GABA A receptors are ligand-gated chloride ion channels that are presumed to be pentamers composed of ␣, ␤, and ␥ subunits. The subunit stoichiometry, however, is controversial, and the subunit arrangement presently is not known. In this study the ratio of subunits in recombinant ␣1␤3␥2 receptors was determined in Western blots from the relative signal intensities of antibodies directed against the N terminus or the cytoplasmic loop of different subunits after the relative reactivity of these antibodies had been determined with GABA A receptor subunit chimeras composed of the N-terminal domain of one and the remaining part of the other subunit. Via this method a subunit stoichiometry of two ␣ subunits, two ␤ subunits, and one ␥ subunit was derived. Similar experiments investigating the composition of ␣1␤3 receptors expressed on the surface of human embryonic kidney (HEK) 293 cells cotransfected with ␣1 and ␤3 subunits resulted in a stoichiometry of two ␣ and three ␤ subunits. Density gradient centrifugation studies indicated that combinations of ␣1␤3␥2 or ␣1␤3 subunits expressed in HEK 293 cells are able to form pentamers, whereas combinations of ␣1␥2 or ␤3␥2 subunits predominantly form heterodimers. These results provide valuable information on the mechanism of GABA A receptor assembly and support the conclusion that GABA A receptors are pentamers in which a total of four alternating ␣ and ␤ subunits are connected by a ␥ subunit. Key words: GABA A receptor; stoichiometry; assembly; subunit arrangement; human embryonic kidney 293 cells; chimeric subunits; density gradient centrifugation; Western blotGABA, the major inhibitory transmitter in the CNS, mediates fast synaptic inhibition by opening the chloride ion channel intrinsic to the GABA A receptor. This receptor is a hetero-oligomeric protein and the site of action of a variety of pharmacologically and clinically important drugs, such as benzodiazepines, barbiturates, steroids, anesthetics, and convulsants (Sieghart, 1995). So far, six ␣, three ␤, three ␥, one ␦, and two subunits of these receptors, as well as several alternatively spliced isoforms of some of these subunits, have been identified in mammalian brain (Macdonald and Olsen, 1994;Sieghart, 1995). Expression studies have indicated that an ␣, a ␤, and a ␥ subunit have to combine to produce GABA A receptors with a pharmacology resembling that of receptors found in the brain and that, depending on the subunits used for transfection of cells, receptors with distinct pharmacological and electrophysiological properties do arise (Sieghart, 1995). Overall it is assumed, however, that a total of five subunits have to combine to form functional GABA A receptors (Nayeem et al., 1994).A variety of subunit-specific antibodies has been raised to investigate the subunit composition of GABA A receptors. Immunocytochemical studies demonstrating the colocalization of subunits in GABA A receptor clusters on neuronal membranes (Fritschy et al., 1992;Caruncho and Costa, 1994;Fritschy and Möhler, 1995;Somogyi et al., 199...

GABA A receptors are the major inhibitory transmitter receptors in the CNS. Recombinant GABA A receptors composed of ␣ 1 ␤ 3 ␥ 2 subunits have been demonstrated to assemble as pentamers consisting of two ␣ 1 , two ␤ 3 , and one ␥ 2 subunit. Using truncated and chimeric ␣ 1 subunits, we identified the ␣ 1 (80-100) sequence as a major binding site for ␥ 2 subunits. In addition, we demonstrated its direct interaction with ␥ 2 (91-104), a sequence that previously has been identified to form the contact to ␣ 1 subunits. The observation that the amino acid residues ␣ 1 P96 and ␣ 1 H101, which can be photolabeled by [ 3 H]flunitrazepam, are located within or adjacent to the ␣ 1 (80-100) sequence, indicates that the benzodiazepine binding site of GABA A receptors is located close to this intersubunit contact. The observation that ␣ 1 (80-100) interacts with ␥ 2 but not with ␤ 3 subunits indicates the existence of an additional ␤ 3 binding site on ␣ 1 subunits. The preferred alternate use of the ␥ 2 and ␤ 3 binding sites in two different ␣ 1 subunits of the same receptor ensures the incorporation of only a single ␥ 2 subunit and thus, determines subunit stoichiometry of ␣ 1 ␤ 3 ␥ 2 receptors. Distinct binding sites and their alternate use can therefore explain how subunits of hetero-oligomeric transmembrane proteins assemble into a defined protein complex.

GABA A receptors are ligand-gated chloride channels composed of five homologous subunits that specifically recognize one another and assemble around an aqueous pore. To identify domains responsible for the specificity of subunit association, we constructed C-terminal truncated ␥ 2 subunits, as well as mutated and chimeric fragments. From their ability to interfere with ␣ 1 ␤ 3 ␥ 2 receptor assembly and to associate with full-length subunits, we concluded that amino acid sequences ␥ 2 -(91-104) and ␥ 2 -(83-90) form the sites mediating assembly with ␣ 1 and ␤ 3 subunits, respectively. Neural network-based secondary structure prediction, Monte Carlo optimization, and hydrophobicity analysis led to the conclusion that these sites also form the intersubunit contacts in the completely assembled receptor and provided important information on the benzodiazepine-binding site and structure of GABA A receptors.

Density gradient centrifugation of native and recombinant ␥-aminobutyric acid, type A (GABA A ) 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 ␣ 1 , ␤ 3 , and ␥ 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 ␣ 1 ␤ 3 ␥ 2 transfected HEK cells cultured at 25°C. In both systems, ␣ 1 , ␤ 3 , and

GABA A receptors are chloride ion channels that can be opened by GABA, the most important inhibitory transmitter in the CNS. In the mammalian brain the majority of these pentameric receptors is composed of two a, two b and one c subunit. To achieve the correct order of subunits around the pore, each subunit must form specific contacts via its plus (+) and minus (-) side. To identify a sequence on the b 3 subunit important for assembly, we generated various full-length or truncated chimeric b 3 constructs and investigated their ability to assemble with a 1 and c 2 subunits. It was demonstrated that replacement of the sequence b 3 (76-89) by the homologous a 1 sequence impaired assembly with a 1 but not with c 2 subunits in a 1 b 3 c 2 -GABA A receptors. Other experiments indicated that assembly was impaired via the b 3 (-) side of the chimeric subunit. Within the sequence b 3 (76-89) the sequence b 3 (85-89) seemed to be of primary importance for assembly with a 1 subunits. A comparison with the structure of the acetylcholinebinding protein supports the conclusion that the sequence b 3 (85-89) is located at the b 3 (-) side and indicates that it contains amino acid residues that might directly interact with the (+) side of the neighbouring a 1 subunit.