Skip to main content
PMC home page
Proceedings of the National Academy of Sciences of the United States of America logoLink to Proceedings of the National Academy of Sciences of the United States of America
. 1992 Jul 15;89(14):6378–6382. doi: 10.1073/pnas.89.14.6378

Stable expression of mammalian type A gamma-aminobutyric acid receptors in mouse cells: demonstration of functional assembly of benzodiazepine-responsive sites.

K L Hadingham 1, P C Harkness 1, R M McKernan 1, K Quirk 1, B Le Bourdellès 1, A L Horne 1, J A Kemp 1, E A Barnard 1, C I Ragan 1, P J Whiting 1
PMCID: PMC49504  PMID: 1321437

Abstract

The differential sensitivity of type A gamma-aminobutyric acid (GABAA) receptors to benzodiazepine ligands seen in the mammalian nervous system is thought to be generated by the existence of a number of different receptor subtypes, assembled from a range of closely related subunits (alpha 1-6, beta 1-3, gamma 1-3, and delta) encoded by discrete genes. The characteristics of a given subtype can be determined by the coexpression of cloned cDNAs encoding the subunits of interest. Two transient expression systems have so far been employed in the study of the ligand-binding characteristics and chloride channel properties of such GABAA receptors--Xenopus oocytes and transfected mammalian cells. Here we report on the use of a steroid-inducible promoter expression system for the production of a permanently transfected clonal cell line expressing the alpha 1 beta 1 gamma 2L GABAA receptor subtype. Using both immunoprecipitation by subunit-specific antisera and gel-exclusion chromatography, we have shown that the alpha 1, beta 1, and gamma 2L subunits coassemble to form receptor macromolecules that are of the same size as native GABAA receptors. Additionally, the recombinant receptors have the same benzodiazepine pharmacology as native alpha 1-containing GABAA receptors and function as GABA-gated chloride channels. Such cell lines expressing individual GABAA receptor subtypes will prove important tools in the study of the structure, function, and pharmacology of GABAA receptors and in the development of subtype-specific drugs.

Full text

PDF
6378

Images in this article

6380Image
on p.6380

Selected References

These references are in PubMed. This may not be the complete list of references from this article.

