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. Author manuscript; available in PMC: 2008 Aug 24.
Published in final edited form as: Brain Res. 2007 Jul 26;1165:15–20. doi: 10.1016/j.brainres.2007.06.051

Low concentrations of ethanol do not affect radioligand binding to the δ-subunit-containing GABAA receptors in the rat brain

Ashok K Mehta 1, CR Marutha Ravindran 1, Maharaj K Ticku 1
PMCID: PMC2034279  NIHMSID: NIHMS29280  PMID: 17662260

Abstract

In the present study, we investigated the co-localization pattern of the δ-subunit with other subunits of GABAA receptors in the rat brain using immunoprecipitation and Western blotting techniques. Further, we investigated whether low concentrations of ethanol affect the δ-subunit-containing GABAA receptor assemblies in the rat brain using radioligand binding to the rat brain membrane homogenates as well as to the immunoprecipitated receptor assemblies. Our results revealed that δ-subunit is not co-localized with γ2-subunit but it is associated with the α1-, α4- or α6-, β2- or/and β3-subunit(s) of GABAA receptors in the rat brain. Ethanol (1–50 mM) neither affected [3H]muscimol (3 nM) binding nor diazepam-insensitive [3H]Ro 15–4513 (2 nM) binding in the rat cerebellum and cerebral cortex membranes. However, a higher concentration of ethanol (500 mM) inhibited the binding of these radioligands to the GABAA receptors partially in the rat cerebellum and cerebral cortex. Similarly, ethanol (up to 50 mM) did not affect [3H]muscimol (15 nM) binding to the immunoprecipitated δ-subunit-containing GABAA receptor assemblies in the rat cerebellum and hippocampus but it inhibited the binding partially at a higher concentration (500 mM). These results suggest that the native δ-subunit-containing GABAA receptors do not play a major role in the pharmacology of clinically relevant low concentrations of ethanol.

Keywords: GABAA receptors, δ-Subunit, Immunoprecipitation, Western blot, Co-localization, Radioligand binding

1. Introduction

GABAA receptors are heteropentameric membrane proteins derived from various subunits such as α1–6, β1–3, γ1–3, δ, ε, θ, π and ρ1–3, and differ in subunit composition, function and sensitivity to various drugs (see Mehta and Ticku, 1999a; Whiting, 2003). The molecular mechanisms underlying the expression of various GABAA receptor subunits and the assembly of subunit combinations to form functional receptors are unknown. To elucidate the cellular location, subunit composition and functional role of native GABAA receptors is important in order to develop better drugs. Majority of the native pentameric GABAA receptor assemblies are believed to contain two α, two β and one γ or δ subunits (Benke et al., 1991; Mertens et al., 1993; Quirk et al., 1994, 1995; Araujo et al., 1998; see Mehta and Ticku, 1999a; Whiting, 2003), and the most common subunit combination for GABAA receptors in the brain is α1β2γ2 (Fritschy et al., 1992; De Blas, 1996). The δ-subunit-containing GABAA receptors are not found in abundance in the brain but the knockout of δ-subunit in mice reduced ethanol consumption, ethanol-withdrawal hyperexcitability as well as anticonvulsant effect of ethanol (Mihalek et al., 2001). However, there were normal anxiolytic, sedative, hypothermic and development of acute/chronic tolerance responses to ethanol in these mice (Mihalek et al., 2001). Further, the discriminatory stimulus effect of ethanol was found to be similar in the δ-subunit knockout versus wild-type mice (Shannon et al., 2004). It has been reported recently by many investigators that the δ-subunit-containing GABAA receptors are involved in the pharmacological actions of clinically relevant low concentrations of ethanol (Sundstrom-Poromaa et al., 2002; Wallner et al., 2003; Wei et al., 2004; Hanchar et al., 2004; Glykys et al., 2007). Further, it has been proposed that ethanol-induced motor impairment is mediated through the extrasynaptic δ-subunit-containing GABAA receptors in the cerebellar granules (Hanchar et al., 2005). However, there are also reports indicating that the δ-subunit of GABAA receptors does not confer sensitivity to clinically relevant low concentrations of ethanol (Borghese et al., 2006; Yamashita et al., 2006; Casagrande et al., 2007). These conflicting reports may be due to differences in the receptor subunits combinations or cell type. Thus we investigated the co-localization pattern of the δ-subunit with major subunits (α1, α4, α6, β2, β3 and γ2) of GABAA receptors in the rat brain under identical conditions using immunoprecipitation and Western blotting techniques prior to using various brain regions for investigating the effect of low concentrations of ethanol on the radioligand binding to the membrane homogenates as well as to the immunoprecipitated δ-subunit-containing GABAA receptor assemblies. Notably, previous studies utilized either behavioral models or patch-clamp technique in recombinant expression system, cultured neurons and tissue slices to explore this aspect.

