Home LiteratureArticle Details
PMID: 8757254 Published · ppublish English Journal Article Research Support, U.S. Gov't, P.H.S.

Stoichiometry of a recombinant GABAA receptor.

The Journal of neuroscience : the official journal of the Society for Neuroscience ·Vol. 16 ·No. 17 ·1996-09-01 ·Pages 5415-24

Chang Y, Wang R, Barot S, Weiss DS

Abstract

GABA is the main inhibitory neurotransmitter in the mammalian brain. The postsynaptic GABAA receptor/pore complex is presumed to be a pentamer typically composed of a combination of alpha, beta, and gamma subunits, although the stoichiometry remains controversial. We probed the stoichiometry of the GABAA receptor by site-directed mutagenesis of a conserved leucine (to serine) in the putative second membrane-spanning domain of the rat alpha 1(alpha L263S), beta 2(alpha L259S), and gamma 2(alpha L274S) subunit isoforms. Coexpression of wild-type and mutant subunits of each class (e.g., alpha and alpha L263S), along with their wild-type counter-parts (e.g., beta and gamma), in Xenopus laevis oocytes resulted in mixed populations of receptors with distinct GABA sensitivities. This is consistent with the interpretation that the leucine mutation increased the GABA sensitivity in proportion to the number of incorporated mutant subunits. The apparent number of incorporated subunits for each class (alpha, beta, and gamma) could then be determined from the number of components comprising the compound GABA dose-response relationships. Using this approach, we conclude that the recombinant alpha 1 beta 2 gamma 2 GABAA receptor is a pentamer composed of two alpha subunits, two beta subunits, and one gamma subunit.

