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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.


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.
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pone.0139072.g004: 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.

Mentions: Consistent with its well-known pharmacological effects on GABAA receptors, the neurosteroid ALLOP had a stimulatory effect on the GABAA receptor response in Xenopus oocytes (Fig 4), with an EC50 of 0.9 ± 0.1 μM. For 1 μM ALLOP, this effect was inhibited 24% by the addition of 10 μM T3 in a sample tracing (Fig 4, lower inset). Further representative traces are in supplementary information (S2 Fig). Similar to the effect of T3 on stimulation by IVM, the addition of a constant concentration of T3 to a dose-response curve for ALLOP shifted the curve to the right. The Schild plot (Fig 4, upper inset) yielded a slope of 0.94±0.03, which was not significantly different from 1 according to 95% confidence intervals. The data therefore support the conclusion that T3 is a competitive inhibitor of the stimulatory effect of ALLOP at the GABAA receptor. A pA2 value of -4.7 ± 0.2 log concentration of T3 was calculated (about 20 μM), suggesting a value for the average affinity of T3 for the site(s) of competition. This value was consistent with the estimated affinity from competition between T3 and IVM, indicating that T3 might compete with both IVM and ALLOP via the same or similar sites; however it is not, in isolation, sufficient to conclude that all three sites overlap.


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 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.
© Copyright Policy
Related In: Results  -  Collection

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

pone.0139072.g004: 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.
Mentions: Consistent with its well-known pharmacological effects on GABAA receptors, the neurosteroid ALLOP had a stimulatory effect on the GABAA receptor response in Xenopus oocytes (Fig 4), with an EC50 of 0.9 ± 0.1 μM. For 1 μM ALLOP, this effect was inhibited 24% by the addition of 10 μM T3 in a sample tracing (Fig 4, lower inset). Further representative traces are in supplementary information (S2 Fig). Similar to the effect of T3 on stimulation by IVM, the addition of a constant concentration of T3 to a dose-response curve for ALLOP shifted the curve to the right. The Schild plot (Fig 4, upper inset) yielded a slope of 0.94±0.03, which was not significantly different from 1 according to 95% confidence intervals. The data therefore support the conclusion that T3 is a competitive inhibitor of the stimulatory effect of ALLOP at the GABAA receptor. A pA2 value of -4.7 ± 0.2 log concentration of T3 was calculated (about 20 μM), suggesting a value for the average affinity of T3 for the site(s) of competition. This value was consistent with the estimated affinity from competition between T3 and IVM, indicating that T3 might compete with both IVM and ALLOP via the same or similar sites; however it is not, in isolation, sufficient to conclude that all three sites overlap.

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.