TY - JOUR

T1 - α4βδ GABA receptors are high-affinity targets for γ-hydroxybutyric acid (GHB)

AU - Absalom, N.

AU - Karim, N.

AU - Eghorn, L.F.

AU - Villumsen, I.S.

AU - Bay, T.

AU - Bräuner-Osborne, H.

AU - Frølund, B.

AU - Clausen, R.P.

AU - Olsen, J.V.

AU - Knudsen, G.M.

AU - Chebib, M.

AU - Wellendorph, P.

N1 - Keywords: gamma-hydroxybutyric acid receptor, gamma-hydroxybutyric acid high-affinity binding sites, alpha4-subunit knockout, photoaffinity ligand

PY - 2012/8/14

Y1 - 2012/8/14

N2 - γ-Hydroxybutyric acid (GHB) binding to brain-specific high-affinity sites is well-established and proposed to explain both physiological and pharmacological actions. However, the mechanistic links between these lines of data are unknown. To identify molecular targets for specific GHB high-affinity binding, we undertook photolinking studies combined with proteomic analyses and identified several GABA receptor subunits as possible candidates. A subsequent functional screening of various recombinant GABA receptors in Xenopus laevis oocytes using the two-electrode voltage clamp technique showed GHB to be a partial agonist at αβδ- but not αβγ-receptors, proving that the δ-subunit is essential for potency and efficacy. GHB showed preference for α4 over α(1,2,6)-subunits and preferably activated α4β1δ (EC = 140 nM) over α4β(2/3)δ (EC = 8.41/1.03 mM). Introduction of a mutation, α4F71L, in α4β 1(δ)-receptors completely abolished GHB but not GABA function, indicating nonidentical binding sites. Radioligand binding studies using the specific GHB radioligand [ H](E,RS)-(6,7,8,9-tetrahydro-5-hydroxy-5H- benzocyclohept-6-ylidene)acetic acid showed a 39% reduction (P = 0.0056) in the number of binding sites in α4 KO brain tissue compared with WT controls, corroborating the direct involvement of the α4-subunit in high-affinity GHB binding. Our data link specific GHB forebrain binding sites with α4-containing GABA receptors and postulate a role for extrasynaptic α4β-containing GABA receptors in GHB pharmacology and physiology. This finding will aid in elucidating the molecular mechanisms behind the proposed function of GHB as a neurotransmitter and its unique therapeutic effects in narcolepsy and alcoholism.

AB - γ-Hydroxybutyric acid (GHB) binding to brain-specific high-affinity sites is well-established and proposed to explain both physiological and pharmacological actions. However, the mechanistic links between these lines of data are unknown. To identify molecular targets for specific GHB high-affinity binding, we undertook photolinking studies combined with proteomic analyses and identified several GABA receptor subunits as possible candidates. A subsequent functional screening of various recombinant GABA receptors in Xenopus laevis oocytes using the two-electrode voltage clamp technique showed GHB to be a partial agonist at αβδ- but not αβγ-receptors, proving that the δ-subunit is essential for potency and efficacy. GHB showed preference for α4 over α(1,2,6)-subunits and preferably activated α4β1δ (EC = 140 nM) over α4β(2/3)δ (EC = 8.41/1.03 mM). Introduction of a mutation, α4F71L, in α4β 1(δ)-receptors completely abolished GHB but not GABA function, indicating nonidentical binding sites. Radioligand binding studies using the specific GHB radioligand [ H](E,RS)-(6,7,8,9-tetrahydro-5-hydroxy-5H- benzocyclohept-6-ylidene)acetic acid showed a 39% reduction (P = 0.0056) in the number of binding sites in α4 KO brain tissue compared with WT controls, corroborating the direct involvement of the α4-subunit in high-affinity GHB binding. Our data link specific GHB forebrain binding sites with α4-containing GABA receptors and postulate a role for extrasynaptic α4β-containing GABA receptors in GHB pharmacology and physiology. This finding will aid in elucidating the molecular mechanisms behind the proposed function of GHB as a neurotransmitter and its unique therapeutic effects in narcolepsy and alcoholism.

UR - http://www.scopus.com/inward/record.url?scp=84865165962&partnerID=8YFLogxK

U2 - 10.1073/pnas.1204376109

DO - 10.1073/pnas.1204376109

M3 - Journal article

C2 - 22753476

VL - 109

SP - 13404

EP - 13409

JO - Proceedings of the National Academy of Sciences of the United States of America

JF - Proceedings of the National Academy of Sciences of the United States of America

SN - 0027-8424

IS - 33

ER -