Molecular and Pharmacological Characterization of Native Cortical γ-Aminobutyric AcidA Receptors Containing Both α1 and α3 Subunits
1996; Elsevier BV; Volume: 271; Issue: 44 Linguagem: Inglês
10.1074/jbc.271.44.27902
ISSN1083-351X
AutoresFrancisco Araujo, Suan Tan, Diego Ruano, Hans E. Schoemaker, Jesús Bénavidès, Javier Vitórica,
Tópico(s)Receptor Mechanisms and Signaling
ResumoWe have investigated the existence, molecular composition, and benzodiazepine binding properties of native cortical α1-α3γ-aminobutyric acidA (GABAA) receptors using subunit-specific antibodies.The co-existence of α1 and α3 subunits in native GABAA receptors was demonstrated by immunoblot analysis of the anti-α1- or anti-α3-immunopurified receptors and by immunoprecipitation experiments of the [3H]zolpidem binding activity. Furthermore, immunodepletion experiments indicated that the α1-α3 GABAA receptors represented 54.7 ± 5.0 and 23.6 ± 3.3% of the α3 and α1 populations, respectively. Therefore, α1 and α3 subunits are associated in the same native GABAA receptor complex, but, on the other hand, these α1-α3 GABAA receptors from the cortex constitute a large proportion of the total α3 population and a relatively minor component of the α1 population.The pharmacological analysis of the α1- or α3-immunopurified receptors demonstrated the presence of two different benzodiazepine binding sites in each receptor population with high (type I binding sites) and low (type II binding sites) affinities for zolpidem and Cl 218,872. These results indicate the existence of native GABAA receptors possessing both α1 and α3 subunits, with α1 and α3 subunits expressing their characteristic benzodiazepine pharmacology.The molecular characterization of the anti-α1-anti-α3 double-immunopurified receptors demonstrated the presence of stoichiometric amounts of α1 and α3 subunits, associated with β2/3, and γ2 subunits. The pharmacological analysis of α1-α3 GABAA receptors demonstrated that, despite the fact that each α subunit retained its benzodiazepine binding properties, the relative proportion between type I and II binding sites or between 51- and 59-61-kDa [3H]Ro15-4513-photolabeled peptides was 70:30. Therefore, the α1 subunit is pharmacologically predominant over the α3 subunit. These results indicate the existence of active and nonactive α subunits in the native α1-α3 GABAA receptors from rat cortex. We have investigated the existence, molecular composition, and benzodiazepine binding properties of native cortical α1-α3γ-aminobutyric acidA (GABAA) receptors using subunit-specific antibodies. The co-existence of α1 and α3 subunits in native GABAA receptors was demonstrated by immunoblot analysis of the anti-α1- or anti-α3-immunopurified receptors and by immunoprecipitation experiments of the [3H]zolpidem binding activity. Furthermore, immunodepletion experiments indicated that the α1-α3 GABAA receptors represented 54.7 ± 5.0 and 23.6 ± 3.3% of the α3 and α1 populations, respectively. Therefore, α1 and α3 subunits are associated in the same native GABAA receptor complex, but, on the other hand, these α1-α3 GABAA receptors from the cortex constitute a large proportion of the total α3 population and a relatively minor component of the α1 population. The pharmacological analysis of the α1- or α3-immunopurified receptors demonstrated the presence of two different benzodiazepine binding sites in each receptor population with high (type I binding sites) and low (type II binding sites) affinities for zolpidem and Cl 218,872. These results indicate the existence of native GABAA receptors possessing both α1 and α3 subunits, with α1 and α3 subunits expressing their characteristic benzodiazepine pharmacology. The molecular characterization of the anti-α1-anti-α3 double-immunopurified receptors demonstrated the presence of stoichiometric amounts