Loss of the major GABA(A) receptor subtype in the brain is not lethal in mice.

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.

Identification of amino acid residues responsible for the alpha5 subunit binding selectivity of L-655,708, a benzodiazepine binding site ligand at the GABA(A) receptor.

L-655,708 is a ligand for the benzodiazepine site of the gamma-aminobutyric acid type A (GABA(A)) receptor that exhibits a 100-fold higher affinity for alpha5-containing receptors compared with alpha1-containing receptors. Molecular biology approaches have been used to determine which residues in the alpha5 subunit are responsible for this selectivity. Two amino acids have been identified, alpha5Thr208 and alpha5Ile215, each of which individually confer approximately 10-fold binding selectivity for the ligand and which together account for the 100-fold higher affinity of this ligand at alpha5-containing receptors. L-655,708 is a partial inverse agonist at the GABA(A) receptor which exhibited no functional selectivity between alpha1- and alpha5-containing receptors and showed no change in efficacy at receptors containing alpha1 subunits where amino acids at both of the sites had been altered to their alpha5 counterparts (alpha1Ser205-Thr,Val212-Ile). In addition to determining the binding selectivity of L-655,708, these amino acid residues also influence the binding affinities 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 first transmembrane domain of the receptor alpha subunit that contributes to this binding site.

Pharmacology of recombinant human GABA(A) receptor subtypes measured using a novel pH-based high-throughput functional efficacy assay.

To facilitate the discovery of novel compounds that modulate human GABA(A) receptor function, we have developed a high throughput functional assay using a fluorescence imaging system. L(tk-) cells expressing combinations of human GABA(A) receptor subunits were incubated with the pH-sensitive dye 2',7'bis-(2-carboxyethyl)-5-(and 6)-carboxyfluorescein, then washed and placed in a 96-well real-time fluorescence plate reader. In buffer adjusted to pH 6.9 there was a robust and persisting acidification response to addition of GABA, which was antagonised by the GABA(A) receptor antagonist bicuculline. The concentration-response relationship for GABA was modulated by allosteric ligands, including benzodiazepine (BZ) site agonists and inverse agonists. The effects of BZ site ligands on the pH response to GABA for receptors containing alpha1beta3gamma2, alpha3beta3gamma2 or alpha5beta3gamma2 subunits were well correlated with results from electrophysiological studies on the same receptor subunit combinations expressed in Xenopus oocytes. Most modulatory compounds tested were found to be relatively unselective across the three subunit combinations tested; however, some showed subtype-dependent efficacy, such as diazepam, which had highest agonist effects on the alpha3beta3gamma2 subtype, substantial but lesser agonism on alpha1beta3gamma2 and still substantial but the least agonism on alpha5beta3gamma2. This indicates that the alpha subunit within the recombinant receptor expressed in L(tk-) cells can affect the efficacy of the response to some BZ compounds. Inhibitors of Na(+)/Cl(-) cotransport, anion/anion exchange and the gastric type of H(+)/K(+) ATPase potently inhibited GABA-evoked acidification, indicating that multiple transporters are involved in the GABA-evoked pH change. This novel fluorescence-based high throughput functional assay allows the rapid characterization of allosteric ligands acting on human GABA(A) receptors.

Alpha subunit isoform influences GABA(A) receptor modulation by propofol.

We have investigated the role of the alpha subunit in the modulation of gamma-aminobutyric acid type A (GABA(A)) receptors by the general anesthetic propofol, using whole-cell patch clamp recordings made from distinct stable fibroblast cell lines which expressed only alpha1beta3gamma2 or alpha6beta3gamma2 GABA(A) receptors. At clinically relevant anesthetic concentrations, propofol potentiated submaximal GABA currents in alpha1beta3gamma2 receptors to a far greater degree than those in alpha6beta3gamma2 receptors. The alpha subunit influenced the efficacy of propofol for modulation, but not its potency. In contrast, direct gating of the ion channel by propofol, in the absence of GABA, was significantly larger in the alpha6 than the alpha1 containing receptors. The potentiation of submaximal GABA by trichloroethanol, and the potentiation and direct gating by methohexital was also studied, and showed the same relative trends as propofol.

