Skip to main content
SpringerLink
Log in

The γ subunits of the native GABAA/benzodiazepine receptors

  • Original Articles
  • Published:
Neurochemical Research Aims and scope Submit manuscript

Abstract

Subunit-specific antibodies to all the γ subunit isoforms described in mammalian brain (γ1, γ2S, γL, and γ3) have been made. The proportion of GABAA receptors containing each γ subunit isoform in various brain regions has been determined by quantitative immunoprecipitation. In all tested regions of the rat brain, the γ1, and γ3 subunits are present in considerable smaller proportion of GABAA receptor than the γ2 subunit. Immunocytochemistry shows that γ1 immunoreactivity concentrates in the stratum oriens and stratum radiatum of the CA1 region of the hippocampus. In the dentate gyrus, γ1 immunoreactivity concentrates on the outer 2/3 of the molecular layer coinciding with the localization of the axospinous synapses of the perforant pathway. In contrast, γ3 immunoreactivity concentrates on the basket cells and other GABAergic local circuit neurons of the hilus. These cells are also rich in γ2S. In the cerebellu, γ1 immunolabeling was localized on the Bergmann glia. The γ2S and γ2L subunits are differentially expressed in various brain regions. Thus the γ2S is highly expressed in the olfactory bulb and hippocampus whereas the γ2L is very abundant in inferior colliculus and cerebellum, particularly in Purkinje cells, as immunocytochemistry, in situ hybridization and immunoprecipitation techniques have revealed. The γ2S and γ2L coexist in some brain areas and cell types. Moreover, the γ2S and γ2L subunits can coexist in the same GABAA receptor pentamer. We have shown that this is the case in some GABAA receptors expressed in cerebellar granule cells. These GABAA receptors also have α and β subunits forming the pentamer. Immunoblots have shown that the rat γ1, γ2S, γ2L and γ3 subunits are peptides of 47, 45, 47 and 44 kDa respectively. Results also indicate that there are aging-related changes in the expression of the γ2S and γ2L subunits in various brain regions which suggest the existence of aging-related changes in the subunit composition of the GABAA receptors which in turn might lead to changes in receptor pharmacology. The results obtained with the various γ subunit isoforms are discussed in terms of the high molecular and binding heterogeneity of the native GABAA receptors in brain.

This is a preview of subscription content, log in via an institution to check access.

Access this article

Log in via an institution

Price excludes VAT (USA)
Tax calculation will be finalised during checkout.

Instant access to the full article PDF.

Institutional subscriptions

Similar content being viewed by others

Refereces

  1. Dunn, S. M. J., Bateson, A. N., and Martin, I. L. 1994. Molecular neurobiology of the GABAA, receptor. International Rev. Neurobiol. 36: 51–96.

    Article  CAS  Google Scholar 

  2. Macdonald, R. L., and Olsen, R. W. 1994. GABAA receptor channels. Ann. Rev. Neurosci. 17: 569–602.

    PubMed  CAS  Google Scholar 

  3. Nayeem, N., Green, T. P., Martin, I. L., and Barnard, E. A. 1994. The quaternary structure of the native GABAA receptors determined by electron microscopic image analysis. J. Neurochem. 62: 815–818.

    Article  PubMed  CAS  Google Scholar 

  4. Bateson, A. N., Lasham, A., and Darlison, M. G. 1991. γ-Aminobutryic acidA receptor heterogeneity is increased by alternative splicing of novel β-subunit gene transcript. J. Neurochem. 56: 1437–1440.

    Article  PubMed  CAS  Google Scholar 

  5. Harvey, R. J., Kim, H.-C., and Darlison, M. G. 1993. Molecular cloning reveals the existence of a fourth γ subunit of the vertebrate brain GABAA receptor. FEBS letters 331: 211–216.

    Article  PubMed  CAS  Google Scholar 

  6. Whiting, P., McKernan, R. M., and Iversen, L. L. 1990). Another mechanism for creating diversity in γ-aminobutyric type A receptors: RNA splicing directs expression of two forms of γ2 subunit, one of which contains a protein kinase C phosphorylation site. Proc. Natl. Acad. Sci. USA 87: 9966–9970.