of α1 and α3 subunits, associated with β2/3, and γ2 subunits. The pharmacological analysis of α1-α3 GABAA receptors demonstrated that, despite the fact that each α subunit retained its benzodiazepine binding properties, the relative proportion between type I and II binding sites or between 51- and 59-61-kDa [3H]Ro15-4513-photolabeled peptides was 70:30. Therefore, the α1 subunit is pharmacologically predominant over the α3 subunit. These results indicate the existence of active and nonactive α subunits in the native α1-α3 GABAA receptors from rat cortex. INTRODUCTIONThe neuropharmacological effects of benzodiazepines are mediated by the benzodiazepine (ω) binding sites associated with the GABAA 1The abbreviations used are: GABAAγ-aminobutyric acidAPBSphosphate-buffered salineFMZflumazenilFNZflunitrazepammAbmonoclonal antibodyCHAPS3-[(3-cholamidopropyl)dimethylammonio]-1-propanesulfonic acid. receptor complex (for reviews, see 1Squires R.F. Lajtha A. Handbook of Neurochemistry. Vol 6. Plenum Press, New York1984,1919: 261-306Crossref Google Scholar and 2Tallman J.F. Gallager D.W. Annu. Rev. Neurosci. 1985; 8: 21-44Crossref PubMed Google Scholar). Based on their affinity for different drugs, two different benzodiazepine binding sites have been identified in the central nervous system. Type I (benzodiazepine receptor 1, ω1) displays high affinity for CL 218,872 (2Tallman J.F. Gallager D.W. Annu. Rev. Neurosci. 1985; 8: 21-44Crossref PubMed Google Scholar), β-carboline derivates (3Nielsen M. Braestrup C. Nature. 1980; 286: 606-607Crossref PubMed Scopus (186) Google Scholar), and the imidazopyridine zolpidem (4Arbilla S. Allen J. Wick A. Langer S.Z. Eur. J. Pharmacol. 1986; 130: 257-263Crossref PubMed Scopus (111) Google Scholar, 5Langer S.Z. Arbilla S. Pharmacol. Biochem. Behav. 1988; 29: 763-767Crossref PubMed Scopus (142) Google Scholar). Type II (benzodiazepine receptor 2, ω2) displays low affinity for these compounds. A third benzodiazepine binding site with very low affinity for zolpidem (type IIL, ω5) has also been identified in isolated rat brain membranes (6Ruano D. Vizuete M. Cano J. Machado A. Vitorica J. J. Neurochem. 1992; 58: 485-493Crossref PubMed Scopus (67) Google Scholar) and sections (7Benavides J. Peny B. Ruano D. Vitorica J. Scatton B. Brain Res. 1993; 604: 240-250Crossref PubMed Scopus (99) Google Scholar).Molecular cloning experiments have demonstrated the existence of five different families of subunits that are components of the GABAA receptor complex. Most of these families comprise several isoforms: α1-α6, β1-β3, γ1-γ3, δ, and ρ1 and ρ2 (for reviews, see 8Olsen R.W. Tobin A.J. FASEB J. 1990; 4: 1469-1480Crossref PubMed Scopus (862) Google Scholar and 9Burt D.R. Kamatchi G.L. FASEB J. 1991; 5: 2916-2923Crossref PubMed Scopus (387) Google Scholar). A minimum of α, β, and γ subunits should be co-expressed in transfected cells to resemble all the pharmacological properties of native GABAA receptors (10Pritchett D.B. Sontheimer H. Shivers B.D. Ymer S. Kettenmann H. Schofield P.R. Seeburg P.H. Nature. 1989; 338: 582-585Crossref PubMed Scopus (1144) Google Scholar). On the other hand, the presence of different α subunits determines the affinity of the different benzodiazepine binding sites. In this sense, the α1-β1-β3-γ2 combination confers type I pharmacology to the recombinant GABAA receptor (i.e. high affinity for, among others, zolpidem and Cl 218,872) (11Pritchett D.B. Seeburg P.H. J. Neurochem. 1990; 54: 1802-1804Crossref PubMed Scopus (453) Google Scholar). Type II properties are conferred by the presence of α2, α3, or α5 subunits (11Pritchett D.B. Seeburg P.H. J. Neurochem. 1990; 54: 1802-1804Crossref PubMed Scopus (453) Google Scholar, 12Pritchett D.B. Luddens H. Seeburg P.H. Science. 