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Volume 16, Number 17, Issue of September 1, 1996 pp. 5415-5424
Copyright ©1996 Society for Neuroscience

Stoichiometry of a Recombinant GABAA Receptor

Received May 1, 1996; revised June 14, 1996; accepted June 18, 1996.

Yongchang Chang, Ruoping Wang, Sonal Barot, and David S. Weiss

Neurobiology Research Center, Department of Physiology and Biophysics, University of Alabama at Birmingham, Birmingham, Alabama 35294

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 counterparts (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 1beta 2gamma 2 GABAA receptor is a pentamer composed of two alpha  subunits, two beta  subunits, and one gamma  subunit.

Key words: GABA; stoichiometry; receptor; ion channel; mutagenesis




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B. X. Carlson, A. C. Engblom, U. Kristiansen, A. Schousboe, and R. W. Olsen
A Single Glycine Residue at the Entrance to the First Membrane-Spanning Domain of the gamma -Aminobutyric Acid Type A Receptor beta 2 Subunit Affects Allosteric Sensitivity to GABA and Anesthetics
Mol. Pharmacol., March 1, 2000; 57(3): 474 - 484.
[Abstract] [Full Text]

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S. Srinivasan, C. J. Nichols, G. M. Lawless, R. W. Olsen, and A. J. Tobin
Two Invariant Tryptophans on the alpha 1 Subunit Define Domains Necessary for GABAA Receptor Assembly
J. Biol. Chem., September 17, 1999; 274(38): 26633 - 26638.
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T. R. Neelands and R. L. Macdonald
Incorporation of the pi Subunit into Functional gamma -Aminobutyric AcidA Receptors
Mol. Pharmacol., September 1, 1999; 56(3): 598 - 610.
[Abstract] [Full Text]

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 Proc. Natl. Acad. Sci. USAHome page
T. P. Bonnert, R. M. McKernan, S. Farrar, B. le Bourdelles, R. P. Heavens, D. W. Smith, L. Hewson, M. R. Rigby, D. J. S. Sirinathsinghji, N. Brown, et al.
theta , a novel gamma -aminobutyric acid type A receptor subunit
PNAS, August 17, 1999; 96(17): 9891 - 9896.
[Abstract] [Full Text] [PDF]

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T. R. Neelands, J. Zhang, and R. L. Macdonald
GABAA Receptors Expressed in Undifferentiated Human Teratocarcinoma NT2 Cells Differ from Those Expressed by Differentiated NT2-N Cells
J. Neurosci., August 15, 1999; 19(16): 7057 - 7065.
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C. Sur, S. J. Farrar, J. Kerby, P. J. Whiting, J. R. Atack, and R. M. McKernan
Preferential Coassembly of alpha 4 and delta  Subunits of the gamma -Aminobutyric AcidA Receptor in Rat Thalamus
Mol. Pharmacol., July 1, 1999; 56(1): 110 - 115.
[Abstract] [Full Text]

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S. J. Farrar, P. J. Whiting, T. P. Bonnert, and R. M. McKernan
Stoichiometry of a Ligand-gated Ion Channel Determined by Fluorescence Energy Transfer
J. Biol. Chem., April 9, 1999; 274(15): 10100 - 10104.
[Abstract] [Full Text] [PDF]

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 Anesth. Analg.Home page
S. Ueno, J. R. Trudell, E. I Eger II, and R. A. Harris
Actions of Fluorinated Alkanols on GABAA Receptors: Relevance to Theories of Narcosis
Anesth. Analg., April 1, 1999; 88(4): 877 - 877.
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A. M L McClellan and R. E Twyman
Receptor system response kinetics reveal functional subtypes of native murine and recombinant human GABAA receptors
J. Physiol., March 15, 1999; 515(3): 711 - 727.
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P. Poisbeau, M. C. Cheney, M. D. Browning, and I. Mody
Modulation of Synaptic GABAA Receptor Function by PKA and PKC in Adult Hippocampal Neurons
J. Neurosci., January 15, 1999; 19(2): 674 - 683.
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T. R. Neelands, J. L. Fisher, M. Bianchi, and R. L. Macdonald
Spontaneous and gamma -Aminobutyric Acid (GABA)-Activated GABAA Receptor Channels Formed by epsilon  Subunit-Containing Isoforms
Mol. Pharmacol., January 1, 1999; 55(1): 168 - 178.
[Abstract] [Full Text]