    Article  PubMed  CAS  Google Scholar 

  7. Kofuji, P., Wang, J. B., Moss, S. J., Huganir, R. L., and Burt, D. R. 1991. Generation of two forms of the γ-aminobutyric acidA receptor γ-subunit in mice by alternative splicing. J. Neurochem. 56: 713–715.

    Article  PubMed  CAS  Google Scholar 

  8. Schofield, P. R., Darlison, M. G., Fujita, M., Burt, D. R., Stephenson, F. A., Rodriguez, H., Rhee, L. M., Ramachandran, J., Reale, V., Glencorse, T. A., Seeburg, P. H., and Barnard, E. A. 1987. Sequence and functional expression of the GABAA receptor shows a ligand-gated receptor super-family. Nature 328: 221–227.

    Article  PubMed  CAS  Google Scholar 

  9. Kellenberger, S., Malherbe, P., and Sigel, E. 1992. Function of the α1β2 γ2S γ-aminobutyric acid Type A receptor is modulated by protein kinase C via multiple phosphorylation sites. J. Biol. Chem. 267: 25660–25663.

    PubMed  CAS  Google Scholar 

  10. Moss, S. J., Doherty, C. A., and Huganir, R. 1992. Identification of the cAMP-dependent protein kinase and protein kinase C phosphorylation sites within the major intracellular' domains of the β1, γ2S and γ2L subunits of the γ-aminobutyric acid type A receptor. J. Biol. Chem. 267: 14470–14476.

    PubMed  CAS  Google Scholar 

  11. Machu, T. K., Firestone, J. A., and Browning, M. D. 1993. Ca2+/Calmodulin-dependent protein kinase II and protein kinase C phosphorylate a synthetic peptide corresponding to a sequence that is specific for the γ2L subunit of the GABAA receptor. J. Neurochem. 61: 375–377.

    Article  PubMed  CAS  Google Scholar 

  12. Wafford, K. A., Burnett, D. M., Leidenheimer, N. J., Burt, D. R., Wang, J. B., Kofuji, P., Dunwiddie, T. V., Harris, R. A., and Sikela, J. M. 1991. Ethanol sensitivity of the GABAA receptor expressed in Xenopus oocytes requires 8 amino acids contained in the γ2L subunit. Neuron. 7: 27–33.

    Article  PubMed  CAS  Google Scholar 

  13. Wafford, K. A., and Whiting, P. J. 1992. Ethanol potentiation of GABAA receptors requires phosphorylation of the alternatively spliced variant of the γ2 subunt. FEBS Letters 313: 113–117.

    Article  PubMed  CAS  Google Scholar 

  14. Pritchett, D. B., Sontheimer, H., Shivers, B. D., Ymer, S., Kettenmann, H., Shofield, P. R., and Seeburg, P. H. 1989. Importance of a novel GABAA receptor subunit for benzodiazepine pharmacology. Nature 338: 582–585.

    Article  PubMed  CAS  Google Scholar 

  15. Verdoorn, T. A., Draguhn, A., Ymer, S., Seeburg, P. H., and Sakmann, B. 1990. Functional properties of recombinant rat GABAA receptors depend upon subunit composition. Neuron. 4: 919–928.

    Article  PubMed  CAS  Google Scholar 

  16. Ymer, S., Draguhn, A., Wisden, W., Werner, P., Keinänen, K., Schofield, P., Sprengel, R., Pritchett, D., and Seeburg, P. 1990. Structural and functional characterization of the γ1 subunit of the GABAA/benzodiazepine receptors. EMBO Journal 9: 3261–3267.

    PubMed  CAS  Google Scholar 

  17. Knoflach, F., Rhyner, T., Villa, M., Kellenberger, S., Drescher, U., Malherbe, P., Sigel, E., and Möhler, H. 1991. The γ3-subunit of the GABAA-receptor confers sensitivity to benzodiazepine receptor ligands, FEBS Lett. 293: 191–194.

