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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

Molecular modeling of ivermectin binding to its subunit interface binding sites in the α1β2γ2L GABAAR. (A) Amino acid sequence alignment of M3 residues of the human α1, β2, and γ2L GABAAR subunits, the human α1 GlyR subunit and the C. elegans α GluClR subunit with their respective 36′ residues highlighted in a box. (B) Molecular structural model of the transmembrane domains of a wild type β2-α1-β2-α1-γ2L GABAARviewed from the extracellular side of the membrane along the pore axis. This receptor accommodates three ivermectin molecules (green) in their subunit interface binding sites. In this and subsequent model structures, the pore location is denoted by the red dashed circle, 36′ sidechains and bound ivermectin molecules (green) are shown in stick form and interfaces that do not bind ivermectin are indicated by a red X. (C) This panel shows the effect of mutagenesis on the ability of ivermectin to bind at individual interfaces. The left column displays single subunit interfaces formed from (top to bottom) α1-β2, α1-γ2L, γ2L-β2, and β2-α1 interfaces, with ivermectin docked where possible. The right column shows the same four subunit interfaces incorporating the indicated mutations (A36′F, S36′F, or M36′A).
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Figure 2: Molecular modeling of ivermectin binding to its subunit interface binding sites in the α1β2γ2L GABAAR. (A) Amino acid sequence alignment of M3 residues of the human α1, β2, and γ2L GABAAR subunits, the human α1 GlyR subunit and the C. elegans α GluClR subunit with their respective 36′ residues highlighted in a box. (B) Molecular structural model of the transmembrane domains of a wild type β2-α1-β2-α1-γ2L GABAARviewed from the extracellular side of the membrane along the pore axis. This receptor accommodates three ivermectin molecules (green) in their subunit interface binding sites. In this and subsequent model structures, the pore location is denoted by the red dashed circle, 36′ sidechains and bound ivermectin molecules (green) are shown in stick form and interfaces that do not bind ivermectin are indicated by a red X. (C) This panel shows the effect of mutagenesis on the ability of ivermectin to bind at individual interfaces. The left column displays single subunit interfaces formed from (top to bottom) α1-β2, α1-γ2L, γ2L-β2, and β2-α1 interfaces, with ivermectin docked where possible. The right column shows the same four subunit interfaces incorporating the indicated mutations (A36′F, S36′F, or M36′A).

Mentions: A previous study showed that the volume of the M3 residue at 36′ position (numbered according to the standard M2 domain residue numbering system) is a crucial determinant of ivermectin sensitivity in both the human α1 GlyR and the H. contortus α3β GluClR (Lynagh and Lynch, 2010). In both receptors, 36′G resulted in exquisite (low nanomolar) ivermectin sensitivity, 36′S and 36′A produced receptors with high nanomolar—low micromolar sensitivity, whereas larger residues (notably 36′F) eliminated ivermectin sensitivity entirely. As the 36′ sidechain lines the mouth of the ivermectin site on the “+” side of the subunit interface (Hibbs and Gouaux, 2011), it is likely that a large volume sidechain at this position sterically hinders ivermectin from entering its site (Lynagh et al., 2011; Lynagh and Lynch, 2012a). A sequence alignment of the α1, β2, and γ2L GABAAR subunits predicts that the α1 and γ2L subunits, which contain 36′A and 36′S, respectively, should support ivermectin binding sites but the β2 subunit, which contains a 36′M, should not (Figure 2A). In support of this, molecular modeling of ivermectin binding to the α1β2γ2L GABAAR reveals that it is able to dock into sites at the α1-β2, α1-γ2L, and γ2L-β2 interfaces but not at the β2-α1 interface (Figure 2B). In agreement with this, a recent study on an insect RDL homomeric GABAAR also found that the G36′M mutation eliminated ivermectin sensitivity (Nakao et al., 2015). We introduced the A36′F and S36′F mutations into the α1 and γ2L subunits, respectively, with the aim of eliminating existing ivermectin sites. We also introduced the M36′A mutation into the β2 subunit which we predicted would decrypt a possible site at the β2-α1 interface. According to our molecular modeling simulations, the A/S36′F mutations eliminated ivermectin binding whereas the M36′A mutations created an ivermectin site (Figure 2B). Of course, these predictions need to be validated by functional analysis.