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 J. Biol. Chem.Home page
J. X. Connor, A. J. Boileau, and C. Czajkowski
A GABAA Receptor alpha 1 Subunit Tagged with Green Fluorescent Protein Requires a beta  Subunit for Functional Surface Expression
J. Biol. Chem., October 30, 1998; 273(44): 28906 - 28911.
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T. Defazio and J. J. Hablitz
Zinc and Zolpidem Modulate mIPSCs in Rat Neocortical Pyramidal Neurons
J Neurophysiol, October 1, 1998; 80(4): 1670 - 1677.
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 Mol. Pharmacol.Home page
R. M. McKernan, S. Farrar, I. Collins, F. Emms, A. Asuni, K. Quirk, and H. Broughton
Photoaffinity Labeling of the Benzodiazepine Binding Site of alpha 1beta 3gamma 2 gamma -Aminobutyric AcidA Receptors with Flunitrazepam Identifies a Subset of Ligands that Interact Directly with His102 of the alpha  Subunit and Predicts Orientation of These within the Benzodiazepine Pharmacophore
Mol. Pharmacol., July 1, 1998; 54(1): 33 - 43.
[Abstract] [Full Text]

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P. J. Groot-Kormelink, W. H. M. L. Luyten, D. Colquhoun, and L. G. Sivilotti
A Reporter Mutation Approach Shows Incorporation of the "Orphan" Subunit beta 3 into a Functional Nicotinic Receptor
J. Biol. Chem., June 19, 1998; 273(25): 15317 - 15320.
[Abstract] [Full Text] [PDF]

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 Pharmacol. Rev.Home page
E. A. Barnard, P. Skolnick, R. W. Olsen, H. Mohler, W. Sieghart, G. Biggio, C. Braestrup, A. N. Bateson, and S. Z. Langer
International Union of Pharmacology. XV. Subtypes of gamma -Aminobutyric AcidA Receptors: Classification on the Basis of Subunit Structure and Receptor Function
Pharmacol. Rev., June 1, 1998; 50(2): 291 - 314.
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 Mol. Pharmacol.Home page
J. Horenstein and M. H. Akabas
Location of a High Affinity Zn2+ Binding Site in the Channel of alpha 1beta 1 gamma -Aminobutyric AcidA Receptors
Mol. Pharmacol., May 1, 1998; 53(5): 870 - 877.
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M. Jechlinger, R. Pelz, V. Tretter, T. Klausberger, and W. Sieghart
Subunit Composition and Quantitative Importance of Hetero-oligomeric Receptors: GABAA Receptors Containing alpha 6 Subunits
J. Neurosci., April 1, 1998; 18(7): 2449 - 2457.
[Abstract] [Full Text] [PDF]

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 J. Pharmacol. Exp. Ther.Home page
M. D. Krasowski, S. E. Finn, Q. Ye, and N. L. Harrison
Trichloroethanol Modulation of Recombinant GABAA, Glycine and GABA rho 1 Receptors
J. Pharmacol. Exp. Ther., March 1, 1998; 284(3): 934 - 942.
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 Mol. Pharmacol.Home page
Y. Chang and D. S. Weiss
Substitutions of the Highly Conserved M2 Leucine Create Spontaneously Opening rho 1 gamma -Aminobutyric Acid Receptors
Mol. Pharmacol., March 1, 1998; 53(3): 511 - 523.
[Abstract] [Full Text]

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 Mol. Pharmacol.Home page
M. D. Krasowski, V. V. Koltchine, C. E. Rick, Q. Ye, S. E. Finn, and N. L. Harrison
Propofol and Other Intravenous Anesthetics Have Sites of Action on the gamma -Aminobutyric Acid Type A Receptor Distinct from That for Isoflurane
Mol. Pharmacol., March 1, 1998; 53(3): 530 - 538.
[Abstract] [Full Text]

