Characterisation of response and binding of GABA and TACA at ρ1 WT and ρ1 T244S receptors.

<p>(A) GABA, TACA and CACA docked in the orthosteric binding site of ρ1 GABA_C 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>i</i>.<i>e</i>. Chi = 91 and Chi2 = –179).</p

Docking of the antagonist in the ρ1 GABA_C homology model based on GluCl in <i>apo</i> state.

<p>Multiple hydrophobic interactions are formed between TPMPA and the loop C residues Tyr241 and Tyr247.</p

Docking studies of isoguvacine in the orthosteric binding site of ρ1 GABA_C homology model.

<p>(A) H-bonds and (B) hydrophobic interactions predicted to be formed by isoguvacine and the ρ1 GABA_C receptor.</p

Activity of GABA, glycine, β-alanine and 5-aminovaleric acid at ρ1 WT and ρ1 T244S receptors.

<p>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).</p

Effect of GABA at mutant receptors.

<p>A. GABA and surrounding residues in the orthosteric binding site of the ρ1 GABA_C homology model. B. Concentration response curves of GABA at ρ1 GABA_C 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.</p

MTSEA produces a reversible response at ρ1 T244C receptors.

<p>(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.</p

Effect of conformational restriction on the efficacy of ligands at GABA receptors.

<p>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.</p

β-alanine is more potent than GABA at ρ1 T244A receptors.

<p>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.</p

Enhancement of the GABA EC₅₀ response by CACA at ρ1 WT and ρ1 T244S receptors.

<p>(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 ρ1GABA_C (B) WT and (C) ρ1 T244S receptors.</p

Docking studies showing interactions between ligands and ρ1 receptors.

<p>(A) GABA and β-alanine docking studies in the orthosteric binding site of GABA_C 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 GABA_C 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 GABA_C homology model based on GluCl in <i>apo</i> 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.</p