2’MeO6MF activity at β3γ2L GABA_ARs.

<p>(A) 2’MeO6MF exhibits a complex spectrum of activity across β3γ2L GABA_ARs expressed at various injection ratios. Representative traces of 3 mM GABA (black) and 100 μM 2’MeO6MF (red) are shown for each ratio. Bars indicate durations of drug application. The holding current values are represented by the dotted lines. (<b>1:1</b>; <i>n</i> = 7), 2’MeO6MF inhibited the constitutive activity of receptors expressed. (<b>1:5</b>; <i>n</i> = 5) and (<b>1:10</b>; <i>n</i> = 10), 2’MeO6MF exhibited mixed agonist and inverse agonist activity. (<b>1:15</b>; <i>n</i> = 30), 2’MeO6MF directly activated receptors expressed efficaciously. (<b>1:20</b>; <i>n</i> = 49), 2’MeO6MF activated β3γ2L (1:20) receptors with variable efficacy. Sample traces of cell 1 and 2 were taken from a simultaneous experiment conducted on two different oocytes injected at the same time. (<b>1:50</b>; <i>n</i> = 15) and (<b>1:100</b>; <i>n</i> = 21), 2’MeO6MF showed activation with low efficacy at these receptors. (B<b>)</b> Mean efficacy of 100 μM 2’MeO6MF direct activation at β3γ2L (1:15), (1:20), (1:50) and (1:100) GABA_ARs. Data are normalised to the 3 mM GABA response. The mean efficacy of 100 μM 2’MeO6MF at various ratios was compared using Tukey’s test, and the significance levels are indicated with n.s. (not significant) and *** (<i>p</i> ≤ 0.001).</p

Characterisation of β3γ2L GABA_ARs expressed at 1:15, 1:50 and 1:100 ratios.

<p>The level of constitutive activity is indicated by (A) holding current of injected oocytes and (B) the inhibition of baseline current by 100 μM Zn²⁺. (A) β3γ2L GABA_ARs expressed at 1:15 ratio showed significantly larger holding current (-260 ± 40 nA; <i>n</i> = 30) than at 1:50 (-28 ± 6.0 nA; <i>n</i> = 15) and 1:100 (-15 ± 5.0 nA; <i>n</i> = 21) ratios (<i>p</i> ≤ 0.001; Tukey’s test). (B<b>)</b> Representative traces demonstrating current responses of 3 mM GABA, 100 μM 2’MeO6MF and 100 μM Zn²⁺. At 1:15 ratio, receptors were sensitive to the inhibition of 100 μM Zn²⁺ (reduction in inward current; <i>n</i> = 10). Zn²⁺ did not have any effects at 1:50 (<i>n</i> = 7) and 1:100 (<i>n</i> = 8) ratios. (C) Representative traces demonstrating 2’MeO6MF’s direct activation from 1 to 300 μM in comparison to 3 mM GABA response. (D) 2’MeO6MF concentration-response curves of β3γ2L (1:15; <i>n</i> = 8), (1:50; <i>n</i> = 7) and (1:100; <i>n</i> = 6) GABA_ARs. Data are normalised to the 3 mM GABA response. (E) Representative traces of GABA current responses from 1 μM to 30 mM at β3γ2L (1:15), (1:50) and (1:100) GABA_ARs. (F) GABA concentration-response curves of β3γ2L (1:15; <i>n</i> = 6), (1:50; <i>n</i> = 8) and (1:100; <i>n</i> = 9) GABA_ARs. Data are presented as mean ± SEM. Bars indicate durations of drug application. The holding current values are represented by the dotted lines.</p

GABA, 2’MeO6MF, etomidate and propofol concentration-response curve parameters at β3γ2L (1:15), (1:50) and (1:100) GABA_ARs derived from curve-fitting procedures.

<p>^<i>a</i> The total amount of mRNA injected per oocyte was adjusted to be approximately 5 ng for each ratio.</p><p>^<i>b</i> Data of 1:15 ratio were better fitted to a monophasic model. Data of 1:50 and 1:100 ratios were significantly better fitter to a biphasic model (<i>p</i> < 0.0001 for both ratios; extra sum-of-squares <i>F</i> test). Concentration-response curves are shown in <a href="http://www.plosone.org/article/info:doi/10.1371/journal.pone.0141359#pone.0141359.g004" target="_blank">Fig 4F</a>.</p><p>^<i>c</i> Current amplitude elicited by 30 mM GABA.</p><p>^<i>d</i> 300 μM 2’MeO6MF-elicited current normalised against 3 mM GABA response.</p><p>^<i>e</i> 30 μM (for 1:15) and 300 μM (for 1:50 and 1:100) etomidate-elicited current normalised against 3 mM GABA response.</p><p>^<i>f</i> 300 μM etomidate-elicited current normalised against 3 mM GABA response.</p><p>^<i>g</i> N.D. = not determined.</p><p>^<i>h</i> 300 μM propofol-elicited current normalised against 3 mM GABA response.</p><p>GABA, 2’MeO6MF, etomidate and propofol concentration-response curve parameters at β3γ2L (1:15), (1:50) and (1:100) GABA_ARs derived from curve-fitting procedures.</p

Characteristics of receptors expressed in various combinations of α2, β3 and γ2L GABA_AR subunits.

<p>^<i>a</i> Expression level is expressed as the current responses ± SEM (nA) elicited by 3 mM GABA, except for homomeric and α2γ2L receptors (60 mM GABA).</p><p>Characteristics of receptors expressed in various combinations of α2, β3 and γ2L GABA_AR subunits.</p

Characterisation of α2β3γ2L GABA_ARs expressed at a 3:1:3 injection ratio.

<p>(A) <i>Left panel</i>, Representative traces of α2β3γ2L (3:1:3) GABA_ARs responses to 3 mM GABA and 100 μM Zn²⁺ alone. <i>Right panel</i>, Mean holding current of oocytes expressing α2β3γ2L (3:1:3) GABA_ARs (-93 ± 19 nA; <i>n</i> = 8). (B) <i>Left panel</i>, Continuous traces demonstrating two consecutive applications of control (100 μM GABA) followed by the co-application of 10 μM Zn²⁺ with control. <i>Right panel</i>, Modulation of 100 μM GABA responses by 10 μM Zn²⁺ (<i>n</i> = 9). (C) <i>Left panel</i>, Continuous traces demonstrating two consecutive applications of control (1 μM GABA) followed by the co-application of 1 μM diazepam with control; 1 μM diazepam and 10 μM flumazenil with control; and control. <i>Right panel</i>, Potentiation by 1 μM diazepam of 1 μM GABA responses (<i>n</i> = 6). Representative traces demonstrating (D) GABA current responses from 1 μM to 10 mM and (E) 2’MeO6MF’s direct activation from 1 to 300 μM (red) in comparison to 3 mM GABA response. Concentration-response curves of GABA (black; <i>n</i> = 6) and 2’MeO6MF (red; <i>n</i> = 5) are shown in (F). Data are presented as mean ± SEM. Bars indicate durations of drug application. The holding current values are represented by the dotted lines.</p

Propofol activates β3γ2L (1:15) and (1:100) receptors with different relative efficacies.

<p>(A) Representative traces of propofol (1–300 μM) direct activation in comparison to 3 mM GABA at β3γ2L (1:15) and (1:100) GABA_ARs. (B) Concentration-response curves of propofol activation at β3γ2L receptors expressed at 1:15 (<i>n</i> = 4) and 1:100 (<i>n</i> = 4) ratios. Data are normalised to the 3 mM GABA response.</p

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

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

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

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