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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 contact residues. (A) This panel shows ivermectin bound at the α1-β2 interface, viewed from within the plane of the membrane from the (−) side of the interface (left panel) and the (+) side of the interface (right panel). Note it only displays interacting residues that are common to all four interface binding sites. (B) This panel shows additional interacting residues, relevant to particular interfaces only. For example, at the β2 (−) interface, ivermectin interacts with a total of 7 residues (4 depicted in A and 3 depicted in B).
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Figure 3: Ivermectin contact residues. (A) This panel shows ivermectin bound at the α1-β2 interface, viewed from within the plane of the membrane from the (−) side of the interface (left panel) and the (+) side of the interface (right panel). Note it only displays interacting residues that are common to all four interface binding sites. (B) This panel shows additional interacting residues, relevant to particular interfaces only. For example, at the β2 (−) interface, ivermectin interacts with a total of 7 residues (4 depicted in A and 3 depicted in B).

Mentions: However, before presenting the functional analysis, we describe in more detail the molecular interactions predicted to exist at each interface site. Table 2 provides a summary of all transmembrane α1β2M36′Aγ2L GABAAR residues predicted to interact with ivermectin in our model structure. Corresponding ivermectin binding residues from the C. elegans α GluClR crystal structure (Hibbs and Gouaux, 2011) are also displayed. Residues in gray are predicted not to bind ivermectin, whereas asterisked residues are predicted to form hydrogen bonds with ivermectin. The spatial orientations of these residues relative to docked ivermectin are depicted in Figure 3. Note that Figure 3A only displays those interacting residues that are common to all four interface binding sites. The additional interacting residues relevant to particular interfaces are shown in Figure 3B. According to Figure 3, the α1-β2 and α1-γ2L interfaces are structurally similar, with ivermectin-interacting residues sharing similar characteristics of polarity and hydrophobicity. On the other hand, the γ2L-β2 interface shows a greater number of residues contributing polar contacts that should result in the pocket forming tighter contacts around the bound ivermectin molecule. The decrypted β2-α1 interface has a similar number of contacts as the α1-β2 and α1-γ2L interfaces, although their spatial distribution patterns differ significantly (Figure 3B).


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

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

Ivermectin contact residues. (A) This panel shows ivermectin bound at the α1-β2 interface, viewed from within the plane of the membrane from the (−) side of the interface (left panel) and the (+) side of the interface (right panel). Note it only displays interacting residues that are common to all four interface binding sites. (B) This panel shows additional interacting residues, relevant to particular interfaces only. For example, at the β2 (−) interface, ivermectin interacts with a total of 7 residues (4 depicted in A and 3 depicted in B).
© Copyright Policy
Related In: Results  -  Collection

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

Figure 3: Ivermectin contact residues. (A) This panel shows ivermectin bound at the α1-β2 interface, viewed from within the plane of the membrane from the (−) side of the interface (left panel) and the (+) side of the interface (right panel). Note it only displays interacting residues that are common to all four interface binding sites. (B) This panel shows additional interacting residues, relevant to particular interfaces only. For example, at the β2 (−) interface, ivermectin interacts with a total of 7 residues (4 depicted in A and 3 depicted in B).
Mentions: However, before presenting the functional analysis, we describe in more detail the molecular interactions predicted to exist at each interface site. Table 2 provides a summary of all transmembrane α1β2M36′Aγ2L GABAAR residues predicted to interact with ivermectin in our model structure. Corresponding ivermectin binding residues from the C. elegans α GluClR crystal structure (Hibbs and Gouaux, 2011) are also displayed. Residues in gray are predicted not to bind ivermectin, whereas asterisked residues are predicted to form hydrogen bonds with ivermectin. The spatial orientations of these residues relative to docked ivermectin are depicted in Figure 3. Note that Figure 3A only displays those interacting residues that are common to all four interface binding sites. The additional interacting residues relevant to particular interfaces are shown in Figure 3B. According to Figure 3, the α1-β2 and α1-γ2L interfaces are structurally similar, with ivermectin-interacting residues sharing similar characteristics of polarity and hydrophobicity. On the other hand, the γ2L-β2 interface shows a greater number of residues contributing polar contacts that should result in the pocket forming tighter contacts around the bound ivermectin molecule. The decrypted β2-α1 interface has a similar number of contacts as the α1-β2 and α1-γ2L interfaces, although their spatial distribution patterns differ significantly (Figure 3B).

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