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Analysis of the Presence and Abundance of GABAA Receptors Containing Two Different Types of α Subunits in Murine Brain Using Point-mutated α Subunits

2004; Elsevier BV; Volume: 279; Issue: 42 Linguagem: Inglês

10.1074/jbc.m407154200

1083-351X

Dietmar Benke, Panagiotis Fakitsas, Christian Roggenmoser, Claudia Michel, Uwe Rudolph, Hanns Möhler,

Receptor Mechanisms and Signaling

γ-Aminobutyric acid, type A (GABAA) receptors are pentameric proteins of which the majority is composed of two α subunits, two β subunits and one γ subunit. It is well documented that two different types of α subunits can exist in a singles GABAA receptor complex. However, information on the abundance of such GABAA receptors is rather limited. Here we tested whether mice containing the His to Arg point mutation in the α1, α2, or α3 subunit at positions 101, 101, and 126, respectively, which render the respective subunits insensitive to diazepam, would be suitable to analyze this issue. Immunodepletion studies indicated that the His to Arg point mutation solely rendered those GABAA receptors totally insensitive to diazepam binding that contain two mutated α subunits in the receptor complex, whereas receptors containing one mutated and one heterologous α subunit not carrying the mutation remained sensitive to diazepam binding. This feature permitted a quantitative analysis of native GABAA receptors containing heterologous α subunits by comparing the diazepam-insensitive binding sites in mutant mouse lines containing one mutated α subunit with those present in mouse lines containing two different mutated α subunits. The data indicate that the α1α1-containing receptors with 61% is the most abundant receptor subtype in brain, whereas the α1α2 (13%), α1α3 (15%), α2α2 (12%), α2α3 (2%), and α3α3 combinations (4%) are considerably less expressed. Only within the α1-containing receptor population does the combination of equal α subunits (84% α1α1, 7% α1α2, and 8% α1α3) prevail, whereas in the α2-containing receptor population (46% α2α2, 36% α2α1, and 19% α2α3) and particularly in the α3-containing receptor population (27% α3α3, 56% α3α1, and 19% α3α2), the receptors with two different types of α subunits predominate. This experimental approach provides the basis for a detailed analysis of the abundance of GABAA receptors containing heterologous α subunits on a brain regional level. γ-Aminobutyric acid, type A (GABAA) receptors are pentameric proteins of which the majority is composed of two α subunits, two β subunits and one γ subunit. It is well documented that two different types of α subunits can exist in a singles GABAA receptor complex. However, information on the abundance of such GABAA receptors is rather limited. Here we tested whether mice containing the His to Arg point mutation in the α1, α2, or α3 subunit at positions 101, 101, and 126, respectively, which render the respective subunits insensitive to diazepam, would be suitable to analyze this issue. Immunodepletion studies indicated that the His to Arg point mutation solely rendered those GABAA receptors totally insensitive to diazepam binding that contain two mutated α subunits in the receptor complex, whereas receptors containing one mutated and one heterologous α subunit not carrying the mutation remained sensitive to diazepam binding. This feature permitted a quantitative analysis of native GABAA receptors containing heterologous α subunits by comparing the diazepam-insensitive binding sites in mutant mouse lines containing one mutated α subunit with those present in mouse lines containing two different mutated α subunits. The data indicate that the α1α1-containing receptors with 61% is the most abundant receptor subtype in brain, whereas the α1α2 (13%), α1α3 (15%), α2α2 (12%), α2α3 (2%), and α3α3 combinations (4%) are considerably less expressed. Only within the α1-containing receptor population does the combination of equal α subunits (84% α1α1, 7% α1α2, and 8% α1α3) prevail, whereas in the α2-containing receptor population (46% α2α2, 36% α2α1, and 19% α2α3) and particularly in the α3-containing receptor population (27% α3α3, 56% α3α1, and 19% α3α2), the receptors with two different types of α subunits predominate. This experimental approach provides the basis for a detailed analysis of the abundance of GABAA receptors containing heterologous α subunits on a brain regional level. GABAA 1The abbreviations used are: GABAA, γ-aminobutyric acid, type A; H/R, His to Arg. receptors mediate the majority of fast inhibitory neurotransmission in the mammalian brain by controlling an integral chloride ion channel. Enhancement of neuronal inhibition by positive allosteric modulation of GABAA receptors via the benzodiazepine-binding site underlies the pharmacotherapy of several neurological and psychiatric disorders such as generalized anxiety disorders, sleep disturbances, and seizure disorders. GABAA receptors are pentameric transmembrane proteins build of different classes of subunits (α1–6, β1–3, γ1–3, ρ1–3, δ, ϵ, and θ) (1Barnard E.A. Skolnick P. Olsen R.W. Mohler H. Sieghart W. Biggio G. Braestrup C. Bateson A.N. Langer S.Z. Pharmacol. Rev. 1998; 50: 291-313PubMed Google Scholar). The vast majority of GABAA receptors are composed of α, β, and γ2 subunits, where the α subunit variant determines the ligand binding characteristics of the benzodiazepine site of the various receptor subtypes. GABAA receptors containing the α1, α2, α3, or α5 subunit in combination with any β subunit and the γ2 subunit are the most abundant subtypes in the brain and mediate the modulatory actions of clinically used benzodiazepine site agonists, such as diazepam, flunitrazepam, and clonazepam (2Mohler H. Benke D. Fritschy J.M. Benson J.A. Martin D.L. Olsen R.W. GABA in the Nervous System: The View at 50 Years. Lippincott Williams & Wilkins, Philadelphia2000: 97-112Google Scholar, 3Mohler H. Fritschy J.M. Luscher B. Rudolph U. Benson J. Benke D. Narahashi T. Ion Channels. 