2. Results

2.1. Co-localization pattern of δ-subunit with other subunits of GABAA receptors in the rat brain

Various amounts of tissue protein (1500 μg, 1000 μg, 500 μg and 100 μg) were immunoprecipitated, dot blotted and immunostained using antibodies for the GABAA receptor subunits (α1, α4, α6, β2, β3, γ2, and δ) as detailed in the experimental procedures. In these experiments, the antibodies for various subunits formed the immunoreaction products so as to yield their respective protein bands (Fig. 1). For subsequent experiments, 1000 μg of the tissue protein was used as this concentration yielded the protein band of satisfactory quality. As a negative control (NC), we performed parallel experiments using non-immune IgG. No protein-band was detected in these control experiments since the non-immune IgG did not react with the polypeptides of the GABAA receptor subunits (Fig. 1). Further, pre-incubation with the peptide (20 μg/ml) for the δ-subunit GABAA receptors antibody blocked the immunoprecipitation in control experiments, thereby indicating the specificity of the antibody for the δ-subunit- containing GABAA receptors.

Fig. 1.

Fig. 1

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Immunoprecipitation, dot blotting and immunostaining using various amounts of the tissue protein (1500 μg, 1000 μg, 500 μg and 100 μg) and antibody for the (a) α1-, (b) α4-, (c) α6-, (d) β2-, (e) β3-, (f) γ2-, or (g) δ-subunit of GABAA receptors. For negative control (NC), affinity-purified non-immune rabbit polyclonal IgG was used. Lane 1: 1500 μg; lane 2: 1000 μg; lane 3: 500 μg and lane 4: 100 μg of the immunoprecipitated protein from the rat cerebellum (α1-, α6-, β2-, β3-, and γ2-subunits) or hippocampus (α4-subunit). Each experiment was performed three times.

In order to investigate the co-localization pattern of δ-subunit with other subunits of GABAA receptors in the rat brain, the δ-subunit of GABAA receptors was immunoprecipitated using antiserum for this subunit, which was followed by Western blot analysis using antibodies for various subunits of GABAA receptors in the rat cerebellum (α1-, α6-, β2-, β3-, γ2-, and δ-subunits) or hippocampus (α4- and δ-subunits) depending on the abundant localization of these subunits (Wisden et al., 1992). Antibody for the γ2-subunit of GABAA receptors did not react with the immunoprecipitated polypeptide of the δ-subunit following Western blotting (Fig. 2), thereby indicating an absence of co-localization of δ-subunit with γ2-subunit in the rat cerebellum. In contrast, antibody for the α1-, α4-, α6-, β2-, or β3-subunit reacted with the immunoprecipitated polypeptides of the δ-subunit following Western blotting (Fig. 2), thereby indicating an association of δ-subunit with α1-, α4- or α6-, β2- or/and β3-subunit(s) of GABAA receptors. We confirmed these results further by immunoprecipitating various subunits of the GABAA receptors using appropriate antibodies, and followed by Western blot analysis using antiserum for the δ-subunit of GABAA receptors. This second set of experiments revealed similar co-localization pattern of δ-subunit with other subunits of GABAA receptors (Fig. 2). These experiments revealed the molecular mass of various subunits of GABAA receptors as follows: α1 = 51 kDa, α4 = 64 kDa, α6 = 58 kDa, β2 = 52 kDa, β3 = 54 kDa, γ2 = 43 kDa and δ = 54 kDa. Notably, there was no protein band in the negative control (NC) experiments with non-immune IgG under identical conditions (Fig. 2). Similarly, there was no protein band when pre-incubated with the peptide (20μg/ml) for the δ-subunit GABAA receptors antibody in control experiments (Fig. 2).

