Abstract
The unique pharmacological properties of δ-containing γ-aminobutyric acid type A receptors (δ-GABAARs) make them an attractive target for selective and persistent modulation of neuronal excitability. However, the availability of selective modulators targeting δ-GABAARs remains limited. AA29504 ([2-amino-4-(2,4,6-trimethylbenzylamino)-phenyl]-carbamic acid ethyl ester), an analog of K+ channel opener retigabine, acts as an agonist and a positive allosteric modulator (Ago-PAM) of δ-GABAARs. Based on electrophysiological studies using recombinant receptors, AA29504 was found to be a more potent and effective agonist in δ-GABAARs than in γ2-GABAARs. In comparison, AA29504 positively modulated the activity of recombinant δ-GABAARs more effectively than γ2-GABAARs, with no significant differences in potency. The impact of AA29504's efficacy- and potency-associated GABAAR subtype selectivity on radioligand binding properties remain unexplored. Using [3H]4'-ethynyl-4-n-propylbicycloorthobenzoate ([3H]EBOB) binding assay, we found no difference in the modulatory potency of AA29504 on GABA- and THIP (4,5,6,7-tetrahydroisoxazolo[5,4-c]pyridin-3-ol)-induced responses between native forebrain GABAARs of wild type and δ knock-out mice. In recombinant receptors expressed in HEK293 cells, AA29504 showed higher efficacy on δ- than γ2-GABAARs in the GABA-independent displacement of [3H]EBOB binding. Interestingly, AA29504 showed a concentration-dependent stimulation of [3H]muscimol binding to γ2-GABAARs, which was absent in δ-GABAARs. This was explained by AA29504 shifting the low-affinity γ2-GABAAR towards a higher affinity desensitized state, thereby rising new sites capable of binding GABAAR agonists with low nanomolar affinity. Hence, the potential of AA29504 to act as a desensitization-modifying allosteric modulator of γ2-GABAARs deserves further investigation for its promising influence on shaping efficacy, duration and plasticity of GABAAR synaptic responses.
Highlights
Introduction γAminobutyric acid type A receptors (GABAAR), members of the Cys-loop ligand-gated ion channels superfamily, are the major sites for fast-acting synaptic inhibition in the mammalian brain [1, 2]
[3H]EBOB binding assay was initially carried out to evaluate AA29504's allosteric modulatory activity on native GABAARs expressed in WT and δ subunit knockout (δKO) forebrain membranes
AA29504 decreased the IC50 of GABA-induced [3H]EBOB displacement from 15.9 ± 5.0 μM to 1.0 ± 0.2 μM in WT mice (p < 0.05) and from 8.4 ± 1.8 μM to 1.2 ± 0.5 μM in δKO mice (p < 0.05)
Summary
Aminobutyric acid type A receptors (GABAAR), members of the Cys-loop ligand-gated ion channels superfamily, are the major sites for fast-acting synaptic inhibition in the mammalian brain [1, 2]. These heteropentameric protein complexes contain an inherent chloride channel that opens upon the binding of GABA, where chloride. Due to the unique functional and pharmacological properties of δ‐GABAARs, they represent an attractive drug target for selective and persistent modulation of neuronal excitability. In comparison to γ2-GABAARs, the availability of selective allosteric modulators targeting δ-GABAARs remains limited
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