4. Plenum Publishing Corp., New York1996: 89-113Google Scholar). The binding of clinical relevant benzodiazepine site ligands to these socalled diazepam-sensitive GABAA receptor subtypes depends on a single amino acid residue, His at position 101 in α1 and α2, position 126 in α3, or position 105 in α5 (4Wieland H.A. Lüdens H. Seeburg P.H. J. Biol. Chem. 1992; 267: 1426-1429Abstract Full Text PDF PubMed Google Scholar, 5Benson J.A. Löw K. Keist R. Mohler H. Rudolph U. FEBS Lett. 1998; 431: 400-404Crossref PubMed Scopus (154) Google Scholar). It is well documented that GABAA receptors exist in brain that contain two different types of subunits in a single receptor complex (6del Rio J.C. Araujo F. Ramos B. Ruano D. Vitorica J. J. Neurochem. 2001; 79: 183-191Crossref PubMed Scopus (22) Google Scholar, 7Araujo F. Ruano D. Vitorica J. J. Pharmacol. Exp. Therap. 1999; 290: 989-997PubMed Google Scholar, 8Araujo F. Tan S. Ruano D. Schoemaker H. Benavides J. Vitorica J. J. Biol. Chem. 1996; 271: 27902-27911Abstract Full Text Full Text PDF PubMed Scopus (54) Google Scholar, 9Benke D. Michel C. Mohler H. J. Neurochem. 1997; 69: 806-814Crossref PubMed Scopus (75) Google Scholar, 10Duggan M.J. Pollard S. Stephenson F.A. J. Biol. Chem. 1991; 266: 24778-24784Abstract Full Text PDF PubMed Google Scholar, 11Endo S. Olsen R.W. J. Neurochem. 1993; 60: 1388-1398Crossref PubMed Scopus (50) Google Scholar, 12Jechlinger M. Pelz R. Tretter V. Klausberger T. Sieghart W. J. Neurosci. 1998; 18: 2449-2457Crossref PubMed Google Scholar, 13Khan Z.U. Gutierrez A. De Blas A.L. J. Neurochem. 1996; 66: 685-691Crossref PubMed Scopus (81) Google Scholar, 14Mertens S. Benke D. Mohler H. J. Biol. Chem. 1993; 268: 5965-5973Abstract Full Text PDF PubMed Google Scholar, 15Pollard S. Duggan J.M. Stephenson F.A. J. Biol. Chem. 1993; 268: 3753-3757Abstract Full Text PDF PubMed Google Scholar). However, it is currently poorly understood to which degree receptors containing two heterologous α subunits contribute to diazepam-sensitive GABAA receptors. So far, the abundance of α1α2- and α1α3-containing receptors in the cerebral cortex and α1α5- and α2α5-containing receptors in the hippocampus have been estimated by sequential steps of immunoprecipitation and quantification of precipitated receptors by radioligand binding (6del Rio J.C. Araujo F. Ramos B. Ruano D. Vitorica J. J. Neurochem. 2001; 79: 183-191Crossref PubMed Scopus (22) Google Scholar, 7Araujo F. Ruano D. Vitorica J. J. Pharmacol. Exp. Therap. 1999; 290: 989-997PubMed Google Scholar, 8Araujo F. Tan S. Ruano D. Schoemaker H. Benavides J. Vitorica J. J. Biol. Chem. 1996; 271: 27902-27911Abstract Full Text Full Text PDF PubMed Scopus (54) Google Scholar). This methodological approach is a demanding task and because of the amount of tissue required is hardly applicable to all brain regions. In addition, immunopurification studies are limited by the fraction of receptors that can be solubilized from the membranes. In the present paper an alternative experimental approach was used to overcome these experimental limitations. This method is based on the analysis by ligand binding studies of mutant mouse lines carrying a point mutation in the diazepam-sensitive α subunits. Because the diazepam sensitivity of GABAA receptors depends on the presence of a histidine residue in the drug-binding domain of the α subunits α1, α2, α3, and α5, they are rendered diazepam-insensitive when this histidine residue is replaced by arginine (4Wieland H.A. Lüdens H. Seeburg P.H. J. Biol. Chem. 1992; 267: 1426-1429Abstract Full Text PDF PubMed Google Scholar, 5Benson J.A. Löw K. Keist R. Mohler H. Rudolph U. FEBS Lett. 1998; 431: 400-404Crossref PubMed Scopus (154) Google Scholar). This feature had been previously used to analyze the contribution of GABAA receptor subtypes to the diverse actions of diazepam. By generating mouse lines that contain the His to Arg (H/R) point mutation in the α1, α2, or α3 subunit, it was shown that sedation, anterograde amnesia, and most of the anticonvulsant activities are mediated by α1-containing receptors (16Rudolph U. Crestani F. Benke D. Brünig I. Benson J.A. Martin J.R. Bluethmann H. Mohler H. Nature. 1999; 401: 796-800Crossref PubMed Scopus (1054) Google Scholar), whereas the anxiolytic like activity is mediated by α2-containing receptors (17Low K. Crestani F. Keist R. Benke D. Brunig I. Benson J.A. Fritschy J.M. Rulicke T. Bluethmann H. Mohler H. Rudolph U. Science. 2000; 290: 131-134Crossref PubMed Scopus (832) Google Scholar). Here we show that the different α(H/R) point-mutated mouse lines provide an opportunity to estimate the abundance of receptor populations containing two different types of α subunits in a receptor complex. Immunodepletion studies indicated that in α(H/R) mutant mouse lines only receptors containing two mutated α subunits fail to bind diazepam (e.g. α1(H101R) α3(H126R)), whereas receptors containing one mutated α subunit and one nonmutated heterologous α subunit (e.g. α1(H101R) α3) remain sensitive to diazepam binding. This permitted a detailed analysis on the presence and abundance of receptors containing either two homologous or two heterologous α subunits by comparing the proportion of diazepam-sensitive and diazepam-insensitive binding sites in wild type mice with the mouse lines containing a single type of mutated α subunit (α1H101R, α2H101R, or α3H126R) and newly crossbred mouse lines containing two different types of mutated α subunits (α1H101R α2H101R, α1H101R α3H126R, or α2H101Rα3H126R). Animals—Mouse lines containing the α1H101R, α2H101R, or α3H126R point mutation (at least five backcrosses to the 129/SvJ background) were generated previously (16Rudolph U. Crestani F. Benke D. Brünig I. Benson J.A. Martin J.R. Bluethmann H. Mohler H. Nature. 