Fig. 2.

Fig. 2

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Co-localization analysis of δ-subunit with other subunits of GABAA receptors in the rat cerebellum (α1-, α6-, β2-, β3-, and γ2-subunits) and hippocampus (α4-subunit) using immunoprecipitation and Western blotting techniques. Information regarding the use of various antibodies for immunoprecipitation is provided at the top of each strip, whereas the information regarding the use of antibodies for detection of the protein band in Western blot analysis is provided at the bottom of each strip. For negative control, affinity-purified non-immune rabbit polyclonal IgG (NC) and/or polypeptide for the GABAA receptors δ-subunit antibody (20 μg/ml; C) was used. Each experiment was performed three times.

2.2. Effect of ethanol on radioligand binding to the rat brain membrane homogenate and immunoprecipitated GABAA receptor assemblies

Ethanol (up to 50 mM) did not elicit any effect on [3H]muscimol (3 nM) and diazepam-insensitive [3H]Ro 15–4513 (2 nM) binding in the membranes from the rat cerebellum and cerebral cortex (Table 1). However, a higher concentration of ethanol (500 mM) caused partial inhibition of [3H]muscimol (3 nM) binding (7–10%) and diazepam-insensitive [3H]Ro 15–4513 (2 nM) binding (18–22%) as shown in Table 1. Similarly, ethanol (up to 50 mM) did not elicit any effect on [3H]muscimol (15 nM) binding to the immunoprecipitated GABAA receptor assemblies derived from δ-subunit in the rat cerebellum or hippocampus, whereas a higher concentration of ethanol (500 mM) caused partial inhibition (12%–14%) as shown in Table 2.

Table 1.

Effect of ethanol on [3H]muscimol (3 nM) binding and diazepam-insensitive [3H]Ro 15–4513 (2 nM) binding in the membranes from the rat cerebellum and cerebral cortex.

Each value is mean ± S.E.M. of number of individual experiments indicated (n), each performed in triplicate.

Group % Inhibition of binding[3H]Muscimol (n) [3H]Ro 15–4513 (n)
Cerebellum
Ethanol (1–50 mM) < 5.0 (8) < 5.0 (4)
Ethanol (100 mM) 2.1 ± 1.6 (6) 5.4 ± 2.3 (4)
Ethanol (200 mM) 5.6 ± 1.1 (6) 6.3 ± 2.1 (5)
Ethanol (500 mM) 6.7 ± 0.9 (6) 17.8 ± 0.8 (5)
Cerebral cortex
Ethanol (1–50 mM) < 5.0 (7) < 5.0 (5)
Ethanol (100 mM) 5.4 ± 2.5 (6) 7.9 ± 0.8 (5)
Ethanol (200 mM) 6.6 ± 1.9 (7) 10.4 ± 0.7 (5)
Ethanol (500 mM) 10.1 ± 2.4 (7) 21.9 ± 1.2 (5)
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Basal [3H]muscimol (3 nM) binding was found to be 1.38 ± 0.08 pmol/mg protein and 0.39 ± 0.05 pmol/mg protein in the membranes from the rat cerebellum and cerebral cortex, respectively. Basal [3H]Ro 15–4513 (2 nM) diazepam-insensitive binding was found to be 0.32 ± 0.02 pmol/mg protein and 0.027 ± 0.002 pmol/mg protein in the membranes from the rat cerebellum and cerebral cortex, respectively.

