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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

Effects of ivermectin on α1A36′Fβ2γ2L GABAARs. (A) Structural model showing the location of the single ivermectin binding site. (B) Sample recording showing the effect of increasing ivermectin concentrations on EC3 GABA-gated currents, with reversible (i.e., GABA-activated) and irreversible current increases indicated. (C) Mean concentration-response relationship of the reversible current component, compared with the corresponding wild type GABAAR data from Figure 1. (D) Mean concentration-response relationship of the irreversible current component, compared with the corresponding wild type GABAAR data from Figure 1. (E) Mean saturating magnitudes of reversible and irreversible ivermectin-modulated currents (defined as indicated in B), and their sum, plotted as a percentage of EC3 GABA-activated current, and compared to corresponding wild type data. *Represents significance of t-test P < 0.05.
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Figure 5: Effects of ivermectin on α1A36′Fβ2γ2L GABAARs. (A) Structural model showing the location of the single ivermectin binding site. (B) Sample recording showing the effect of increasing ivermectin concentrations on EC3 GABA-gated currents, with reversible (i.e., GABA-activated) and irreversible current increases indicated. (C) Mean concentration-response relationship of the reversible current component, compared with the corresponding wild type GABAAR data from Figure 1. (D) Mean concentration-response relationship of the irreversible current component, compared with the corresponding wild type GABAAR data from Figure 1. (E) Mean saturating magnitudes of reversible and irreversible ivermectin-modulated currents (defined as indicated in B), and their sum, plotted as a percentage of EC3 GABA-activated current, and compared to corresponding wild type data. *Represents significance of t-test P < 0.05.

Mentions: According to our molecular modeling, the α1A36′F mutation blocks ivermectin binding at the α1-β and α1-γ2L interfaces, leaving the γ2L-β2 interface as the only available ivermectin site (Figure 5A). A sample recording, showing the effect of increasing ivermectin concentrations on EC3 GABA-gated currents on α1A36′Fβ2γ2L GABAARs, reveals both reversible (i.e., potentiating GABA-activated currents) and irreversible current increases (Figure 5B). The mean concentration-response relationship for the reversible current revealed no change in ivermectin sensitivity relative to the wild type receptor, although the saturating magnitude of the potentiation was significantly reduced (Figure 5C). The mean concentration-response of the irreversible current revealed a significantly larger peak current relative to that observed the wild type receptor (Figure 5D). Plotting the saturating reversible and irreversible current magnitudes as a percentage of the mean saturating GABA current supported these findings (Figure 5E). The main result is a significant increase in the ivermectin-induced irreversible current component in α1A36′Fβ2γ2L GABAARs relative to wild type GABAARs (Figure 5E).


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

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

Effects of ivermectin on α1A36′Fβ2γ2L GABAARs. (A) Structural model showing the location of the single ivermectin binding site. (B) Sample recording showing the effect of increasing ivermectin concentrations on EC3 GABA-gated currents, with reversible (i.e., GABA-activated) and irreversible current increases indicated. (C) Mean concentration-response relationship of the reversible current component, compared with the corresponding wild type GABAAR data from Figure 1. (D) Mean concentration-response relationship of the irreversible current component, compared with the corresponding wild type GABAAR data from Figure 1. (E) Mean saturating magnitudes of reversible and irreversible ivermectin-modulated currents (defined as indicated in B), and their sum, plotted as a percentage of EC3 GABA-activated current, and compared to corresponding wild type data. *Represents significance of t-test P < 0.05.
© Copyright Policy
Related In: Results  -  Collection

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

Figure 5: Effects of ivermectin on α1A36′Fβ2γ2L GABAARs. (A) Structural model showing the location of the single ivermectin binding site. (B) Sample recording showing the effect of increasing ivermectin concentrations on EC3 GABA-gated currents, with reversible (i.e., GABA-activated) and irreversible current increases indicated. (C) Mean concentration-response relationship of the reversible current component, compared with the corresponding wild type GABAAR data from Figure 1. (D) Mean concentration-response relationship of the irreversible current component, compared with the corresponding wild type GABAAR data from Figure 1. (E) Mean saturating magnitudes of reversible and irreversible ivermectin-modulated currents (defined as indicated in B), and their sum, plotted as a percentage of EC3 GABA-activated current, and compared to corresponding wild type data. *Represents significance of t-test P < 0.05.
Mentions: According to our molecular modeling, the α1A36′F mutation blocks ivermectin binding at the α1-β and α1-γ2L interfaces, leaving the γ2L-β2 interface as the only available ivermectin site (Figure 5A). A sample recording, showing the effect of increasing ivermectin concentrations on EC3 GABA-gated currents on α1A36′Fβ2γ2L GABAARs, reveals both reversible (i.e., potentiating GABA-activated currents) and irreversible current increases (Figure 5B). The mean concentration-response relationship for the reversible current revealed no change in ivermectin sensitivity relative to the wild type receptor, although the saturating magnitude of the potentiation was significantly reduced (Figure 5C). The mean concentration-response of the irreversible current revealed a significantly larger peak current relative to that observed the wild type receptor (Figure 5D). Plotting the saturating reversible and irreversible current magnitudes as a percentage of the mean saturating GABA current supported these findings (Figure 5E). The main result is a significant increase in the ivermectin-induced irreversible current component in α1A36′Fβ2γ2L GABAARs relative to wild type GABAARs (Figure 5E).

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