Microtubule depolymerization inhibits ethanol-induced enhancement of GABAA responses in stably transfected cells.

We studied whether microtubule organization is important for actions of ethanol on GABAA ergic responses by testing the effects of microtubule depolymerization on ethanol enhancement of GABA action in mouse L(tk-) cells stably transfected with GABAA receptor alpha 1 beta 1 gamma 2L subunits. The microtubule-disrupting agents colchicine, taxol, and vinblastine completely blocked ethanol-induced enhancement of muscimol-stimulated chloride uptake. beta-Lumicolchicine, a colchicine analogue that does not disrupt microtubules, had no effect on ethanol action. Colchicin did not alter the potentiating actions of flunitrazeparn or pentobarbital on muscimol-stimulated chloride uptakes. Thus, colchicine specifically inhibited the potentiating action of ethanol. From these findings, we conclude that intact microtubules are required for ethanol-induced enhancement of GABAA responses and suggest that a mechanism involving microtubules produces posttranslational modifications that are necessary for ethanol sensitivity in this cell system.

Cloning of cDNAs encoding the human gamma-aminobutyric acid type A receptor alpha 6 subunit and characterization of the pharmacology of alpha 6-containing receptors.

A cDNA encoding the human gamma-aminobutyric acidA (GABAA) receptor alpha 6 subunit has been cloned and sequenced. The deduced amino acid sequence of this cDNA shows 91.4% identity with the published rat alpha 6 subunit. In situ hybridization histochemistry reveals the alpha 6 mRNA to be located within the granule cell layer of the human cerebellar cortex. Recombinant human alpha 6 beta gamma 2S GABAA receptors have been expressed in both stably transfected cells and Xenopus oocytes, and the pharmacology of the benzodiazepine binding site has been determined. The recombinant receptor has a diazepam-insensitive pharmacology, with negligible affinity for a number of classic benzodiazepines. A number of compounds that bind to the benzodiazepine site potentiated the GABA response of alpha 6 beta 2 gamma 2 receptors. Most importantly, the classic benzodiazepine antagonist ethyl-8-fluoro-5,6-dihydro-5-methyl-6-oxo-4H-imidazo[1,5-a] [1,4]benzodiazepine-3-carboxylate (Ro 15-1788) and the partial inverse agonist ethyl-8-azido-5,6-dihydro-5-methyl-6-oxo-4H-imidazo[1,5-a] [1,4]benzodiazepine-3-carboxylate (Ro 15-4513) both acted as agonists at the alpha 6 containing receptor. This observation demonstrates definitively that efficacy of benzodiazepine compounds cannot be generalized across receptor subtypes and may also help explain some of the behavioral effects that have been reported for these compounds.

Identification of domains in human recombinant GABAA receptors that are photoaffinity labelled by [3H]flunitrazepam and [3H]Ro15-4513.

We have used [3H]flunitrazepam and [3H]Ro15-4513 as photoaffinity labelling agents in combination with a chemical cleavage technique to localize the benzodiazepine recognition sites of specific human recombinant alpha 1 beta 1 gamma 2, alpha 1 beta 3 gamma 2 and alpha 6 beta 3 gamma 2 GABAA receptor subtypes. The chemical agent utilized was hydroxylamine, whose substrate is a relatively rare asparagine-glycine amide bond that occurs only in the alpha subunits of the receptors examined in this study. Cleavage products were resolved using sodium dodecyl sulphate-polyacrylamide gel electrophoresis (SDS-PAGE). The results of these experiments show that, in the alpha 1 subunit-containing receptors, incorporation of [3H]flunitrazepam occurs within residues 1-103 of the alpha 1 subunit, while incorporation of [3H]Ro15-4513 occurs within the region of the alpha 1 subunit that lies between residue 104 and the C-terminus. Photolabelling of membranes prepared from the alpha 6 beta 3 gamma 2-expressing cell line with [3H]Ro15-4513 resulted in the incorporation of radiolabel into two major protein species of M(r) 56,000 and M(r) 48,000, indicating incorporation into the alpha 6 subunit and possibly also the gamma 2 subunit. Hydroxylamine cleavage of alpha 6-containing receptors labelled with [3H]Ro15-4513 produced a gel profile consistent with the incorporation of the label occurring between residue 125 and the C-terminal. Thus, we have shown that the recognition sites for the agonist [3H]flunitrazepam and the inverse agonist [3H]Ro15-4513 occur within distinct domains of the human GABAA receptor.

