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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

Picrotoxin block of currents directly activated by ivermectin. (A) Examples of ivermectin activation and picrotoxin block of GABAARs comprising the indicated subunits. (B) Averaged picrotoxin block data from (A) compared with averaged ivermectin irreversible activation data from Figure 5E. All results were averaged from at least 4 cells. This confirms that direct ivermectin activation requires receptors with intact γ2L-β2 interfaces. *Represents significance of t-test P < 0.05.
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Figure 9: Picrotoxin block of currents directly activated by ivermectin. (A) Examples of ivermectin activation and picrotoxin block of GABAARs comprising the indicated subunits. (B) Averaged picrotoxin block data from (A) compared with averaged ivermectin irreversible activation data from Figure 5E. All results were averaged from at least 4 cells. This confirms that direct ivermectin activation requires receptors with intact γ2L-β2 interfaces. *Represents significance of t-test P < 0.05.

Mentions: In a final set of experiments, we sought to confirm the relative magnitudes of the irreversible ivermectin-gated currents in α1β2γ2L, α1A36′Fβ2γ2L, and α1β2γ2LS36′F GABAARs using picrotoxin block. Our approach involved applying 30 μM ivermectin until maximal activation was achieved, and then blocking this current with 100 μM picrotoxin (Figure 9A). The mean picrotoxin-blocked current was then expressed as a percentage of the saturating GABA-activated current, and compared to the irreversible ivermectin-activated current magnitude replotted from Figure 5E. The averaged results (Figure 9B), confirm that ivermectin activates irreversible currents in the α1β2γ2L and α1A36′Fβ2γ2L GABAARs but not in the α1β2γ2LS36′F GABAAR.


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

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

Picrotoxin block of currents directly activated by ivermectin. (A) Examples of ivermectin activation and picrotoxin block of GABAARs comprising the indicated subunits. (B) Averaged picrotoxin block data from (A) compared with averaged ivermectin irreversible activation data from Figure 5E. All results were averaged from at least 4 cells. This confirms that direct ivermectin activation requires receptors with intact γ2L-β2 interfaces. *Represents significance of t-test P < 0.05.
© Copyright Policy
Related In: Results  -  Collection

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

Figure 9: Picrotoxin block of currents directly activated by ivermectin. (A) Examples of ivermectin activation and picrotoxin block of GABAARs comprising the indicated subunits. (B) Averaged picrotoxin block data from (A) compared with averaged ivermectin irreversible activation data from Figure 5E. All results were averaged from at least 4 cells. This confirms that direct ivermectin activation requires receptors with intact γ2L-β2 interfaces. *Represents significance of t-test P < 0.05.
Mentions: In a final set of experiments, we sought to confirm the relative magnitudes of the irreversible ivermectin-gated currents in α1β2γ2L, α1A36′Fβ2γ2L, and α1β2γ2LS36′F GABAARs using picrotoxin block. Our approach involved applying 30 μM ivermectin until maximal activation was achieved, and then blocking this current with 100 μM picrotoxin (Figure 9A). The mean picrotoxin-blocked current was then expressed as a percentage of the saturating GABA-activated current, and compared to the irreversible ivermectin-activated current magnitude replotted from Figure 5E. The averaged results (Figure 9B), confirm that ivermectin activates irreversible currents in the α1β2γ2L and α1A36′Fβ2γ2L GABAARs but not in the α1β2γ2LS36′F GABAAR.

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