    Article  PubMed  CAS  Google Scholar 

  18. Pritchett, D. B., and Seeburg, P. H. 1991. γ-Aminobutyric Acid type A receptor point mutation increases the affinity of compounds for the benzodiazepine site. Proc. Natl. Acad. Sci. USA 88: 1421–1425.

    Article  PubMed  CAS  Google Scholar 

  19. Herb, A., Wisden, W., Lüddens, H., Puia, G., Vicini, S., Seeburg, P. H. 1992. The third γ subunit of the γ-aminobutyric acid type A receptor family. Proc Natl. Acad. Sci. U.S.A. 89: 1433–1437.

    Article  PubMed  CAS  Google Scholar 

  20. Korpi, E. R., and Lüddens, H. 1993. Regional γ-aminobutyric acid sensitivity of t-Butylbicyclophosphoro[35S]thionate binding depends on γ-aminobutyric acidA receptor α subunit. Mol. Pharmaco. 44: 87–92.

    CAS  Google Scholar 

  21. Wafford, K. A., Bain, C. J., Whiting, P. J., and Kemp, J. A. 1993. Functional comparison of the role of γ subunits in recombinant human γ-aminobutyric acidA/benzodiazepine receptors.Mol. Pharmacol. 44: 437–442.

    PubMed  CAS  Google Scholar 

  22. Mihic, S. J., Whiting, P. J., Klein, R. L., Wafford, K. A., and Harris, R. A. 1994. A single amino acid of the human γ-aminobutyric acid type A receptor γ2 subunit determines benzodiazepine efficacy. J. Biol. Chem. 269: 32768–32773.

    PubMed  CAS  Google Scholar 

  23. Khan, Z., Fernando, L., Escribá, P., Busquets, X., Mallet, J., Miralles, C., Filla, M., and De Blas, A. L. 1993. Antibodies to the human γ2 subunit of the GABAA receptor. J. Neurochem. 60: 961–971.

    Article  PubMed  CAS  Google Scholar 

  24. Khan, Z. U., Gutiérrez, A., and De Blas, A. L. 1994. The subunit composition of a GABAA/benzodiazepine receptor from rat cerebellum. J. Neurochem. 63: 371–374.

    Article  PubMed  CAS  Google Scholar 

  25. Khan, Z. U., Gutiérrez, A., and De Blas, A. L. 1994. Short and long from γ2 subunits of the GABAA/benzodiazepine receptors. J. Neurochem. 63: 1466–1476.

    Article  PubMed  CAS  Google Scholar 

  26. Miralles, C. P., Gutiérrez, A. Khan, Z. U., Vitorica, J., and De Blas, A. L. 1994. Differential expression of the short and long forms of the γ2 subunit of the GABAA/benzodiazepine receptors. Mol. Brain Res. 24: 129–139.

    Article  PubMed  CAS  Google Scholar 

  27. Gutiérrez, A., Khan, Z. U., and De Blas, A. L. 1994. Immunocytochemical localization of γ2 short and γ2 long subunits of the GABAA receptor in the rat brain. J. Neurosci. 14: 7168–7179.

    PubMed  Google Scholar 

  28. Gutiérrez, A., Khan, Z. U., Miralles, C. P., and De Blas, A. L. 1996. Altered expression of γ2S and γ2L receptor subunits in the aging rat brain. Mol. Brain Res. (in press).

  29. Fernando, L. P., Khan, Z. U., McKernan, R. M., and De Blas, A. L. 1995. Monoclonal antibodies to the human γ2 subunit of the GABAA/benzodiazepine receptors. J. Neurochem. 64: 1305–1311.

    Article  PubMed  CAS  Google Scholar 

  30. Shivers, B. D., Killisch, I., Sprengel, R., Sontheimer, H., Köhler, M., Schofield, P. R., and Seeburg, P. H. 1989. Two novel GABAA receptor subunits exist in distinct neuronal subpopulations. Neuron 3: 327–337.

    Article  PubMed  CAS  Google Scholar 

  31. De Blas, A. L., Vitorica, J., and Friedrich, P. 1988. Localization of GABAA receptor in the rat brain with a monoclonal antibody to the 57,000 Mr peptide of GABAA receptor/benzodiazepine receptor/Cl channel complex. J. Neurosci. 8: 602–614.

