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

N. Absalom; N. Karim; L.F. Eghorn; I.S. Villumsen; T. Bay; H. Bräuner-Osborne; B. Frølund; R.P. Clausen; J.V. Olsen; G.M. Knudsen; M. Chebib; P. Wellendorph

doi:10.1073/pnas.1204376109

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

N. Absalom, N. Karim, L.F. Eghorn, I.S. Villumsen, T. Bay, H. Bräuner-Osborne, B. Frølund, R.P. Clausen, J.V. Olsen, G.M. Knudsen, M. Chebib, P. Wellendorph

Novo Nordisk Foundation Center for Protein Research

70 Citations (Scopus)

Abstract

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 _A receptor subunits as possible candidates. A subsequent functional screening of various recombinant GABA _A 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 ₅₀ = 140 nM) over α4β(2/3)δ (EC ₅₀ = 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 [ ³H](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 _A receptors and postulate a role for extrasynaptic α4β-containing GABA _A 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.

Original language	English
Journal	Proceedings of the National Academy of Sciences USA (PNAS)
Volume	109
Issue number	33
Pages (from-to)	13404-13409
Number of pages	6
ISSN	0027-8424
DOIs	https://doi.org/10.1073/pnas.1204376109
Publication status	Published - 14 Aug 2012

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Absalom, N., Karim, N., Eghorn, L. F., Villumsen, I. S., Bay, T., Bräuner-Osborne, H., Frølund, B., Clausen, R. P., Olsen, J. V., Knudsen, G. M., Chebib, M., & Wellendorph, P. (2012). α4βδ GABA receptors are high-affinity targets for γ-hydroxybutyric acid (GHB). Proceedings of the National Academy of Sciences USA (PNAS), 109(33), 13404-13409. https://doi.org/10.1073/pnas.1204376109

Absalom, N, Karim, N, Eghorn, LF, Villumsen, IS, Bay, T, Bräuner-Osborne, H , Frølund, B , Clausen, RP , Olsen, JV , Knudsen, GM, Chebib, M & Wellendorph, P 2012, 'α4βδ GABA receptors are high-affinity targets for γ-hydroxybutyric acid (GHB)', Proceedings of the National Academy of Sciences USA (PNAS), vol. 109, no. 33, pp. 13404-13409. https://doi.org/10.1073/pnas.1204376109

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title = "α4βδ GABA receptors are high-affinity targets for γ-hydroxybutyric acid (GHB)",

abstract = "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 A receptor subunits as possible candidates. A subsequent functional screening of various recombinant GABA A 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 50 = 140 nM) over α4β(2/3)δ (EC 50 = 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 [ 3H](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 A receptors and postulate a role for extrasynaptic α4β-containing GABA A 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.",

author = "N. Absalom and N. Karim and L.F. Eghorn and I.S. Villumsen and T. Bay and H. Br{\"a}uner-Osborne and B. Fr{\o}lund and R.P. Clausen and J.V. Olsen and G.M. Knudsen and M. Chebib and P. Wellendorph",

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

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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 A receptor subunits as possible candidates. A subsequent functional screening of various recombinant GABA A 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 50 = 140 nM) over α4β(2/3)δ (EC 50 = 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 [ 3H](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 A receptors and postulate a role for extrasynaptic α4β-containing GABA A 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 A receptor subunits as possible candidates. A subsequent functional screening of various recombinant GABA A 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 50 = 140 nM) over α4β(2/3)δ (EC 50 = 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 [ 3H](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 A receptors and postulate a role for extrasynaptic α4β-containing GABA A 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.

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

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