1989; 245: 1389-1392Crossref PubMed Scopus (636) Google Scholar).Several approaches have been taken to identify which subunits co-exist in the native GABAA receptor complex. However, the subunit composition of the different native GABAA receptor complexes remains unsolved. Immunoprecipitations or immunoaffinity purifications using anti-α subunit antibodies (anti-α1, -α2, -α3, -α5, and -α6 subunits) indicated that a significant proportion of native receptors are made by the association of two different α subunits (such as α1α2, α1α3, α1α5, or α1α6) (13Duggan M.J. Pollard S. Stephenson F.A. J. Biol. Chem. 1991; 266: 24778-24784Abstract Full Text PDF PubMed Google Scholar, 14Pollard S. Duggan M.J. Stephenson F.A. J. Biol. Chem. 1993; 268: 3753-3757Abstract Full Text PDF PubMed Google Scholar, 15Mertens S. Benke D. Mohler H. J. Biol. Chem. 1993; 268: 5965-5973Abstract Full Text PDF PubMed Google Scholar, 16Pollard S. Thompson C.L. Stephenson F.A. J. Biol. Chem. 1995; 270: 21285-21290Abstract Full Text Full Text PDF PubMed Scopus (63) Google Scholar, 17Khan Z.U. Gutierrez A. Deblas A.L. J. Neurochem. 1996; 66: 685-691Crossref PubMed Scopus (80) Google Scholar) in a single receptor complex. However, other authors have indicated the absence of association between different α subunits (18McKernan R.M. Quirk K. Prince R. Cox P.A. Gillard N.P. Ragan C.I. Whiting P. Neuron. 1991; 7: 667-676Abstract Full Text PDF PubMed Scopus (177) Google Scholar, 19Quirk K. Gillard N.P. Ragan C.I. Whiting P.J. McKernan R.M. J. Biol. Chem. 1994; 269: 16020-16028Abstract Full Text PDF PubMed Google Scholar). On the other hand, the pharmacological properties of these GABAA receptors are also unknown.In the present article we have addressed these questions by determining the molecular and pharmacological properties of the immunopurified receptors using subunit-specific antibodies to the major α subunits expressed in the rat cerebral cortex, the α1 and α3 subunits.DISCUSSIONThe molecular composition of native GABAA receptors is unknown. Evidence is accumulating for the existence of different α subunit combinations (such as α1α3, α1α2, α1α5, and α1α6) co-assembled in single native GABAA receptor complexes. However, other studies also indicated the absence of co-existence between different α subtypes (18McKernan R.M. Quirk K. Prince R. Cox P.A. Gillard N.P. Ragan C.I. Whiting P. Neuron. 1991; 7: 667-676Abstract Full Text PDF PubMed Scopus (177) Google Scholar, 19Quirk K. Gillard N.P. Ragan C.I. Whiting P.J. McKernan R.M. J. Biol. Chem. 1994; 269: 16020-16028Abstract Full Text PDF PubMed Google Scholar). On the other hand, it is currently accepted that the benzodiazepine binding properties of the GABAA receptors are mainly determined by the α subunits (10Pritchett D.B. Sontheimer H. Shivers B.D. Ymer S. Kettenmann H. Schofield P.R. Seeburg P.H. Nature. 1989; 338: 582-585Crossref PubMed Scopus (1144) Google Scholar, 11Pritchett D.B. Seeburg P.H. J. Neurochem. 1990; 54: 1802-1804Crossref PubMed Scopus (453) Google Scholar, 12Pritchett D.B. Luddens H. Seeburg P.H. Science. 1989; 245: 1389-1392Crossref PubMed Scopus (636) Google Scholar). Therefore, if two different α subunit subtypes are co-assembled in a single GABAA receptor, two pharmacologically different benzodiazepine binding sites could co-exist in a single complex. In the present article we have investigated the possible existence and the pharmacological properties of native α1-α3 GABAA receptors from the rat cortex.The presence of α1 and α3 subunits in the same GABAA receptor complex was demonstrated by immunoprecipitation and immunopurification experiments. It has been described that, in transfected GABAA receptors, the high affinity binding sites for zolpidem (type I benzodiazepine binding sites) are determined by the presence of α1 subunits. Other α subtypes (such as α2, α3, and α5) confer low affinity for this ligand (type II benzodiazepine receptors) (11Pritchett D.B. Seeburg P.H. J. Neurochem. 