The alpha1beta2gamma2 is the most abundant subtype of the GABA(A) receptor and is localized in many regions of the brain. To gain more insight into the role of this receptor subtype in the modulation of inhibitory neurotransmission, we generated mice lacking either the alpha1 or beta2 subunit. In agreement with the reported abundance of this subtype, >50% of total GABA(A) receptors are lost in both alpha1-/- and beta2-/- mice. Surprisingly, homozygotes of both mouse lines are viable, fertile, and show no spontaneous seizures. Initially half of the alpha1-/- mice died prenatally or perinatally, but they exhibited a lower mortality rate in subsequent generations, suggesting some phenotypic drift and adaptive changes. Both adult alpha1-/- and beta2-/- mice demonstrate normal performances on the rotarod, but beta2-/- mice displayed increased locomotor activity. Purkinje cells of the cerebellum primarily express alpha1beta2gamma2 receptors, and in electrophysiological recordings from alpha1-/- mice GABA currents in these neurons are dramatically reduced, and residual currents have a benzodiazepine pharmacology characteristic of alpha2- or alpha3-containing receptors. In contrast, the cerebellar Purkinje neurons from beta2-/- mice have only a relatively small reduction of GABA currents. In beta2-/- mice expression levels of all six alpha subunits are reduced by approximately 50%, suggesting that the beta2 subunit can coassemble with alpha subunits other than just alpha1. Our data confirm that alpha1beta2gamma2 is the major GABA(A) receptor subtype in the murine brain and demonstrate that, surprisingly, the loss of this receptor subtype is not lethal.

No abstract

A novel series of 3-heteroaryl-5,6-bis(aryl)-1-methyl-2-pyridones were developed with high affinity for the benzodiazepine (BZ) binding site of human gamma-aminobutyric acid (GABA(A)) receptor ion channels, low binding selectivity for alpha 2- and/or alpha 3- over alpha 1-containing GABA(A) receptor subtypes and high binding selectivity over alpha 5 subtypes. High affinity appeared to be associated with a coplanar conformation of the pyridone and sulfur-containing 3-heteroaryl rings resulting from an attractive S.O intramolecular interaction. Functional selectivity (i.e., selective efficacy) for alpha 2 and/or alpha 3 GABA(A) receptor subtypes over alpha1 was observed in several of these compounds in electrophysiological assays. Furthermore, an alpha 3 subtype selective inverse agonist was proconvulsant and anxiogenic in rodents while an alpha 2/alpha 3 subtype selective partial agonist was anticonvulsant and anxiolytic, supporting the hypothesis that subtype selective BZ site agonists may provide new anxiolytic therapies.

L-655,708 is a ligand for the benzodiazepine site of the g-aminobutyric acid type A (GABA A ) receptor that exhibits a 100-fold higher af®nity for a5-containing receptors compared with a1-containing receptors. Molecular biology approaches have been used to determine which residues in the a5 subunit are responsible for this selectivity. Two amino acids have been identi®ed, a5Thr208 and a5Ile215, each of which individually confer approximately 10-fold binding selectivity for the ligand and which together account for the 100-fold higher af®nity of this ligand at a5-containing receptors. L-655,708 is a partial inverse agonist at the GABA A receptor which exhibited no functional selectivity between a1-and a5-containing receptors and showed no change in ef®cacy at receptors containing a1 subunits where amino acids at both of the sites had been altered to their a5 counterparts (a1DSer205-Thr,Val212-Ile). In addition to determining the binding selectivity of L-655,708, these amino acid residues also in¯uence the binding af®nities of a number of other benzodiazepine (BZ) site ligands. They are thus important elements of the BZ site of the GABA A receptor, and further delineate a region just N-terminal to the ®rst transmembrane domain of the receptor a subunit that contributes to this binding site. The mammalian g-aminobutyric acid type A (GABA A ) receptor is a pentameric structure containing different combinations of a1±6, b1±3, g1±3, d, 1 and u subunits (for reviews, see Barnard et al. 1998;Mehta and Ticku 1999). Among the many classes of drug which interact with the receptor are the benzodiazepines (BZs) that are used as anxiolytics, anticonvulsants and hypnotics. Immunoprecipitation with subunit speci®c antibodies, and recombinant receptor studies have demonstrated that high af®nity BZ-binding sites are found on receptors of abg 2 composition. The differential af®nity of drugs such as diazepam for such receptors in which the a1 subunit has been substituted by a6, demonstrating that the a subunit is a major contributor to the BZ binding site present on the receptor (Lu Èddens et al. 1990). Site-directed mutagenesis studies have been shown in a number of cases (e.g. Pritchett and Seeburg 1991;Wieland et al. 1992;Amin et al. 1997;Buhr et al. 1997) to identify residues within the a subunit which are critical for the drug±receptor interaction. Although the diazepam insensitivity of receptors containing a4 and a6 is well documented (e.g. Lu Èddens et al. 1990;Wisden et al. 1991), few clinically ef®cacious drugs other than zolpidem (which is 10-fold selective for a1-containing receptors over a2-and a3-containing receptors, with negligible af®nity for a4-, a5-and a6-containing receptors) discriminate between receptors containing other a subunits. Recently, however, the identi®cation of a ligand (L-655,708) with selectivity for the a5-containing receptor has been reported (Quirk et al.

Nonselective inverse agonists at the benzodiazepine binding site on the GABA-A chloride ion channel enhance cognitive performance in animals but cannot be used in the treatment of cognitive disorders because of anxiogenic and convulsant side effects. We have identified a novel series of GABA-A alpha5 receptor ligands during our search for alpha5 receptor inverse agonists as potential cognition enhancers. In particular, 6,6-dimethyl-3-(2-hydroxyethyl)thio-1-(thiazol-2-yl)-6,7-dihydro-2-benzothiophen-4(5H)-one (26) has been identified as a functionally selective GABA-A alpha5 inverse agonist.