1999; 401: 796-800Crossref PubMed Scopus (1054) Google Scholar, 17Low K. Crestani F. Keist R. Benke D. Brunig I. Benson J.A. Fritschy J.M. Rulicke T. Bluethmann H. Mohler H. Rudolph U. Science. 2000; 290: 131-134Crossref PubMed Scopus (832) Google Scholar). The double homozygous mutant mouse lines α1H101R α2H101R, α1H101R α3H126R, and α2H101Rα3H126R were generated by cross-breeding the single-mutant mouse lines. Mice were kept under normal 12-h day-night cycle conditions with food and water ad libitum. Antibodies—Antibodies selectively recognizing the α1 (18Benke D. Cicin-Sain A. Mertens S. Mohler H. J. Recep. Res. 1991; 11: 407-424Crossref PubMed Scopus (72) Google Scholar), α2 (19Marksitzer R. Benke D. Fritschy J.M. Trezciak A. Bannwarth W. Mohler H. J. Recep. Res. 1993; 13: 467-477Crossref PubMed Scopus (72) Google Scholar), or α3 subunit (18Benke D. Cicin-Sain A. Mertens S. Mohler H. J. Recep. Res. 1991; 11: 407-424Crossref PubMed Scopus (72) Google Scholar) had been previously generated against short amino acid sequences present only in the subunit of interest (α1, amino acids 421–429; α2, amino acids 418–424; and α3, amino acids 1–15). The antibodies had been thoroughly analyzed for subunit specificity by Western blotting, immunoprecipitation, and immunohistochemistry (for details see Refs. 9Benke D. Michel C. Mohler H. J. Neurochem. 1997; 69: 806-814Crossref PubMed Scopus (75) Google Scholar and 18Benke D. Cicin-Sain A. Mertens S. Mohler H. J. Recep. Res. 1991; 11: 407-424Crossref PubMed Scopus (72) Google Scholar, 19Marksitzer R. Benke D. Fritschy J.M. Trezciak A. Bannwarth W. Mohler H. J. Recep. Res. 1993; 13: 467-477Crossref PubMed Scopus (72) Google Scholar, 20Benke D. Honer M. Michel C. Mohler H. Neuropharmacology. 1996; 35: 1413-1423Crossref PubMed Scopus (112) Google Scholar). Membrane Preparation—Brain tissue from 8–12-week-old mice was homogenized in 10 volumes of 10 mm Tris-HCl, pH 7.4, 0.32 m sucrose, 5 mm EDTA, 0.1 mm phenylmethylsulfonyl fluoride and centrifuged at 1000 × g for 10 min. Following recentrifugation of the supernatant for 20 min at 12,000 × g, the crude membrane pellet was washed three times with buffer and stored at –30 °C until used. Solubilization and Immunoprecipitation—For solubilization, the membranes were thawed and washed once with 10 mm Tris-HCl, pH 8, 150 mm NaCl, 1 mm EDTA, 200 mg/liter bacitracin, 0.1 mm phenylmethylsulfonyl fluoride, 2.3 mg/liter aprotinin, 1 mm benzamidine and resuspended in the same buffer to a protein concentration of 5–7 mg/ml followed by addition of sodium deoxycholate to a final concentration of 0.5%. After incubation for 30 min at 4 °C, insoluble material was removed by centrifugation for 60 min at 100,000 × g. The supernatant was immediately used for immunoprecipitation experiments. For immunoprecipitation, aliquots (0.2–0.5 ml) of the deoxycholate extract were incubated with subunit-selective antiserum at concentrations leading to the maximum immunoprecipitation of the corresponding receptors overnight at 4 °C, followed by incubation with 50 μl of Pansorbin (suspension of 10% Staphylococcus aureus; Calbiochem) for 30–60 min at room temperature. After extensive washing, the precipitates were resuspended in 10 mm Tris-HCl, pH 8, 150 mm NaCl, 1 mm EDTA, 200 mg/liter bacitracin, 0.1 mm phenylmethylsulfonyl fluoride, 2.3 mg/liter aprotinin, 1 mm benzamidine, 0.2% Triton X-100 for [3H]Ro 15-4513 binding. The specificity of immunoprecipitation was verified in competition experiments using the respective peptide antigen (50 μg/ml extract). No specific [3H]Ro 15-4513 binding was observed in the immunoprecipitate after co-incubation of the respective antiserum with the corresponding peptide antigen (data not shown). [3H]Ro 15-4513 Binding Experiments—For Scatchard analysis, crude mouse brain membranes were thawed and washed once with 50 mm Tris-HCl, pH 7.5, and aliquots containing 100 μg of protein were incubated with increasing concentrations (0.5–70 nm) of [3H]Ro 15-4513 (24.3 Ci/mmol; PerkinElmer Life Sciences) in a total volume of 0.2 ml for 90 min on ice. Diazepam-insensitive binding sites were detected in parallel by inclusion of 10 μm diazepam in the incubation. Nonspecific [3H]Ro 15-4513 binding was assessed at each radioligand concentration by the addition of 10 μm flumazenil to the reaction. Incubation was stopped by rapid vacuum filtration on Whatman GF/C filters using a semiautomatic cell harvester (Skatron Instruments) and subjected to liquid scintillation counting. Diazepam displacement studies using immunoprecipitated GABAA receptors were performed by incubating aliquots of the washed immunoprecipitates with serial dilutions of diazepam at a fixed concentration of [3H]Ro 15-4513 (10 nm). After incubation for 90 min on ice, the reaction was stopped by addition of 4 ml of 50 mm Tris-HCl, pH 7.5, followed immediately by rapid vacuum filtration on Whatman GF/C filters. The filters were washed three times with 4 ml of buffer and subjected to liquid scintillation counting. Nonspecific radioligand binding was determined by including 10 μm of flumazenil in parallel incubations. The binding data were analyzed using the program'KELL for Windows 6.0.5′ (Biosoft, UK). Diazepam Binding to GABAA Receptors in Point-mutated Mice Containing α1(H101R), α2(H101R), or α3(H126R) Subunits— Generation of mouse lines containing the H/R point mutation in the α1, α2, or α3 subunit resulted in an increase of diazepam-insensitive [3H]Ro 15-4513-binding sites with a brain regional distribution that corresponded to the expression pattern of the respective α subunit in wild type mice (16Rudolph U. Crestani F. Benke D. Brünig I. Benson J.A. Martin J.R. Bluethmann H. Mohler H. Nature. 