Naffaa MM et al. (2016), Investigating the Role of Loop C Hydrophilic Re...

XB-ART-53851

PLoS One 2016 Jan 01;115:e0156618. doi: 10.1371/journal.pone.0156618.

Show Gene links Show Anatomy links

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

Naffaa MM , Absalom N , Solomon VR , Chebib M , Hibbs DE , Hanrahan JR .

???displayArticle.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.

???displayArticle.pubmedLink??? 27244450
???displayArticle.pmcLink??? PMC4887073
???displayArticle.link??? PLoS One

Species referenced: Xenopus laevis
Genes referenced: aopep crygdl2.2

???attribute.lit??? ???displayArticles.show???

	Fig 1. Chemical structures of ligands used in this study.
	Fig 2. Effect of GABA at mutant receptors.A. GABA and surrounding residues in the orthosteric binding site of the ρ1 GABAC homology model. B. Concentration response curves of GABA at ρ1 GABAC WT, ρ1 T244S, ρ1 T244A and ρ1 T244C receptors, (Data = Mean ± SEM, n = 5). Note: GABA elicited sub-maximal efficacy compared to β-alanine and MTSEA at ρ1 T244A and ρ1 T244C mutant receptors, respectively.
	Fig 3. Effect of conformational restriction on the efficacy of ligands at GABA receptors.Concentration response curves of GABA and the unsaturated analogues, TACA and CACA at (A) ρ1 WT and (B) ρ1 T244S receptors, (Data = Mean ± SEM, n = 5). (C) Sample traces of the maximal responses of GABA and TACA at ρ1 WT and ρ1 T244S receptors.
	Fig 4. Characterisation of response and binding of GABA and TACA at ρ1 WT and ρ1 T244S receptors.(A) GABA, TACA and CACA docked in the orthosteric binding site of ρ1 GABAC homology model based on GluCl in open conformation. (B) Poses of GABA and TACA in their binding conformations showing distances between the two poles. (C) Various contacts of the side chain of GABA with the side chain of serine at Thr244 site. (D) Various contacts of the side chain of TACA with the side chain of serine at Thr244 site. The rotamer of serine shown in D and E has similar Chi1 and Chi2 (i.e. first and second dihedral angles of the side chain) values to the predicted conformation of Thr244 at this site (i.e. Chi = 91 and Chi2 = –179).
	Fig 5. Enhancement of the GABA EC50 response by CACA at ρ1 WT and ρ1 T244S receptors.(A) Concentration response curve of the co-application of increasing concentrations of CACA in the presence of GABA EC50 at ρ1 WT and ρ1 T244S receptors, (Data = Mean ± SEM, n = 5). Sample traces of CACA co-applied with GABA EC50 at ρ1GABAC (B) WT and (C) ρ1 T244S receptors.
	Fig 6. Activity of GABA, glycine, β-alanine and 5-aminovaleric acid at ρ1 WT and ρ1 T244S receptors.Concentration response curves of GABA, glycine, β-alanine and 5-aminovaleric acid at ρ1 WT (A) and ρ1 T244S (B) receptors, (Data = Mean ± SEM, n = 5).
	Fig 7. Effect of glycine, β-alanine and 5-aminovaleric acid on the GABA EC50 response.Concentration response curves of GABA EC50 in the presence of (A) glycine, (B) β-alanine and (C) 5-aminovaleric acid at ρ1 WT and ρ1 T244S receptors, (Data = Mean ± SEM, n = 5).
	Fig 8. 5-Aminovaleric acid fits in the ρ1 GABAC orthosteric binding site in a folded conformation.GABA (white) and 5-aminovaleric acid (red) docked in the orthosteric binding site of ρ1 GABAC homology model based on GluCl in apo state.
	Fig 9. Characterisation of the effect of isoguvacine at ρ1 T244S mutant receptors.(A) Concentration response curves of GABA and isoguvacine at ρ1 WT receptors, (Data = Mean ± SEM, n = 5). (B) Concentration response curves of the co-application of isoguvacine with GABA EC50 at ρ1 GABAC WT and ρ1 T244S receptors, (Data = Mean ± SEM, n = 5). Sample response traces of isoguvacine when co-applied with GABA EC50 at (C) ρ1 GABAC WT receptors and (D) at ρ1 T244S receptors.
	Fig 10. Docking studies of isoguvacine in the orthosteric binding site of ρ1 GABAC homology model.(A) H-bonds and (B) hydrophobic interactions predicted to be formed by isoguvacine and the ρ1 GABAC receptor.
	Fig 11. β-alanine is more potent than GABA at ρ1 T244A receptors.Concentration response curves of β-alanine and GABA at ρ1 T244A mutant receptors, (Data = Mean ± SEM, n = 15). (B) Sample response traces of β-alanine and GABA at ρ1 T244A mutant receptors. (C) Sample response traces of β-alanine, GABA and TPMPA at ρ1 T244A receptors.
	Fig 12. Docking studies showing interactions between ligands and ρ1 receptors.(A) GABA and β-alanine docking studies in the orthosteric binding site of GABAC homology model based on GluCl in open conformation. (Left) hydrogen bonds between the side chains of GABA and β-alanine with side chains of Thr244 and Glu196 residues. (Right) various hydrophobic interactions between the side chains of GABA and β-alanine with side chains of Thr244 and Glu196. (B) GABA and β-alanine docking studies in the orthosteric binding site of GABAC homology model based on GluCl in open conformation. (Left) hydrophobic interactions between GABA side chain and alanine residues at the site of Thr244. (Right) hydrophobic interactions between β-alanine side chain and alanine residue at the site of Thr244. (C) TPMPA docking studies in the orthosteric binding site of GABAC homology model based on GluCl in apo conformation. (Left) hydrophobic interactions between the side chain of TPMPA and the side chain of Thr244. (Middle) hydrophobic interactions between the side chain of TPMPA and the side chain of serine residue at the site of Thr244. (Right) hydrophobic interactions between the side chain of TPMPA and the side chain of alanine residue at the site of Thr244.
	Fig 13. MTSEA produces a reversible response at ρ1 T244C receptors.(A) Concentration response curves of MTSEA and GABA at ρ1 T244C mutant receptors, (Data = Mean ± SEM, n = 15). (B) Sample response traces of MTSEA and GABA at ρ1 WT receptors. (C) Sample response traces of MTSEA at ρ1 T244C receptors.
	Fig 14. Docking studies of MSTEA and GABA (A) in the WT ρ1 GABAC homology model based on GluCl in open conformation. (B) Docking studies of GABA (white) and MTSEA (yellow) with ρ1 T244C homology model based on GluCl where thiol group of Cys244 is oriented away from the binding site (Chi1 = -64°).
	Fig 15. Docking of the antagonist in the ρ1 GABAC homology model based on GluCl in apo state.Multiple hydrophobic interactions are formed between TPMPA and the loop C residues Tyr241 and Tyr247.

References [+] :

Albuquerque, Mammalian nicotinic acetylcholine receptors: from structure to function. 2009, Pubmed