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Interactions of L-3,5,4'-Triiodothyronine, Allopregnanolone, and Ivermectin with the GABAA Receptor: Evidence for Overlapping Intersubunit Binding Modes.

Westergard T, Salari R, Martin JV, Brannigan G - PLoS ONE (2015)

Bottom Line: 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.

View Article: PubMed Central - PubMed

Affiliation: Department of Neuroscience, Thomas Jefferson University, Philadelphia, Pennsylvania, United States of America; Center for Computational and Integrative Biology, Rutgers University-Camden, Camden, New Jersey, United States of America.

ABSTRACT
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.

No MeSH data available.


Related in: MedlinePlus

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).
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pone.0139072.g002: 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).

Mentions: The inhibiting effect of T3 (0.1 μM- 100 μM) on GABAA receptor stimulation by 10 μM GABA is shown in Fig 2A, with a representative trace indicating a significant reduction in the response of GABA in the presence of 10 μM T3 (Fig 2A, inset). Further representative traces are in supplementary information (S2 Fig). Apparent-maximal concentrations of T3 (between 50–100 μM) reduced response to GABA to 60 ± 3% of control, with an IC50 of 8 ± 2 μM (Fig 2A). These results are similar to previously reported findings using a α1β2γ2 construct expressed in Xenopus oocytes [17], indicating low sensitivity of T3 response to β subunit sequence, as also observed in neurosteroid response [38].


Interactions of L-3,5,4'-Triiodothyronine, Allopregnanolone, and Ivermectin with the GABAA Receptor: Evidence for Overlapping Intersubunit Binding Modes.

Westergard T, Salari R, Martin JV, Brannigan G - PLoS ONE (2015)

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).
© Copyright Policy
Related In: Results  -  Collection

License
Show All Figures
getmorefigures.php?uid=PMC4589331&req=5

pone.0139072.g002: 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).
Mentions: The inhibiting effect of T3 (0.1 μM- 100 μM) on GABAA receptor stimulation by 10 μM GABA is shown in Fig 2A, with a representative trace indicating a significant reduction in the response of GABA in the presence of 10 μM T3 (Fig 2A, inset). Further representative traces are in supplementary information (S2 Fig). Apparent-maximal concentrations of T3 (between 50–100 μM) reduced response to GABA to 60 ± 3% of control, with an IC50 of 8 ± 2 μM (Fig 2A). These results are similar to previously reported findings using a α1β2γ2 construct expressed in Xenopus oocytes [17], indicating low sensitivity of T3 response to β subunit sequence, as also observed in neurosteroid response [38].

Bottom Line: 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.

View Article: PubMed Central - PubMed

Affiliation: Department of Neuroscience, Thomas Jefferson University, Philadelphia, Pennsylvania, United States of America; Center for Computational and Integrative Biology, Rutgers University-Camden, Camden, New Jersey, United States of America.

ABSTRACT
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.

No MeSH data available.


Related in: MedlinePlus