Table 2.

Effect of ethanol on [3H]muscimol (15 nM) binding to the immunoprecipitated δ-subunit-containing GABAA receptor assemblies in the rat cerebellum and hippocampus.

Each value is mean ± S.E.M. of three individual experiments, each performed in triplicate.

Treatment % Inhibition of binding
Cerebellum Hippocampus
Ethanol (5–50 mM) < 5.0 <5.0
Ethanol (200 mM) 7.8 ± 0.8 10.5 ± 1.7
Ethanol (500 mM) 12.4 ± 0.9 13.9 ± 2.1
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[3H]muscimol (15 nM) binding to the solubilized GABAA receptors from the rat cerebellum and hippocampus was found to be 0.12 ± 0.01 pmol/mg protein (n=3) and 0.09 ± 0.01 pmol/mg protein (n=3), respectively.

3. Discussion

δ-Subunit is thought to co-localize with either α4 or α6 subunit in the native GABAA receptor assemblies (Jones et al., 1997; Sur et al., 1999). A small population of the GABAA receptors in the mouse cerebellum is also reported to have both δ- and α1-subunits (Poltl et al., 2003). GABAA receptors derived from the δ-subunit co-localized with α1-subunit are reported to form functional receptors in expression systems(Wohlfarth et al., 2002; Bianchi et al., 2002) as well as in the mouse hippocampus (Glykys et al., 2007). However, δ-subunit does not co-localize with γ-subunit in the native GABAA receptor assemblies (Quirk et al., 1994, 1995; Araujo et al., 1998). Various conflicting reports in the literature regarding the involvement of δ-subunit-containing GABAA receptors in the pharmacology of ethanol may be due to differences in the receptor subunits combination or cell type. Our present study revealed that the δ-subunit of GABAA receptors does not co-localize with γ2-subunit but it is associated with α1-, α4- or α6-, β2- or/and β3-subunit(s) in the rat brain.