1990; 54: 1802-1804Crossref PubMed Scopus (453) Google Scholar, 12Pritchett D.B. Luddens H. Seeburg P.H. Science. 1989; 245: 1389-1392Crossref PubMed Scopus (636) Google Scholar). Therefore, the association of an α1 with other α subunits could be estimated by immunoprecipitation of the [3H]zolpidem binding activity. Our immunoprecipitation experiments (Fig. 2A and 21Ruano D. Araujo F. Machado A. De Blas A.L. Vitorica J. Mol. Brain Res. 1994; 25: 225-233Crossref PubMed Scopus (25) Google Scholar and 22Ruano D. Khan Z.U. De Blas A.L. Machado A. Vitorica J. Eur. J. Pharmacol Mol. Pharmacol. Sect. 1994; 267: 123-128Crossref PubMed Scopus (24) Google Scholar) demonstrate that most, if not all (90.0 ± 5.4%), of the high affinity binding sites for zolpidem are due to the presence of an α1 subunit in the GABAA receptor. Importantly, 25-30% of these [3H]zolpidem binding sites were immunoprecipitated by anti-α3 antibody, thus suggesting an α1-α3 association. Consistently, immunodepletion of the α1 subunits suppress the immunoprecipitation of [3H]zolpidem binding by the anti-α3 antibody (Table II). Thus, α1 binding properties could be immunoprecipitated in association with α3 subunits, suggesting the presence of both α1 and α3 subunits in the same receptor complex.The association between both subunits was confirmed by immunopurification experiments. The immunoblot analysis of the anti-α1- or anti-α3-immunopurified receptors (Fig. 2B) revealed the presence of α3 immunoreaction product in the anti-α1-immunopurified receptors and, reciprocally, the presence of α1 in anti-α3-immunopurified receptors. Furthermore, the association between both α subunits was not due to interactions with cytoskeletal elements. Taken together, these results demonstrated the existence of α1-α3 GABAA receptors from the rat cortex. Immunodepletion experiments indicated that the α1-α3 GABAA receptors constituted a relatively minor proportion of the total α1-containing GABAA receptors (20-25% of this population) but 50-55% of the α3 containing GABAA receptors. Thus, and in partial agreement with previous reports (13Duggan M.J. Pollard S. Stephenson F.A. J. Biol. Chem. 1991; 266: 24778-24784Abstract Full Text PDF PubMed Google Scholar, 14Pollard S. Duggan M.J. Stephenson F.A. J. Biol. Chem. 1993; 268: 3753-3757Abstract Full Text PDF PubMed Google Scholar, 15Mertens S. Benke D. Mohler H. J. Biol. Chem. 1993; 268: 5965-5973Abstract Full Text PDF PubMed Google Scholar), the association between two different α subunits represents a minor population from the total α1-containing receptors but a high proportion of other α subunits, such as α3.The presence of different α subtypes, in combination with β1-β3 and γ2 subunits, determines the benzodiazepine binding properties of recombinant GABAA receptors (10Pritchett D.B. Sontheimer H. Shivers B.D. Ymer S. Kettenmann H. Schofield P.R. Seeburg P.H. Nature. 1989; 338: 582-585Crossref PubMed Scopus (1144) Google Scholar, 11Pritchett D.B. Seeburg P.H. J. Neurochem. 1990; 54: 1802-1804Crossref PubMed Scopus (453) Google Scholar, 12Pritchett D.B. Luddens H. Seeburg P.H. Science. 1989; 245: 1389-1392Crossref PubMed Scopus (636) Google Scholar). As mentioned above, the α1 subunit confers type I benzodiazepine binding properties (high affinity for zolpidem and Cl 218,872), whereas the α3 subunit confers type II binding properties (low affinity for these ligands). Therefore, if two different α subunits, such as α1 and α3, are co-assembled in the same receptor complex, and both α1 and α3 subunits are pharmacologically active, two different benzodiazepine binding subtypes should be discriminated in either anti-α1- and anti-α3-immunopurified receptors. As shown in Table III, in anti-α1- and anti-α3-immunopurified receptors, two different binding sites were identified. The affinities for zolpidem (determined by Scatchard and displacement experiments) or Cl 218,872 (determined by displacement experiments) were similar in both immunopurified receptors and similar to those reported for type I and II benzodiazepine binding sites in cortical membranes (6Ruano D. Vizuete M. Cano J. Machado A. Vitorica J. J. Neurochem. 