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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 α1β2γ2LS36′F and α1A36′Fβ2γ2LS36′F GABAARs. (A) Structural model of α1β2γ2LS36′F showing the location of the ivermectin binding sites. (B) Sample recording showing the effect of increasing ivermectin concentrations on EC3 GABA-gated currents in α1 β2γ2LS36′F GABAARs. (C) Structural model of α1A36′Fβ2γ2LS36′F showing the lack of ivermectin binding sites. (D) Sample recording showing the effect of increasing ivermectin concentrations on EC10 GABA-gated currents in α1A36′Fβ2γ2LS36′F 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 6: Effects of ivermectin on α1β2γ2LS36′F and α1A36′Fβ2γ2LS36′F GABAARs. (A) Structural model of α1β2γ2LS36′F showing the location of the ivermectin binding sites. (B) Sample recording showing the effect of increasing ivermectin concentrations on EC3 GABA-gated currents in α1 β2γ2LS36′F GABAARs. (C) Structural model of α1A36′Fβ2γ2LS36′F showing the lack of ivermectin binding sites. (D) Sample recording showing the effect of increasing ivermectin concentrations on EC10 GABA-gated currents in α1A36′Fβ2γ2LS36′F GABAARs. (E) Mean concentration-response data for the experiments as shown in (B,D). *Represents significance of t-test P < 0.05.

Mentions: According to our modeling, α1β2γ2LS36′F GABAARs should contain ivermectin sites at the α1-β2 and α1-γ2L interfaces only (Figure 6A). As shown in Figure 6B, ivermectin enhanced GABA-gated currents but induced no irreversible activation of these GABAARs. The averaged concentration-response relationship of the reversible potentiation revealed a reduction in its peak magnitude relative to the wild type receptor (Figure 6E), but no significant difference to that observed at the α1A36′Fβ2γ2L GABAAR (P>0.05, unpaired t-test). This similarity in potentiating efficacy may mean that the γ2L-β2 interface is functionally equivalent to the α1-β2 and α1-γ2L sites combined. However, the results may also be explained by the α1A36′F mutation being ineffective in preventing ivermectin binding to the α1-β2 or α1-γ2L sites. We investigated this possibility in two-ways. First, we expressed α1A36′Fβ2γ2LS36′F GABAARs which should contain no functional ivermectin sites (Figure 6C). If, on the other hand, the α1-β2 or α1-γ2L sites retain some residual functionality then this receptor should exhibit detectable ivermectin sensitivity. However, as shown in Figures 6D,E, no effect of ivermectin was observed at any concentration at α1A36′Fβ2γ2LS36′F 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 α1β2γ2LS36′F and α1A36′Fβ2γ2LS36′F GABAARs. (A) Structural model of α1β2γ2LS36′F showing the location of the ivermectin binding sites. (B) Sample recording showing the effect of increasing ivermectin concentrations on EC3 GABA-gated currents in α1 β2γ2LS36′F GABAARs. (C) Structural model of α1A36′Fβ2γ2LS36′F showing the lack of ivermectin binding sites. (D) Sample recording showing the effect of increasing ivermectin concentrations on EC10 GABA-gated currents in α1A36′Fβ2γ2LS36′F 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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Related In: Results  -  Collection

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

Figure 6: Effects of ivermectin on α1β2γ2LS36′F and α1A36′Fβ2γ2LS36′F GABAARs. (A) Structural model of α1β2γ2LS36′F showing the location of the ivermectin binding sites. (B) Sample recording showing the effect of increasing ivermectin concentrations on EC3 GABA-gated currents in α1 β2γ2LS36′F GABAARs. (C) Structural model of α1A36′Fβ2γ2LS36′F showing the lack of ivermectin binding sites. (D) Sample recording showing the effect of increasing ivermectin concentrations on EC10 GABA-gated currents in α1A36′Fβ2γ2LS36′F GABAARs. (E) Mean concentration-response data for the experiments as shown in (B,D). *Represents significance of t-test P < 0.05.
Mentions: According to our modeling, α1β2γ2LS36′F GABAARs should contain ivermectin sites at the α1-β2 and α1-γ2L interfaces only (Figure 6A). As shown in Figure 6B, ivermectin enhanced GABA-gated currents but induced no irreversible activation of these GABAARs. The averaged concentration-response relationship of the reversible potentiation revealed a reduction in its peak magnitude relative to the wild type receptor (Figure 6E), but no significant difference to that observed at the α1A36′Fβ2γ2L GABAAR (P>0.05, unpaired t-test). This similarity in potentiating efficacy may mean that the γ2L-β2 interface is functionally equivalent to the α1-β2 and α1-γ2L sites combined. However, the results may also be explained by the α1A36′F mutation being ineffective in preventing ivermectin binding to the α1-β2 or α1-γ2L sites. We investigated this possibility in two-ways. First, we expressed α1A36′Fβ2γ2LS36′F GABAARs which should contain no functional ivermectin sites (Figure 6C). If, on the other hand, the α1-β2 or α1-γ2L sites retain some residual functionality then this receptor should exhibit detectable ivermectin sensitivity. However, as shown in Figures 6D,E, no effect of ivermectin was observed at any concentration at α1A36′Fβ2γ2LS36′F 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