The δ-subunit-containing GABAA receptors are known to have high affinity for the GABA binding site agonist muscimol but lack affinity for the classical benzodiazepines (Shivers et al., 1989; Saxena and Macdonald, 1994, 1996; Storustovu and Ebert, 2006). Further, the substitution of the γ-subunit by δ-subunit is reported to produce GABAA receptors that display high affinity for the GABA agonist muscimol but no benzodiazepine binding sites (Saxena and MacDonald 1996). However, Ro 15–4513 is reported to have high affinity for the δ-subunit-containing GABAA receptors (Araujo et al., 1998; Hanchar et al., 2006; Yamashita et al., 2006). In view of conflicting reports regarding the involvement of δ-subunit-containing GABAA receptors in the pharmacology of clinically relevant low concentrations of ethanol based on behavioral models (Mihalek et al., 2001; Shannon et al., 2004; Hanchar et al., 2005) or patch-clamp technique in recombinant expression system (Sundstrom-Poromaa et al., 2002; Wallner et al., 2003; Hanchar et al., 2005; Borghese et al., 2006; Yamashita et al., 2006), cultured cerebellar granule cells (Yamashita et al., 2006; Casagrande et al., 2007), cerebellar slices (Hanchar et al., 2005) and hippocampal slices (Wei et al., 2004; Borghese et al., 2006; Glykys et al., 2007), we investigated this aspect using [3H]muscimol and [3H]Ro 15–4513 binding to the rat brain membrane homogenates (cerebral cortex and cerebellum) as well as [3H]muscimol binding to the immunoprecipitated δ-subunit-containing GABAA receptor assemblies from the rat cerebellum and hippocampus. However, we did not investigate the effect of ethanol on [3H]Ro 15–4513 binding to the immunoprecipitated δ-subunit-containing GABAA receptors because of difficulty in quantifying it accurately due to low binding sites, which is about 6% of the [3H]Ro 15–4513 binding sites in cerebellum (Hanchar et al., 2006) and far less in hippocampus and cerebral cortex in contrast to [3H]muscimol binding to the δ-subunit-containing GABAA receptors in the rat cerebellum (≈20%, Araujo et al., 1998; see Mehta and Ticku, 1999a). Further, we did not investigate the effect of ethanol on [3H]muscimol or [3H]Ro 15–4513 binding in the rat hippocampal membrane homogenate due to (a) small amount of available tissue, (b) negative data in the immunoprecipitated experiments with [3H]muscimol/hippocampus in the present study, and (c) negative data in cerebral cortex membrane homogenate which has similar δ-subunit-containing GABAA receptor assemblies (δ- and α4/α1-subunits combination). Our data indicate that ethanol does not affect the native δ-subunit-containing GABAA receptors at clinically relevant low concentrations in the rat cerebellum (δ- and α6/α1-subunits combination), hippocampus (δ- and α4/α1-subunits combination) and cerebral cortex (δ- and α4/α1-subunits combination), thereby supporting the view that the native δ-subunit-containing GABAA receptors do not play a major role in the pharmacology of clinically relevant low concentrations of ethanol (Borghese et al., 2006; Yamashita et al., 2006; Casagrande et al., 2007). However, we are unable to answer the question why some investigators (Sundstrom-Poromaa et al., 2002; Wallner et al., 2003; Wei et al., 2004; Hanchar et al., 2004; Glykys et al., 2007) are able to demonstrate the effect of clinically relevant low concentrations of ethanol on the δ-subunit-containing GABAA receptors since ethanol did not elicit any effect on the δ-subunit-containing GABAA receptor assemblies in the rat cerebellum, cerebral cortex, or hippocampus in our present study. It is possible that differences in the unknown intracellular factors/components may be responsible for the conflicting effects of ethanol on the δ-subunit-containing GABAA receptors in different experimental models.

4. Experimental procedures

Adult male Sprague-Dawley rats (Harlan, Indianapolis IN, U.S.A.) weighing 200–250 g were maintained at a constant room temperature (22°C) on a 12-h light/12-h dark cycle. All experiments were conducted in accordance with the Declaration of Helsinki and/or with the Guide for the Care and Use of Laboratory Animals, as adopted and promulgated by the National Institutes of Health. Adequate measures were taken to minimize pain or discomfort to the animals. Food and water were available ad libitum. Different regions of the rat brain were dissected, and tissues were stored at −80°C until use.

