Novel Radioiodinated γ-Hydroxybutyric Acid Analogues for Radiolabeling and Photolinking of High-Affinity γ-Hydroxybutyric Acid Binding Sites

Abstract

␥-Hydroxybutyric acid (GHB) is a therapeutic drug, a drug of abuse, and an endogenous substance that binds to low-and high-affinity sites in the mammalian brain. I]BnOPh-GHB) binds to one site in rat brain cortical membranes with low nanomolar affinity (K d , 7 nM; B max , 61 pmol/mg protein). The binding is inhibited by GHB and selected analogs, but not by ␥-aminobutyric acid. Autoradiography using horizontal slices from rat brain demonstrates the highest density of binding in hippocampus and cortical regions and the lowest density in the cerebellum. Altogether, the findings correlate with the labeling and brain regional distribution of highaffinity GHB sites or [

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

“…Using an engineered GHB photoaffinity ligand (28), we previously determined the high-affinity GHB binding protein to be ∼50 kDa (8). In attempt to provide better validity in the identifications made by the proteomics analysis, we also subjected samples to limited proteolysis by timedependent treatment with proteinase K, which resulted in minor bands of ∼28, ∼21, and ∼18 kDa ( Fig.…”

“…The reaction was terminated by rapid filtration through GF/ C unifilters, and radioactivity was measured on a Packard TopCount NXT Microplate Scintillation Counter (PerkinElmer). Autoradiography was performed as previously reported (8), except that the incubation buffer was 50 mM Tris·HCl buffer (pH 7.4). In brief, horizontal brain slices (20 μm; bregma = 4.5-4.8 mm) (51) from male Sprague-Dawley rats (∼250 g; Charles River) were preincubated for 30 min in buffer and then incubated at room temperature for 30 min with 100 pM [ 125 I]BnOPh-GHB in the absence (total binding) or presence of competing compound.…”

“…The photoaffinity radiolabeling with [ 125 I]azido-BnOPh-GHB of rat membrane was carried out exactly as previously described (8,28) and treated with proteinase K (Sigma-Aldrich) and denaturing gel electrophoresis. The isolated gel bands were prepared for liquid chromatography-MS. Additional details in SI Materials and Methods.…”

“…In addition to the validated GABA B receptor effects and other suggested receptors (7), GHB binds with nanomolar to micromolar affinity to a remarkably abundant protein of distinct spatial distribution and ontogenesis (4), representing an additional functional target. Interestingly, this high-affinity binding protein is preserved in GABA B(1) KO mice (6) and can be specifically probed with BnOPh-GHB) (8). Furthermore, several reports point to GHBinduced effects that cannot be consequences of GABA B receptor activation alone: Fos expression studies with GHB indicate a unique pattern of neuronal activation, which in several ways, is different from the pattern produced by the GABA B receptor agonist baclofen (9).…”

“…In this study, we have exploited an in-house-developed, highaffinity, and selective GHB photoligand (8,27,28) to cross-link and partially purify the high-affinity GHB binding protein from rat brain cortex, with several GABA A subunits emerging as candidate proteins. Functional studies in Xenopus laevis oocytes and radioligand binding studies in KO mouse brain tissue verified α4βδ-receptors as high-affinity targets for GHB.…”