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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 α1β2M36′Aγ2L and α1A36′Fβ2M36′Aγ2LS36′F GABAARs. (A) Structural model of α1β2M36′Aγ2L showing the location of the ivermectin binding sites. (B) Sample recording showing the effect of increasing ivermectin concentrations on EC3 GABA-gated currents in α1β2M36′Aγ2L GABAARs. (C) Structural model of α1A36′Fβ2M36′Aγ2LS36′F showing the location of the single ivermectin binding site. (D) Sample recording showing the inhibitory effect of increasing ivermectin concentrations on EC10 GABA-gated currents in α1A36′Fβ2M36′Aγ2LS36′F GABAARs. (E) Mean concentration-response data for the experiments as shown in (B). (F) Mean concentration-response data for the experiments as shown in (D). *Represents significance of t-test P < 0.05.
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Figure 8: Effects of ivermectin on α1β2M36′Aγ2L and α1A36′Fβ2M36′Aγ2LS36′F GABAARs. (A) Structural model of α1β2M36′Aγ2L showing the location of the ivermectin binding sites. (B) Sample recording showing the effect of increasing ivermectin concentrations on EC3 GABA-gated currents in α1β2M36′Aγ2L GABAARs. (C) Structural model of α1A36′Fβ2M36′Aγ2LS36′F showing the location of the single ivermectin binding site. (D) Sample recording showing the inhibitory effect of increasing ivermectin concentrations on EC10 GABA-gated currents in α1A36′Fβ2M36′Aγ2LS36′F GABAARs. (E) Mean concentration-response data for the experiments as shown in (B). (F) Mean concentration-response data for the experiments as shown in (D). *Represents significance of t-test P < 0.05.

Mentions: To help validate the role of 36′ sidechains in forming ivermectin sites, we investigated the functional properties of GABAARs incorporating the β2M36′A mutation. We first characterized the ivermectin sensitivity of the α1β2M36′Aγ2L GABAAR which should incorporate five ivermectin sites per pentamer (Figure 8A). The effects of increasing ivermectin concentrations on EC3 GABA-gated currents in this receptor revealed significant potentiation of GABA-gated currents but no irreversible activation (Figure 8B). The averaged ivermectin concentration-response relationship demonstrates that the magnitude of the reversible potentiation to be significantly smaller than that observed at the wild type receptor (Figure 8E) although it was not significantly different to that observed at α1β2γ2LS36′F or α1A36′Fβ2γ2L GABAARs (P>0.05 unpaired t-tests). This result was unexpected for two reasons. First, despite a presumably intact γ2L-β2 binding site, it exhibited no irreversible ivermectin activation, apparently contradicting our conclusion from the previous section. Second, it seems surprising that receptors with five ivermectin sites should yield a reduced ivermectin efficacy relative to wild type receptors which have only three sites. We then investigated the α1A36′Fβ2M36′Aγ2LS36′ GABAAR, which should incorporate ivermectin sites at the two β2-α1 interfaces only (Figure 8C). As shown in the sample recording (Figure 8D) and in the averaged concentration-response data (Figure 8E), EC10 GABA-gated currents were strongly inhibited by ivermectin. This unexpected result provides a possible explanation as to why the ivermectin potentiating and direct activation efficacy at α1β2M36′Aγ2L GABAARs was reduced relative to wild type GABAARs.


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

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

Effects of ivermectin on α1β2M36′Aγ2L and α1A36′Fβ2M36′Aγ2LS36′F GABAARs. (A) Structural model of α1β2M36′Aγ2L showing the location of the ivermectin binding sites. (B) Sample recording showing the effect of increasing ivermectin concentrations on EC3 GABA-gated currents in α1β2M36′Aγ2L GABAARs. (C) Structural model of α1A36′Fβ2M36′Aγ2LS36′F showing the location of the single ivermectin binding site. (D) Sample recording showing the inhibitory effect of increasing ivermectin concentrations on EC10 GABA-gated currents in α1A36′Fβ2M36′Aγ2LS36′F GABAARs. (E) Mean concentration-response data for the experiments as shown in (B). (F) Mean concentration-response data for the experiments as shown in (D). *Represents significance of t-test P < 0.05.
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Figure 8: Effects of ivermectin on α1β2M36′Aγ2L and α1A36′Fβ2M36′Aγ2LS36′F GABAARs. (A) Structural model of α1β2M36′Aγ2L showing the location of the ivermectin binding sites. (B) Sample recording showing the effect of increasing ivermectin concentrations on EC3 GABA-gated currents in α1β2M36′Aγ2L GABAARs. (C) Structural model of α1A36′Fβ2M36′Aγ2LS36′F showing the location of the single ivermectin binding site. (D) Sample recording showing the inhibitory effect of increasing ivermectin concentrations on EC10 GABA-gated currents in α1A36′Fβ2M36′Aγ2LS36′F GABAARs. (E) Mean concentration-response data for the experiments as shown in (B). (F) Mean concentration-response data for the experiments as shown in (D). *Represents significance of t-test P < 0.05.
Mentions: To help validate the role of 36′ sidechains in forming ivermectin sites, we investigated the functional properties of GABAARs incorporating the β2M36′A mutation. We first characterized the ivermectin sensitivity of the α1β2M36′Aγ2L GABAAR which should incorporate five ivermectin sites per pentamer (Figure 8A). The effects of increasing ivermectin concentrations on EC3 GABA-gated currents in this receptor revealed significant potentiation of GABA-gated currents but no irreversible activation (Figure 8B). The averaged ivermectin concentration-response relationship demonstrates that the magnitude of the reversible potentiation to be significantly smaller than that observed at the wild type receptor (Figure 8E) although it was not significantly different to that observed at α1β2γ2LS36′F or α1A36′Fβ2γ2L GABAARs (P>0.05 unpaired t-tests). This result was unexpected for two reasons. First, despite a presumably intact γ2L-β2 binding site, it exhibited no irreversible ivermectin activation, apparently contradicting our conclusion from the previous section. Second, it seems surprising that receptors with five ivermectin sites should yield a reduced ivermectin efficacy relative to wild type receptors which have only three sites. We then investigated the α1A36′Fβ2M36′Aγ2LS36′ GABAAR, which should incorporate ivermectin sites at the two β2-α1 interfaces only (Figure 8C). As shown in the sample recording (Figure 8D) and in the averaged concentration-response data (Figure 8E), EC10 GABA-gated currents were strongly inhibited by ivermectin. This unexpected result provides a possible explanation as to why the ivermectin potentiating and direct activation efficacy at α1β2M36′Aγ2L GABAARs was reduced relative to wild type GABAARs.

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