Positioning of the alpha-subunit isoforms confers a functional signature to gamma-aminobutyric acid type A receptors.

Fast synaptic inhibitory transmission in the CNS is mediated by gamma-aminobutyric acid type A (GABA(A)) 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 alpha-subunit isoforms, alpha(1) and alpha(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 gamma(2)beta(2)alpha(1)beta(2)alpha(1), gamma(2)beta(2)alpha(6)beta(2)alpha(6), gamma(2)beta(2)alpha(1)beta(2)alpha(6), and gamma(2)beta(2)alpha(6)beta(2)alpha(1) GABA(A) 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 alpha(6)-subunit is sufficient to confer high furosemide sensitivity, and that the diazepam efficacy is determined exclusively by the alpha-subunit neighboring the gamma(2)-subunit. By using this diagnostic tool, it should become possible to determine the subunit arrangement of receptors expressed in vivo that contain alpha(1)- and alpha(6)-subunits. This method may also be applied to the study of other ion channels.