Expression and pharmacology of human GABAA receptors containing gamma 3 subunits.

A cDNA encoding the gamma 3 subunit of the human GABAA receptor has been obtained by molecular cloning. Its deduced amino acid sequence shows a high level of sequence identity with the published mouse and rat sequences (96%). The ligand binding pharmacology of the benzodiazepine site formed by stably-expressed human alpha 5 beta 3 gamma 2S and alpha 5 beta 3 gamma 3 GABAA receptor subtypes have been compared for a number of ligands, Benzodiazepine site ligands were found to be either non-selective or gamma 2-selective, with the exception of CL218,872, which was found to be 10-fold selective for the alpha 5 beta 3 gamma 3-containing subtype Two benzodiazepine site ligands. Ro15-4513 and FG8205 were more efficacious at alpha 5 beta 3 gamma 3 receptors than alpha 5 beta 3 gamma 2 receptors expressed in Xenopus oocytes, CL218,872, which is a partial agonist at alpha 1 containing receptors, had no intrinsic activity at either alpha 5 beta 3 gamma 2 or alpha 5 beta 3 gamma 3, alpha 1 beta 2 gamma 2S and alpha 1 beta 2 gamma 3 human GABAA receptors were also expressed in Xenopus oocytes and their benzodiazepine pharmacology investigated. Both the EC50 and efficacy of benzodiazepine site ligands were influenced by the type of gamma subunit coexpressed with alpha 1 and beta 2.

Regulation of allosteric coupling and function of stably expressed gamma-aminobutyric acid (GABA)A receptors by chronic treatment with GABAA and benzodiazepine agonists.

Chronic treatments with drugs that stimulate or potentiate gamma-aminobutyric acid (GABA)A receptor function cause uncoupling of allosteric sites and downregulation of the GABAA receptors expressed in neurons. To study these effects on receptors having a defined subunit composition, we treated stably transfected mouse Ltk- cells (PA3 cells) with drugs known to uncouple GABAA receptors. Because dexamethasone controls the expression of bovine alpha-1, beta-1 and gamma 2L GABAA receptor subunit genes in PA3 cells, this expression system provides a way to study receptors in the absence of neuronal subunit gene promoters. We assayed binding of [3H]flunitrazepam to measure allosteric coupling and uptake of 36Cl- to measure GABAA receptor function. Chronic (4 day) treatment of PA3 cells with muscimol, GABA or flunitrazepam reduced the GABA enhancement of binding of [3H]flunitrazepam to PA3 cells. Chronic flurazepam or muscimol treatments also caused downregulation of benzodiazepine potentiation of muscimol-stimulated 36Cl- uptake. Chronic treatment with muscimol did not affect levels of subunit mRNAS and alpha 1- or beta 1-subunit protein of GABAA receptors and chronic flunitrazepam did not affect subunit mRNAs or alpha 1 protein. We conclude that chronic drug treatments regulate allosteric coupling and function of GABAA receptors in stably transfected cells and this regulation cannot be attributed to decreases in the expression of receptor subunits or to expression of subunits other than alpha 1 beta 1 or gamma 2L.

