γ-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. To target the specific GHB binding sites, we have developed a 125I-labeled GHB analog and characterized its binding in rat brain homogenate and slices. Our data show that [125I]4-hydroxy-4-[4-(2-iodobenzyloxy)phenyl]butanoate ([125I]BnOPh-GHB) binds to one site in rat brain cortical membranes with low nanomolar affinity (Kd, 7 nM; Bmax, 61 pmol/mg protein). The binding is inhibited by GHB and selected analogs, but not by γ-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 high-affinity GHB sites or [3H](E,RS)-(6,7,8,9-tetrahydro-5-hydroxy-5H-benzocyclohept-6-ylidene)acetic acid ([3H]NCS-382) binding sites. Using a 125I-labeled photoaffinity derivative of the new GHB ligand, we have performed denaturing protein electrophoresis and detected one major protein band with an apparent mass of 50 kDa from cortical and hippocampal membranes. [125I]BnOPh-GHB is the first reported 125I-labeled GHB radioligand and is a useful tool for in vitro studies of the specific high-affinity GHB binding sites. The related photoaffinity linker [125I]4-hydroxy-4-[4-(2-azido-5-iodobenzyloxy)phenyl]butanoate can be used as a probe for isolation of the elusive GHB binding protein.
γ-Hydroxybutyric acid (GHB) is a structural analog and naturally occurring metabolite of γ-aminobutyric acid (GABA) (Fig. 1). Clinically, the drug is used for treatment of narcolepsy (Robinson and Keating, 2007) or alcohol-withdrawal symptoms (Caputo et al., 2009), but it is also a popular recreational drug occasionally used as a “date rape drug” because of its euphoric, prosocial, relaxing, and sleep-inducing effects (Anderson et al., 2006; Drasbek et al., 2006).
In the central nervous system, GHB possesses weak affinity for GABAB receptors (Mathivet et al., 1997) and high affinity for GHB sites that are distinct from the GABAB receptor (Kaupmann et al., 2003; Wu et al., 2004). Although many of the reported pharmacological and clinical effects of exogenously administered GHB seem to be mediated through GABAB receptors (Carai et al., 2008), the presence of [3H]GHB high-affinity binding sites with distinct ontogenesis (Snead, 1994) and distribution (Castelli et al., 2000; Gould et al., 2003) argues in favor of a specific GHB molecular site of action (sometimes referred to as the GHB receptor). There is notable interest in elucidating the functional correlates of this GHB-interacting protein.
The weak partial agonistic effect of GHB at the GABAB receptor was reported more than 10 years ago (Lingenhoehl et al., 1999). The unequivocal role for GABAB receptors in mediating important pharmacological GHB effects has since been underlined by the lack of effects of GHB on behavior, temperature regulation, locomotion, and dopamine synthesis in mice lacking functional GABAB receptors and otherwise produced in wild-type mice (Kaupmann et al., 2003; Quéva et al., 2003; Carter et al., 2004) and by the notion that GABAB receptor antagonists can block many GHB-induced behavioral effects in normal rats (Carai et al., 2008). However, clear differences also exist between the effects of GHB and the prototypical GABAB receptor agonist baclofen [4-amino-3-(4-chlorophenyl)-butanoic acid], e.g., in relation to the abuse potential and in terms of efficacy of GHB in patients with narcolepsy versus baclofen. Unlike GHB, baclofen is not reported to produce euphoric effects or physical dependence despite widespread clinical use (Nicholson and Balster, 2001), and rats can learn to discriminate GHB from baclofen (Carter et al., 2004; Wu et al., 2004). In narcolepsy, GHB, but not baclofen, reduces daytime sleepiness and cataplexy in humans (Huang and Guilleminault, 2009), and selective GHB ligands have been reported to reduce ataxia and cataplexy in rats by a mechanism partly unrelated to the activation of GABAB receptors (Carter et al., 2005). Altogether, this suggests that the high-affinity GHB binding sites contribute to the biological effects of GHB. Further support of a mechanism of action of GHB unrelated to GABAB receptors, and convincing proof that GHB and GABAB binding sites are separate entities, comes from studies showing that [3H]GHB and [3H](E,RS)-(6,7,8,9-tetrahydro-5-hydroxy-5H-benzocyclohept-6-ylidene)acetic acid ([3H]NCS-382) binding sites are preserved in the brains of GABAB1 receptor knockout mice (Kaupmann et al., 2003; Wu et al., 2004).
