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 -