The pharmacology of the benzodiazepine site of the GABA-A receptor is dependent on the type of gamma-subunit present.

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.

Role of the beta subunit in determining the pharmacology of human gamma-aminobutyric acid type A receptors.

A cDNA encoding the human gamma-aminobutyric acid (GABA)A receptor beta 2 subunit has been cloned and sequenced. The deduced amino acid sequence of this cDNA shows only a single amino acid change from the rat sequence (Asn-347 in rat, serine in human). Using polymerase chain reaction amplification of human-specific products from human x rodent somatic cell hybrid DNAs, the gene has been assigned to human chromosome 6. By expressing recombinant human GABAA receptors containing different beta subunits (beta 1, beta 2 or beta 3) in both transfected cells and Xenopus oocytes, we have been able to determine the influence of the beta subunit on the pharmacology of the receptor. For a number of benzodiazepine binding site compounds, a barbiturate, and several neurosteroids, neither the affinity nor the efficacy of the compounds is influenced by the type of beta subunit present in the receptor molecule. These data suggest that the beta subunit does not significantly influence the benzodiazepine, barbiturate, or steriod site pharmacologies of human GABAA receptor subtypes.

Cloning of cDNA sequences encoding human alpha 2 and alpha 3 gamma-aminobutyric acidA receptor subunits and characterization of the benzodiazepine pharmacology of recombinant alpha 1-, alpha 2-, alpha 3-, and alpha 5-containing human gamma-aminobutyric acidA receptors.

cDNAs encoding human alpha 2 and alpha 3 gamma-aminobutyric acidA receptor subunits have been cloned. Their deduced amino acid sequences show much sequence identity with the published bovine sequences (98.2% and 97.0% for alpha 2 and alpha 3, respectively). Human alpha 1 beta 1 gamma 2, alpha 2 beta 1 gamma 2, alpha 3 beta 1 gamma 2, and alpha 5 beta 1 gamma 2 subunit combinations were expressed in transiently transfected cells and their pharmacologies were characterized using a series of benzodiazepine (BZ) binding site ligands. Human alpha 1-containing receptors exhibited a BZ1-type pharmacology, and alpha 2-, alpha 3-, and alpha 5-containing receptors exhibited a broadly BZ2-type pharmacology. The partial inverse agonist Ro15-4513 showed an approximately 10-15-fold higher affinity for alpha 5-containing than for alpha 1-, alpha 2-, or alpha 3-containing receptors and is thus the first compound shown to have a significantly higher affinity for another receptor subtype than for alpha 1 beta 1 gamma 2.

The influence of the gamma 2L subunit on the modulation of responses to GABAA receptor activation.

1. Whole-cell patch clamp recordings were made from L-cells transfected with 2 combinations of subunits of the GABAA receptor. Log concentration-response curves were constructed to gamma-aminobutyric acid (GABA) on alpha 1,beta 1,gamma 2L containing cells and compared to those from alpha 1,beta 1 containing cells. The effects of flunitrazepam, pentobarbitone and alphaxalone on the concentration-response relationships were also examined. 2. From the log concentration-response curves, GABA had a mean (+/- s.e. mean) pEC50 = 5.2 +/- 0.09 and slope factor = 1.7 +/- 0.02 on alpha 1,beta 1,gamma 2L cells which were significantly different from the values obtained from alpha 1,beta 1 cells where the pEC50 = 5.6 +/- 0.02 and the slope = 1.5 +/- 0.02. 3. Flunitrazepam produced a parallel leftward shift of GABA concentration-response curves on alpha 1,beta 1,gamma 2L cells. The EC50 for flunitrazepam = 6.3 +/- 2.7 nM. No increase in the maxima of the GABA concentration-response curves was found in the presence of flunitrazepam. Flunitrazepam did not potentiate responses from alpha 1,beta 1 cells. 4. The log concentration-response curves from both populations of cells were shifted to the left by equal amounts by pentobarbitone. A significant increase in the maximal response to GABA was also produced by pentobarbitone. This occurred at lower concentrations of pentobarbitone on alpha 1,beta 1 cells. 5. Alphaxalone produced leftward shifts of GABA log concentration-response curves of similar magnitudes in both populations of cells. Significant increases in the maxima were found at 100 nM in alpha 1, beta 1 cells but not up to 1 microM in alpha 1,beta 1,gamma 2L cells.6. These results provide further evidence of the modulatory role of the gamma 2L subunit of the GABAA receptor containing alpha 1 and beta 2 subunits. As well as influencing the apparent affinity of GABA and conferring benzodiazepine modulation, it also appeared to regulate the increase in maximal response produced in the presence of barbiturates and steroids. This latter effect may imply that barbiturates and steroids increase the channel open-state probability in the presence of GABA and that this effect is diminished by the presence of the gamma 2L subunit.

