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Cloning and functional characterization of a gamma-hydroxybutyrate receptor identified in the human brain

Institut de Chimie Biologique and INSERM U-575, Faculty of Medicine, Strasbourg, France

¹Correspondence: Faculty of Medicine and INSERM U-575, 11, rue Humann, 67085, Strasbourg Cedex, France. E-mail: maitre{at}neurochem.u-strasbg.fr

Two parent clones of a -hydroxybutyrate (GHB) receptor, C12K32 and GHBh1, were isolated from a human frontal cortex cDNA library. The two clones differ by a deleted cytosine in C12K32. CHO cells transfected with either C12K32 or GHBh1 responded positively to submicromolar GHB stimulation. However, unlike C12K32, GHBh1 desensitizes rapidly on application of low concentrations of GHB. GHB receptor properties were then studied on C12K32 expressed in CHO cells. C12K32 bound GHB with a K_d of 114 nM and has no affinity for GABA or glutamate. GHB and NCS-382 displaced [³H]GHB with an IC₅₀ of 53 ± 8 and 120 ± 18 nM, respectively. In patch-clamp experiments, GHB induced a dose-dependent response with an EC₅₀ of 130 nM. This response was antagonized by NCS-382, was not reproduced by GABA, and was sensitive to the addition of GTP--S in the recording pipette. CHO cells transfected with C12K32 exhibited GTP-³⁵S binding with an EC₅₀ of 462 nM for GHB and an IC₅₀ of 2.9 µM for NCS-382. The present data led to the conclusion that both C12K32 and GHBh1 are two closely related isoforms of a human GHB receptor, GHBh1, that is described in the databank as the GPCR 172A—Andriamampandry, C., Taleb, O., Kemmel, V., Humbert, J.-P., Aunis, D., Maitre, M. Cloning and functional characterization of a gamma-hydroxybutyrate receptor identified in the human brain.

Key Words: G-protein coupled receptors • electrophysiological characterization • functional expression • GHB