Investigating the Role of Loop C Hydrophilic Residue 'T244' in the Binding Site of ρ1 GABAC Receptors via Site Mutation and Partial Agonism.

Moawiah M Naffaa,Nathan Absalom,Jane R Hanrahan,Mary Chebib,V Raja Solomon,David E Hibbs,Elena Papaleo

doi:10.1371/journal.pone.0156618

Abstract

The loop C hydrophilic residue, threonine 244 lines the orthosteric binding site of ρ1 GABAC receptors was studied by point mutation into serine, alanine and cysteine, and tested with GABA, some representative partial agonists and antagonists. Thr244 has a hydroxyl group essential for GABA activity that is constrained by the threonine methyl group, orienting it toward the binding site. Significant decreases in activation effects of the studied ligands at ρ1 T244S mutant receptors, suggests a critical role for this residue. Results of aliphatic and heteroaromatic partial agonists demonstrate different pharmacological effects at ρ1 T244S mutant receptors when co-applied with GABA EC50 responses. ρ1 T244A and ρ1 T244C mutant receptors have minimal sensitivity to GABA at high mM concentrations, whereas, the ρ1 WT partial agonists, β-alanine and MTSEA demonstrate more efficacy and potency, respectively, than GABA at these mutant receptors. This study explores the role of Thr244 in the binding of agonists as an initial step during channel gating by moving loop C towards the ligand.

Highlights

The γ-aminobutyric acid type C (GABAC) receptors, known as γ-aminobutyric acid type A rho (ρ GABAA) receptors are ligand gated ion channel receptors that activated by GABA, the most inhibitory neurotransmitter substance in the vertebrate CNS [1]
The moderate decrease in GABA potency that occurs at ρ1 T244S mutant receptors suggests the importance of the threonine methyl group in restricting the position of the hydroxyl group doi:10.1371/journal.pone.0156618.g002
When the partial agonists imidazole-4-acetic acid and muscimol were tested at ρ1 T244S mutant receptors, their agonist effects were eliminated by the mutation and they acted only as competitive antagonists

Summary

Introduction

The γ-aminobutyric acid type C (GABAC) receptors, known as γ-aminobutyric acid type A rho (ρ GABAA) receptors are ligand gated ion channel receptors that activated by GABA, the most inhibitory neurotransmitter substance in the vertebrate CNS (central nervous system) [1]. The least efficacious compound at ρ1 WT receptors, 5-aminovaleric acid showed a large decrease in potency, shifting the curve to the right, the ligand was still able to weakly activate the mutant receptors at very high concentrations (6000μM, 3% efficacy relative to the GABA maximal response at 30 mM) (Fig 6B).

Results

Conclusion