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 GABAA 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 δ-containing extrasynaptic GABAA 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 α4β3δ GABAA receptors. α4/δ-Containing GABAA receptors displayed high sensitivity to GABA, with mid-nanomolar concentrations activating α4β1δ (EC50 = 24 nM) and α4β3δ (EC50 = 12 nM) receptors. In the majority of oocytes expressing α4β3δ 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 μM). At α4β2δ, GABA had low micromolar activity (EC 50 = 1 μM). An analysis of 10 N-terminal singly mutated α4β3δ receptors shows that GABA interacts with amino acids different to those reported for α1β2γ2 GABAA receptors. Residues Y205 and R207 of the β3-subunit significantly affected GABA potency, while the residue F71 of the α4- and the residue Y97 of the β3-subunit did not significantly affect GABA potency. Mutating the residue R218 of the δ-subunit, equivalent to the GABA binding residue R207 of the β2-subunit, reduced the potency of GABA by 670-fold, suggesting a novel GABA binding site at the δ-subunit interface. Taken together, GABA may have different binding modes for extrasynaptic δ-containing GABA A receptors compared to their synaptic counterparts.
AB - Ionotropic GABAA 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 δ-containing extrasynaptic GABAA 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 α4β3δ GABAA receptors. α4/δ-Containing GABAA receptors displayed high sensitivity to GABA, with mid-nanomolar concentrations activating α4β1δ (EC50 = 24 nM) and α4β3δ (EC50 = 12 nM) receptors. In the majority of oocytes expressing α4β3δ 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 μM). At α4β2δ, GABA had low micromolar activity (EC 50 = 1 μM). An analysis of 10 N-terminal singly mutated α4β3δ receptors shows that GABA interacts with amino acids different to those reported for α1β2γ2 GABAA receptors. Residues Y205 and R207 of the β3-subunit significantly affected GABA potency, while the residue F71 of the α4- and the residue Y97 of the β3-subunit did not significantly affect GABA potency. Mutating the residue R218 of the δ-subunit, equivalent to the GABA binding residue R207 of the β2-subunit, reduced the potency of GABA by 670-fold, suggesting a novel GABA binding site at the δ-subunit interface. Taken together, GABA may have different binding modes for extrasynaptic δ-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
SN - 0006-2952
VL - 84
SP - 549
EP - 557
JO - Biochemical Pharmacology
JF - Biochemical Pharmacology
IS - 4
ER -