Skip to main content
PMC home page
The EMBO Journal logoLink to The EMBO Journal
. 1992 Jun;11(6):2017–2023. doi: 10.1002/j.1460-2075.1992.tb05258.x

Point mutations affecting antagonist affinity and agonist dependent gating of GABAA receptor channels.

E Sigel 1, R Baur 1, S Kellenberger 1, P Malherbe 1
PMCID: PMC556666  PMID: 1376242

Abstract

Two variant amino acid sequences, which differ in a single amino acid residue, have been reported for the alpha 1-subunit of the rat brain GABAA receptor. We separately co-expressed these two variants in Xenopus oocytes, in combination with beta 2 and gamma 2. This experiment showed that substitution of alpha 1-Phe64 by Leu strongly decreases the apparent affinity for GABA dependent channel gating from 6 microM to 1260 microM. Starting from this observation, we used in vitro mutagenesis to obtain information relevant for the localization of the agonist/antagonist binding site in the GABAA receptor. Homologous mutation in alpha 5 had similar consequences for alpha 5 beta 2 gamma 2. Homologous mutation in beta 2 and gamma 2 resulted in intermediate and small shifts in EC50, respectively. The apparent affinities of the competitive antagonists bicuculline methiodide and SR95531, the latter sharing close structural similarity with the agonist GABA, were decreased 60- to 200-fold by these mutations in alpha-subunits. Interestingly, these affinities remained nearly unaffected upon introduction of the homologous mutations in beta 2 and gamma 2, or upon mutation of the neighbouring amino acid in alpha 1, Phe65 to Leu. These results suggest close functional and structural association of alpha-subunits with the agonist/antagonist binding site, and involvement of N-terminal portions of the extracellular domains of all subunits in the gating of the channel.

