Westergard T et al. (2015), Interactions of L-3,5,3'-Triiodothyronine [corr...

XB-ART-51273

PLoS One 2015 Sep 04;109:e0139072. doi: 10.1371/journal.pone.0139072.

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Interactions of L-3,5,3'-Triiodothyronine [corrected], Allopregnanolone, and Ivermectin with the GABAA Receptor: Evidence for Overlapping Intersubunit Binding Modes.

Westergard T , Salari R , Martin JV , Brannigan G .

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Structural mechanisms of modulation of γ-aminobutyric acid (GABA) type A receptors by neurosteroids and hormones remain unclear. The thyroid hormone L-3,5,3'-triiodothyronine (T3) inhibits GABAA receptors at micromolar concentrations and has common features with neurosteroids such as allopregnanolone (ALLOP). Here we use functional experiments on α2β1γ2 GABAA receptors expressed in Xenopus oocytes to detect competitive interactions between T3 and an agonist (ivermectin, IVM) with a crystallographically determined binding site at subunit interfaces in the transmembrane domain of a homologous receptor (glutamate-gated chloride channel, GluCl). T3 and ALLOP also show competitive effects, supporting the presence of both a T3 and ALLOP binding site at one or more subunit interfaces. Molecular dynamics (MD) simulations over 200 ns are used to investigate the dynamics and energetics of T3 in the identified intersubunit sites. In these simulations, T3 molecules occupying all intersubunit sites (with the exception of the α-β interface) display numerous energetically favorable conformations with multiple hydrogen bonding partners, including previously implicated polar/acidic sidechains and a structurally conserved deformation in the M1 backbone.

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Species referenced: Xenopus
Genes referenced: gabarap gnl3

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	Fig 2. Inhibition of GABA response by T3.(A) Dose-response curve for the effects of T3 on the GABA-stimulated current as a percent of the maximal GABA response in the absence of T3. The values are expressed as a mean of three separate determinations, with error bars representing the standard error of the mean (S.E.M.). (A, Inset) Representative tracings for the effect of 10 μM GABA with or without added 10 μM T3. The solid lines above the tracing indicate the time of superfusion of the oocyte with the indicated solutions. (B) Evaluation of T3 inhibition of GABA response. Dose-response curves for the effects of GABA were constructed separately in the presence of 0, 5, 10, or 20 μM T3. The data are represented as means ± S.E.M. for triplicate determinations. For each data point, n = 3–5. (B, Inset) Schild plot of the data from (B). “Dr” stands for dose-ratio. The slope of the line was 0.04 ± 0.02, which was significantly different from unity according to 95% confidence levels (shown in dotted lines).
	Fig 3. Inhibition of IVM response by T3.(A) Dose-response curve for the effects of T3 on the IVM-stimulated current as a percent of the maximal IVM response in the absence of T3. The values are expressed as a mean of three separate determinations ± S.E.M. (A, Inset) Representative tracings for the effect of 10 μM IVM with or without added 10 μM T3. The solid lines above the tracing indicate the time of superfusion of the oocyte with the indicated solutions. (B) Evaluation of T3 inhibition of the IVM response. Dose-response curves for the effects of IVM were constructed separately in the presence of 0, 5, 10, or 20 μM T3. The data are represented as means ± S.E.M. for triplicate determinations. For each data point, n = 3–5. (B, Inset) Schild plot of the data from (B). “Dr” stands for dose-ratio. The slope of the line was 1.2 ± 0.1, which was not significantly different from unity according to 95% confidence intervals, shown in dotted lines.
	Fig 4. Inhibition of ALLOP response by T3.Dose-response curves for the effects of ALLOP were constructed separately in the presence of 0, 5, 10, or 20 μM T3. The data are represented as means ± S.E.M. for triplicate determinations. For each data point, n = 3–5. (Top Inset) Schild plot of the data from Fig 4. “Dr” stands for dose-ratio. The slope of the line was 1.2 ± 0.1, which was not significantly different from unity according to 95% confidence levels (shown in dotted line). (Bottom Inset) Representative tracings for the effect of 10 μM ALLOP with or without added 10 μM T3. The solid lines above the tracing indicate the time of superfusion of the oocyte with the indicated solutions.
	Fig 5. Poses indicated by automated docking of IVM (A), T3 (B), and ALLOP (C) to the GABAA receptor model.Five runs generating twenty poses each were conducted for all three ligands. Poses are colored according to docking score rank within a single run with strong scores in red, intermediate scores in white, and the weakest scores in blue, indicating that although poses for ALLOP and T3 are confined to subunit interfaces, multiple docking runs yield significant dispersion in orientation and ranking of individual interfaces (average scores in S1 Table). Poses located in the ion channel pore were excluded. The GABAA receptor transmembrane domain is shown and is colored by subunit: α-silver, β-green, γ-blue. In (B) and (C) the ligand hydroxyl is shown as a space-filling sphere to indicate orientation. Residues implicated by mutagensis for activation by ALLOP are shown in orange (34) and those photolabeled by etomidate (35) are shown in black.
	Fig 6. Molecular Dynamics Simulations of GABAA receptors with T3 bound.A) GABAA receptor-membrane complex, shown in cartoon with surface overlay, from a view parallel to the membrane. Lipids in the membrane are in licorice with POPC molecules colored by atom name and cholesterol molecules in pink. T3 molecules colored by atom name are shown at the interfaces between the α (silver) and β (green) subunits. B) Initial (gray) and final (orange) poses of T3 from 200 ns MD simulation. C) Representative frame showing interactions between T3 at one of the β-α interfaces, corresponding to the first column of S1–S3 Tables. Numbers shown in parentheses in residue labels represent the contribution of hydrogen bonds with the residue to the total number of hydrogen bonds observed in the second (equilibrated) half of the MD simulation (listed for all interfaces in S3 Table). T3 frequently formed simultaneous hydrogen bonds to αM1 I228 and βM2 S265, as shown here. D) Total energy of T3 molecules across trajectory, relative to that of the β-α interface shown in first column and in Panel C, and in left to right order of increasing average energy/decreasing favorability. Energy trajectories were smoothed with a 20 ns window, with the initial and final 20 ns removed due to distortion from the windowing process. Solid region of the trajectory curve indicates the equilibrated regions used in calculating the average listed in S2 Table, and represented here by the horizontal solid line. Decomposition of the total energies can be found in S2 Table and S4 Fig
	Fig 1. Chemical analogy among modulators and receptors.(A) Comparisons of structures of the neurosteroid ALLOP and thyroid hormone T3. Neuroactive steroids and T3 share common features, including molecular dimensions, placement of hydrogen-bond accepting groups, and multiple rings. (B) The GluCl IVM binding site. Residues identical to those at the GABAA β-α interface are red, similar residues are white, and residues with no similarity are blue.

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