The GABA A receptors are the major inhibitory neurotransmitter receptors in mammalian brain. Each isoform consists of five homologous or identical subunits surrounding a central chloride ion-selective channel gated by GABA. How many isoforms of the receptor exist is far from clear. GABA A receptors located in the postsynaptic membrane mediate neuronal inhibition that occurs in the millisecond time range; those located in the extrasynaptic membrane respond to ambient GABA and confer long-term inhibition. GABA A receptors are responsive to a wide variety of drugs, e.g. benzodiazepines, which are often used for their sedative/hypnotic and anxiolytic effects.

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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 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 to be pentameric (16 -18), the subunit stoichiometry of receptors composed of ␣ and ␤ is still controversial. Recombinantly expressed receptors have been reported as possibly tetrameric (19,20) as well as pentameric (18,21). Unitary dose-response curves for ␣␤ receptors, single IC 50 values for Zn 2ϩ inhibition, and unitary single channel properties (1) provide evidence against the formation of two populations of receptors, e.g. 2␣3␤ and 3␣2␤. A tetrameric rather than a pentameric structure has been proposed as one of several explanations for the lower average...

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Two variant amino acid sequences, which differ in a single amino acid residue, have been reported for the alpha 1‐subunit of the rat brain GABAA receptor. We separately co‐expressed these two variants in Xenopus oocytes, in combination with beta 2 and gamma 2. This experiment showed that substitution of alpha 1‐Phe64 by Leu strongly decreases the apparent affinity for GABA dependent channel gating from 6 microM to 1260 microM. Starting from this observation, we used in vitro mutagenesis to obtain information relevant for the localization of the agonist/antagonist binding site in the GABAA receptor. Homologous mutation in alpha 5 had similar consequences for alpha 5 beta 2 gamma 2. Homologous mutation in beta 2 and gamma 2 resulted in intermediate and small shifts in EC50, respectively. The apparent affinities of the competitive antagonists bicuculline methiodide and SR95531, the latter sharing close structural similarity with the agonist GABA, were decreased 60‐ to 200‐fold by these mutations in alpha‐subunits. Interestingly, these affinities remained nearly unaffected upon introduction of the homologous mutations in beta 2 and gamma 2, or upon mutation of the neighbouring amino acid in alpha 1, Phe65 to Leu. These results suggest close functional and structural association of alpha‐subunits with the agonist/antagonist binding site, and involvement of N‐terminal portions of the extracellular domains of all subunits in the gating of the channel.

Ligands of the benzodiazepine binding site of the GABAA receptor come in three flavors: positive allosteric modulators, negative allosteric modulators and antagonists, all of which can bind with high affinity. The GABA(A) receptor is a pentameric protein which forms a chloride selective ion channel and ligands of the benzodiazepine binding site stabilize three different conformations of this receptor channel. Classical benzodiazepines exert a positive allosteric effect by increasing the affinity of channel opening by the agonist gamma-aminobutyric acid (GABA). We concentrate here on the major adult isoform, the alpha1beta2gamma2 GABA(A) receptor. The binding pocket for benzodiazepines is located in a subunit cleft between gamma2 and alpha1 subunits in a position homologous to the agonist binding site for GABA that is located between alpha1 and beta2 subunits. It is reviewed here how we arrived at this picture. In particular, point mutations were performed in combination with subsequent analysis of the expressed mutant proteins using either electrophysiological techniques or radioactive ligand binding assays. The predictive power of these methods is assessed by comparing the results with the predictions that can be made on the basis of the recently published crystal structure of the acetylcholine binding protein that shows homology to the N-terminal, extracellular domain of the GABA(A) receptor.

The major isoform of the ␥-aminobutyric acid type A (GABA A ) receptor is thought to be composed of 2␣ 1 , 2␤ 2 , and 1␥ 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 ␥ 2 ␤ 2 ␣ 1 ␤ 2 ␣ 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 ␣ 1 ␤ 2 ␥ 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 ␥ 2 ␤ 2 ␣ 1 ␤ 2 ␣ 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.

GABA A receptors are the major ionotropic inhibitory neurotransmitter receptors. The endocannabinoid system is a lipid signaling network that modulates different brain functions. Here we show a direct molecular interaction between the two systems. The endocannabinoid 2-arachidonoyl glycerol (2-AG) potentiates GABA A receptors at low concentrations of GABA. Two residues of the receptor located in the transmembrane segment M4 of β 2 confer 2-AG binding. 2-AG acts in a superadditive fashion with the neurosteroid 3α, 21-dihydroxy-5α-pregnan-20-one (THDOC) and modulates δ-subunit-containing receptors, known to be located extrasynaptically and to respond to neurosteroids. 2-AG inhibits motility in CB 1 /CB 2 cannabinoid receptor double-KO, whereas β 2 -KO mice show hypermotility. The identification of a functional binding site for 2-AG in the GABA A receptor may have far-reaching consequences for the study of locomotion and sedation.retrograde signaling | electrophysiology | allosteric modulation