MeSH Terms
Amino Acid Sequence Animals Base Sequence Conserved Sequence Leucine/genetics Molecular Sequence Data Mutagenesis, Site-Directed Oligonucleotide Probes/genetics Rats Receptors, GABA-A/chemistry,genetics Recombinant Proteins Xenopus laevis gamma-Aminobutyric Acid/metabolism
Chemicals
Oligonucleotide Probes Receptors, GABA-A Recombinant Proteins gamma-Aminobutyric Acid Leucine
Authors & Affiliations
4 authors, click to expand affiliations / ORCID
Chang Y
Department of Physiology and Biophysics, University of Alabama at Birmingham 35294, USA.
Wang R
Barot S
Weiss D S
References (60)
60 references, click to expand
  1. gamma-Aminobutyric acid type A receptors in the rat brain can contain both gamma 2 and gamma 3 subunits, but gamma 1 does not exist in combination with another gamma subunit.
    Mol Pharmacol. 1994 Jun;45(6):1061-70 PMID: 8022401
  2. Gating scheme for single GABA-activated Cl- channels determined from stability plots, dwell-time distributions, and adjacent-interval durations.
    J Neurosci. 1989 Apr;9(4):1314-24 PMID: 2539443
  3. Molecular basis of the two nonequivalent ligand binding sites of the muscle nicotinic acetylcholine receptor.
    Neuron. 1989 Sep;3(3):349-57 PMID: 2642001
  4. Molecular characterization of type I GABAA receptor complex from rat cerebral cortex and hippocampus.
    Brain Res Mol Brain Res. 1994 Sep;25(3-4):225-33 PMID: 7808221
  5. Insights into the activation mechanism of rho1 GABA receptors obtained by coexpression of wild type and activation-impaired subunits.
    Proc Biol Sci. 1996 Mar 22;263(1368):273-82 PMID: 8920250
  6. Quaternary structure of the native GABAA receptor determined by electron microscopic image analysis.
    J Neurochem. 1994 Feb;62(2):815-8 PMID: 7507518
  7. Biophysical and pharmacological properties of cloned GABAA receptor subunits expressed in Xenopus oocytes.
    Neuron. 1988 Nov;1(9):773-81 PMID: 2856103
  8. Single channels activated by high concentrations of GABA in superior cervical ganglion neurones of the rat.
    J Physiol. 1991 Jan;432:203-33 PMID: 1715917
  9. GABAA receptors display association of gamma 2-subunit with alpha 1- and beta 2/3-subunits.
    J Biol Chem. 1991 Mar 5;266(7):4478-83 PMID: 1847922
  10. A novel alpha subunit in rat brain GABAA receptors.
    Neuron. 1989 Dec;3(6):745-53 PMID: 2561977
  11. Biochemical properties of acteylcholine receptor subunits from Torpedo californica.
    Biochemistry. 1979 Oct 16;18(21):4465-70 PMID: 497150
  12. Primer-directed enzymatic amplification of DNA with a thermostable DNA polymerase.
    Science. 1988 Jan 29;239(4839):487-91 PMID: 2448875
  13. Chloride channel expression with the tandem construct of alpha 6-beta 2 GABAA receptor subunit requires a monomeric subunit of alpha 6 or gamma 2.
    J Biol Chem. 1995 Nov 3;270(44):26063-6 PMID: 7592805
  14. A single histidine in GABAA receptors is essential for benzodiazepine agonist binding.
    J Biol Chem. 1992 Jan 25;267(3):1426-9 PMID: 1346133
  15. Mutations in the channel domain alter desensitization of a neuronal nicotinic receptor.
    Nature. 1991 Oct 31;353(6347):846-9 PMID: 1719423
  16. Single amino acid substitution affects desensitization of the 5-hydroxytryptamine type 3 receptor expressed in Xenopus oocytes.
    Proc Natl Acad Sci U S A. 1993 Jun 1;90(11):5030-3 PMID: 8506347
  17. Cloning and expression of a novel rat GABAA receptor.
    FEBS Lett. 1989 Mar 27;246(1-2):145-8 PMID: 2540033
  18. Functional properties of nicotinic acetylcholine receptor subunits expressed in various combinations.
    FEBS Lett. 1987 Apr 20;214(2):253-8 PMID: 2436944
  19. Activation of ion channels in the frog end-plate by high concentrations of acetylcholine.
    J Physiol. 1988 Jan;395:131-59 PMID: 2457675
  20. GABAA receptor needs two homologous domains of the beta-subunit for activation by GABA but not by pentobarbital.
    Nature. 1993 Dec 9;366(6455):565-9 PMID: 7504783
  21. The agonist binding site of the gamma-aminobutyric acid type A channel is not formed by the extracellular cysteine loop.
    Mol Pharmacol. 1994 Feb;45(2):317-23 PMID: 7509443
  22. Activation of acetylcholine receptors on clonal mammalian BC3H-1 cells by high concentrations of agonist.
    J Physiol. 1987 Apr;385:325-59 PMID: 2443668
  23. Point mutations affecting antagonist affinity and agonist dependent gating of GABAA receptor channels.
    EMBO J. 1992 Jun;11(6):2017-23 PMID: 1376242
  24. Intraburst kinetic properties of the GABAA receptor main conductance state of mouse spinal cord neurones in culture.
    J Physiol. 1990 Apr;423:193-220 PMID: 2167362
  25. Agonist binding site of Torpedo electric tissue nicotinic acetylcholine receptor. A negatively charged region of the delta subunit within 0.9 nm of the alpha subunit binding site disulfide.
    J Biol Chem. 1991 Nov 25;266(33):22603-12 PMID: 1939274
  26. Functional domains of GABAA receptors.
    Trends Pharmacol Sci. 1995 May;16(5):162-8 PMID: 7624971
  27. Determination of NMDA NR1 subunit copy number in recombinant NMDA receptors.
    Proc Biol Sci. 1995 Nov 22;262(1364):205-13 PMID: 8524912
  28. Conserved quaternary structure of ligand-gated ion channels: the postsynaptic glycine receptor is a pentamer.
    Proc Natl Acad Sci U S A. 1988 Oct;85(19):7394-8 PMID: 2459705
  29. Pentameric structure and subunit stoichiometry of a neuronal nicotinic acetylcholine receptor.
    Nature. 1991 Mar 21;350(6315):235-8 PMID: 2005979
  30. d-Tubocurarine binding sites are located at alpha-gamma and alpha-delta subunit interfaces of the nicotinic acetylcholine receptor.
