Forced Subunit Assembly in α1β2γ2 GABAAReceptors

• Published in: The Journal of biological chemistry Vol. 277; no. 48; p. 46020
• Main Authors: Sabine W. Baumann, Roland Baur, Erwin Sigel
• Format: Journal Article
• Language: English
• Published: American Society for Biochemistry and Molecular Biology 29.11.2002
• ISSN: 0021-9258; 1083-351X
• DOI: 10.1074/jbc.M207663200

The major isoform of the γ-aminobutyric acid type A (GABA A ) receptor is thought to be composed of 2α 1 , 2β 2 , and 1γ 2 subunit(s), which surround the ion pore. Definite evidence for the subunit arrangement is lacking. We show here that GABA A receptor subunits can be concatenated to a trimer that can be functionally expressed upon combination with a dimer. Many combinations did not result in the functional expression. In contrast, four different combinations of triple subunits with dual subunit constructs, all resulting in the identical pentameric receptor γ 2 β 2 α 1 β 2 α 1 , could be successfully expressed in Xenopus oocytes. We characterized the functional properties of these receptors in respect to agonist, competitive antagonist, and diazepam sensitivity. All properties were similar to those of wild type α 1 β 2 γ 2 GABA A receptors. Thus, together with information on the crystal structure of the homologous acetylcholine-binding protein (Brejc, K., van Dijk, W. J., Klaassen, R. V., Schuurmans, M., van Der Oost, J., Smit, A. B., and Sixma, T. K., (2001) Nature 411, 269–276, we provide evidence for an arrangement γ 2 β 2 α 1 β 2 α 1 , counterclockwise when viewed from the synaptic cleft. Forced subunit assembly will also allow receptors containing different subunit isoforms or mutant subunits to be expressed, each in a desired position. The methods established here should be applicable to the entire ion channel family comprising nicotinic acetylcholine, glycine, and 5HT 3 receptors.