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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 α1β2 and α1A36′Fβ2 GABAARs. (A) Structural model of α1β2 showing the location of the ivermectin binding sites. (B) Sample recording showing the effect of increasing ivermectin concentrations on EC10 GABA-gated currents in α1β2 GABAARs. (C) Structural model of α1A36′Fβ2 showing the lack of ivermectin binding sites. (D) Sample recording showing the effect of increasing ivermectin concentrations on EC10 GABA-gated currents α1A36′Fβ2 GABAARs. (E) Mean concentration-response data for the experiments as shown in (B,D). *Represents significance of t-test P < 0.05.
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Figure 7: Effects of ivermectin on α1β2 and α1A36′Fβ2 GABAARs. (A) Structural model of α1β2 showing the location of the ivermectin binding sites. (B) Sample recording showing the effect of increasing ivermectin concentrations on EC10 GABA-gated currents in α1β2 GABAARs. (C) Structural model of α1A36′Fβ2 showing the lack of ivermectin binding sites. (D) Sample recording showing the effect of increasing ivermectin concentrations on EC10 GABA-gated currents α1A36′Fβ2 GABAARs. (E) Mean concentration-response data for the experiments as shown in (B,D). *Represents significance of t-test P < 0.05.

Mentions: The second control experiment was to investigate the binary α1β2 GABAAR which exists in an obligatory α1-β2-α1-β2-β2 stoichiometry (Baumann et al., 2001). This receptor should thus have two functional α1-β2 ivermectin sites per pentamer (Figure 7A). As indicated in the sample recording in Figure 7B and in the averaged data plotted below, these receptors exhibited significant ivermectin-mediated potentiation of GABA-gated currents with a concentration-response profile indistinguishable from that of α1β2γ2LS36′F GABAARs, but exhibited no irreversible ivermectin activation. As a negative control for this experiment we also tested α1A36′Fβ2 GABAARs, which like α1A36′Fβ2γ2LS36′F GABAARs, should contain no functional ivermectin sites (Figure 7C). As predicted, this receptor exhibited no ivermectin sensitivity at all, even at concentrations up to 30 μM (Figures 7D, E). Taken together, results from Figures 6, 7 indicate that the α1A36′F mutation completely eliminates ivermectin binding at the α1-β2 and α1-γ2L interface sites. Thus, we conclude that the maximum efficacy with which ivermectin potentiates GABA-gated currents is similar for receptors incorporating a single γ2L-β2 interface site as it is in receptors incorporating α1-β2 and α1-γ2L interface sites combined. We also conclude that irreversible ivermectin activation requires a functional γ2L-β2 interface site.


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

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

Effects of ivermectin on α1β2 and α1A36′Fβ2 GABAARs. (A) Structural model of α1β2 showing the location of the ivermectin binding sites. (B) Sample recording showing the effect of increasing ivermectin concentrations on EC10 GABA-gated currents in α1β2 GABAARs. (C) Structural model of α1A36′Fβ2 showing the lack of ivermectin binding sites. (D) Sample recording showing the effect of increasing ivermectin concentrations on EC10 GABA-gated currents α1A36′Fβ2 GABAARs. (E) Mean concentration-response data for the experiments as shown in (B,D). *Represents significance of t-test P < 0.05.
© Copyright Policy
Related In: Results  -  Collection

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

Figure 7: Effects of ivermectin on α1β2 and α1A36′Fβ2 GABAARs. (A) Structural model of α1β2 showing the location of the ivermectin binding sites. (B) Sample recording showing the effect of increasing ivermectin concentrations on EC10 GABA-gated currents in α1β2 GABAARs. (C) Structural model of α1A36′Fβ2 showing the lack of ivermectin binding sites. (D) Sample recording showing the effect of increasing ivermectin concentrations on EC10 GABA-gated currents α1A36′Fβ2 GABAARs. (E) Mean concentration-response data for the experiments as shown in (B,D). *Represents significance of t-test P < 0.05.
Mentions: The second control experiment was to investigate the binary α1β2 GABAAR which exists in an obligatory α1-β2-α1-β2-β2 stoichiometry (Baumann et al., 2001). This receptor should thus have two functional α1-β2 ivermectin sites per pentamer (Figure 7A). As indicated in the sample recording in Figure 7B and in the averaged data plotted below, these receptors exhibited significant ivermectin-mediated potentiation of GABA-gated currents with a concentration-response profile indistinguishable from that of α1β2γ2LS36′F GABAARs, but exhibited no irreversible ivermectin activation. As a negative control for this experiment we also tested α1A36′Fβ2 GABAARs, which like α1A36′Fβ2γ2LS36′F GABAARs, should contain no functional ivermectin sites (Figure 7C). As predicted, this receptor exhibited no ivermectin sensitivity at all, even at concentrations up to 30 μM (Figures 7D, E). Taken together, results from Figures 6, 7 indicate that the α1A36′F mutation completely eliminates ivermectin binding at the α1-β2 and α1-γ2L interface sites. Thus, we conclude that the maximum efficacy with which ivermectin potentiates GABA-gated currents is similar for receptors incorporating a single γ2L-β2 interface site as it is in receptors incorporating α1-β2 and α1-γ2L interface sites combined. We also conclude that irreversible ivermectin activation requires a functional γ2L-β2 interface site.

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