1999; 401: 796-800Crossref PubMed Scopus (1054) Google Scholar, 17Low K. Crestani F. Keist R. Benke D. Brunig I. Benson J.A. Fritschy J.M. Rulicke T. Bluethmann H. Mohler H. Rudolph U. Science. 2000; 290: 131-134Crossref PubMed Scopus (832) Google Scholar). In α1(H101R) mice the increase in diazepam-insensitive binding sites, determined by Scatchard analysis (61 ± 3%) (16Rudolph U. Crestani F. Benke D. Brünig I. Benson J.A. Martin J.R. Bluethmann H. Mohler H. Nature. 1999; 401: 796-800Crossref PubMed Scopus (1054) Google Scholar), corresponded roughly to the abundance of α1-containing receptors in whole brain (60–80%, as previously analyzed by immunoprecipitation) (11Endo S. Olsen R.W. J. Neurochem. 1993; 60: 1388-1398Crossref PubMed Scopus (50) Google Scholar, 14Mertens S. Benke D. Mohler H. J. Biol. Chem. 1993; 268: 5965-5973Abstract Full Text PDF PubMed Google Scholar, 18Benke D. Cicin-Sain A. Mertens S. Mohler H. J. Recep. Res. 1991; 11: 407-424Crossref PubMed Scopus (72) Google Scholar, 21Benke D. Mertens S. Mohler H. Mol. Neuropharmacol. 1991; 1: 103-110Google Scholar, 22McKernan R.M. Quirk K. Prince R. Cox P.A. Gillard N.A. Ragan C.I. Whiting P. Neuron. 1991; 7: 667-676Abstract Full Text PDF PubMed Scopus (178) Google Scholar). However, the increase was considerably smaller than expected in the α2(H101R) and α3(H126R) mice. In α2(H101R) and α3(H126R) mice the number of diazepam-insensitive binding sites increased by 12 ± 0.1 and 4 ± 2% (17Low K. Crestani F. Keist R. Benke D. Brunig I. Benson J.A. Fritschy J.M. Rulicke T. Bluethmann H. Mohler H. Rudolph U. Science. 2000; 290: 131-134Crossref PubMed Scopus (832) Google Scholar), respectively, whereas the abundance of α2-containing receptors was immunobiochemically estimated to be 20–28% (11Endo S. Olsen R.W. J. Neurochem. 1993; 60: 1388-1398Crossref PubMed Scopus (50) Google Scholar, 19Marksitzer R. Benke D. Fritschy J.M. Trezciak A. Bannwarth W. Mohler H. J. Recep. Res. 1993; 13: 467-477Crossref PubMed Scopus (72) Google Scholar, 22McKernan R.M. Quirk K. Prince R. Cox P.A. Gillard N.A. Ragan C.I. Whiting P. Neuron. 1991; 7: 667-676Abstract Full Text PDF PubMed Scopus (178) Google Scholar) and that for α3-containing receptors to 18–24% (8Araujo F. Tan S. Ruano D. Schoemaker H. Benavides J. Vitorica J. J. Biol. Chem. 1996; 271: 27902-27911Abstract Full Text Full Text PDF PubMed Scopus (54) Google Scholar, 14Mertens S. Benke D. Mohler H. J. Biol. Chem. 1993; 268: 5965-5973Abstract Full Text PDF PubMed Google Scholar, 18Benke D. Cicin-Sain A. Mertens S. Mohler H. J. Recep. Res. 1991; 11: 407-424Crossref PubMed Scopus (72) Google Scholar, 22McKernan R.M. Quirk K. Prince R. Cox P.A. Gillard N.A. Ragan C.I. Whiting P. Neuron. 1991; 7: 667-676Abstract Full Text PDF PubMed Scopus (178) Google Scholar). This mismatch between the increase of diazepam-insensitive binding sites in the α2(H101R) and α3(H126R) mouse lines and the abundance of α2- and α3-containing receptor populations suggested that the receptors that contained the respective point-mutated subunit were not rendered fully insensitive to diazepam binding. To analyze this assumption, the respective α1(H101R)-, α2(H101R)-, and α3(H126R)-containing receptor populations were isolated by immunoprecipitation from deoxycholate extracts of whole brain membranes using subunit-selective antisera. The proportion of diazepam-sensitive and diazepam-insensitive [3H]Ro 15-4513-binding sites in the immunoprecipitated receptor populations was then determined by diazepam displacement of [3H]Ro 15-4513 binding. Interestingly, all of the receptor populations containing the point mutation displayed a high affinity diazepam-binding component corresponding to that of wild type receptors (Fig. 1). Whereas in α1(H101R)-containing receptors only 16 ± 1% remained diazepam-sensitive, 54 ± 10% of α2(H101R)-containing receptors and 73 ± 4% of α3(H126R)-containing receptors displayed diazepam-sensitive binding sites (Fig. 1 and Table II). This result demonstrates that at least some subpopulations of GABAA receptors containing the H/R mutation had retained diazepam-sensitive binding sites.Table IIProportion of diazepam-sensitive and diazepam-insensitive [3H]Ro 15-4513-binding sites in GABAA receptor populations immunoprecipitated with α subunit-selective antisera from brain extracts of the various single- and double-mutant mouse linesGenotypeDiazepam displacement of [3H]Ro 15-4513 binding to immunoprecipitation GABAA receptorsAntibody used for IPDiazepam-sensitive binding sitesDiazepam-insensitive binding sites% total% total% increaseaThe values are compared to the respective single mutants. % increase refers to the diazepam-insensitive binding sites present in the double-mutant receptor (e.g. α1(H101R) α2(H101R), precipitated with α1 antiserum: 91%) minus the diazepam-insensitive binding sites of the corresponding single-mutant receptors (α1(H101R): 84%). In this example, the difference in diazepam-insensitive binding sites (7%) corresponds to the abundance of α1α2 receptors in the α1 receptor population. The Ki values of diazepam-sensitive and diazepam-insensitive [3H]Ro 15-4513-binding sites were in the range of 30-100 nm and >60,000 nm, respectively. The data represent the means ± S.D. of three to six independent experiments.