The GABA A receptors are ligand-gated chloride channels. The subunit stoichiometry of the receptors is controversial; four, five, or six subunits per receptor molecule have been proposed for ␣␤ receptors, whereas ␣␤␥ receptors are assumed to be pentamers. In this study, ␣-␤ and ␤-␣ tandem cDNAs from the ␣1 and ␤2 subunits of the GABA A receptor were constructed. We determined the minimal length of the linker that is required between the two subunits for functional channel expression for each of

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... the tandem constructs. 10-and 23-amino acid residues are required for ␣-␤ and ␤-␣, respectively. The tandem constructs either alone or in combination with each other failed to express functional channels in Xenopus oocytes. Therefore, we can exclude tetrameric or hexameric ␣␤ GABA A receptors. We can also exclude proteolysis of the tandem constructs. In addition, the tandem constructs were combined with single ␣, ␤, or ␥ subunits to allow formation of pentameric arrangements. In contrast to the combination with ␣ subunits, the combination with either ␤ or ␥ subunits led to expression of functional channels. Therefore, a pentameric arrangement containing two ␣1 and three ␤2 subunits is proposed for the receptor composed of ␣ and ␤ subunits. Our findings also favor an arrangement ␤␣␥␤␣ for the receptor composed of ␣, ␤, and ␥ subunits. GABA A 1 receptors mediate fast synaptic inhibition in the mammalian brain. They are believed to form heterooligomers composed of subunits from six classes with several isoforms (␣1-6, ␤1-3, ␥1-3, ⑀, ␦, , ) (1-5). These subunits belong to the gene superfamily of ligand-gated ion channels, which includes nicotinic acetylcholine receptors, GABA A receptors, glycine receptors, and the serotonin type 3 (5HT 3 ) receptor. The major GABA A receptor isoform is likely to be composed of ␣1, ␤2, and ␥2 subunits (1, 2, 6, 7). Heterologous expression demonstrated that the combination of ␣ and ␤ subunits produces GABA-gated currents, but coexpression of a ␥ subunit is required for benzodiazepine sensitivity of the expressed receptors (8). GABA A receptors composed of ␣ and ␤ subunits differ from receptors that additionally contain the ␥ subunit in regard to Zn 2ϩ and benzodiazepine sensitivity and to single channel conductance (9 -13). Some populations of neuronal GABA A receptors show high Zn 2ϩ sensitivity coupled with low single-channel conductance as described for ␣␤ receptors (14, 15). Although receptors made from ␣, ␤, and ␥ subunits are thought