TY - JOUR

T1 - Low nanomolar GABA effects at extrasynaptic a4ß1/ß3delta GABAA receptor subtypes indicate a different binding mode for GABA at these receptors

AU - Karim, Nasiara

AU - Wellendorph, Petrine

AU - Absalom, Nathan

AU - Bang, Line Haunstrup

AU - Jensen, Marianne Lerbech

AU - Hansen, Maja Michelle

AU - Lee, Ho Joon

AU - Johnston, Graham A R

AU - Hanrahan, Jane R

AU - Chebib, Mary

N1 - Keywords: GABA, Extrasynaptic GABAA receptors, Site-directed mutagenesis, GABA binding site, delta-Subunit, d-Subunit

PY - 2012/8/15

Y1 - 2012/8/15

N2 - 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 d-containing extrasynaptic GABA(A) receptors expressed in Xenopus oocytes using the two-electrode voltage clamp technique, and to investigate, using site-directed mutagenesis, the molecular determinants for GABA potency at a4ß3d GABA(A) receptors. a4/d-Containing GABA(A) receptors displayed high sensitivity to GABA, with mid-nanomolar concentrations activating a4ß1d (EC(50)=24nM) and a4ß3d (EC(50)=12nM) receptors. In the majority of oocytes expressing a4ß3d subtypes, GABA produced a biphasic concentration-response curve, and activated the receptor with low and high concentrations (EC(50)(1)=16nM; EC(50)(2)=1.2µM). At a4ß2d, GABA had low micromolar activity (EC(50)=1µM). An analysis of 10 N-terminal singly mutated a4ß3d receptors shows that GABA interacts with amino acids different to those reported for a1ß2¿2 GABA(A) receptors. Residues Y205 and R207 of the ß3-subunit significantly affected GABA potency, while the residue F71 of the a4- and the residue Y97 of the ß3-subunit did not significantly affect GABA potency. Mutating the residue R218 of the d-subunit, equivalent to the GABA binding residue R207 of the ß2-subunit, reduced the potency of GABA by 670-fold, suggesting a novel GABA binding site at the d-subunit interface. Taken together, GABA may have different binding modes for extrasynaptic d-containing GABA(A) receptors compared to their synaptic counterparts.

AB - 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 d-containing extrasynaptic GABA(A) receptors expressed in Xenopus oocytes using the two-electrode voltage clamp technique, and to investigate, using site-directed mutagenesis, the molecular determinants for GABA potency at a4ß3d GABA(A) receptors. a4/d-Containing GABA(A) receptors displayed high sensitivity to GABA, with mid-nanomolar concentrations activating a4ß1d (EC(50)=24nM) and a4ß3d (EC(50)=12nM) receptors. In the majority of oocytes expressing a4ß3d subtypes, GABA produced a biphasic concentration-response curve, and activated the receptor with low and high concentrations (EC(50)(1)=16nM; EC(50)(2)=1.2µM). At a4ß2d, GABA had low micromolar activity (EC(50)=1µM). An analysis of 10 N-terminal singly mutated a4ß3d receptors shows that GABA interacts with amino acids different to those reported for a1ß2¿2 GABA(A) receptors. Residues Y205 and R207 of the ß3-subunit significantly affected GABA potency, while the residue F71 of the a4- and the residue Y97 of the ß3-subunit did not significantly affect GABA potency. Mutating the residue R218 of the d-subunit, equivalent to the GABA binding residue R207 of the ß2-subunit, reduced the potency of GABA by 670-fold, suggesting a novel GABA binding site at the d-subunit interface. Taken together, GABA may have different binding modes for extrasynaptic d-containing GABA(A) receptors compared to their synaptic counterparts.

U2 - 10.1016/j.bcp.2012.05.017

DO - 10.1016/j.bcp.2012.05.017

M3 - Journal article

C2 - 22658986

VL - 84

SP - 549

EP - 557

JO - Biochemical Pharmacology

JF - Biochemical Pharmacology

SN - 0006-2952

IS - 4

ER -