α1(H101R)α116 ± 184 ± 1α2(H101R)α254 ± 1046 ± 10α3(H126R)α373 ± 427 ± 4α1(H101R) α2(H101R)α19 ± 791 ± 77α218 ± 782 ± 736α1(H101R) α3(H126R)α18 ± 1092 ± 108α317 ± 283 ± 256α2(H101R) α3(H126R)α235 ± 865 ± 819α354 ± 1346 ± 1319a The values are compared to the respective single mutants. % increase refers to the diazepam-insensitive binding sites present in the double-mutant receptor (e.g. α1(H101R) α2(H101R), precipitated with α1 antiserum: 91%) minus the diazepam-insensitive binding sites of the corresponding single-mutant receptors (α1(H101R): 84%). In this example, the difference in diazepam-insensitive binding sites (7%) corresponds to the abundance of α1α2 receptors in the α1 receptor population. The Ki values of diazepam-sensitive and diazepam-insensitive [3H]Ro 15-4513-binding sites were in the range of 30-100 nm and >60,000 nm, respectively. The data represent the means ± S.D. of three to six independent experiments. Open table in a new tab Because GABAA receptors exist in brain that contain two different types of α subunits in the same receptor complex (6del Rio J.C. Araujo F. Ramos B. Ruano D. Vitorica J. J. Neurochem. 2001; 79: 183-191Crossref PubMed Scopus (22) Google Scholar, 7Araujo F. Ruano D. Vitorica J. J. Pharmacol. Exp. Therap. 1999; 290: 989-997PubMed Google Scholar, 8Araujo F. Tan S. Ruano D. Schoemaker H. Benavides J. Vitorica J. J. Biol. Chem. 1996; 271: 27902-27911Abstract Full Text Full Text PDF PubMed Scopus (54) Google Scholar, 9Benke D. Michel C. Mohler H. J. Neurochem. 1997; 69: 806-814Crossref PubMed Scopus (75) Google Scholar, 10Duggan M.J. Pollard S. Stephenson F.A. J. Biol. Chem. 1991; 266: 24778-24784Abstract Full Text PDF PubMed Google Scholar, 11Endo S. Olsen R.W. J. Neurochem. 1993; 60: 1388-1398Crossref PubMed Scopus (50) Google Scholar, 12Jechlinger M. Pelz R. Tretter V. Klausberger T. Sieghart W. J. Neurosci. 1998; 18: 2449-2457Crossref PubMed Google Scholar, 13Khan Z.U. Gutierrez A. De Blas A.L. J. Neurochem. 1996; 66: 685-691Crossref PubMed Scopus (81) Google Scholar, 14Mertens S. Benke D. Mohler H. J. Biol. Chem. 1993; 268: 5965-5973Abstract Full Text PDF PubMed Google Scholar, 15Pollard S. Duggan J.M. Stephenson F.A. J. Biol. Chem. 1993; 268: 3753-3757Abstract Full Text PDF PubMed Google Scholar), the presence of high affinity diazepam binding in the mutated receptor populations is likely to be caused by the subpopulation of GABAA receptors containing a nonmutated heterologous α subunit (which is not α4 or α6) in the receptor complex that confers high affinity diazepam binding. To verify this hypothesis, brain extracts were immunodepleted in a sequential fashion to initially remove receptors containing the nonmutated α subunit (Fig. 2). For instance, from the brain extract of α2(H101R) mice α1-containing receptors were removed by quantitative immunoprecipitation followed by precipitation of the receptor population containing the α2 subunit and determination of diazepam-sensitive and diazepam-insensitive binding sites. If the diazepam-sensitive binding component in the mutated receptor population is due to a heterologous nonmutated α subunit in the receptor complex (e.g. α1 in α1α2(H101R)), the proportion of diazepam-insensitive binding sites will be increased after immunodepletion of these receptors because diazepam-sensitive binding sites are removed from the mutated receptor population. Indeed, in the brain extract from α2(H101R) mice immunodepletion of α1-containing receptors increased diazepam-insensitive binding in the α2(H101R)-containing receptor population from 46 ± 10 to 80 ± 6%, and immunodepletion of α3-containing receptors increased diazepam-insensitive binding to 62 ± 5% (Fig. 2). Likewise, depletion of α1- or α2-containing receptors increased diazepam-insensitive binding in α3(H126R)-containing GABAA receptors from 27 ± 4 to 76 ± 3% and 49 ± 14%, respectively (Fig. 2). Thus, the immunodepletion experiments demonstrate that the diazepam-sensitive binding sites present in the point-mutated receptor populations are due to the presence of a subfraction of receptors that contain a nonmutated heterologous α subunit. Proportion of Diazepam-insensitive [3H]Ro 15-4513 Binding in Double-mutant Mouse Lines—The observation that the remaining diazepam-sensitive binding in receptors containing a point-mutated α subunit is due to the presence of a nonmutated heterologous α subunit provides a means to estimate the abundance of receptors containing two different types of α subunits. As a prerequisite, mouse lines were generated by cross-breeding that contained the point mutation in two different α subunits, i.e. α1(H101R)α2(H101R), α1(H101R)α3(H126R), and α2(H101R)α3(H126R). The abundance of receptor subpopulations containing two heterologous α subunits was then estimated by comparing the proportion of diazepam-insensitive binding present in the membrane fractions of brains of double-mutant mice with the sum of diazepam-insensitive binding present in the corresponding single-mutant mice. Because in the double-mutant mice GABAA receptors containing the two distinct point-mutated α subunits (e.g. α1(H101R)α2(H101R)) are rendered in addition insensitive to diazepam binding (compared with the respective single-mutant mice), the amount of diazepam-insensitive binding sites is expected to be greater than the sum calculated from the corresponding single-mutant mice. Scatchard analysis of diazepam-sensitive and diazepam-insensitive [3H]Ro 15-4513 binding to crude membrane preparations of brains from the three double-mutant mouse lines revealed that the diazepam-insensitive binding sites exceeded the sum of diazepam-insensitive sites in the corresponding single-mutant mice (Table I). Whereas the sum of diazepam-insensitive sites in the single-mutant α1(H101R) and α2(H101R) mice together amounted to 73% of total [3H]Ro 15-4513 binding sites, the corresponding α1(H101R)α2(H101R) double-mutant mice expressed 86 ± 2% diazepam-insensitive binding sites. This fraction of 13% additional diazepam-insensitive binding present in the α1(H101R)α2(H101R) double-mutant mice compared with the sum of the respective