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

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

Molecular modeling of ivermectin binding to its subunit interface binding sites in the α1β2γ2L GABAAR. (A) Amino acid sequence alignment of M3 residues of the human α1, β2, and γ2L GABAAR subunits, the human α1 GlyR subunit and the C. elegans α GluClR subunit with their respective 36′ residues highlighted in a box. (B) Molecular structural model of the transmembrane domains of a wild type β2-α1-β2-α1-γ2L GABAARviewed from the extracellular side of the membrane along the pore axis. This receptor accommodates three ivermectin molecules (green) in their subunit interface binding sites. In this and subsequent model structures, the pore location is denoted by the red dashed circle, 36′ sidechains and bound ivermectin molecules (green) are shown in stick form and interfaces that do not bind ivermectin are indicated by a red X. (C) This panel shows the effect of mutagenesis on the ability of ivermectin to bind at individual interfaces. The left column displays single subunit interfaces formed from (top to bottom) α1-β2, α1-γ2L, γ2L-β2, and β2-α1 interfaces, with ivermectin docked where possible. The right column shows the same four subunit interfaces incorporating the indicated mutations (A36′F, S36′F, or M36′A).
© Copyright Policy
Related In: Results  -  Collection

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Figure 2: Molecular modeling of ivermectin binding to its subunit interface binding sites in the α1β2γ2L GABAAR. (A) Amino acid sequence alignment of M3 residues of the human α1, β2, and γ2L GABAAR subunits, the human α1 GlyR subunit and the C. elegans α GluClR subunit with their respective 36′ residues highlighted in a box. (B) Molecular structural model of the transmembrane domains of a wild type β2-α1-β2-α1-γ2L GABAARviewed from the extracellular side of the membrane along the pore axis. This receptor accommodates three ivermectin molecules (green) in their subunit interface binding sites. In this and subsequent model structures, the pore location is denoted by the red dashed circle, 36′ sidechains and bound ivermectin molecules (green) are shown in stick form and interfaces that do not bind ivermectin are indicated by a red X. (C) This panel shows the effect of mutagenesis on the ability of ivermectin to bind at individual interfaces. The left column displays single subunit interfaces formed from (top to bottom) α1-β2, α1-γ2L, γ2L-β2, and β2-α1 interfaces, with ivermectin docked where possible. The right column shows the same four subunit interfaces incorporating the indicated mutations (A36′F, S36′F, or M36′A).
Mentions: A previous study showed that the volume of the M3 residue at 36′ position (numbered according to the standard M2 domain residue numbering system) is a crucial determinant of ivermectin sensitivity in both the human α1 GlyR and the H. contortus α3β GluClR (Lynagh and Lynch, 2010). In both receptors, 36′G resulted in exquisite (low nanomolar) ivermectin sensitivity, 36′S and 36′A produced receptors with high nanomolar—low micromolar sensitivity, whereas larger residues (notably 36′F) eliminated ivermectin sensitivity entirely. As the 36′ sidechain lines the mouth of the ivermectin site on the “+” side of the subunit interface (Hibbs and Gouaux, 2011), it is likely that a large volume sidechain at this position sterically hinders ivermectin from entering its site (Lynagh et al., 2011; Lynagh and Lynch, 2012a). A sequence alignment of the α1, β2, and γ2L GABAAR subunits predicts that the α1 and γ2L subunits, which contain 36′A and 36′S, respectively, should support ivermectin binding sites but the β2 subunit, which contains a 36′M, should not (Figure 2A). In support of this, molecular modeling of ivermectin binding to the α1β2γ2L GABAAR reveals that it is able to dock into sites at the α1-β2, α1-γ2L, and γ2L-β2 interfaces but not at the β2-α1 interface (Figure 2B). In agreement with this, a recent study on an insect RDL homomeric GABAAR also found that the G36′M mutation eliminated ivermectin sensitivity (Nakao et al., 2015). We introduced the A36′F and S36′F mutations into the α1 and γ2L subunits, respectively, with the aim of eliminating existing ivermectin sites. We also introduced the M36′A mutation into the β2 subunit which we predicted would decrypt a possible site at the β2-α1 interface. According to our molecular modeling simulations, the A/S36′F mutations eliminated ivermectin binding whereas the M36′A mutations created an ivermectin site (Figure 2B). Of course, these predictions need to be validated by functional analysis.

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