To specifically target the high-affinity GHB sites, we have generated analogs of GHB that lack affinity at GABAB receptors (Wellendorph et al., 2005; Høg et al., 2008). These features have been evaluated in rat cortical homogenate binding assays using the commercially available radiolabeled NCS-382 (Fig. 1) and at GABA receptors. [3H]NCS-382 is used to specifically label GHB binding sites over GABA sites because it displays nanomolar affinity for GHB binding sites but has no affinity for GABAB receptors (Mehta et al., 2001; Castelli et al., 2002; Kaupmann et al., 2003). Furthermore, [3H]NCS-382 displays a regional distribution of binding sites in the brain that is convincingly overlapping with high-affinity [3H]GHB sites, characterized by a particularly high density in the hippocampus and cortex and a very low density in the cerebellum, which is also markedly different from the distribution of GABAB receptors (Snead, 1996; Castelli et al., 2000; Gould et al., 2003; Kaupmann et al., 2003; Wu et al., 2004).
With the aim of isolating the elusive GHB binding sites we here report studies on novel radiolabeled GHB analogs with optimized properties for effective radiolabeling and photolinking, i.e., containing high specific radioactivity from the presence of the 125I radioisotope and displaying single-digit nanomolar affinity. Such criteria have previously been proven crucial in the design of radioligands used for the isolation of their respective binding proteins, such as in the cloning of the GABAB receptor (Kaupmann et al., 1997; Froestl et al., 2003).
Materials and Methods
Compounds and Radioligands.
Compounds were purchased from Sigma-Aldrich (St. Louis, MO), except for NCS-382, which was from Tocris Bioscience (Bristol, UK). Unlabeled lithium 4-hydroxy-4-[4-(2-iodobenzyloxy)phenyl]butanoate (BnOPh-GHB) was prepared in-house as described previously (compound 17b in Høg et al., 2008). The radioligands [125I]4-hydroxy-4-[4-(2-iodobenzyloxy)phenyl]butanoate ([125I]BnOPh-GHB) and [125I]4-hydroxy-4-[4-(2-azido-5-iodobenzyloxy)phenyl]butanoate ([125I]azido-BnOPh-GHB) were synthesized from their corresponding tributylstannyl precursors by direct iodination as described previously (Sabbatini et al., 2010). Both radioligands had radioactive purities more than 95%. Whereas [125I]BnOPh-GHB had an estimated specific activity of 2000 Ci/mmol, [125I]azido-BnOPh-GHB contained traces of cold [127I]azido-BnOPh-GHB and thus had a moderate specific activity of approximately 2 Ci/mmol. Each radioligand batch was used within 120 days (two half-lives).
Binding assays were performed by using synaptic membranes of cerebral cortex from adult male Sprague-Dawley rats with tissue preparation as described previously (Ransom and Stec, 1988). On the day of the assay, the membrane preparation was quickly thawed, suspended in 40 volumes of ice-cold incubation buffer (50 mM KH2PO4 buffer, pH 6.0) using an UltraTurrax homogenizer (IKA Works, Inc., Wilmington, NC), and centrifuged at 48,000g for 10 min at 4°C. This washing step was repeated four times to remove endogenous GHB. The final pellet was resuspended in the incubation buffer.
[125I]BnOPh-GHB Binding Assay.