single-mutant mice is likely to be accounted for by the α1(H101R)α2(H101R)-containing receptor population present in the double-mutant α1(H101R)α2(H101R) mice. Likewise, diazepam-insensitive sites in α1(H101R) and α3(H126R) mutant mice sum up to 65%, whereas 80 ± 5% were present in the α1(H101R)α3(H126R) double-mutant mice. This finding indicates that 15% of all diazepam-sensitive receptors in mouse brain contain the α subunit combination α1α3. However, in α2(H101R)α3(H126R) mice diazepam-insensitive binding sites were only marginally increased (2%) over the sum of the corresponding single mutants (α2(H101R) and α3(H126R)), indicating that receptors containing the α2α3 subunit combination are rarely expressed in mouse brain (Table I).Table IProportion of diazepam-insensitive [3H]Ro 15-4513 binding in crude membrane preparations of brains from the various single- and double-mutant mouse linesGenotypeDiazepam-insensitive [3H]Ro 15-4513 bindingPercentage of totalPercentage of increaseaPercentage of increase refers to the increase of diazepam-insensitive binding sites introduced by the point mutation (i.e. percentage of total mutant mice — percentage of total of wild type mice).Sum of increase in corresponding single mutantsbSum of increased diazepam-insensitive binding in the corresponding single-mutant mice.Fraction in double mutants exceeding sum of corresponding single mutantscFraction of diazepam-insensitive binding in double-mutant mice exceeding the sum of the corresponding single-mutant mice. This fraction likely represents the receptor subpopulation containing the respective heterologous α subunits (e.g. α1α2-containing receptors in the α1(H101R) α2(H101R) mice).%%Wild type5 ± 1α1(H101R)66 ± 3dThe data were taken from Ref. 16.61 ± 3α2(H101R)17 ± 0.1eThe data were taken from Ref. 17.12 ± 0.1α3(H126R)9 ± 2eThe data were taken from Ref. 17.4 ± 2α1(H101R) α2(H101R)91 ± 286 ± 27313α1(H101R) α3(H126R)85 ± 580 ± 56515α2(H101R) α3(H126R)23 ± 418 ± 4162a Percentage of increase refers to the increase of diazepam-insensitive binding sites introduced by the point mutation (i.e. percentage of total mutant mice — percentage of total of wild type mice).b Sum of increased diazepam-insensitive binding in the corresponding single-mutant mice.c Fraction of diazepam-insensitive binding in double-mutant mice exceeding the sum of the corresponding single-mutant mice. This fraction likely represents the receptor subpopulation containing the respective heterologous α subunits (e.g. α1α2-containing receptors in the α1(H101R) α2(H101R) mice).d The data were taken from Ref. 16Rudolph U. Crestani F. Benke D. Brünig I. Benson J.A. Martin J.R. Bluethmann H. Mohler H. Nature. 1999; 401: 796-800Crossref PubMed Scopus (1054) Google Scholar.e The data were taken from Ref. 17Low K. Crestani F. Keist R. Benke D. Brunig I. Benson J.A. Fritschy J.M. Rulicke T. Bluethmann H. Mohler H. Rudolph U. Science. 2000; 290: 131-134Crossref PubMed Scopus (832) Google Scholar. Open table in a new tab [3H]Ro 15-4513 Binding in Immuno-isolated GABAA Receptor Populations of Double-mutant Mice—To estimate the relative abundance of GABAA receptors containing two different types of α subunits within the α1-, α2-, or α3-containing receptor population, GABAA receptors were immunoprecipitated with the respective subunit-selective antisera from deoxycholate extracts of crude brain membranes derived from single-mutant mice (α1(H101R), α2(H101R), and α3(H126R)) as well as from double-mutant mice (α1(H101R)α2(H101R), α1(H101R)α3(H126R), and α2(H101R)α3(H126R)) and analyzed for the proportion of diazepam-sensitive and diazepam-insensitive binding. In all cases the diazepam-insensitive binding sites in the immunoprecipitates from double-mutant mice exceeded the sum of diazepam-insensitive sites in the immunoprecipitates of the corresponding single-mutant mice (Table II). Immunoprecipitation of α1-containing receptors from brain extracts of α1(H101R)α2(H101R) mice yielded 91 ± 7% diazepam-insensitive binding sites, which is 7% larger than the diazepam-insensitive sites from the corresponding single-mutant α1(H101R) (84 ± 1%; Table II). Likewise, immunoprecipitation of α2-containing receptors from brain extracts of α1(H101R)α2(H101R) mice resulted in 82 ± 7% diazepam-insensitive binding sites, which is 36% larger than the diazepam-insensitive sites from the corresponding single-mutant α2(H101R). This result suggests that the α1 receptor population comprises 7% α1α2-containing receptors, whereas the α2-containing receptor population includes 36% α1α2-containing receptors. A similar analysis on α1(H101R)α3(H126R) and α2(H101R)α3(H126R) mice indicated the presence of 8% of α1α3-containing receptors in the α1-containing receptor population, whereas the contribution of the α1α3 combination to the α3-containing receptor population was 56% (Table II). In addition, the proportion of α2α3-containing receptors was 19% in both the α2- and α3-containing GABAA receptor population. GABAA receptors are known to contain two α subunits that can be homologous or heterologous. If they are distinct they represent a different class of receptor subtype. In the present paper we studied the presence and abundance of GABAA receptors containing two different types of α subunits based on an analysis of mice containing the H/R point mutation in the α1, α2, or α3 subunit. This point mutation rendered the respective subunits insensitive to diazepam binding. Basis and Prerequisites—There are two prerequisites for using mouse lines containing the α(H/R) mutation for a quantitative analysis of GABAA receptors containing two different types of α subunits. First, the introduction of the point mutation should not affect the expression levels and the distribution of the mutated subunit as well as those of the nonmutated subunits. It was shown previously that expression and targeting of receptor subunits appear not to be affected by the point mutation. Mice containing the H/R mutation in the α1, α2, or α3 subunit expressed the mutated subunit at normal levels with unaltered expression patterns and did not induce up- or down-regulation of other GABAA receptor subunits to an appreciable extent (16Rudolph U. Crestani F. Benke D. Brünig I. Benson J.A. Martin J.R. Bluethmann H. Mohler H. Nature. 