  1. Benke D., Mertens S., Trzeciak A., Gillessen D., Mohler H. GABAA receptors display association of gamma 2-subunit with alpha 1- and beta 2/3-subunits. J Biol Chem. 1991 Mar 5;266(7):4478–4483. [PubMed] [Google Scholar]
  2. Buchstaller A., Adamiker D., Fuchs K., Sieghart W. N-deglycosylation and immunological identification indicates the existence of beta-subunit isoforms of the rat GABAA receptor. FEBS Lett. 1991 Aug 5;287(1-2):27–30. doi: 10.1016/0014-5793(91)80008-q. [DOI] [PubMed] [Google Scholar]
  3. Chen C. A., Okayama H. Calcium phosphate-mediated gene transfer: a highly efficient transfection system for stably transforming cells with plasmid DNA. Biotechniques. 1988 Jul-Aug;6(7):632–638. [PubMed] [Google Scholar]
  4. Duggan M. J., Stephenson F. A. Bovine gamma-aminobutyric acidA receptor sequence-specific antibodies: identification of two epitopes which are recognised in both native and denatured gamma-aminobutyric acidA receptors. J Neurochem. 1989 Jul;53(1):132–139. doi: 10.1111/j.1471-4159.1989.tb07304.x. [DOI] [PubMed] [Google Scholar]
  5. Kemp J. A., Priestley T. Effects of (+)-HA-966 and 7-chlorokynurenic acid on the kinetics of N-methyl-D-aspartate receptor agonist responses in rat cultured cortical neurons. Mol Pharmacol. 1991 May;39(5):666–670. [PubMed] [Google Scholar]
  6. Khrestchatisky M., MacLennan A. J., Chiang M. Y., Xu W. T., Jackson M. B., Brecha N., Sternini C., Olsen R. W., Tobin A. J. A novel alpha subunit in rat brain GABAA receptors. Neuron. 1989 Dec;3(6):745–753. doi: 10.1016/0896-6273(89)90243-2. [DOI] [PubMed] [Google Scholar]
  7. Kofuji P., Wang J. B., Moss S. J., Huganir R. L., Burt D. R. Generation of two forms of the gamma-aminobutyric acidA receptor gamma 2-subunit in mice by alternative splicing. J Neurochem. 1991 Feb;56(2):713–715. doi: 10.1111/j.1471-4159.1991.tb08209.x. [DOI] [PubMed] [Google Scholar]
  8. Levitan E. S., Schofield P. R., Burt D. R., Rhee L. M., Wisden W., Köhler M., Fujita N., Rodriguez H. F., Stephenson A., Darlison M. G. Structural and functional basis for GABAA receptor heterogeneity. Nature. 1988 Sep 1;335(6185):76–79. doi: 10.1038/335076a0. [DOI] [PubMed] [Google Scholar]
  9. Lüddens H., Pritchett D. B., Köhler M., Killisch I., Keinänen K., Monyer H., Sprengel R., Seeburg P. H. Cerebellar GABAA receptor selective for a behavioural alcohol antagonist. Nature. 1990 Aug 16;346(6285):648–651. doi: 10.1038/346648a0. [DOI] [PubMed] [Google Scholar]
  10. Malherbe P., Sigel E., Baur R., Persohn E., Richards J. G., Mohler H. Functional characteristics and sites of gene expression of the alpha 1, beta 1, gamma 2-isoform of the rat GABAA receptor. J Neurosci. 1990 Jul;10(7):2330–2337. doi: 10.1523/JNEUROSCI.10-07-02330.1990. [DOI] [PMC free article] [PubMed] [Google Scholar]
  11. Mamalaki C., Barnard E. A., Stephenson F. A. Molecular size of the gamma-aminobutyric acidA receptor purified from mammalian cerebral cortex. J Neurochem. 1989 Jan;52(1):124–134. doi: 10.1111/j.1471-4159.1989.tb10906.x. [DOI] [PubMed] [Google Scholar]
  12. McKernan R. M., Biggs C. S., Gillard N., Quirk K., Ragan C. I. Molecular size of the 5-HT3 receptor solubilized from NCB 20 cells. Biochem J. 1990 Aug 1;269(3):623–628. doi: 10.1042/bj2690623. [DOI] [PMC free article] [PubMed] [Google Scholar]
  13. McKernan R. M., Quirk K., Prince R., Cox P. A., Gillard N. P., Ragan C. I., Whiting P. GABAA receptor subtypes immunopurified from rat brain with alpha subunit-specific antibodies have unique pharmacological properties. Neuron. 1991 Oct;7(4):667–676. doi: 10.1016/0896-6273(91)90379-e. [DOI] [PubMed] [Google Scholar]
  14. Moss S. J., Smart T. G., Porter N. M., Nayeem N., Devine J., Stephenson F. A., Macdonald R. L., Barnard E. A. Cloned GABA receptors are maintained in a stable cell line: allosteric and channel properties. Eur J Pharmacol. 1990 Jul 31;189(1):77–88. doi: 10.1016/0922-4106(90)90232-m. [DOI] [PubMed] [Google Scholar]
  15. Pritchett D. B., Lüddens H., Seeburg P. H. Type I and type II GABAA-benzodiazepine receptors produced in transfected cells. Science. 1989 Sep 22;245(4924):1389–1392. doi: 10.1126/science.2551039. [DOI] [PubMed] [Google Scholar]
  16. Pritchett D. B., Sontheimer H., Gorman C. M., Kettenmann H., Seeburg P. H., Schofield P. R. Transient expression shows ligand gating and allosteric potentiation of GABAA receptor subunits. Science. 1988 Dec 2;242(4883):1306–1308. doi: 10.1126/science.2848320. [DOI] [PubMed] [Google Scholar]