A binding assay for [125I]BnOPh-GHB in rat cerebral cortical homogenate was modified from the [3H]NCS-382 binding assay protocol described previously (Mehta et al., 2001; Wellendorph et al., 2005). In brief, well washed membrane suspensions (approximately 5 μg of protein/aliquot) were incubated with [125I]BnOPh-GHB in 50 mM phosphate buffer, pH 6, with or without competing ligand for 1 h at room temperature in a total volume of 2 ml. Protein amount and volume were adjusted so that total bound radioligand accounted for less than 10% of totally added radioactivity in the reaction. Nonspecific binding was defined by inclusion of 1 mM unlabeled GHB. The binding reaction was terminated by rapid filtration through Whatman GF/C filter (PerkinElmer Life and Analytical Sciences, Waltham, MA) using a Brandel Inc. (Gaithersburg, MD) M-48R cell harvester, followed by washing with 3 × 3 ml of ice-cold incubation buffer. For dissociation experiments, the radioligand was allowed to bind to the membranes for 1 h, and dissociation was initiated by the addition of 20 μl of GHB (final concentration of 1 mM). The amount of filter-bound radioactivity was quantified by adding 3 ml of scintillation fluid (Opti-Fluor; PerkinElmer Life and Analytical Sciences) to the dried filters and counting in a Tri-Carb 2100 liquid scintillation counter (PerkinElmer Life and Analytical Sciences) (energy range 0 to 70 kEV) (Horrocks, 1976), and expressed as disintegrations per minute (dpm).
For determination of IC50 values, 100 pM [125I]BnOPh-GHB was used in the absence and presence of varying concentrations of displacer, whereas saturation isotherms (Kd and Bmax) were measured by means of radioisotope dilution by addition of varying amounts of unlabeled BnOPh-GHB ranging from 2 to 80 nM in the incubation mixture containing [125I]BnOPh-GHB (1 nM). The nonspecific binding was determined by using 1 mM GHB, and the obtained linear relationship was extrapolated to calculate the nonspecific binding at higher radioligand concentrations. Typical competition binding assays yielded 95 to 97% specific binding with approximately 25,000 dpm total binding and 1000 dpm nonspecific binding. For protein content determination we used bovine serum albumin as a standard; the Bradford protein assay was used according to the protocol of the supplier (Bio-Rad Laboratories, Hercules, CA).
Cryostat Sectioning and Autoradiography.
Brains from male Sprague-Dawley rats (250–300 g; Charles River, Sulzfeld, Germany) were sectioned on a cryostat in 20-μm horizontal sections at −25°C (corresponding to bregma 4.5–4.8 mm) (Paxinos and Watson, 1998), thaw-mounted onto gelatinized glass slides, allowed to dry, and stored at −80°C until analyzed. [125I]BnOPh-GHB autoradiography was carried out by using 50 mM phosphate buffer as in the homogenate binding studies. Frozen tissue sections were warmed to room temperature, then preincubated for 30 min at room temperature in phosphate buffer under constant gentle shaking. Sections were incubated in the same buffer containing 100 pM [125I]BnOPh-GHB for 30 min at room temperature to determine total binding. Sections used for nonspecific binding were incubated in the presence of GHB, NCS-382, BnOPh-GHB, or GABA at the concentrations specified. Sections were washed for 2 × 20 s in ice-cold incubation buffer followed by 20 s in ice-cold double-distilled H2O. Slides were dried at room temperature in a gentle airstream for 1 h. They were then fixed in paraformaldehyde vapor overnight at 4°C. The sections were dried for 3 h in a dessicator at room temperature, followed by exposure to a 125I-sensitive BAS-2040 phosphor imaging plate (Science Imaging Scandinavia AB, Nacka, Sweden) for 24 to 48 h at 4°C. The imaging plate was scanned on a BAS-2500 bioimaging analyzer (Fujifilm Europe GmbH, Düsseldorf, Germany). Autoradiograms were analyzed with ImageJ V.1.38j (http://rsbweb.nih.gov/ij).
Photoaffinity Labeling with [125I]azido-BnOPh-GHB, SDS-Polyacrylamide Gel Electrophoresis, and Autoradiography.