1992; 58: 485-493Crossref PubMed Scopus (67) Google Scholar, 7Benavides J. Peny B. Ruano D. Vitorica J. Scatton B. Brain Res. 1993; 604: 240-250Crossref PubMed Scopus (99) Google Scholar). Furthermore, the affinities for both ligands corresponded to those reported for recombinant receptors containing α1 subunits (high affinity binding sites) and α3 subunits (low affinity binding sites) (11Pritchett D.B. Seeburg P.H. J. Neurochem. 1990; 54: 1802-1804Crossref PubMed Scopus (453) Google Scholar, 12Pritchett D.B. Luddens H. Seeburg P.H. Science. 1989; 245: 1389-1392Crossref PubMed Scopus (636) Google Scholar). In consequence, these results suggest the presence of benzodiazepine binding sites in both α1 and α3 subunits co-assembled in a single GABAA receptor complex (also see 17Khan Z.U. Gutierrez A. Deblas A.L. J. Neurochem. 1996; 66: 685-691Crossref PubMed Scopus (80) Google Scholar).To discern whether both α1 and α3 subunits, co-assembled in a single complex, display benzodiazepine binding activity, the GABAA receptor was immunopurified by anti-α1 and anti-α3 affinity columns in series; therefore, the whole population of the isolated GABAA receptors should contain two different α subunits. It is noteworthy that anti-α3 immunoaffinity columns retained 20-25% of the α1-immunopurified GABAA receptors, corroborating the proportion of α1 to α3 GABAA receptors calculated by depletion experiments (compare Fig. 3A and Table II). Immunoblot analysis (Fig. 3B) indicates that α1 and α3 subunits are mainly associated with β2/3 and γ2 in the same receptor complex, consistent with previous experiments (21Ruano D. Araujo F. Machado A. De Blas A.L. Vitorica J. Mol. Brain Res. 1994; 25: 225-233Crossref PubMed Scopus (25) Google Scholar, 22Ruano D. Khan Z.U. De Blas A.L. Machado A. Vitorica J. Eur. J. Pharmacol Mol. Pharmacol. Sect. 1994; 267: 123-128Crossref PubMed Scopus (24) Google Scholar). The β1 subunits are a relatively minor component of the receptor (41Benke D. Fritschy J.M. Trzeciak A. Bannwarth W. Mohler H. J. Biol. Chem. 1994; 269: 27100-27107Abstract Full Text PDF PubMed Google Scholar), and, on the other hand, it has been demonstrated that γ1 is not associated with γ2-containing GABAA receptors (42Quirk K. Gillard N.P. Ragan C.I. Whiting P.J. McKernan R.M. Mol. Pharmacol. 1994; 45: 1061-1070PubMed Google Scholar). Thus, we propose a molecular composition of α1, α3, β2/3 and γ2 for these native GABAA receptor complexes from rat cortex.A relevant question to ascertain the pharmacological activity of the α subunits, co-assembled in a single native GABAA receptor, is the stoichiometry between both subunits in the complex. Thus, we have estimated the stoichiometry between both α subunits by quantifying the immunoreaction products of anti-α1 and anti-α3 antibodies in immunoblots. We are aware that immunoblots are only semiquantitative. However, within the limitations of the technique, the results (Fig. 4) indicated the presence of stoichiometric amounts of each α subunit (ratio 1:1; also see 16Pollard S. Thompson C.L. Stephenson F.A. J. Biol. Chem. 1995; 270: 21285-21290Abstract Full Text Full Text PDF PubMed Scopus (63) Google Scholar for discussion). The stoichiometry of γ2, β2, and β3 subunits was not determined.If both α subunits display benzodiazepine binding sites, the double-immunopurified receptors should display type I and II binding properties in similar proportions, and two peptides should be photoaffinity labeled by [3H]Ro15-4513 to a similar extent. Indeed, the pharmacological analysis of the α1-α3 GABAA receptors indicated the presence of two different benzodiazepine binding sites. Both Cl 218,872 and zolpidem discriminated between two different binding sites with high (type I) and low affinities (type II). The calculated Ki values for either ligand were similar to those of immunopurified α1 or α3 receptors (compare Table III, Table IV) and to cerebral membranes (6Ruano D. Vizuete M. Cano J. Machado A. Vitorica J. J. Neurochem. 