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Originally published In Press as doi:10.1074/jbc.M009508200 on February 23, 2001

J. Biol. Chem., Vol. 276, Issue 19, 16024-16032, May 11, 2001
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Detection and Binding Properties of GABAA Receptor Assembly Intermediates*

Thomas Klausberger, Noosha Ehya, Karoline Fuchs, Thomas Fuchs, Veronika Ebert, Isabella Sarto, and Werner SieghartDagger

From the Section of Biochemical Psychiatry, University Clinic for Psychiatry, A-1090 Vienna, Austria and the Brain Research Institute, University of Vienna, Division of Biochemistry and Molecular Biology, A-1090 Vienna, Austria

Density gradient centrifugation of native and recombinant gamma -aminobutyric acid, type A (GABAA) receptors was used to detect assembly intermediates. No such intermediates could be identified in extracts from adult rat brain or from human embryonic kidney (HEK) 293 cells transfected with alpha 1, beta 3, and gamma 2 subunits and cultured at 37 °C. However, subunit dimers, trimers, tetramers, and pentamers were found in extracts from the brain of 8-10-day-old rats and from alpha 1beta 3gamma 2 transfected HEK cells cultured at 25 °C. In both systems, alpha 1, beta 3, and gamma 2 subunits could be identified in subunit dimers, indicating that different subunit dimers are formed during GABAA receptor assembly. Co-transfection of HEK cells with various combinations of full-length and C-terminally truncated alpha 1 and beta 3 or alpha 1 and gamma 2 subunits and co-immunoprecipitation with subunit-specific antibodies indicated that even subunits containing no transmembrane domain can assemble with each other. Whereas alpha 1gamma 2, alpha 1Ngamma 2, alpha 1gamma 2N, and alpha 1Ngamma 2N, combinations exhibited specific [3H]Ro 15-1788 binding, specific [3H]muscimol binding could only be found in alpha 1beta 3 and alpha 1beta 3N, but not in alpha 1Nbeta 3 or alpha 1Nbeta 3N combinations. This seems to indicate that a full-length alpha 1 subunit is necessary for the formation of the muscimol-binding site and for the transduction of agonist binding into channel gating.

* This work was supported by Grant 7835 of the Jubiläumsfonds of the Austrian National Bank and by Grant P12637-Med of the Austrian Science Foundation.The costs of publication of this article were defrayed in part by the payment of page charges. The article must therefore be hereby marked "advertisement" in accordance with 18 U.S.C. Section 1734 solely to indicate this fact.

Dagger To whom correspondence should be addressed: Brain Research Inst., University of Vienna, Div. of Biochemistry and Molecular Biology, Spitalgasse 4, A-1090 Vienna, Austria. Tel.: 43-1-4277-62950; Fax: 43-1-4277-62959; E-mail: Werner.Sieghart@univie.ac.at.

Copyright © 2001 by The American Society for Biochemistry and Molecular Biology, Inc.
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Copyright © 2001 by the American Society for Biochemistry and Molecular Biology.