4.1. Immunoprecipitation and immunoblotting

Tissues were homogenized in ice-cold lysis buffer (50 mM HEPES, pH 7.4, 150 mM sodium chloride, 1% Triton X 100, 10 mM sodium fluoride, 1 mg/ml bacitracin, 50 mM β-glycerophosphate, 1 mM phenylmethylsulfonyl fluoride (PMSF), 1 mM N-ethyl maleimide, 1 mM Na3VO4, 10 μg/ml leupeptin). These samples were centrifuged (2300 g, 10 min, 4°C) so as to remove the supernatant containing protein. Immunoprecipitation experiments were performed as detailed earlier (Kanakura et al., 1991; Marutha Ravindran and Ticku, 2006a, 2006b; Marutha Ravindran et al., 2007). Earlier reports from our laboratory and preliminary experiments revealed that 1:1000 dilution (α1-, β2-, β3-, and γ2-subunits), 1:100 dilution (δ- and α6-subunits), or 1:500 dilution (α4-subunit) of antiserum for the GABAA receptor subunits yields the protein band of satisfactory quality (Marutha Ravindran and Ticku, 2006a, 2006b; Marutha Ravindran et al., 2007). Antibodies for α4- (catalog # OPA1-04102), β2- (catalog # OPA1-04107), and β3-subunits (catalog # OPA1-04108) of GABAA receptors were obtained from Affinity Bio-Reagents (Golden CO, U.S.A.). These antibodies were fusion protein of MBP with the aminoacid sequence from the cytosolic loop of rat GABAA receptors. Polyclonal antibodies specific for the α1 (AA 1–15), α6 (AA 1–13), γ2 (AA 1–29) and δ (AA 1–11) subunits of GABAA receptors were procured from Alpha Diagnostics (San Antonio TX, U.S.A.). For dot blotting analysis, various amounts of the tissue protein (1500, 1000, 500, and 100 μg) were diluted with phosphate-buffered saline 1X (PBS) to 1 μg/μl, boiled in a boiling water-bath for 5 minutes to denature the proteins followed by immunoprecipitation using 30μl of the diluted antiserum for the GABAA receptor subunits (α1, α4, α6, β2, β3, γ2, or δ) and 150 μl of Protein A agarose beads (Upstate, Charlottesville VA, U.S.A.). For the negative control, 1500 μg of protein samples were treated with 7.5 μl (7.5 μg) of the affinity-purified non-immune rabbit polyclonal IgG (Santa Cruz Biotechnology, Santa Cruz CA, U.S.A.) and 150 μl of Protein A agarose beads as detailed earlier (Marutha Ravindran and Ticku, 2006a, 2006b; Marutha Ravindran et al., 2007). To test the specificity of antibody for the GABAA receptors δ-subunit, the peptide for δ-subunit antibody (20 μg/ml) was added prior to incubation with the antibody so as to check whether it blocks the detection of δ-subunit. The immunoprecipitated proteins were dot blotted to the PVDF membrane (Bio-Rad, Chicago IL, U.S.A.) as described in the literature (Araujo et al., 1998). For subsequent experiments, 1000 μg of the tissue protein was used so as to obtain a satisfactory quality of the protein bands. To study the association of δ-subunit with the other subunits of the GABAA receptors, 1000 μg of the immunoprecipitated protein of the GABAA receptors δ-subunit following electrophoresis was transferred to the membrane and was incubated with primary antibody for various subunits (α1, α4, α6, β2, β3, or γ2) for Western blot analysis. Then the membrane was washed several times, and peroxidase-coupled secondary antibody (anti-mouse IgG/anti-rabbit IgG; New England Biolabs, U.S.A.) was added and incubated for 1h. The membrane was washed and specific bands were visualized using super signal west pico chemiluminescent substrate kit (Pierce Biotechnology, Rockford IL, U.S.A.) as described earlier (Marutha Ravindran and Ticku, 2006a, 2006b; Marutha Ravindran et al., 2007). Association pattern of the δ-subunit with other subunits of GABAA receptors was also investigated in a reversible manner, i.e., 1000 μg of the immunoprecipitated proteins of various subunits (α1, α4, α6, β2/3, or γ2) of the GABAA receptors were transferred to the membrane and were incubated with primary antibody for the δ-subunit of the GABAA receptors and then with the peroxidase-coupled secondary antibody as detailed earlier. Negative control experiments were performed in parallel as described above.