1992; 58: 485-493Crossref PubMed Scopus (67) Google Scholar, 7Benavides J. Peny B. Ruano D. Vitorica J. Scatton B. Brain Res. 1993; 604: 240-250Crossref PubMed Scopus (99) Google Scholar). However, the proportion between both binding sites (70:30 for high and low affinity, respectively) demonstrates that the α1 subunits are predominantly active over the α3 subunits. It could be argued that the different proportions between both binding sites, determined by displacement experiments, is due to differences in the KD values of α1 and α3 subunits for the 3H-labeled ligand ([3H]FNZ or [3H]FMZ). However, these results were confirmed by [3H]Ro15-4513 photoaffinity-labeling experiments at three different degrees of saturation. As expected, in the double-immunopurified receptors, two photolabeled peptides of 51 kDa (corresponding to α1 subunits) and 59-61 kDa (α3 subunits) were identified. However, despite the fact that both α subunits are assembled in stoichiometric amounts in the same receptor complex, the proportion between both photolabeled peptides (at all three concentrations) was 70:30 for 51 and 59-61 kDa, respectively (Fig. 5). Thus, α1 subunits are pharmacologically predominant over the α3 subunits. It should be noted that [3H]Ro15-4513 photolabeled most, if not all, the benzodiazepine binding sites from cerebral membranes (90% in this work; also see 43Sieghart W. Eichinger A. Richards J.G. Mohler H. J. Neurochem. 1987; 48: 46-52Crossref PubMed Scopus (178) Google Scholar).Our data could be explained by the existence of at least two pharmacologically different populations of α1-α3 GABAA receptors (see Fig. 6 for a model). As shown in Fig. 6A, 70% of the α1-α3 GABAA receptors may be assembled by a functional α1 subunit associated with an inactive α3 subunit. The remaining 30% of the population may be constituted by a functional α3 subunit associated with inactive α1 subunits. Nevertheless, we cannot completely exclude the existence of α1-α3 GABAA receptors containing two benzodiazepine binding sites (Fig. 6B). In such a model, in which two functional α1 and α3 subunits are co-localized in the same receptor complex, 60% of the benzodiazepine binding sites should be conferred by GABAA receptors containing two functional α subunits and 30% by functional α1 subunits associated with inactive α3 subunits. Our results do not allow discrimination between these two models.The presence or absence of active benzodiazepine binding sites could be determined by the distribution of the α and γ2 subunits in the pentameric GABAA receptor complex (10Pritchett D.B. Sontheimer H. Shivers B.D. Ymer S. Kettenmann H. Schofield P.R. Seeburg P.H. Nature. 1989; 338: 582-585Crossref PubMed Scopus (1144) Google Scholar, 44Stephenson F.A. Duggan M.J. Pollard S. J. Biol. Chem. 1990; 265: 21160-21165Abstract Full Text PDF PubMed Google Scholar). It has been proposed that both α and γ2 subunits are implicated in the benzodiazepine binding sites (44Stephenson F.A. Duggan M.J. Pollard S. J. Biol. Chem. 1990; 265: 21160-21165Abstract Full Text PDF PubMed Google Scholar, 45Sieghart W. Pharmacol. Rev. 1995; 47: 181-234PubMed Google Scholar), and, on the other hand, the GABAA receptors may contain two α subunits, two β subunits, and a single γ2 subunit (46Backus K.H. Arigoni M. Drescher U. Scheurer L. Malherbe P. Mohler H. Benson J.A. Neuroreport. 