The specific mechanisms underlying general anesthesia are primarily unknown. The intravenous general anesthetic etomidate acts by potentiating GABA(A) receptors, with selectivity for beta2 and beta3 subunit-containing receptors determined by a single asparagine residue. We generated a genetically modified mouse containing an etomidate-insensitive beta2 subunit (beta2 N265S) to determine the role of beta2 and beta3 subunits in etomidate-induced anesthesia. Loss of pedal withdrawal reflex and burst suppression in the electroencephalogram were still observed in the mutant mouse, indicating that loss of consciousness can be mediated purely through beta3-containing receptors. The sedation produced by subanesthetic doses of etomidate and during recovery from anesthesia was present only in wild-type mice, indicating that the beta2 subunit mediates the sedative properties of anesthetics. These findings show that anesthesia and sedation are mediated by distinct GABA(A) receptor subtypes.

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

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.

The differential sensitivity of type A -aminobutyric acid (GABAA) receptors to benzodiazepine lgands seen in the malian nervous system is thought to be generated by the existence of a number of different receptor subtypes, assembled from a range of closely related subunits (a1-6, P1-3, V1-3, and 6) encoded by discrete genes. The characteristics of a given subtype can be determined by the coexpression ofcloned cDNAs encoding the subunits ofinterest. Two transient expression systems have so far been employed in the study of the ligand-binding characteristics and chloride channel properties of such GABAA receptors-Xenopus oocytes and transfected mammalian cells. Here we report on the use of a steroid-inducible promoter expression system for the production of a permanently transfected clonal cell line expressing the aj.8Vy2L GABAA receptor subtype. Using both immunoprecipitation by subunit-specific antisera and gelexclusion chromatography, we have shown that the ai, 13i, and 'Y2L subunits coassemble to form receptor macromolecules that are of the same size as native GABAA receptors. Additionally, the recombinant receptors have the same bendiazepine pharmacology as native a1-containing GABAA receptors and function as GABA-gated chloride channels. Such cell lines expressing individual GABAA receptor subtypes will prove important tools in the study of the structure, function, and pharmacology ofGABAA receptors and in the development of subtype-specific drugs.In the mammalian brain, type A y-aminobutyric acid (GABAA) receptors are the major mediators of inhibitory neurotransmission. They constitute GABA-gated chloride channels, whose activity may be allosterically modulated by a number of drugs, including the barbiturates and benzodiazepines (BZs) (1). Heterogeneity in the responses of the mammalian receptors to BZ-type ligands has been attributed to the differential assembly of GABAA receptor subtypes from a family of subunits (al16, 11-3, y1-3, and 8) that have been identified by recombinant DNA approaches (2-14).As individual GABAA receptor subtypes are not known to be expressed in any transformed cell lines, it has proved necessary to employ other means for their in vitro analysis. Transient expression of various subunit combinations in Xenopus oocytes (2-7) and human embryonic kidney 293 cells (7)(8)(9)(10)(11)(15)(16)(17) have been used to investigate their functional and ligand-binding properties. These approaches have suggested that, while there is a requirement for an a subunit, a 13 subunit, and the Y2 subunit in order to allow BZ binding (8), the relative affinities for various ligands, as classically defined by BZ-1 or BZ-2 pharmacology (18) (2) were then subcloned into the modified pMSGneo. The bovine 'Y2L subunit cDNA (12) was modified for transfection by addition of 5' untranslated-region sequence derived from the bovine a, clone. Briefly, oligonucleotides 5'-GCGGAGC-

The pharmacology of native and recombinant GABA-A receptors containing either gamma1, gamma2 or gamma3 subunits has been investigated. The pharmacology of native receptors has been investigated by immunoprecipitating receptors from solubilised preparations of rat brain with antisera specific for individual gamma-subunits and analysing their radioligand binding characteristics. Receptors containing a gamma1-subunit do not bind benzodiazepine radioligands with high affinity. Those containing either a gamma2 or gamma3 subunit bind [3H]flumazenil with high affinity. Some compounds compete for these binding sites with multiple affinities, reflecting the presence of populations of receptors containing several different types of alpha-subunit. Photoaffinity-labelling of GABA-A receptors from a cell line stably expressing GABA-A receptors of composition alpha1beta3gamma2 followed by immunoprecipitation of individual subunits revealed that the alpha and gamma but not the beta-subunit could be irreversibly labelled by [3H]flunitrazepam. The properties of recombinant receptors have been investigated in oocytes expressing gamma1, gamma2, or gamma3 subunits in combination with an alpha and a beta-subunit. Some compounds such as zolpidem, DMCM and flunitrazepam show selectivity for receptors containing different gamma-subunits. Others such as CL 218,872 show no selectivity between receptors containing different gamma-subunits but exhibit selectivity for receptors containing different alpha-subunits. These data taken together suggest that the benzodiazepine site of the GABA-A receptor is formed with contributions from both the alpha and gamma-subunits.