The photoaffinity radiolabeling with [125I]azido-BnOPh-GHB was carried out by using a protocol similar to that for the [125I]BnOPh-GHB binding assay and directed by initially performed investigations on the photolinking properties as described elsewhere (Sabbatini et al., 2010). Washed rat brain cortical membranes (30 μg per assay point) were incubated in the dark with 100 nM total concentration [125I]azido-BnOPh-GHB ± displacer in a total volume of 200 μl for 1 h at room temperature with shaking. To minimize nonspecific binding, Protein LoBind tubes (Eppendorf AG, Hamburg, Germany) were used. To remove unbound from bound radiolabel and limit dimerization of the photoligand, the suspension was centrifuged at 21,000g for 2 min at 4°C, and the pellet was washed twice in ice-cold buffer using two extra centrifugation steps. The pellet was resuspended in 200 μl of ice-cold buffer and illuminated with UV light (302 nm, 25 W) for 1 min followed by centrifugation for 2 min at 4°C. Pelleted, photolinked membrane homogenate was washed three times with ice-cold buffer by repeated centifugations at 4°C for 2 min each. After the final washing step, the supernatant was carefully removed. The pellet was suspended in 8 μl of 4× Nupage LDS sample buffer (Invitrogen, Paisley, UK), 1 μl of 10× Nupage sample reducing agent (Invitrogen), and 1 μl of dithiothreitol (final concentration 100 mM). Samples were not boiled but simply mixed by pipetting at room temperature. Proteins were separated on a 4 to 12% gradient gel (Invitrogen) by eluting at 180 V for 50 min along with 14C-methylated molecular mass markers (CFA.626; GE Healthcare, Little Chalfont, Buckinghamshire, UK). The gel was fixed and dried overnight at room temperature. The dried gel was exposed to a BAS-2040 imaging plate (Science Imaging Scandinavia AB) for 2 to 3 h and scanned by phosphorimaging in a BAS 2500 scanner (Fujifilm Europe).
The data are expressed as mean ± S.E.M. The binding data were analyzed by nonlinear regression curve-fitting using Prism 5 (GraphPad Software Inc., San Diego, CA). To determine the maximal number of binding sites, Bmax, and the dissociation constant of the radioligand Kd, saturation data were fitted to a one-site model: specific bound = Bmax/[1+ (Kd/[L])], where [L] is the concentration of free radioligand. Kinetic data were fitted by using models for exponential association and dissociation, yielding the on rate and off rate, respectively, as the time for specific binding of the radioligand to reach 50% of its equilibrium level or for 50% of the bound radioligand to dissociate. Competition data were fitted to the equation: % bound = 100% bound/ (1 + ([I]/IC50), where [I] is the concentration of the inhibitor. Ki values were determined by using the equation Ki = IC50/(1 + [L]/Kd), where [L] is the actually used radioligand concentration.
Generation of [125I]BnOPh-GHB.
In the development of [125I]BnOPh-GHB, we have investigated previously a vast number of GHB analogs for affinity to [3H]NCS-382 sites (Wellendorph et al., 2005, 2009; Høg et al., 2008), aimed at generating a low nanomolar affinity ligand lacking affinity for GABAA and GABAB receptors. The iodine-substituted compound 4-hydroxy-4-[4-(2-iodobenzyloxy)phenyl]butanoate (17b in Høg et al., 2008) fulfilled these criteria best and, consequently, the corresponding 125I-labeled analog referred to as [125I]BnOPh-GHB (Fig. 1) was therefore chosen.
Development of a [125I]BnOPh-GHB Binding Assay.
The protocol for [125I]BnOPh-GHB binding to rat membranes was based on a previously established filtration protocol for the commercially available radioligand [3H]NCS-382 (Wellendorph et al., 2005). Initially, different concentrations of [125I]BnOPh-GHB and protein were investigated. Binding of 100 pM [125I]BnOPh-GHB was linear with increasing protein concentrations of the tissue membranes up to 25 μg/assay point (data not shown). We observed no binding to filters or plastic. To avoid ligand depletion, the protein amount had to be adjusted to 5 μg of membrane protein per assay tube.