    PubMed  Google Scholar 

  32. Mernoff, R., Cherwinski, H. M., Becker, J. W., and De Blas, A. L. 1983. Solubilization of brain benzodiazepine receptors with a zwitterionic detergent: optimal preservation of their functional interaction with GABA receptors. J. Neurochem. 41: 752–758.

    Article  PubMed  CAS  Google Scholar 

  33. De Blas, A. L., and Cherwinski, H. M. 1983. Detection of antigens on nitrocellulose paper immunoblots with monoclonal antibodies. Anal. Biochem. 133: 214–219.

    Article  PubMed  Google Scholar 

  34. Young, W. S., III, Kuhar, M. J. 1979. Autoradiographic localization of benzodiazepine receptors in the brains of humans and animals. Nature 280: 393–395.

    Article  Google Scholar 

  35. Palacios, J. M., Young, W. S., III, and Kuhar, M. J. 1980. GABA and benzodiazepine receptors in rat and human brain; autoradiographic localization by a novel technique. Pages 573–583,in Usdin E, Sourkes TL, Youdi MBH, (eds) Enzymes and Neurotransmitters in Mental Disease, New York: John Wiley and Sons.

    Google Scholar 

  36. Gutiérrez, A., Khan, Z. U., Morris, S. J., and De Blas, A. L. 1994. Age-related decrease of GABAA receptor subunits and glutamic acid decarboxylase in the rat inferior colliculus. J. Neurosci. 14: 7469–7477.

    PubMed  Google Scholar 

  37. Laurie, D. J. Seeburg, P. H., and Wisden, W. 1992. The distribution of 13 GABAA receptor subunit mRNAs in he rat brain II olfactory bulb and cerebellum. J. Neurosci. 12: 1063–1076.

    PubMed  CAS  Google Scholar 

  38. Persohn, E., Mahlerbe, P., and Richards, J. G. 1992. Comparative molecular neuroanatomy of cloned GABAA receptors subunits in the rat CNS. J. Comp. Neurol. 326: 193–216.

    Article  PubMed  CAS  Google Scholar 

  39. Wisden, W., Laurie, D. J., Monyer, H., and Seeburg, P. H. 1992. The distribution of 13 GABAA receptor subunit in mRNA in the rat brain. I. telencephalon, diencephalon mesencephalon, J. Neurosci. 12: 1040–1062.

    PubMed  CAS  Google Scholar 

  40. Mossier, B., Tögel, M., Fuch, K., and Sieghart, W. 1994. Immunoaffinity purification of γ-aminobutyric acidA (GABAA) receptors containing γ1 subunits. J. Biol. Chem. 269: 25777–25782.

    PubMed  CAS  Google Scholar 

  41. Stephenson, F. A., Duggan, M. J., and Pollard, S. 1990. The γ2 subunit is an integral component of the γ-aminobutyric acidA receptor but the α1 polypeptide is the principal site of the agonist benzodiazepine photoaffinity labeling reactions. J. Biol. Chem. 265: 21160–21165.

    PubMed  CAS  Google Scholar 

  42. Benke, D., Mertens, S., Trzeciak, A., Gillessen, D., and Möhler, H. 1991. GABAA receptors display association of γ2-subunit with α1- and γ2 3 subunits. J. Biol. Chem. 266: 4478–4483.

    PubMed  CAS  Google Scholar 

  43. Araki, T., Kiyama, H., Maeno, H., and Tohyama, M. 1993. Differential immunocytochemical localization of GABAA, receptor γ1 and γ2 subunits in the rat brain. Mol. Brain Res. 20: 263–266.

    Article  PubMed  CAS  Google Scholar 

  44. Nadler, L. S., Guirguis, E. R., and Siegel, R. E. 1994. GABAA receptor subunit polypeptides increase in parallel but exhibit distinct distributions in the developing rat cerebellum. J. Neurobiol. 25: 1533–1544.