  17. Pritchett D. B., Sontheimer H., Shivers B. D., Ymer S., Kettenmann H., Schofield P. R., Seeburg P. H. Importance of a novel GABAA receptor subunit for benzodiazepine pharmacology. Nature. 1989 Apr 13;338(6216):582–585. doi: 10.1038/338582a0. [DOI] [PubMed] [Google Scholar]
  18. Schofield P. R., Darlison M. G., Fujita N., Burt D. R., Stephenson F. A., Rodriguez H., Rhee L. M., Ramachandran J., Reale V., Glencorse T. A. Sequence and functional expression of the GABA A receptor shows a ligand-gated receptor super-family. Nature. 1987 Jul 16;328(6127):221–227. doi: 10.1038/328221a0. [DOI] [PubMed] [Google Scholar]
  19. Shivers B. D., Killisch I., Sprengel R., Sontheimer H., Köhler M., Schofield P. R., Seeburg P. H. Two novel GABAA receptor subunits exist in distinct neuronal subpopulations. Neuron. 1989 Sep;3(3):327–337. doi: 10.1016/0896-6273(89)90257-2. [DOI] [PubMed] [Google Scholar]
  20. Squires R. F., Benson D. I., Braestrup C., Coupet J., Klepner C. A., Myers V., Beer B. Some properties of brain specific benzodiazepine receptors: new evidence for multiple receptors. Pharmacol Biochem Behav. 1979 May;10(5):825–830. doi: 10.1016/0091-3057(79)90341-1. [DOI] [PubMed] [Google Scholar]
  21. Stephenson F. A., Duggan M. J., Pollard S. The gamma 2 subunit is an integral component of the gamma-aminobutyric acidA receptor but the alpha 1 polypeptide is the principal site of the agonist benzodiazepine photoaffinity labeling reaction. J Biol Chem. 1990 Dec 5;265(34):21160–21165. [PubMed] [Google Scholar]
  22. Tricklebank M. D., Honoré T., Iversen S. D., Kemp J. A., Knight A. R., Marshall G. R., Rupniak N. M., Singh L., Tye S., Watjen F. The pharmacological properties of the imidazobenzodiazepine, FG 8205, a novel partial agonist at the benzodiazepine receptor. Br J Pharmacol. 1990 Nov;101(3):753–761. doi: 10.1111/j.1476-5381.1990.tb14152.x. [DOI] [PMC free article] [PubMed] [Google Scholar]
  23. Verdoorn T. A., Draguhn A., Ymer S., Seeburg P. H., Sakmann B. Functional properties of recombinant rat GABAA receptors depend upon subunit composition. Neuron. 1990 Jun;4(6):919–928. doi: 10.1016/0896-6273(90)90145-6. [DOI] [PubMed] [Google Scholar]
  24. Whiting P., McKernan R. M., Iversen L. L. Another mechanism for creating diversity in gamma-aminobutyrate type A receptors: RNA splicing directs expression of two forms of gamma 2 phosphorylation site. Proc Natl Acad Sci U S A. 1990 Dec;87(24):9966–9970. doi: 10.1073/pnas.87.24.9966. [DOI] [PMC free article] [PubMed] [Google Scholar]
  25. Whiting P., Schoepfer R., Lindstrom J., Priestley T. Structural and pharmacological characterization of the major brain nicotinic acetylcholine receptor subtype stably expressed in mouse fibroblasts. Mol Pharmacol. 1991 Oct;40(4):463–472. [PubMed] [Google Scholar]
  26. Wilson-Shaw D., Robinson M., Gambarana C., Siegel R. E., Sikela J. M. A novel gamma subunit of the GABAA receptor identified using the polymerase chain reaction. FEBS Lett. 1991 Jun 24;284(2):211–215. doi: 10.1016/0014-5793(91)80687-x. [DOI] [PubMed] [Google Scholar]
  27. Wisden W., Herb A., Wieland H., Keinänen K., Lüddens H., Seeburg P. H. Cloning, pharmacological characteristics and expression pattern of the rat GABAA receptor alpha 4 subunit. FEBS Lett. 1991 Sep 9;289(2):227–230. doi: 10.1016/0014-5793(91)81076-k. [DOI] [PubMed] [Google Scholar]
  28. Ymer S., Draguhn A., Wisden W., Werner P., Keinänen K., Schofield P. R., Sprengel R., Pritchett D. B., Seeburg P. H. Structural and functional characterization of the gamma 1 subunit of GABAA/benzodiazepine receptors. EMBO J. 1990 Oct;9(10):3261–3267. doi: 10.1002/j.1460-2075.1990.tb07525.x. [DOI] [PMC free article] [PubMed] [Google Scholar]
  29. Ymer S., Schofield P. R., Draguhn A., Werner P., Köhler M., Seeburg P. H. GABAA receptor beta subunit heterogeneity: functional expression of cloned cDNAs. EMBO J. 1989 Jun;8(6):1665–1670. doi: 10.1002/j.1460-2075.1989.tb03557.x. [DOI] [PMC free article] [PubMed] [Google Scholar]
  30. Zezula J., Sieghart W. Isolation of type I and type II GABAA-benzodiazepine receptors by immunoaffinity chromatography. FEBS Lett. 1991 Jun 17;284(1):15–18. doi: 10.1016/0014-5793(91)80750-w. [DOI] [PubMed] [Google Scholar]

Articles from Proceedings of the National Academy of Sciences of the United States of America are provided here courtesy of National Academy of Sciences

RESOURCES