    Proc Natl Acad Sci U S A. 1990 Apr;87(7):2785-9 PMID: 2320589
  31. More than one alpha variant may exist in a GABAA/benzodiazepine receptor complex.
    J Recept Res. 1991;11(1-4):535-51 PMID: 1653350
  32. Quantitative characterization of alpha 6 and alpha 1 alpha 6 subunit-containing native gamma-aminobutyric acidA receptors of adult rat cerebellum demonstrates two alpha subunits per receptor oligomer.
    J Biol Chem. 1995 Sep 8;270(36):21285-90 PMID: 7673164
  33. Assembly of the inhibitory glycine receptor: identification of amino acid sequence motifs governing subunit stoichiometry.
    Neuron. 1993 Dec;11(6):1049-56 PMID: 8274276
  34. Nicotinic acetylcholine receptor at 9 A resolution.
    J Mol Biol. 1993 Feb 20;229(4):1101-24 PMID: 8445638
  35. Short and long form gamma 2 subunits of the GABAA/benzodiazepine receptors.
    J Neurochem. 1994 Oct;63(4):1466-76 PMID: 7931299
  36. GABAA receptor channels.
    Annu Rev Neurosci. 1994;17:569-602 PMID: 7516126
  37. Molecular dissection of subunit interfaces in the acetylcholine receptor: identification of residues that determine curare selectivity.
    Proc Natl Acad Sci U S A. 1993 Oct 15;90(20):9436-40 PMID: 8415719
  38. Acetylcholine receptor channel imaged in the open state.
    Nature. 1995 Jan 5;373(6509):37-43 PMID: 7800037
  39. Formation of heteromeric gamma-aminobutyric acid type A receptors containing two different alpha subunits.
    Mol Pharmacol. 1994 Mar;45(3):475-80 PMID: 8145733
  40. Importance of a novel GABAA receptor subunit for benzodiazepine pharmacology.
    Nature. 1989 Apr 13;338(6216):582-5 PMID: 2538761
  41. gamma-Aminobutyric acid type A receptor point mutation increases the affinity of compounds for the benzodiazepine site.
    Proc Natl Acad Sci U S A. 1991 Feb 15;88(4):1421-5 PMID: 1847522
  42. Molecular cloning reveals the existence of a fourth gamma subunit of the vertebrate brain GABAA receptor.
    FEBS Lett. 1993 Oct 4;331(3):211-6 PMID: 8397108
  43. Sequence and functional expression of the GABA A receptor shows a ligand-gated receptor super-family.
    Nature. 1987 Jul 16-22;328(6127):221-7 PMID: 3037384
  44. GABAA receptor beta subunit heterogeneity: functional expression of cloned cDNAs.
    EMBO J. 1989 Jun;8(6):1665-70 PMID: 2548852
  45. DNA sequencing with chain-terminating inhibitors.
    Proc Natl Acad Sci U S A. 1977 Dec;74(12):5463-7 PMID: 271968
  46. Stoichiometry of a recombinant GABAA receptor deduced from mutation-induced rectification.
    Neuroreport. 1993 Dec 13;5(3):285-8 PMID: 7507726
  47. Molecular weight in detergent solution of acetylcholine receptor from Torpedo californica.
    Biochemistry. 1978 May 30;17(11):2035-8 PMID: 667008
  48. The effect of subunit composition of rat brain GABAA receptors on channel function.
    Neuron. 1990 Nov;5(5):703-11 PMID: 1699569
  49. Acetylcholine receptor: complex of homologous subunits.
    Science. 1980 Jun 27;208(4451):1454-6 PMID: 7384786
  50. Neuronal nicotinic acetylcholine receptors expressed in Xenopus oocytes have a pentameric quaternary structure.
    J Biol Chem. 1991 Jun 15;266(17):11192-8 PMID: 2040627
  51. Two novel GABAA receptor subunits exist in distinct neuronal subpopulations.
    Neuron. 1989 Sep;3(3):327-37 PMID: 2561970
  52. Channel gating governed symmetrically by conserved leucine residues in the M2 domain of nicotinic receptors.
    Nature. 1995 Aug 10;376(6540):514-6 PMID: 7637783
  53. Distribution, prevalence, and drug binding profile of gamma-aminobutyric acid type A receptor subtypes differing in the beta-subunit variant.
    J Biol Chem. 1994 Oct 28;269(43):27100-7 PMID: 7929453
  54. 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):2330-7 PMID: 2165521
  55. The role of conserved leucines in the M2 domain of the acetylcholine receptor in channel gating.
    Mol Pharmacol. 1995 Sep;48(3):379-84 PMID: 7565616
  56. Functional properties of recombinant rat GABAA receptors depend upon subunit composition.
    Neuron. 1990 Jun;4(6):919-28 PMID: 1694446
  57. Identification of channel-lining residues in the M2 membrane-spanning segment of the GABA(A) receptor alpha1 subunit.
    J Gen Physiol. 1996 Feb;107(2):195-205 PMID: 8833341
  58. 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):24778-84 PMID: 1662216
  59. Gamma- and delta-subunits regulate the affinity and the cooperativity of ligand binding to the acetylcholine receptor.
    J Biol Chem. 1991 Oct 15;266(29):19369-77 PMID: 1680865
  60. The distribution of 13 GABAA receptor subunit mRNAs in the rat brain. I. Telencephalon, diencephalon, mesencephalon.
    J Neurosci. 1992 Mar;12(3):1040-62 PMID: 1312131
Article Info
Journal
The Journal of neuroscience : the official journal of the Society for Neuroscience
Abbr.
J Neurosci
ISSN
0270-6474
Published
1996-09-01
Pages
5415-24
Language
English
Region
United States
NLM ID
8102140
PMCID
PMC6578878
Subset
IM
Grants
NINDS NIH HHS · R01 NS035291 · United States
NINDS NIH HHS · R37 NS035291 · United States
NINDS NIH HHS · NS35291 · United States
NIAAA NIH HHS · AA09212 · United States
External Links
PubMed source PMC Full Text View journal details
Analysis Services
Analysis Services

Contact

No. 2 Wenbo Road, Zhangqiu District, Jinan, Shandong

Qilu Normal University · Genelibs Bioinformatics Lab

750 Shunhua Rd, Jinan

2F, Bldg F, University Science Park

Tel: 0531-88819269

WeChat Official Account

Follow our WeChat subscription account for real-time updates and the latest in medical and biological research.


Business Email

E-mail: product@genelibs.com

Copyright © 2026.Genelibs Jinan Honghui Biotechnology Co., Ltd. 鲁ICP备20007159号-2