No abstract

Fast synaptic inhibitory transmission in the CNS is mediated by ␥-aminobutyric acid type A (GABAA) receptors. They belong to the ligand-gated ion channel receptor superfamily, and are constituted of five subunits surrounding a chloride channel. Their clinical interest is highlighted by the number of therapeutic drugs that act on them. It is well established that the subunit composition of a receptor subtype determines its pharmacological properties. We have investigated positional effects of two different ␣-subunit isoforms, ␣1 and ␣6, in a single pentamer. For this purpose, we used concatenated subunit receptors in which subunit arrangement is predefined. The resulting receptors were expressed in Xenopus oocytes and analyzed by using the two-electrode voltage-clamp technique. Thus, we have characterized ␥2␤2␣1␤2␣1, ␥2␤2␣6␤2␣6, ␥2␤2␣1␤2␣6, and ␥2␤2␣6␤2␣1 GABAA receptors. We investigated their response to the agonist GABA, to the partial agonist piperidine-4-sulfonic acid, to the noncompetitive inhibitor furosemide and to the positive allosteric modulator diazepam. Each receptor isoform is characterized by a specific set of properties. In this case, subunit positioning provides a functional signature to the receptor. We furthermore show that a single ␣6-subunit is sufficient to confer high furosemide sensitivity, and that the diazepam efficacy is determined exclusively by the ␣-subunit neighboring the ␥2-subunit. By using this diagnostic tool, it should become possible to determine the subunit arrangement of receptors expressed in vivo that contain ␣1-and ␣6-subunits. This method may also be applied to the study of other ion channels.T he ␥-aminobutyric acid type A (GABA A ) receptors are the major inhibitory neuronal receptors in the mammalian brain. They belong to the family of pentameric Cys-loop ligand-gated ion channels that includes nicotinic acetylcholine, glycine, and serotonin type 3 receptors. In human, several GABA A receptor subunit isoforms have been cloned: ␣ 1-6 , ␤ 1-3 , ␥ 1-3 , ␦, , , and(1-7). Subunit composition of a GABA A receptor determines its pharmacological properties (8). The five subunits are arranged pseudosymmetrically around a central Cl Ϫ -selective channel (1). The major receptor subtype of the GABA A receptor in the brain most probably consists of ␣ 1 -, ␤ 2 -, and ␥ 2 -subunits (1, 2, 9-11). The most likely stoichiometry is two ␣ subunits, two ␤-subunits, and one ␥-subunit (12-16). Previous studies (16, 17) on the ␣ 1 ␤ 2 ␥ 2 GABA A receptor have shown that the subunit arrangement of a functional receptor is ␥␤␣␤␣, counterclockwise when viewed from the synaptic cleft.A specific GABA A receptor subtype is only then defined properly when it is known where each subunit is positioned in the receptor pentamer. For the characterization of the pharmacological properties of a specific GABA A receptor subtype, it is desirable to be able to specifically locate subunit isoforms. In addition, it is useful to be able to target point mutations to only one of two identical subunits. However, re...

We show that the five subunits of a c-aminobutyric acid type A receptor (GABA A receptor) can be concatenated to yield a functional receptor. This concatenated receptor a 1 -b 2 -a 1 -c 2 -b 2 has the advantage of a known subunit arrangement. Most of its functional properties are not significantly different from a receptor formed by individual subunits. Extent of expression amounted to about 40% of that of non-concatenated receptors in Xenopus oocytes, after injection of oocytes with comparable amounts of cRNA coding for concatenated and non-concatenated receptors. The ability to express receptors consisting of five subunits enables detailed studies of GABA A receptor subtype selective compounds.