The time course of [125I]BnOPh-GHB association to membrane-bound receptors is shown in Fig. 2 A. At room temperature, 50% of total binding occurred within 3 min and equilibrium was reached after 15 to 20 min. At 2 h the binding had decreased by approximately 30% (Fig. 2A). All subsequent incubations were carried out for 1 h to ensure full equilibrium. Dissociation of bound [125I]BnOPh-GHB was fast with 50% dissociation taking place within 1 to 1.5 min (Fig. 2A). To avoid unbinding during termination of the binding reaction, we therefore used fast filtration with ice-cold buffer. With these optimized conditions, we obtained a robust binding assay for [125I]BnOPh-GHB with more than 95% specific binding when using 1 mM GHB for determination of nonspecific binding.
Determination of Kd and Bmax Values.
Saturation analysis of [125I]BnOPh-GHB binding in cortical membranes gave a Kd of 7 nM and a Bmax of 61 pmol/mg protein (Fig. 2B; Table 1). Under our experimental conditions, the saturation isotherm for [125I]BnOPh-GHB was only reliably fitted by using a one-site model. This is also consistent with previous data using [3H]NCS-382 binding to either rat or mouse brain membranes (Kaupmann et al., 2003; Wellendorph et al., 2005). All generated inhibition curves were therefore fitted by using a one-site model.
Inhibition of [125I]BnOPh-GHB Binding.
We next tested the ability of several GHB ligands to displace [125I]BnOPh-GHB binding from cortical membranes. GHB, NCS-382, and BnOPh-GHB (Fig. 1) all inhibited [125I]BnOPh-GHB binding in a concentration-dependent manner, and the rank order of affinity was BnOPh-GHB > NCS-382 > GHB (Fig. 2C), with derived Ki values given in Table 2. Other reported GHB ligands trans-4-hydroxycrotonic acid (Hechler et al., 1990) and succinate (Molnár et al., 2006; Wellendorph et al., 2009) inhibited more than 90% of the [125I]BnOPh-GHB specific binding at 100 μM, whereas addition of GABA and (R)-baclofen had negligible effect, up to a concentration of 1 mM (data not shown).
Brain Regional Distribution Using Autoradiography.
The regional distribution of [125I]BnOPh-GHB binding was assessed qualitatively by autoradiography in rat brain slices, primarily to confirm the known distribution of cerebral binding: a high density of binding sites in cerebral cortex and hippocampus compared with cerebellum, horizontal sections of rat brains at a level (bregma 4.5–4.8 mm) that would allow such an overview (Paxinos and Watson, 1998). As illustrated in Fig. 3 , it is evident that [125I]BnOPh-GHB labels all layers of the frontal cortex and hippocampus with high intensity and the cerebellum with low intensity. Specific radioligand binding was not displaced by GABA but it was clearly decreased in the presence of GHB, NCS-382, or BnOPh-GHB (Fig. 3).
Photoaffinity Labeling of the Specific GHB Binding Sites with [125I]azido-BnOPh-GHB.
To allow for cross-linking of the specific GHB binding site, we designed a photoaffinity ligand, [125I]azido-BnOPh-GHB, based on the scaffold structure of [125I]BnOPh-GHB (Fig. 1). We initially examined the kinetics and photolinking properties of the radioligand and found an unchanged kinetic profile compared with [125I]BnOPh-GHB and an ability of the ligand to become covalently linked to protein upon irradiation with UV light of 302 nm for 1 min (Sabbatini et al., 2010).
We then performed photolinking studies by using rat membrane homogenate followed by SDS-polyacrylamide gel electrophoresis and autoradiography to examine the selectivity of [125I]azido-BnOPh-GHB for the specific GHB binding sites and determine the molecular masses of any labeled proteins. In Fig. 4A, a representative gel autoradiogram is presented showing that [125I]azido-BnOPh-GHB strongly labels a band at approximately 50 kDa that is unaffected by the presence of GABA or the prototypical GABAB receptor-selective agonist, baclofen, but is inhibited by either GHB, BnOPh-GHB, or NCS-382. Next, we examined the regional selectivity for the ∼50-kDa protein and, as expected, found that it is reproducibly photoaffinity-labeled in cortex and hippocampus but not in cerebellum (Fig. 4B).
