Low nanomolar GABA effects at extrasynaptic α4β1/β3δ GABAA receptor subtypes indicate a different binding mode for GABA at these receptors

Ionotropic GABA(A) receptors are a highly heterogenous population of receptors assembled from a combination of multiple subunits. The aims of this study were to characterize the potency of GABA at human recombinant delta-containing extrasynaptic GABA(A) receptors expressed in Xeno pus oocytes using the two-electrode voltage clamp technique, and to investigate, using site-directed mutagenesis, the molecular determinants for GABA potency at alpha 4 beta 3 delta GABA(A) receptors. alpha 4/delta-Containing GABA(A) receptors displayed high sensitivity to GABA, with mid-nanomolar concentrations activating alpha 4 beta 1 delta (EC50 = 24 nM) and alpha 4 beta 3 delta (EC50 = 12 nM) receptors. In the majority of oocytes expressing alpha 4 beta 3 delta subtypes, GABA produced a biphasic concentration-response curve, and activated the receptor with low and high concentrations (EC50(1) = 16 nM; EC50(2) = 1.2 mu M). At alpha 4 beta 2 delta, GABA had low micromolar activity (EC50 = 1 mu M). An analysis of 10 N-terminal singly mutated alpha 4 beta 3 delta receptors shows that GABA interacts with amino acids different to those reported for alpha 1 beta 2 gamma 2 GABA(A) receptors. Residues Y205 and R207 of the beta 3-subunit significantly affected GABA potency, while the residue F71 of the alpha 4- and the residue Y97 of the beta 3-subunit did not significantly affect GABA potency. Mutating the residue R218 of the delta-subunit, equivalent to the GABA binding residue R207 of the beta 2-subunit, reduced the potency of GABA by 670-fold, suggesting a novel GABA binding site at the delta-subunit interface. Taken together, GABA may have different binding modes for extrasynaptic delta-containing GABA(A) receptors compared to their synaptic counterparts. (C) 2012 Elsevier Inc. All rights reserved.