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

Nasiara Karim, Petrine Wellendorph, Nathan Absalom, Line Haunstrup Bang, Marianne Lerbech Jensen, Maja Michelle Hansen, Ho Joon Lee, Graham A R Johnston, Jane R Hanrahan, Mary Chebib

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    36 Citationer (Scopus)

    Abstract

    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.
    OriginalsprogEngelsk
    TidsskriftBiochemical Pharmacology
    Vol/bind84
    Udgave nummer4
    Sider (fra-til)549-557
    ISSN0006-2952
    DOI
    StatusUdgivet - 15 aug. 2012

    Bibliografisk note

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

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