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Functional characterization of ivermectin binding sites in α1β2γ2L GABA(A) receptors.

Estrada-Mondragon A, Lynch JW - Front Mol Neurosci (2015)

Bottom Line: When it binds to α1-β2 sites it elicits potentiation of GABA-gated currents but has no irreversible activating effect.Molecular docking simulations reveal that the γ2L-β2 interface forms more contacts with ivermectin than the other interfaces, possibly explaining why ivermectin appears to bind irreversibly at this interface.This study demonstrates unexpectedly stark pharmacological differences among GABAAR ivermectin binding sites.

View Article: PubMed Central - PubMed

Affiliation: Queensland Brain Institute, The University of Queensland Brisbane, QLD, Australia.

ABSTRACT
GABAA receptors (GABAARs) are the major inhibitory neurotransmitter receptors in the brain and are therapeutic targets for many indications including sedation, anesthesia and anxiolysis. There is, however, considerable scope for the development of new therapeutics with improved beneficial effects and reduced side-effect profiles. The anthelminthic drug, ivermectin, activates the GABAAR although its binding site is not known. The molecular site of action of ivermectin has, however, been defined by crystallography in the homologous glutamate-gated chloride channel. Resolving the molecular mechanisms of ivermectin binding to α1β2γ2L GABAARs may provide insights into the design of improved therapeutics. Given that ivermectin binds to subunit interfaces, we sought to define (1) which subunit interface sites it binds to, (2) whether these sites are equivalent in terms of ivermectin sensitivity or efficacy, and (3) how many must be occupied for maximal efficacy. Our approach involved precluding ivermectin from binding to particular interfaces by introducing bulky M3 domain 36'F sidechains to the "+" side of those interfaces. We thereby demonstrated that ivermectin produces irreversible channel activation only when it binds to the single γ2L-β2 interface site. When it binds to α1-β2 sites it elicits potentiation of GABA-gated currents but has no irreversible activating effect. Ivermectin cannot bind to the β2-α1 interface site due to its endogenous bulky 36' methionine. Replacing this with an alanine creates a functional site at this interface, but surprisingly it is inhibitory. Molecular docking simulations reveal that the γ2L-β2 interface forms more contacts with ivermectin than the other interfaces, possibly explaining why ivermectin appears to bind irreversibly at this interface. This study demonstrates unexpectedly stark pharmacological differences among GABAAR ivermectin binding sites.

No MeSH data available.


Related in: MedlinePlus

Ivermectin modulation of wild type α1β2γ2L GABAARs. In this and subsequent figures, all displayed traces were recorded from HEK293 cells expressing WT or mutated α1β2γ2L receptors using whole cell recording, and the durations of ivermectin and GABA applications are indicated by gray and black bars, respectively. (A) Sample recording showing the effect of repeated applications of EC3 GABA together with increasing concentrations of ivermectin at 1 min intervals as indicated. A current response to saturating GABA from the same cell is also shown. (B) Averaged ivermectin concentration-response relationships from 4 to 5 cells, with error bars shown as SEM. Ivermectin-induced reversible and irreversible currents, measured as shown in (B), are plotted separately as circles and squares, respectively. (C) Mean saturating magnitudes of reversible and irreversible ivermectin-modulated currents (defined as indicated in A), and their sum, plotted as a percentage of the saturating GABA-activated current. Mean GABA EC50, nH and Imax values for this and all other constructs are provided in Table 1.
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Figure 1: Ivermectin modulation of wild type α1β2γ2L GABAARs. In this and subsequent figures, all displayed traces were recorded from HEK293 cells expressing WT or mutated α1β2γ2L receptors using whole cell recording, and the durations of ivermectin and GABA applications are indicated by gray and black bars, respectively. (A) Sample recording showing the effect of repeated applications of EC3 GABA together with increasing concentrations of ivermectin at 1 min intervals as indicated. A current response to saturating GABA from the same cell is also shown. (B) Averaged ivermectin concentration-response relationships from 4 to 5 cells, with error bars shown as SEM. Ivermectin-induced reversible and irreversible currents, measured as shown in (B), are plotted separately as circles and squares, respectively. (C) Mean saturating magnitudes of reversible and irreversible ivermectin-modulated currents (defined as indicated in A), and their sum, plotted as a percentage of the saturating GABA-activated current. Mean GABA EC50, nH and Imax values for this and all other constructs are provided in Table 1.

Mentions: Ivermectin concentration-response relationships were determined by applying ivermectin at progressively increasing concentrations (0.1, 0.3, 1, 3, 10, and 30 μM) for 10 s periods at 1 min intervals. An EC3 concentration of GABA was co-applied with ivermectin for the second 5 s period of every 10 s application. A sample recording from α1β2γ2L GABAARs using this protocol is shown in Figure 1A. Under these conditions, ivermectin induced both a potentiation of the reversible GABA-gated current plus an irreversibly-activated current component. Mean concentration-response relationships for both responses were normalized to the saturating GABA-gated current in the same cell and plotted separately (Figure 1B). In addition, the mean saturating magnitudes of the reversible and irreversible current components relative to the saturating GABA-gated current magnitude in the same cell are plotted in Figure 1C. Throughout the rest of this study we quantitated the effects of ivermectin as illustrated in Figure 1B.


Functional characterization of ivermectin binding sites in α1β2γ2L GABA(A) receptors.