GABAA receptors composed of human alpha 1 beta 2 gamma 2L, alpha 1 beta 2 gamma 2S, alpha 1 beta 3 gamma 2S, alpha 6 beta 3 gamma 2S, and alpha 5 beta 3 gamma 3 subunits as well as bovine alpha 1 beta 1 gamma 2L and alpha 1 beta 1 subunits were stably expressed in mammalian L(tk-) cells and transiently expressed in Xenopus oocytes. Effects of muscimol, ethanol, flunitrazepam, and pentobarbital on receptor function were compared for the two expression systems using a 36Cl- flux assay for cells and an electrophysiological assay for oocytes. Muscimol activated all receptors in both expression systems but was more potent for L(tk-) cells than oocytes; this difference ranged from 2.6-to 26-fold, depending upon subunit composition. The most pronounced differences between receptors and expression systems were found for ethanol. In L(tk-) cells, low (5-50 mM) concentrations of ethanol potentiated muscimol responses only with receptors containing the gamma 2L subunit. In oocytes, concentrations of 30-100 mM produced small enhancements for most subunit combinations. Flunitrazepam enhanced muscimol responses for all receptors except alpha 6 beta 3 gamma 2S and alpha 1 beta 1, and this enhancement was similar for both expression systems. Pentobarbital also enhanced muscimol responses for all receptors, and this enhancement was similar for L(tk-) cells and oocytes, except for alpha 6 beta 3 gamma 2S where the pentobarbital enhancement was much greater in oocytes than cells. The alpha 6 beta 3 gamma 2S receptors were also distinct in that pentobarbital produced direct activation of chloride channels in both expression systems. Thus, the type of expression/assay system markedly affects the actions of ethanol on GABAA receptors and also influences the actions of muscimol and pentobarbital on this receptor. Differences between these expression systems may reflect posttranslational modifications of receptor subunits.

GPCRs are one of the most popular classes of therapeutic drug targets. It is therefore important to design specific assay formats to readily identify ligands at these receptors. CypHer 5 technology utilizes the general ability of GPCRs to be internalized into the endosomal pathway of a cell in response to agonist ligands. The CypHer 5 dye is fluorescent in acidic environments, but nonfluorescent at neutral pH. When CypHer 5 is bound to a receptor on the extracellular surface of the cell, it is essentially nonfluorescent. On internalization into a cell, it displays a significant increase in fluorescence. Here we demonstrate the detection of agonist activation of two GPCRs in stably transfected live cells using CypHer 5 technology. The G(q)-coupled TRHR-1 and the G(s)-coupled beta(2)-adrenoceptor were both N-terminally tagged with VSV-G. Following addition of CypHer 5-labeled anti-VSV-G antibodies to HEK 293 cells stably expressing the beta(2)-adrenoceptor or CHO-K1 cells stably expressing the TRHR-1, the cells were treated with agonists and then imaged on Amersham Biosciences' IN Cell Analyzer 3000. Data were quantified using a granularity analysis module. Concentration-response curves were obtained with signal-to-background ratios of 7:1 for both receptors. An EC(50) of 0.52 nM was observed on TRH stimulation of the TRHR-1, and an EC(50) of 30 nM was obtained on isoprenaline stimulation of the beta(2)-adrenoceptor. These results demonstrated that the CypHer technology was capable of measuring high-potency agonist responses. The beta(2)-adrenoceptor antagonist, alprenolol, competed for isoprenaline with an IC(50) of 30 nM, indicating that a high-potency antagonist inhibition curve could also be observed using CypHer. CypHer 5 provides a generic tool to measure GPCR activation in a live cell, homogeneous assay format, and may be equally suitable for detecting activation of other classes of cell surface receptors.