1999; 401: 796-800Crossref PubMed Scopus (1054) Google Scholar, 17Low K. Crestani F. Keist R. Benke D. Brunig I. Benson J.A. Fritschy J.M. Rulicke T. Bluethmann H. Mohler H. Rudolph U. Science. 2000; 290: 131-134Crossref PubMed Scopus (832) Google Scholar). Second, in receptors containing one mutated and one nonmutated α subunit, the nonmutated α subunit should confer diazepam-sensitive binding to the receptor complex. The first hint that mouse lines carrying the α(H/R) mutation fulfill this second requirement came from the observation that subfractions in all of the mutated GABAA receptor populations displayed at least some high affinity diazepam binding (Fig. 1). Subsequent immunodepletion studies indicated that the presence of a heterologous α subunit in the receptor complex that is not mutated (e.g. α1 in α1α3(H126R) or α2 in α2α3(H126R)) conferred sensitivity to diazepam binding (Fig. 2). This feature permitted an assessment of GABAA receptors containing two different α subunits using the various mouse lines carrying the a(H/R) mutation. However, the effect of the presence of a mutated α subunit together with a nonmutated α subunit in a single receptor complex on GABAA receptor function, i.e. pharmacology, is currently unknown. In particular, it is unclear whether the diazepam sensitivity observed in radioligand binding experiments to a mutated α subunit and a nonmutated α subunit reflects the ability of those receptors to functionally respond to diazepam. Abundance of GABAA Receptors Containing Two Different α Subunits—The data indicate that the α(H/R) point mutation solely rendered GABAA receptors fully insensitive to diazepam binding that contain two homologous mutated α subunits, whereas receptors containing one mutated α subunit and one nonmutated α subunit retained diazepam binding. This feature permitted an assessment of GABAA receptors containing two different types of α subunits by comparing the extent of diazepam-insensitive binding in the double-mutant mouse lines with that present in the corresponding single-mutant mice. The different receptor subpopulations are calculated by subtracting the sum of diazepam-insensitive binding sites present in the respective single mutated receptors (e.g. α1(H101R) plus α2(H101R)) from diazepam-insensitive binding sites present in the corresponding double-mutant mice (α1(H101R)α2(H101R)). This difference in diazepam-insensitive sites represents the amount of the respective receptors containing two different α subunits (e.g. α1α2-containing receptors). Two types of information were obtained from the experimental data: 1) Determination of the proportion of diazepam-sensitive and diazepam-insensitive binding to crude membrane preparations permitted the estimation of the size of GABAA receptor populations in brain containing the different α subunit combinations (Table I). This analysis is restricted to the diazepam-sensitive α1-, α2-, and α3-containing receptors expressed in brain, which, however, represent the vast majority of GABAA receptors (summarized in Table III). 2) Data on the presence of diazepam-sensitive and diazepam-insensitive binding sites in immuno-isolated GABAA receptors allowed an estimate on the abundance of the different α subunit combinations present within a particular GABAA receptor population (e.g. the α1α3 receptor combination in α1-containing receptors; Table II). The data obtained from this analysis are summarized in Table IV. Because the experimental data exhibit partially considerable standard deviation (Table II), the data are considered to represent a rough estimate of the receptor populations.Table IIISummary of the estimated abundance of diazepam-sensitive GABAA receptors containing different types of α subunits inwhole mouse brain membranesSubunit combinationTotal diazepam-sensitive receptor population%α1α161α2α212α3α34α1α213α1α315α2α32 Open table in a new tab Table IVSummary of the estimated abundance of GABAA receptors containing heterologous α subunits within the α 1, α 2, and α 3 receptor populations immunoprecipitated from whole brain extractsTotal receptor populationReceptor populationSubunit combinationCalculated from diazepam competition experimentsCalculated from immunodepletion experiments%α1-Containing receptor populationα1α184NDα1α27NDα1α38NDα2-Containing receptor populationα2α246α2α13634α2α31916α3-Containing receptor populationα3α327α3α15649α3α21922 Open table in a new tab With about 61%, the α1α1-containing receptor is by far the most abundant GABAA receptor subtype in brain, whereas α2α2- (12%) and α3α3-containing receptors (4%) are considerably less expressed (Table III). Interestingly, levels of α1α2-containing receptors (13%) are similar to that of α2α2-containing receptors and levels of α1α3-containing receptors (15%) are considerably higher than that of α3α3-containing receptors (4%; Table III). However, receptors containing the α2 and α3 subunit in a single receptor complex appear to be rarely expressed in relation to the total diazepam-sensitive GABAA receptor population (2%; Table III). Correspondingly, a similar distribution of the prevalence of the various α subunit combinations was detected within the α1-, α2-, and α3-containing receptor populations (Table IV). Again, the α1α1 combination (84%) predominated the α1α2 (7%) and α1α3 combinations (8%) in the α1-containing receptor population. Within the α2 receptor population the α2α2 (46%) and the α1α2 combination (36%) are expressed to a similar extent, whereas the α2α3 combination (19%) is less abundant. Finally, within the α3-containing receptor population, the α2α3 (19%) and α3α3 combinations (27%) are clearly dominated by the α1α3 combination (56%). These values are in good agreement with the data derived from the immunodepletion experiments (Table IV). In addition, the data fit nicely to values published so far on the abundance of the α1α2 combination in the α2-containing receptor population of rat hippocampus and cerebral cortex (36% versus 36 and 39%, respectively) (6del Rio J.C. Araujo F. Ramos B. Ruano D. Vitorica J. J. Neurochem. 