1993; 5: 285-288Crossref PubMed Scopus (118) Google Scholar, 47Im W.B. Pregenzer J.F. Binder J.A. Dillon G.H. Alberts G.L. J. Biol. Chem. 1995; 270: 26063-26066Abstract Full Text Full Text PDF PubMed Scopus (81) Google Scholar). Thus, the predominance of α1 pharmacology (type I benzodiazepine binding sites or the 51-kDa photolabeled peptides) could be interpreted by the presence of a single γ2 subunit properly associated with the α1 subunit in the α1-α3 GABAA receptor complex (Fig. 6A). In these receptors, the α3 subunits should lack the benzodiazepine binding sites (see Fig. 6). On the other hand, two different γ2 subunits could also co-exist in the same receptor complex (46Backus K.H. Arigoni M. Drescher U. Scheurer L. Malherbe P. Mohler H. Benson J.A. Neuroreport. 1993; 5: 285-288Crossref PubMed Scopus (118) Google Scholar, 48Khan Z.U. Gutierrez A. De Blas A.L. J. Neurochem. 1994; 63: 1466-1476Crossref PubMed Scopus (60) Google Scholar). If this is the case, both α subunits could display benzodiazepine binding properties (Fig. 6B).The physiological significance of GABAA receptors containing two different α subtypes, such as an α1-α3 combination, is unknown. The α1 subunit is highly and uniformly expressed in all cortical layers, whereas the expression of the α3 subunit is localized in layers V and VI (49Fritschy J.M. Mohler H. J. Comp. Neurol. 1995; 359: 154-194Crossref PubMed Scopus (1044) Google Scholar). Therefore, the α1- and α3-containing GABAA receptors should be restricted to these cortical layers. Co-localization of α1 and α3 subunits has been also observed in other discrete brain regions (such as mitral cells of the olfactory bulb and the medial septum; 49Fritschy J.M. Mohler H. J. Comp. Neurol. 1995; 359: 154-194Crossref PubMed Scopus (1044) Google Scholar). On the other hand, in recombinant GABAA receptors, the co-expression of α1, α3, β2, and γ2 subunits confers unique functional properties, distinct from GABAA receptors containing a single α subtype (50Verdoorn T.A. Mol. Pharmacol. 1994; 45: 475-480PubMed Google Scholar, 51Ebert B. Wafford K.A. Whiting P.J. Krogsgaardlarsen P. Kemp J.A. Mol. Pharmacol. 1994; 46: 957-963PubMed Google Scholar). Therefore, the presence and pharmacological activity of two different α subunit subtypes in native receptor complexes, localized in discrete brain areas and/or cellular regions, could influence the functional and pharmacological properties of the GABAA receptor. The existence and pharmacological properties of α1α3-containing receptors increase the heterogeneity of the native GABAA receptor complex in the central nervous system.In summary, our results demonstrate the existence of cortical GABAA receptors containing both α1 and α3 subunits in stoichiometric amounts. Furthermore, both α subunits retained their benzodiazepine binding properties. However, the α1 subunit is pharmacologically predominant over α3 subunits, indicating the existence of active and nonactive benzodiazepine binding sites associated with these α subunits. INTRODUCTIONThe neuropharmacological effects of benzodiazepines are mediated by the benzodiazepine (ω) binding sites associated with the GABAA 1The abbreviations used are: GABAAγ-aminobutyric acidAPBSphosphate-buffered salineFMZflumazenilFNZflunitrazepammAbmonoclonal antibodyCHAPS3-[(3-cholamidopropyl)dimethylammonio]-1-propanesulfonic acid. receptor complex (for reviews, see 1Squires R.F. Lajtha A. Handbook of Neurochemistry. Vol 6. Plenum Press, New York1984,1919: 261-306Crossref Google Scholar and 2Tallman J.F. Gallager D.W. Annu. Rev. Neurosci. 1985; 8: 21-44Crossref PubMed Google Scholar). Based on their affinity for different drugs, two different benzodiazepine binding sites have been identified in the central nervous system. Type I (benzodiazepine receptor 1, ω1) displays high affinity for CL 218,872 (2Tallman J.F. Gallager D.W. Annu. Rev. Neurosci. 