Full text

PDF

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. Browning M. D., Bureau M., Dudek E. M., Olsen R. W. Protein kinase C and cAMP-dependent protein kinase phosphorylate the beta subunit of the purified gamma-aminobutyric acid A receptor. Proc Natl Acad Sci U S A. 1990 Feb;87(4):1315–1318. doi: 10.1073/pnas.87.4.1315. [DOI] [PMC free article] [PubMed] [Google Scholar]
  3. Casalotti S. O., Stephenson F. A., Barnard E. A. Separate subunits for agonist and benzodiazepine binding in the gamma-aminobutyric acidA receptor oligomer. J Biol Chem. 1986 Nov 15;261(32):15013–15016. [PubMed] [Google Scholar]
  4. Cutting G. R., Lu L., O'Hara B. F., Kasch L. M., Montrose-Rafizadeh C., Donovan D. M., Shimada S., Antonarakis S. E., Guggino W. B., Uhl G. R. Cloning of the gamma-aminobutyric acid (GABA) rho 1 cDNA: a GABA receptor subunit highly expressed in the retina. Proc Natl Acad Sci U S A. 1991 Apr 1;88(7):2673–2677. doi: 10.1073/pnas.88.7.2673. [DOI] [PMC free article] [PubMed] [Google Scholar]
  5. Duggan M. J., Pollard S., Stephenson F. A. Immunoaffinity purification of GABAA receptor alpha-subunit iso-oligomers. Demonstration of receptor populations containing alpha 1 alpha 2, alpha 1 alpha 3, and alpha 2 alpha 3 subunit pairs. J Biol Chem. 1991 Dec 25;266(36):24778–24784. [PubMed] [Google Scholar]
  6. Galzi J. L., Revah F., Bessis A., Changeux J. P. Functional architecture of the nicotinic acetylcholine receptor: from electric organ to brain. Annu Rev Pharmacol Toxicol. 1991;31:37–72. doi: 10.1146/annurev.pa.31.040191.000345. [DOI] [PubMed] [Google Scholar]
  7. Gambarana C., Beattie C. E., Rodríguez Z. R., Siegel R. E. Region-specific expression of messenger RNAs encoding GABAA receptor subunits in the developing rat brain. Neuroscience. 1991;45(2):423–432. doi: 10.1016/0306-4522(91)90238-j. [DOI] [PubMed] [Google Scholar]
  8. Gambarana C., Pittman R., Siegel R. E. Developmental expression of the GABAA receptor alpha 1 subunit mRNA in the rat brain. J Neurobiol. 1990 Dec;21(8):1169–1179. doi: 10.1002/neu.480210803. [DOI] [PubMed] [Google Scholar]
  9. Grenningloh G., Rienitz A., Schmitt B., Methfessel C., Zensen M., Beyreuther K., Gundelfinger E. D., Betz H. The strychnine-binding subunit of the glycine receptor shows homology with nicotinic acetylcholine receptors. Nature. 1987 Jul 16;328(6127):215–220. doi: 10.1038/328215a0. [DOI] [PubMed] [Google Scholar]
  10. 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]
  11. Knoflach F., Rhyner T., Villa M., Kellenberger S., Drescher U., Malherbe P., Sigel E., Möhler H. The gamma 3-subunit of the GABAA-receptor confers sensitivity to benzodiazepine receptor ligands. FEBS Lett. 1991 Nov 18;293(1-2):191–194. doi: 10.1016/0014-5793(91)81184-a. [DOI] [PubMed] [Google Scholar]
  12. Kuhse J., Schmieden V., Betz H. A single amino acid exchange alters the pharmacology of neonatal rat glycine receptor subunit. Neuron. 1990 Dec;5(6):867–873. doi: 10.1016/0896-6273(90)90346-h. [DOI] [PubMed] [Google Scholar]
  13. 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]
  14. Lo D. C., Pinkham J. L., Stevens C. F. Influence of the gamma subunit and expression system on acetylcholine receptor gating. Neuron. 1990 Dec;5(6):857–866. doi: 10.1016/0896-6273(90)90345-g. [DOI] [PubMed] [Google Scholar]
  15. Lo D. C., Pinkham J. L., Stevens C. F. Role of a key cysteine residue in the gating of the acetylcholine receptor. Neuron. 1991 Jan;6(1):31–40. doi: 10.1016/0896-6273(91)90119-k. [DOI] [PubMed] [Google Scholar]
  16. Lolait S. J., O'Carroll A. M., Kusano K., Muller J. M., Brownstein M. J., Mahan L. C. Cloning and expression of a novel rat GABAA receptor. FEBS Lett. 1989 Mar 27;246(1-2):145–148. doi: 10.1016/0014-5793(89)80271-6. [DOI] [PubMed] [Google Scholar]
  17. 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]
  18. Malherbe P., Draguhn A., Multhaup G., Beyreuther K., Möhler H. GABAA-receptor expressed from rat brain alpha- and beta-subunit cDNAs displays potentiation by benzodiazepine receptor ligands. Brain Res Mol Brain Res. 1990 Aug;8(3):199–208. doi: 10.1016/0169-328x(90)90017-8. [DOI] [PubMed] [Google Scholar]
  19. 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]
  20. Malherbe P., Sigel E., Baur R., Persohn E., Richards J. G., Möhler H. Functional expression and sites of gene transcription of a novel alpha subunit of the GABAA receptor in rat brain. FEBS Lett. 1990 Jan 29;260(2):261–265. doi: 10.1016/0014-5793(90)80118-3. [DOI] [PubMed] [Google Scholar]
  21. 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]
  22. Middleton R. E., Cohen J. B. Mapping of the acetylcholine binding site of the nicotinic acetylcholine receptor: [3H]nicotine as an agonist photoaffinity label. Biochemistry. 1991 Jul 16;30(28):6987–6997. doi: 10.1021/bi00242a026. [DOI] [PubMed] [Google Scholar]
  23. Olsen R. W., Tobin A. J. Molecular biology of GABAA receptors. FASEB J. 1990 Mar;4(5):1469–1480. doi: 10.1096/fasebj.4.5.2155149. [DOI] [PubMed] [Google Scholar]
  24. Pritchett D. B., Seeburg P. H. Gamma-aminobutyric acidA receptor alpha 5-subunit creates novel type II benzodiazepine receptor pharmacology. J Neurochem. 1990 May;54(5):1802–1804. doi: 10.1111/j.1471-4159.1990.tb01237.x. [DOI] [PubMed] [Google Scholar]
  25. 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]
  26. Revah F., Bertrand D., Galzi J. L., Devillers-Thiéry A., Mulle C., Hussy N., Bertrand S., Ballivet M., Changeux J. P. Mutations in the channel domain alter desensitization of a neuronal nicotinic receptor. Nature. 1991 Oct 31;353(6347):846–849. doi: 10.1038/353846a0. [DOI] [PubMed] [Google Scholar]
  27. 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]
  28. 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]
  29. Sigel E., Barnard E. A. A gamma-aminobutyric acid/benzodiazepine receptor complex from bovine cerebral cortex. Improved purification with preservation of regulatory sites and their interactions. J Biol Chem. 1984 Jun 10;259(11):7219–7223. [PubMed] [Google Scholar]
  30. Sigel E., Baur R. Allosteric modulation by benzodiazepine receptor ligands of the GABAA receptor channel expressed in Xenopus oocytes. J Neurosci. 1988 Jan;8(1):289–295. doi: 10.1523/JNEUROSCI.08-01-00289.1988. [DOI] [PMC free article] [PubMed] [Google Scholar]
  31. Sigel E., Baur R., Trube G., Möhler H., Malherbe P. The effect of subunit composition of rat brain GABAA receptors on channel function. Neuron. 1990 Nov;5(5):703–711. doi: 10.1016/0896-6273(90)90224-4. [DOI] [PubMed] [Google Scholar]
  32. Sigel E. Properties of single sodium channels translated by Xenopus oocytes after injection with messenger ribonucleic acid. J Physiol. 1987 May;386:73–90. doi: 10.1113/jphysiol.1987.sp016523. [DOI] [PMC free article] [PubMed] [Google Scholar]
  33. Sigel E., Stephenson F. A., Mamalaki C., Barnard E. A. A gamma-aminobutyric acid/benzodiazepine receptor complex of bovine cerebral cortex. J Biol Chem. 1983 Jun 10;258(11):6965–6971. [PubMed] [Google Scholar]
  34. Séquier J. M., Richards J. G., Malherbe P., Price G. W., Mathews S., Möhler H. Mapping of brain areas containing RNA homologous to cDNAs encoding the alpha and beta subunits of the rat GABAA gamma-aminobutyrate receptor. Proc Natl Acad Sci U S A. 1988 Oct;85(20):7815–7819. doi: 10.1073/pnas.85.20.7815. [DOI] [PMC free article] [PubMed] [Google Scholar]
  35. Tomaselli G. F., McLaughlin J. T., Jurman M. E., Hawrot E., Yellen G. Mutations affecting agonist sensitivity of the nicotinic acetylcholine receptor. Biophys J. 1991 Sep;60(3):721–727. doi: 10.1016/S0006-3495(91)82102-6. [DOI] [PMC free article] [PubMed] [Google Scholar]
  36. 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]
  37. Ymer S., Draguhn A., Köhler M., Schofield P. R., Seeburg P. H. Sequence and expression of a novel GABAA receptor alpha subunit. FEBS Lett. 1989 Nov 20;258(1):119–122. doi: 10.1016/0014-5793(89)81630-8. [DOI] [PubMed] [Google Scholar]
  38. 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]

Articles from The EMBO Journal are provided here courtesy of Nature Publishing Group

RESOURCES