Despite convincing identification of a high-affinity binding site for GHB in the mammalian brain, inherently distinct from the GABAB receptor (Kaupmann et al., 2003; Wu et al., 2004), the molecular nature and function of this protein still remains to be elucidated. Reports have emerged identifying GHB receptors in rat and human (Andriamampandry et al., 2003, 2007), but because these proteins are not homologous, on either a protein or functional level, and do not convincingly link the high-affinity GHB binding sites to function, the identity of these clones has been reviewed critically (Bettler et al., 2004; Drasbek et al., 2006; Castelli, 2008). Our current study describes the first 125I-labeled radioligand and the first 125I-labeled photoaffinity ligand for targeting the non-GABAB high-affinity specific GHB binding site. Our developed radioligands have been especially targeted to have affinity for the protein corresponding to earlier profiled [3H]NCS-382 binding sites (Mehta et al., 2001; Gould et al., 2003; Kaupmann et al., 2003; Wellendorph et al., 2005; Høg et al., 2008), thus clearly not equivalent to the reported GHB receptor in rat, which lacks NCS-382 sensitivity (Andriamampandry et al., 2003).
[125I]BnOPh-GHB was found to bind reversibly, saturably, and with high affinity (Kd 7 nM) to specific GHB binding sites in rat cortical homogenate. The specific signal was extremely robust (> 95% of the total binding) at concentrations commonly used in the present study (100 pM–1 nM). Kinetic analyses for the association of the radiolabel confirmed that [125I]BnOPh-GHB bound in a reversible and time-dependent manner. Equilibrium was reached within 15 to 20 min and remained stable for more than 1 h. At equilibrium, GHB was able to rapidly induce dissociation of bound [125I]BnOPh-GHB (t1/2 of approximately 1–1.5 min) from the GHB binding sites. As we have previously performed binding studies with [3H]NCS-382 using the same buffer and same membrane preparation, the saturation isotherms are directly comparable. Whereas the Bmax values are quite similar, suggesting that the two radioligands indeed label the same protein, [125I]BnOPh-GHB binds with a Kd value 60 times lower than [3H]NCS-382 (Table 1) and is therefore the highest-affinity radioligand described so far for the specific GHB binding sites. Thus, [125I]BnOPh-GHB exhibits superior binding characteristics to the commercially available [3H]NCS-382, both in terms of affinity and sensitivity, the latter caused by the very high specific activity of the 125I label.
In [125I]BnOPh-GHB competition binding, GHB and NCS-382 gave similar inhibition values to those published with [3H]NCS-382 (Mehta et al., 2001; Høg et al., 2008; Wellendorph et al., 2009) (Table 2), indicating that [125I]BnOPh-GHB labels the same site as [3H]NCS-382. However displacement with cold BnOPh-GHB yielded a 7- to 8-fold lower affinity for [125I]BnOPh-GHB compared with [3H]NCS-382.
Preliminary nonquantitative experiments addressing the regional distribution of [125I]BnOPh-GHB binding sites in rat brain slices showed specific and a high density of binding to cortical layers and hippocampus. This binding was not displaceable by GABA, but by GHB, NCS-382, and unlabeled BnOPh-GHB. As expected, cerebellum displayed a negligible degree of specific binding. High cortical and hippocampal binding and low binding in cerebellum is in accordance with other autoradiographic studies using either low concentrations of [3H]GHB (Hechler et al., 1992; Castelli et al., 2000; Kaupmann et al., 2003; Wu et al., 2004) or [3H]NCS-382 (Gould et al., 2003; Wu et al., 2004), and it further corroborates that [125I]BnOPh-GHB is labeling the GHB sites.