1985; 8: 21-44Crossref PubMed Google Scholar), β-carboline derivates (3Nielsen M. Braestrup C. Nature. 1980; 286: 606-607Crossref PubMed Scopus (186) Google Scholar), and the imidazopyridine zolpidem (4Arbilla S. Allen J. Wick A. Langer S.Z. Eur. J. Pharmacol. 1986; 130: 257-263Crossref PubMed Scopus (111) Google Scholar, 5Langer S.Z. Arbilla S. Pharmacol. Biochem. Behav. 1988; 29: 763-767Crossref PubMed Scopus (142) Google Scholar). Type II (benzodiazepine receptor 2, ω2) displays low affinity for these compounds. A third benzodiazepine binding site with very low affinity for zolpidem (type IIL, ω5) has also been identified in isolated rat brain membranes (6Ruano D. Vizuete M. Cano J. Machado A. Vitorica J. J. Neurochem. 1992; 58: 485-493Crossref PubMed Scopus (67) Google Scholar) and sections (7Benavides J. Peny B. Ruano D. Vitorica J. Scatton B. Brain Res. 1993; 604: 240-250Crossref PubMed Scopus (99) Google Scholar).Molecular cloning experiments have demonstrated the existence of five different families of subunits that are components of the GABAA receptor complex. Most of these families comprise several isoforms: α1-α6, β1-β3, γ1-γ3, δ, and ρ1 and ρ2 (for reviews, see 8Olsen R.W. Tobin A.J. FASEB J. 1990; 4: 1469-1480Crossref PubMed Scopus (862) Google Scholar and 9Burt D.R. Kamatchi G.L. FASEB J. 1991; 5: 2916-2923Crossref PubMed Scopus (387) Google Scholar). A minimum of α, β, and γ subunits should be co-expressed in transfected cells to resemble all the pharmacological properties of native GABAA receptors (10Pritchett D.B. Sontheimer H. Shivers B.D. Ymer S. Kettenmann H. Schofield P.R. Seeburg P.H. Nature. 1989; 338: 582-585Crossref PubMed Scopus (1144) Google Scholar). On the other hand, the presence of different α subunits determines the affinity of the different benzodiazepine binding sites. In this sense, the α1-β1-β3-γ2 combination confers type I pharmacology to the recombinant GABAA receptor (i.e. high affinity for, among others, zolpidem and Cl 218,872) (11Pritchett D.B. Seeburg P.H. J. Neurochem. 1990; 54: 1802-1804Crossref PubMed Scopus (453) Google Scholar). Type II properties are conferred by the presence of α2, α3, or α5 subunits (11Pritchett D.B. Seeburg P.H. J. Neurochem. 1990; 54: 1802-1804Crossref PubMed Scopus (453) Google Scholar, 12Pritchett D.B. Luddens H. Seeburg P.H. Science. 1989; 245: 1389-1392Crossref PubMed Scopus (636) Google Scholar).Several approaches have been taken to identify which subunits co-exist in the native GABAA receptor complex. However, the subunit composition of the different native GABAA receptor complexes remains unsolved. Immunoprecipitations or immunoaffinity purifications using anti-α subunit antibodies (anti-α1, -α2, -α3, -α5, and -α6 subunits) indicated that a significant proportion of native receptors are made by the association of two different α subunits (such as α1α2, α1α3, α1α5, or α1α6) (13Duggan M.J. Pollard S. Stephenson F.A. J. Biol. Chem. 1991; 266: 24778-24784Abstract Full Text PDF PubMed Google Scholar, 14Pollard S. Duggan M.J. Stephenson F.A. J. Biol. Chem. 1993; 268: 3753-3757Abstract Full Text PDF PubMed Google Scholar, 15Mertens S. Benke D. Mohler H. J. Biol. Chem. 1993; 268: 5965-5973Abstract Full Text PDF PubMed Google Scholar, 16Pollard S. Thompson C.L. Stephenson F.A. J. Biol. 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On the other hand, the pharmacological properties of these GABAA receptors are also unknown.In the present article we have addressed these questions by determining the molecular and pharmacological properties of the immunopurified receptors using subunit-specific antibodies to the major α subunits expressed in the rat cerebral cortex, the α1 and α3 subunits.
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