2001; 79: 183-191Crossref PubMed Scopus (22) Google Scholar, 8Araujo F. Tan S. Ruano D. Schoemaker H. Benavides J. Vitorica J. J. Biol. Chem. 1996; 271: 27902-27911Abstract Full Text Full Text PDF PubMed Scopus (54) Google Scholar) and the α1α3 combination in the α3-containing receptor population in the cerebral cortex (56% versus 55%) (6del Rio J.C. Araujo F. Ramos B. Ruano D. Vitorica J. J. Neurochem. 2001; 79: 183-191Crossref PubMed Scopus (22) Google Scholar, 8Araujo F. Tan S. Ruano D. Schoemaker H. Benavides J. Vitorica J. J. Biol. Chem. 1996; 271: 27902-27911Abstract Full Text Full Text PDF PubMed Scopus (54) Google Scholar). However, for the α1α3 combination in the α1-containing receptor population in cerebral cortex, an abundance of 24% was reported (6del Rio J.C. Araujo F. Ramos B. Ruano D. Vitorica J. J. Neurochem. 2001; 79: 183-191Crossref PubMed Scopus (22) Google Scholar, 8Araujo F. Tan S. Ruano D. Schoemaker H. Benavides J. Vitorica J. J. Biol. Chem. 1996; 271: 27902-27911Abstract Full Text Full Text PDF PubMed Scopus (54) Google Scholar), whereas the estimate of the present report yielded a value of 8% for whole mouse brain extracts. It is currently unclear whether this mismatch is due to experimental reasons, the brain areas used (cerebral cortex versus whole brain), or the species analyzed (rat versus mouse). Interestingly, only in the α1-containing receptor population the α1α1 combination is the most prevalent one, whereas in the α2 and particularly in the α3 receptor population receptors with two different types of α subunits predominate. Because GABAA receptors containing two different types of α subunits appear to be expressed at considerable levels, it is conceivable that the inclusion of a second type of α subunit may influence GABAA receptor function. However, the consequences of two different types of α subunits within a single receptor complex on GABAA receptor function are largely unknown. In particular, it is not clear how the expression of heterologous α subunits in a receptor complex affects functional benzodiazepine receptor pharmacology. So far, electrophysiological experiments on recombinant α1α3β2γ2, α1α5β2γ2, and α1α6β2γ2 indicate that receptors containing two different α subunit variants display unique kinetics and pharmacological properties (23Ebert B. Wafford K.A. Whiting P.J. Krogsgaard-Larsen P. Kemp J.A. Mol. Pharmacol. 1994; 46: 957-963PubMed Google Scholar, 24Tia S. Wang J.F. Kotchabhakdi N. Vicini S. Neuropharmacol. 1996; 35: 1375-1382Crossref PubMed Scopus (106) Google Scholar, 25Verdoorn T.A. Mol. Pharmacol. 1994; 45: 475-480PubMed Google Scholar, 26Sigel E. Baur R. J. Neurochem. 2000; 74: 2590-2596Crossref PubMed Scopus (39) Google Scholar). All of these studies inherently lack the ultimate certainty that indeed predominantly the subunit combination containing two heterologous α subunits was measured and not a mixture of different subunit combinations containing only a single α subunit variant. However, recent successful approaches linking the subunits covalently together to assess the subunit arrangement within the receptor complex and the functional consequences of subunit position in the receptor pentamer provide now the basis for analyzing GABAA receptor subtypes of predefined subunit arrangement (27Baumann S.W. Baur R. Sigel E. J. Biol. Chem. 2002; 277: 46020-46025Abstract Full Text Full Text PDF PubMed Scopus (229) Google Scholar, 28Minier F. Sigel E. Proc. Natl. Acad. Sci. U. S. A. 2004; 101: 7769-7774Crossref PubMed Scopus (101) Google Scholar, 29Baumann S. Baur R. Sigel E. J. Neurosci. 2003; 23: 11158-11166Crossref PubMed Google Scholar). A first study on the functional consequences of the position of α1 and α6 subunits in the same receptor pentamer clearly demonstrated that only receptors with the α1 subunit next to the γ2 subunit (γ2-β2-α6-β2-α1) displayed modulation by diazepam, whereas receptors with the α6 subunit next to the γ2 subunit (γ2-β2-α1-β2-α6) were insensitive to diazepam (28Minier F. Sigel E. Proc. Natl. Acad. Sci. U. S. A. 2004; 101: 7769-7774Crossref PubMed Scopus (101) Google Scholar). These data support the results of the present study indicating that only receptors containing two mutated α subunits are insensitive to diazepam binding. In addition, the data suggest that the nonmutated, diazepam-sensitive α subunit preferentially assembles with the γ2 subunit. In conclusion, the results of this study indicate that the mouse lines containing the α(H/R) mutation represent a valuable model for analyzing the abundance of receptor populations containing two different types of α subunits. This approach allows the analysis to be performed on membrane fractions, tissue homogenates, or even tissue sections without solubilization and immunoprecipitation of the receptors. A detailed analysis of receptor subtypes containing two different types of α subunits on a brain regional level by quantitative receptor autoradiography on brain sections is now feasible.