Differentiating Enantioselective Actions of GABOB: A Possible Role for Threonine 244 in the Binding Site of GABAC 蟻1 Receptors

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• 作者：Izumi Yamamoto ; Nathan Absalom ; Jane E. Carland ; Munikumar R. Doddareddy ; Navnath Gavande ; Graham A. R. Johnston ; Jane R. Hanrahan ; Mary Chebib
• 刊名：ACS Chemical Neuroscience
• 出版年：2012
• 出版时间：September 19, 2012
• 年：2012
• 卷：3
• 期：9
• 页码：665-673
• 全文大小：411K
• 年卷期：v.3,no.9(September 19, 2012)
• ISSN：1948-7193

文摘

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 蟻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 蟻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 蟻₁T244 to serine (蟻₁T244S) produced a GABA responsive receptor, albeit 39-fold less sensitive to GABA than 蟻₁wild-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 蟻₁T244S mutant receptors (Muscimol: 蟻₁wild-type, EC₅₀ = 1.4 渭M; 蟻₁T244S, IC₅₀ = 32.8 渭M. I4AA: 蟻₁wild-type, EC₅₀ = 8.6 渭M; 蟻₁T244S, IC₅₀ = 21.4 渭M). This indicates that T244 is predominantly involved in channel gating. R-(鈭?-GABOB and S-(+)-GABOB are full agonists at 蟻₁wild-type receptors. In contrast, R-(鈭?-GABOB was a weak partial agonist at 蟻₁T244S (1 mM activates 26% of the current produced by GABA EC₅₀ versus 蟻₁wild-type, EC₅₀ = 19 渭M; I_max 100%), and S-(+)-GABOB was a competitive antagonist at 蟻₁T244S receptors (蟻₁wild-type, EC₅₀ = 45 渭M versus 蟻₁T244S, IC₅₀ = 417.4 渭M, K_B = 204 渭M). 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-(鈭?-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