The pharmacology of recombinant GABAA receptors containing bovine alpha 1, beta 1, gamma 2L sub-units stably transfected into mouse fibroblast L-cells.

1. Responses to gamma-aminobutyric acid (GABA) were evoked in mouse fibroblast L-cells stably transfected with bovine, alpha 1, beta 1, gamma 2L sub-units of the GABAA receptor. Expression was stimulated via a steroid-inducible promoter system. 2. In near symmetrical intracellular and extracellular chloride concentrations, GABA evoked inward currents at negative holding potentials that reversed at +5 mV and displayed slight outward rectification. Concentration-response curves were fitted well by the logistic equation. GABA had a pEC50 = 5.1 +/- 0.1 and the curves had a slope of 1.9 +/- 0.1. 3. Responses to GABA were antagonized by bicuculline, picrotoxin and penicillin. The action of bicuculline was competitive (pA2 = 6.4) whilst the block by picrotoxin was uncompetitive and strongly agonist-dependent. 4. Benzodiazepine receptor agonists potentiated responses to 3 microM GABA. The rank order of potency was FG 8205 > flunitrazepam > zolpidem > C1218872. FG 8205 and C1218872 produced markedly lower maximal potentiations with efficacies 0.4 and 0.6 x that of flunitrazepam, respectively. The potencies of zolpidem and C1218872 observed are in agreement with the BZ1 type pharmacology of this sub-unit combination. The potentiation of GABA by flunitrazepam was antagonized by flumazenil with a Ki of 3.8 nM. 5. GABA responses were potentiated in the presence of pentobarbitone and alphaxalone. The response was also noticeably broadened by these compounds due to a decrease in the response decay rate. Concentrations of pentobarbitone of 100 microM and above evoked an inward current in the absence of GABA. Alphaxalone up to 10 microM did not evoke a direct response. 6. This expression system produced functional receptors that behaved in a fashion analogous to those found endogenously in other preparations. Thus, this system appears to provide a useful and versatile preparation for the analysis of sub-unit regulation of GABAA receptor pharmacology.

Stable expression of mammalian type A gamma-aminobutyric acid receptors in mouse cells: demonstration of functional assembly of benzodiazepine-responsive sites.

The differential sensitivity of type A gamma-aminobutyric acid (GABAA) receptors to benzodiazepine ligands seen in the mammalian 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 (alpha 1-6, beta 1-3, gamma 1-3, and delta) encoded by discrete genes. The characteristics of a given subtype can be determined by the coexpression of cloned cDNAs encoding the subunits of interest. 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 alpha 1 beta 1 gamma 2L GABAA receptor subtype. Using both immunoprecipitation by subunit-specific antisera and gel-exclusion chromatography, we have shown that the alpha 1, beta 1, and gamma 2L subunits coassemble to form receptor macromolecules that are of the same size as native GABAA receptors. Additionally, the recombinant receptors have the same benzodiazepine pharmacology as native alpha 1-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 of GABAA receptors and in the development of subtype-specific drugs.