Amin and Weiss, 1993; 1994). Recently, a T244S mutation in the GABA C receptor was found to have a more dramatic effect on molecules such as IMA, R (−)‐GABOB and S (+)‐GABOB, nearly eliminating sensitivity or causing them to shift from agonist to antagonist (Yamamoto et al ., ). Interestingly, in the analogous position (215) of the UNC‐49B subunit, the naturally occurring residue is a serine (Figure A).…”

“…Previous research has shown that a threonine residue in loop C is important for the ability of several agonists to activate the GABA C receptor (Yamamoto et al ., ). T244S mutagenesis yielded a functional receptor but with a significant increase in the EC 50 of GABA compared with wild type receptors.…”

“…T244S mutagenesis yielded a functional receptor but with a significant increase in the EC 50 of GABA compared with wild type receptors. R (−)‐GABOB failed to achieve maximal current gating at 1 mM and S (+)‐GABOB displayed antagonist properties (Yamamoto et al ., ). Interestingly, Hco‐UNC‐49 has a serine in the analogous position (S215).…”

“…However, this mutation did result in a moderate twofold increase in the efficacy of both enantiomers of GABOB and significantly enhanced the maximal response of the partial agonist DAVA. These results are consistent with other studies suggesting that this position is important for channel gating (Amin and Weiss, ; Yamamoto et al ., ). It is possible that in Hco‐UNC‐49, the replacement of serine to a threonine at position 215 is somehow changing the position of DAVA allowing a more favourable interaction with key residues.…”

GABA‐ρ receptors: distinctive functions and molecular pharmacology

“…Mutation of the ρ1 Thr 244 residue in loop C, which has its hydroxyl group oriented toward the GABA binding site (Figure A), found that only the T244S mutation resulted in functional receptors, which were 35‐fold less sensitive to GABA (Amin and Weiss, ). A range of agonists studied at the T244S mutant receptor demonstrated many‐fold decreases in potency, while antagonist activity remained unaffected by the mutation (Yamamoto et al, ). Thr 244 is proposed to be essential for the formation of an H‐bond with agonists initiating conformational changes through movement of Loop C to open the channel (Naffaa et al, ).…”