4.2. Radioligand binding to the brain membrane homogenate

Membranes from the rat cerebral cortex and cerebellum were prepared as described previously (Mehta and Ticku, 1998). Briefly, the frozen tissue was thawed and homogenized in ice-cold 0.32 M sucrose, pH 7.4 (20 ml/g of tissue). The mixture was then centrifuged at 1000 g for 10 min at 4°C so as to discard the P1 fraction. This step was omitted in the case of cerebellum. The supernatant or the homogenized tissue was then centrifuged at 140,000 g for 30 min at 4°C to obtain the mitochondrial plus microsomal fraction. This fraction was dispersed in ice-cold purified water, and homogenized by a Brinkman Polytron at a setting of six for two 10-sec bursts, 10 sec apart. The suspension was centrifuged at 140,000 g for 30 min at 4°C. The pellet was then resuspended in ice-cold Tris-HCl buffer (50 mM, pH 7.4), and centrifuged at 140,000 g for 30 min at 4°C. The pellet was resuspended in Tris-HCl buffer, and kept frozen overnight at −80°C. After thawing, Tris-HCl buffer was added to the tissue, and the mixture was centrifuged at 140,000 g for 30 min at 4°C three times. The membranes were then suspended in Tris-HCl (50 mM, pH 7.4), distributed in aliquots, and kept frozen at −80°C until use. On the day of assay, the tissue was thawed and washed one time with buffer as before (140,000 g for 30 min at 4°C) and then resuspended in assay buffer. [3H]Muscimol and [3H]Ro 15–4513 binding were measured using a filtration method as described previously (Mehta and Shank, 1995; Mehta and Ticku, 1998). Briefly, aliquots (0.1–0.2 mg protein) of membrane preparation in Tris-HCl buffer (50 mM, pH 7.4) were incubated with either [3H]Ro 15–4513 (2 nM) at 4°C for 90 min or [3H]muscimol (3 nM) at 4°C for 45 min in the absence and presence of different concentrations of ethanol. In the case of [3H]Ro 15–4513 binding, all the assay tubes also contained diazepam (10 μM) so as to displace the diazepam-sensitive binding. Non-specific binding was determined using Ro 15–4513 (10 μM) or GABA (100 μM) for [3H]Ro 15–4513 and [3H]muscimol binding, respectively. Radioligand binding to the GABAA receptors was terminated by separating the membrane material from the incubation medium by vacuum filtration membrane harvester (Brandel MB-48L membrane harvester). The samples were washed twice with 2 ml of ice-cold Tris-HCl buffer (50 mM, pH 7.4). Radioactivity was quantified by liquid scintillation spectrometry. The data are expressed as mean ± S.E.M.

4.3. Radioligand binding to the immunoprecipitated receptor assemblies

Membranes from the rat cerebellum and hippocampus were prepared as described above (Mehta and Ticku, 1998). The GABAA receptors solubilization, immunoprecipitation and [3H]muscimol binding assays were performed as described by us earlier (Mehta and Ticku, 1999b, 2005). Briefly, GABAA receptors were solubilized in modified radioimmune precipitation assay buffer (RIPA), i.e., solubilization buffer (pH 7.4) containing sodium chloride (0.137 M), sodium deoxycholate (1% w/v), Triton X-100 (1% v/v), sodium dodecyl sulfate, i.e, SDS (0.1% w/v), Tris (10 mM) and a cocktail of protease inhibitors containing EDTA (1 mM), EGTA (1 mM), benzamidine HCl (2 mM), trypsin inhibitor type 1-S (0.1 mg/ml), bacitracin (0.1 mg/ml) and phenylmethylsulfonyl fluoride (0.3 mM). After incubation for 1 h at 4°C, insoluble material was removed by centrifugation (100,000 g for 1 h at 4°C). A sample of 400 μl (≈300 μg protein) of the solubilized receptors was incubated overnight at 4°C with 30 μl of the antiserum for the rat GABAA receptors δ-subunit. The receptor-antibody complexes were recovered by incubation with protein A-agarose suspension (60 μl of 40% v/v) followed by centrifugation. Immunoprecipitation was quantified by determining the binding of [3H]muscimol (15 nM) to the immunoprecipitated pellet and supernatant. Non-specific radioligand binding was determined with GABA (100 μM). The data are expressed as mean ± S.E.M.

Acknowledgments

This research work was supported by the National Institute on Alcohol and Alcohol Abuse (NIAAA) grant AA10552.

Abbreviations

PMSF

phenylmethylsulfonyl fluoride

PBS

phosphate-buffered saline

SDS

sodium dodecyl sulfate

Footnotes

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