Low nanomolar GABA effects at extrasynaptic α4β1/β3δ GABA(A) 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 δ-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 α4β3δ GABA(A) receptors. α4/δ-Containing GABA(A) 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 (EC₅₀=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 GABA(A) 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.
Faculty of Pharmacy, Department of Pharmacology, University of Sydney, Sydney, NSW 2006, Australia. nasiara.karim@sydney.edu.au
Nasiara
N
Karim
Petrine
P
Wellendorph
Nathan
N
Absalom
Line Haunstrup
LH
Bang
Marianne Lerbech
ML
Jensen
Maja Michelle
MM
Hansen
Ho Joon
HJ
Lee
Graham A R
GA
Johnston
Jane R
JR
Hanrahan
Mary
M
Chebib
D000595:Amino Acid Sequence; D000818:Animals; D005260:Female; D006801:Humans; D008969:Molecular Sequence Data; D016297:Mutagenesis, Site-Directed; D009154:Mutation; D009865:Oocytes / Q000378:metabolism; D018408:Patch-Clamp Techniques; D021122:Protein Subunits / Q000235:genetics / Q000378:metabolism; D011963:Receptors, GABA-A / Q000235:genetics / Q000378:metabolism*; D011994:Recombinant Proteins / Q000235:genetics / Q000378:metabolism; D013569:Synapses / Q000378:metabolism*; D014982:Xenopus laevis; D005680:gamma-Aminobutyric Acid / Q000378:metabolism*
D021122:Protein Subunits; D011963:Receptors, GABA-A; D011994:Recombinant Proteins; D005680:gamma-Aminobutyric Acid