    Article  PubMed  CAS  Google Scholar 

  45. Tögel, M., Mossier, B., Fuch, K., and Sieghart, W. 1994. γ-Aminobutyric acidA receptor displaying association of γ3-subunit with β2 3 and different α-subunits exhibit unique pharmacological properties. J. Biol. Chem. 269: 12993–12998.

    PubMed  Google Scholar 

  46. Quirk, K., Guillard, N. P., Ragan, C. I., Whiting, P. J., and McKernan, R. M. 1994. Model of subunit composition of γ-aminobutyric acid A receptor subtypes expressed in rat cerebellum with respect to their α and γ/δ subunits. J. Biol. Chem. 269: 16020–16028.

    PubMed  CAS  Google Scholar 

  47. Quirk, K., Gillard, N. P., Ragan, C. I., Whiting, P. J., and McKernan, R. M. 1994. γ-aminobutyric acid type A receptors in the rat brain can contain both γ2 and γ3 subunits but γ1 does not exist in combination with another γ subunit. Mol. Pharmacol. 45: 1061–1070.

    PubMed  CAS  Google Scholar 

  48. Mhatre, M. C., Fernandes, G., Ticku, M. K. 1991. Aging reduces the mRNA of α1 subunit in rat cerebral cortex. Eur. J. Pharmacol. 208: 171–174.

    Article  PubMed  CAS  Google Scholar 

  49. Mhatre, M. C., Ticku, M. K. 1992. Aging related alterations in GABAA receptor subunit mRNA levels in Fischer rats. Mol. Brain Res. 14: 71–78.

    Article  PubMed  CAS  Google Scholar 

  50. Concas, A., Toffano, G., Biggio, G. 1988. Aging reduces the FABA dependent36Cl-flux in rat brain membrane vesicles. Life Sci. 43: 1761–1771.

    Article  PubMed  CAS  Google Scholar 

  51. Erdö, S. L., Wolff, J.R. 1989. Age related loss of t-[35S]butylbicyclophosphoro-thionate binding to the γ-aminobutyric acidA receptor-coupled chloride ionophore in rat cerebral cortex. J. Neurochem. 53:648–651.

    Article  PubMed  Google Scholar 

  52. Ruano, D., Cano, J., Machado, A., Vitorica, J. 1991. Pharmacologic characterization of GABAA/benzodiazepine receptor in rat hippocampus during aging. J. Pharmacol. Exp. Ther. 256:902–908.

    PubMed  CAS  Google Scholar 

  53. Ruano, D., Benavides, J., Machado, A., and Vitorica, J. 1995. Aging-associated changes in the pharmacological properties of the benzodiazepine (ω) receptor isotypes in the rat hippocampus. J. Neurochem. 64:225–233.

    Google Scholar 

  54. Barnhill, J. G., Greenblatt, D. J., Miller, L. G., Gaver, A., Harmatz, J. S., Shader, R. I. 1990. Kinetic and dynamic components of increased benzodiazepine sensitivity in aging animals, J. Pharmacol. Exp. Ther. 325:1153–1161.

    Google Scholar 

  55. Greenblatt, D. J., Shader, R. I., and Harmatz, J. S. (1989). Implications of altered drug disposition in the elderly: studies of benzodiazepines. J. Clin. Pharmacol. 29:866–872.

    PubMed  CAS  Google Scholar 

  56. Nikaido, A. M., Ellinwood, E. H., Jr., Heatherly, D. G., Gupta, S. K. 1990. Age-related increase in CNS sensitivity to benzodiazepines as assessed by task difficulty. Psychopharmacol. 100:90–97.

    CAS  Google Scholar 

  57. Ruano, D., Khan, Z., De Blas, A. L., Machado, A., and Vitorica, J. 1994. Molecular heterogeneity of the type I GABAA/benzodiazepine receptor complex. Eur. J. Pharmacol. 267:123–128.

    Article  PubMed  CAS  Google Scholar 

  58. Ruano, D., Araujo, F., Machado, A., De Blas, A. L., and Vitorica, J. 1994. Molecular characterization of type I GABAA receptor complex from rat cerebral cortex and hippocampus. Mol. Brain Res. 25:225–233.

