Biochem Pharmacol 2018 Apr 01;150:305-319. doi: 10.1016/j.bcp.2018.02.015.

Delineation of the functional properties and the mechanism of action of AA29504, an allosteric agonist and positive allosteric modulator of GABAA receptors.

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The retigabine analog 2-amino-4-[(2,4,6-trimethylbenzylamino)-phenyl]-carbamic acid ethyl ester (AA29504) is a positive allosteric modulator (PAM) of γ-aminobutyric acidA receptors (GABAARs), and the modulator has been used in ex vivo/in vivo studies to probe the physiological roles of native δ-containing GABAARs. In this study, the functional properties and mode of action of AA29504 were investigated at human GABAARs expressed in Xenopus oocytes by two-electrode voltage clamp electrophysiology. AA29504 was found to be an allosteric GABAAR agonist displaying low intrinsic activities at 3-30 μM. AA29504 was essentially equipotent as a PAM at the 13 GABAAR subtypes tested (EC50: 0.45-5.2 μM), however GABA EC5-evoked currents through αβδ subtypes were modulated to substantially higher levels than those through αβγ2S subtypes (relative to GABA Imax). While the δ/γ2S-difference clearly was key for this differential GABA efficacy modulation, studies of the AA29504-mediated modulation of different α4,5,6-containing αβ, αβγ2S and αβδ GABAARs revealed the α-subunit identity to be another important determinant. Based on its functional properties at numerous mutant GABAARs and on in silico analysis of its low-energy conformations, AA29504 is proposed to act through an allosteric site in the transmembrane β(+)/α(-) interface in the GABAAR also targeted by etomidate and several other modulators. In contrast to these modulators, however, AA29504 did not display substantial β2/β3-over-β1 GABAAR preference, which challenges the notion of ligands targeting this site always possessing this subtype-selectivity profile. Hence, the detailed pharmacological profiling of AA29504 both highlights the complexity of allosteric GABAAR modulation and provides valuable information about this modulator as a pharmacological tool.

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Species referenced: Xenopus laevis
Genes referenced: gabarap pam