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 Mol. Pharmacol.Home page
B. Ebert, S. A. Thompson, K. Saounatsou, R. McKernan, P. Krogsgaard-Larsen, and K. A. Wafford
Differences in Agonist/Antagonist Binding Affinity and Receptor Transduction Using Recombinant Human gamma -Aminobutyric Acid Type A Receptors
Mol. Pharmacol., December 1, 1997; 52(6): 1150 - 1156.
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P. Skolnick, R. J. Hu, C. M. Cook, S. D. Hurt, J. D. Trometer, R. Liu, Q. Huang, and J. M. Cook
[3H]RY 80: A High-Affinity, Selective Ligand for gamma -Aminobutyric AcidA Receptors Containing Alpha-5 Subunits,
J. Pharmacol. Exp. Ther., November 1, 1997; 283(2): 488 - 493.
[Abstract] [Full Text]

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 Mol. Pharmacol.Home page
P. B. Wingrove, S. A. Thompson, K. A. Wafford, and P. J. Whiting
Key Amino Acids in the gamma  Subunit of the gamma -Aminobutyric AcidA Receptor that Determine Ligand Binding and Modulation at the Benzodiazepine Site
Mol. Pharmacol., November 1, 1997; 52(5): 874 - 881.
[Abstract] [Full Text]

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 Proc. Natl. Acad. Sci. USAHome page
E. Palma, L. Maggi, F. Eusebi, and R. Miledi
Neuronal nicotinic threonine-for-leucine 247 alpha 7 mutant receptors show different gating kinetics when activated by acetylcholine or by the noncompetitive agonist 5-hydroxytryptamine
PNAS, September 2, 1997; 94(18): 9915 - 9919.
[Abstract] [Full Text] [PDF]

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G. H. Gorrie, Y. Vallis, A. Stephenson, J. Whitfield, B. Browning, T. G. Smart, and S. J. Moss
Assembly of GABAA Receptors Composed of alpha 1 and beta 2 Subunits in Both Cultured Neurons and Fibroblasts
J. Neurosci., September 1, 1997; 17(17): 6587 - 6596.
[Abstract] [Full Text] [PDF]

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 J. Biol. Chem.Home page
M. Li and A. L. De Blas
Coexistence of Two beta  Subunit Isoforms in the Same gamma -Aminobutyric Acid Type A Receptor
J. Biol. Chem., June 27, 1997; 272(26): 16564 - 16569.
[Abstract] [Full Text] [PDF]

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 J. Neurosci.Home page
V. Tretter, N. Ehya, K. Fuchs, and W. Sieghart
Stoichiometry and Assembly of a Recombinant GABAA Receptor Subtype
J. Neurosci., April 15, 1997; 17(8): 2728 - 2737.
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 J. Biol. Chem.Home page
S. M. O'Shea and N. L. Harrison
Arg-274 and Leu-277 of the gamma -Aminobutyric Acid Type A Receptor alpha 2 Subunit Define Agonist Efficacy and Potency
J. Biol. Chem., July 21, 2000; 275(30): 22764 - 22768.
[Abstract] [Full Text] [PDF]

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 J. Biol. Chem.Home page
A. Buhr, C. Wagner, K. Fuchs, W. Sieghart, and E. Sigel
Two Novel Residues in M2 of the gamma -Aminobutyric Acid Type A Receptor Affecting Gating by GABA and Picrotoxin Affinity
J. Biol. Chem., March 9, 2001; 276(11): 7775 - 7781.
[Abstract] [Full Text] [PDF]

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 J. Biol. Chem.Home page
T. Klausberger, N. Ehya, K. Fuchs, T. Fuchs, V. Ebert, I. Sarto, and W. Sieghart
Detection and Binding Properties of GABAA Receptor Assembly Intermediates
J. Biol. Chem., May 4, 2001; 276(19): 16024 - 16032.
[Abstract] [Full Text] [PDF]

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 J. Biol. Chem.Home page
S. W. Baumann, R. Baur, and E. Sigel
Subunit Arrangement of gamma -Aminobutyric Acid Type A Receptors
J. Biol. Chem., September 21, 2001; 276(39): 36275 - 36280.
[Abstract] [Full Text] [PDF]

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 J. Biol. Chem.Home page
A. J. Boileau, J. G. Newell, and C. Czajkowski
GABAA Receptor beta 2 Tyr97 and Leu99 Line the GABA-binding Site. INSIGHTS INTO MECHANISMS OF AGONIST AND ANTAGONIST ACTIONS
J. Biol. Chem., January 18, 2002; 277(4): 2931 - 2937.
[Abstract] [Full Text] [PDF]


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