The Influence of AA29504 on GABA(A) Receptor Ligand Binding Properties and Its Implications on Subtype Selectivity

The unique pharmacological properties of δ-containing γ-aminobutyric acid type A receptors (δ-GABA(A)Rs) make them an attractive target for selective and persistent modulation of neuronal excitability. However, the availability of selective modulators targeting δ-GABA(A)Rs 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-GABA(A)Rs. Based on electrophysiological studies using recombinant receptors, AA29504 was found to be a more potent and effective agonist in δ-GABA(A)Rs than in γ2-GABA(A)Rs. In comparison, AA29504 positively modulated the activity of recombinant δ-GABA(A)Rs more effectively than γ2-GABA(A)Rs, with no significant differences in potency. The impact of AA29504's efficacy- and potency-associated GABA(A)R subtype selectivity on radioligand binding properties remain unexplored. Using [(3)H]4'-ethynyl-4-n-propylbicycloorthobenzoate ([(3)H]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 GABA(A)Rs of wild type and δ knock-out mice. In recombinant receptors expressed in HEK293 cells, AA29504 showed higher efficacy on δ- than γ(2)-GABA(A)Rs in the GABA-independent displacement of [(3)H]EBOB binding. Interestingly, AA29504 showed a concentration-dependent stimulation of [(3)H]muscimol binding to γ(2)-GABA(A)Rs, which was absent in δ-GABA(A)Rs. This was explained by AA29504 shifting the low-affinity γ(2)-GABA(A)R towards a higher affinity desensitized state, thereby rising new sites capable of binding GABA(A)R agonists with low nanomolar affinity. Hence, the potential of AA29504 to act as a desensitization-modifying allosteric modulator of γ2-GABA(A)Rs deserves further investigation for its promising influence on shaping efficacy, duration and plasticity of GABA(A)R synaptic responses.