Estrada-Mondragon A, Lynch JW - Front Mol Neurosci (2015)

Ivermectin modulation of wild type α1β2γ2L GABAARs. In this and subsequent figures, all displayed traces were recorded from HEK293 cells expressing WT or mutated α1β2γ2L receptors using whole cell recording, and the durations of ivermectin and GABA applications are indicated by gray and black bars, respectively. (A) Sample recording showing the effect of repeated applications of EC3 GABA together with increasing concentrations of ivermectin at 1 min intervals as indicated. A current response to saturating GABA from the same cell is also shown. (B) Averaged ivermectin concentration-response relationships from 4 to 5 cells, with error bars shown as SEM. Ivermectin-induced reversible and irreversible currents, measured as shown in (B), are plotted separately as circles and squares, respectively. (C) Mean saturating magnitudes of reversible and irreversible ivermectin-modulated currents (defined as indicated in A), and their sum, plotted as a percentage of the saturating GABA-activated current. Mean GABA EC50, nH and Imax values for this and all other constructs are provided in Table 1.
© Copyright Policy
Related In: Results  -  Collection

License
Show All Figures
getmorefigures.php?uid=PMC4585179&req=5

Figure 1: Ivermectin modulation of wild type α1β2γ2L GABAARs. In this and subsequent figures, all displayed traces were recorded from HEK293 cells expressing WT or mutated α1β2γ2L receptors using whole cell recording, and the durations of ivermectin and GABA applications are indicated by gray and black bars, respectively. (A) Sample recording showing the effect of repeated applications of EC3 GABA together with increasing concentrations of ivermectin at 1 min intervals as indicated. A current response to saturating GABA from the same cell is also shown. (B) Averaged ivermectin concentration-response relationships from 4 to 5 cells, with error bars shown as SEM. Ivermectin-induced reversible and irreversible currents, measured as shown in (B), are plotted separately as circles and squares, respectively. (C) Mean saturating magnitudes of reversible and irreversible ivermectin-modulated currents (defined as indicated in A), and their sum, plotted as a percentage of the saturating GABA-activated current. Mean GABA EC50, nH and Imax values for this and all other constructs are provided in Table 1.
Mentions: Ivermectin concentration-response relationships were determined by applying ivermectin at progressively increasing concentrations (0.1, 0.3, 1, 3, 10, and 30 μM) for 10 s periods at 1 min intervals. An EC3 concentration of GABA was co-applied with ivermectin for the second 5 s period of every 10 s application. A sample recording from α1β2γ2L GABAARs using this protocol is shown in Figure 1A. Under these conditions, ivermectin induced both a potentiation of the reversible GABA-gated current plus an irreversibly-activated current component. Mean concentration-response relationships for both responses were normalized to the saturating GABA-gated current in the same cell and plotted separately (Figure 1B). In addition, the mean saturating magnitudes of the reversible and irreversible current components relative to the saturating GABA-gated current magnitude in the same cell are plotted in Figure 1C. Throughout the rest of this study we quantitated the effects of ivermectin as illustrated in Figure 1B.

Bottom Line: When it binds to α1-β2 sites it elicits potentiation of GABA-gated currents but has no irreversible activating effect.Molecular docking simulations reveal that the γ2L-β2 interface forms more contacts with ivermectin than the other interfaces, possibly explaining why ivermectin appears to bind irreversibly at this interface.This study demonstrates unexpectedly stark pharmacological differences among GABAAR ivermectin binding sites.

View Article: PubMed Central - PubMed

Affiliation: Queensland Brain Institute, The University of Queensland Brisbane, QLD, Australia.

ABSTRACT
GABAA receptors (GABAARs) are the major inhibitory neurotransmitter receptors in the brain and are therapeutic targets for many indications including sedation, anesthesia and anxiolysis. There is, however, considerable scope for the development of new therapeutics with improved beneficial effects and reduced side-effect profiles. The anthelminthic drug, ivermectin, activates the GABAAR although its binding site is not known. The molecular site of action of ivermectin has, however, been defined by crystallography in the homologous glutamate-gated chloride channel. Resolving the molecular mechanisms of ivermectin binding to α1β2γ2L GABAARs may provide insights into the design of improved therapeutics. Given that ivermectin binds to subunit interfaces, we sought to define (1) which subunit interface sites it binds to, (2) whether these sites are equivalent in terms of ivermectin sensitivity or efficacy, and (3) how many must be occupied for maximal efficacy. Our approach involved precluding ivermectin from binding to particular interfaces by introducing bulky M3 domain 36'F sidechains to the "+" side of those interfaces. We thereby demonstrated that ivermectin produces irreversible channel activation only when it binds to the single γ2L-β2 interface site. When it binds to α1-β2 sites it elicits potentiation of GABA-gated currents but has no irreversible activating effect. Ivermectin cannot bind to the β2-α1 interface site due to its endogenous bulky 36' methionine. Replacing this with an alanine creates a functional site at this interface, but surprisingly it is inhibitory. Molecular docking simulations reveal that the γ2L-β2 interface forms more contacts with ivermectin than the other interfaces, possibly explaining why ivermectin appears to bind irreversibly at this interface. This study demonstrates unexpectedly stark pharmacological differences among GABAAR ivermectin binding sites.

No MeSH data available.


Related in: MedlinePlus