    Article  PubMed  CAS  Google Scholar 

  59. Sigel, E., Baur, R., Trube, G., Möhler, H., and Malherbe, P. 1990. The effect of subnit composition of rat brain GABA-A receptors on channel function. Neuron 5:703–711.

    Article  PubMed  CAS  Google Scholar 

  60. Wong, G., Sei, Y., and Skolnick, P. 1992. Stable expression of type I γ-aminobutyric acidA/benzodiazepine receptors in a transfected cell line. Mol. Pharmacol. 42:996–1003.

    PubMed  CAS  Google Scholar 

  61. Im, H. K., Im, W. B., Hamilton, B. J., Carter, D. B., Vonvoigtalnder, P. F. 1993. Potentiation of γ-aminobutyric acid-induced chloride currents by various benzodiazepine site agonists with α1 α2, β2 γ2 and α1 β2 γ2 subtypes of cloned γ-aminobutyric acid type A receptors. Mol. Pharmacol. 44:866–870.

    PubMed  CAS  Google Scholar 

  62. Amin, J., and Weiss, D. S. 1993. GABAA receptor needs two homolgous domains of the β-subunit for activation by GABA but not by pentobarbital. Nature 366:565–569.

    Article  PubMed  CAS  Google Scholar 

  63. Smith, G. B., and Olsen, R. W. 1994. Identification of a [3H]muscimol photoaffinity/substrate in the bovine γ-aminobutyric acidA receptor α subunit. J. Biol. Chem. 269:20380–20387.

    PubMed  CAS  Google Scholar 

  64. Backus, K. H., Arigoni, M., Drescher, U., Scheurer, L., Malherbe, P., Möhler, H., and Benson, J. A. 1993. Stoichiometry of a recombinant GABAA receptor deduced from mutation-induced rectification. NeuroReport 5:285–288.

    Article  PubMed  CAS  Google Scholar 

  65. Khrestchatisky, M., MacLennon, A.J., Chiang, M. Y., Xu, W., Jackson, M. B., Brecha, N., Sternini, C., Olsen, R. W., and Tobin, A. J. 1989. A novel α subunit in rat brain GABAA receptors. Neuron 3:745–753.

    Article  PubMed  CAS  Google Scholar 

  66. Gutiérrez, A., Khan, Z. U., Ruano, D., Miralles, C. P., Vitorica, J., and De Blas, A. L. 1995. Aging-related subunit expression changes of the GABAA receptor in the rat hippocampus. (submitted).

  67. Khan, Z. U., Gutiérrez, A., and De Blas, A. J. (1995). The α1 and α6 subunits can coexist in the same cerebellar GABAA receptor maintaining their individual binding specificities. J. Neurochem. 66:685–691.

    Article  Google Scholar 

Download references

Author information

Authors and Affiliations

  1. Division of Molecular Bilogy and Biochemistry, School of Biological Sciences, University of Missouri-Kansas City, 64110-2499, Kansas City, MO

    Zafar U. Khan, Antonia Gutiérrez, Celia P. Miralles & Angel L. De Blas

Authors
  1. Zafar U. Khan
    View author publications

    You can also search for this author in PubMed Google Scholar

  2. Antonia Gutiérrez
    View author publications

    You can also search for this author in PubMed Google Scholar

  3. Celia P. Miralles
    View author publications

    You can also search for this author in PubMed Google Scholar

  4. Angel L. De Blas
    View author publications

    You can also search for this author in PubMed Google Scholar

Additional information

Special issue dedicated to Dr. Kinya Kuriyama

Rights and permissions

Reprints and permissions

About this article

Cite this article

Khan, Z.U., Gutiérrez, A., Miralles, C.P. et al. The γ subunits of the native GABAA/benzodiazepine receptors. Neurochem Res 21, 147–159 (1996). https://doi.org/10.1007/BF02529132

Download citation

  • Accepted:

  • Issue Date:

  • DOI: https://doi.org/10.1007/BF02529132

Key Words

Search

Navigation