Our data show that [125I]BnOPh-GHB is a good candidate as a highly potent and specific marker of GHB binding sites in vitro. BnOPh-GHB seems to be selective for GHB sites over GABA sites because it does not compete for GABAA and GABAB receptors in binding (Høg et al., 2008). The high percentage of specific binding, and the presence of only one band when photolinking the azido-derivative, clearly argues in favor of the specificity of BnOPh-GHB for the high-affinity GHB binding site when applying nanomolar concentrations and thus renders [125I]BnOPh-GHB as a good candidate as a radiolabeled tracer for imaging studies. Because there is accumulating evidence that GHB plays an important role in the sleeping disorder narcolepsy (Robinson and Keating, 2007), and hypotheses have emerged about involvement of GHB in alcoholism (Caputo et al., 2009) and neurodegenerative disorders and depression (Mamelak, 2007, 2009), we propose that suitably radiolabeled BnOPh-GHB derivatives could provide important tools in future in vivo positron emission tomography or single photon emission computed tomography imaging for the characterization of GHB binding sites, their distribution in the human brain, and the relevance for substance abuse and neurological disorders.
Photoaffinity labeling of rat cortical membranes with [125I]BnOPh-GHB revealed a protein of an approximate molecular mass of 50 kDa. The fact that the band is inhibited by preincubation with cold GHB ligands but not GABA receptor ligands, and that the regional profile matches the autoradiography (Fig. 3), confirms that the intended protein is labeled. The molecular mass supplies very little information as to the possible nature of the GHB binding protein but does rule out similarity to GABAB receptors that are of a much larger size (100 to 130 kDa) (Kaupmann et al., 1997).
In summary, we have developed and characterized a novel radioligand with a desired high affinity for the specific, yet elusive, GHB/NCS-382 binding site in the rat brain. Compared with [3H]NCS-382, our radioligand has the added advantage of a 125I isotope and a single-digit nanomolar affinity, making it an important addition in the toolbox for better understanding the physiological relevance of GHB action in the central nervous system and the therapeutic potential of targeting GHB-sensitive proteins. The additional incorporation of a photoreactive group into the high-affinity 125I-labeled ligand presents a major step in attempting to isolate and clone GHB interacting protein and might be accomplished either by expression cloning or proteomics. The latter will require further purification of the 50-kDa protein by means of two-dimensional gel electrophoresis or affinity column chromatography. Undoubtedly, the photoaffinity linker will be useful for identifying the amino acid residues participating in binding of GHB ligand binding site, at least when a recombinant protein is at hand, and ultimately this knowledge will facilitate long-sought understanding of the structure and function of the molecular targets for GHB.
We thank Drs. Bernhard Bettler and Klemens Kaupmann for fruitful discussions; Mr. Hans Jørgen Jensen and Dr. Cecilie Löe Licht for help with autoradiography; Mr. Shahrokh Padrah for help with synthesis of compounds; Mrs. Birgitte Nielsen for help with UV spectrometry; and Drs. Osman Mirza and Bente Vestergaard for helpful advice regarding protein chemistry.
This work was supported by the Danish Medical Research Council (P.W.), Fonden til Lægevidenskabens Fremme (P.W.), Carl and Ellen Hertz Legat for Dansk Natur-og Lægevidenskab (P.W.), the Alfred Benzon Foundation (P.W.), a grant for women in science from L'Oréal, United Nations Educational, Scientific and Cultural Organization, and The Royal Danish Academy of Science and Letters (P.W.).
Article, publication date, and citation information can be found at http://jpet.aspetjournals.org.
- γ-hydroxybutyric acid
- γ-aminobutyric acid
- (E,RS)-(6,7,8,9-tetrahydro-5-hydroxy-5H-benzocyclohept-6-ylidene)acetic acid
- lithium 4-hydroxy-4-[4-(2-iodobenzyloxy)phenyl]butanoate
- Received May 26, 2010.
- Accepted August 5, 2010.
- Copyright © 2010 by The American Society for Pharmacology and Experimental Therapeutics