Rat and human hippocampal alpha5 subunit-containing gamma-aminobutyric AcidA receptors have alpha5 beta3 gamma2 pharmacological characteristics

The gamma-aminobutyric acid (GABA)A receptor is a hetero-oligomer consisting of five subunits, the combination of which confers unique pharmacological properties to the receptor. To understand the physiological role of native GABAA receptors, it is critical to determine their subunit compositions. The pharmacological characteristics of human alpha5 beta3 gamma2 and alpha5beta3gamma3 GABAA receptors stably expressed in L(tk-) cells were characterized with the alpha5-selective ligand [3H]L-655,708 and compared with the pharmacological characteristics of [3H]L-655,708 binding sites from rat and human hippocampus. Saturation analyses revealed a 9-fold selective affinity of [3H]L-655,708 for alpha5 beta3 gamma2 receptors (Kd = 1.7 +/- 0.4 nM), compared with alpha5 beta3 gamma3 receptors (Kd = 15 +/- 3 nM). Rat and human hippocampal [3H]L-655,708 binding sites had affinities of 2.2 +/- 0.6 and 1.0 +/- 0.2 nM, respectively, comparable to the affinity of alpha5 beta3 gamma2 receptors. Pharmacological analysis of [3H]L-655,708 binding sites in rat and human hippocampi revealed a strong correlation with the affinities of seven benzodiazepine site ligands for alpha5 beta3 gamma2 but not alpha5 beta3 gamma3 receptors. Immunoprecipitation of [3H]L-655,708 binding sites from rat hippocampus with a gamma2-selective antibody yielded 19 +/- 4% of total benzodiazepine binding sites measured using [3H]Ro15-1788, whereas no specific binding was measured after immunoprecipitation with an anti-gamma3 antibody. Combinatorial immunoprecipitations of [3H]muscimol binding sites with anti-alpha5 and anti-gamma2 or anti-alpha5 and anti-gamma3 antibodies established the preferential expression of alpha5 gamma2 receptors, accounting for 22 +/- 2% of total rat hippocampal GABAA receptors. These observations provide pharmacological and structural evidence for the prevalence of alpha5 beta3 gamma2 GABAA receptors in rat hippocampus, despite the clustering of alpha5 and gamma3 loci on the same chromosome.     

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Ligands of the benzodiazepine binding site allosterically modulate gamma-aminobutyric acidA receptors. Their binding pocket is made up of amino acid residues located on both alpha and gamma subunits. We transiently expressed wild-type alpha1beta2gamma2 and mutant GABAA receptors in human embryonic kidney 293 cells and determined their binding properties. Receptors containing the mutant alphaY209A showed approximately 40-fold decrease in affinity for [3H]Ro 15-1788 and diazepam, whereas zolpidem displayed no measurable affinity. Receptors containing the mutant alphaY209F showed a small-to-moderate decrease in affinity for [3H]Ro 15-1788, diazepam, zolpidem, methyl-6,7-dimethoxy-4-ethyl-beta-carboline-3-carboxylate, and Cl 218872, amounting to 2-8-fold. Receptors containing the mutant alphaY209Q appeared in the surface membrane of transfected cells, bound [3H]muscimol with wild-type affinity, but failed to bind [3H]Ro 15-1788 or [3H]flunitrazepam with detectable affinity. If these mutant receptors were expressed in Xenopus laevis oocytes, the apparent affinity for GABA was only slightly decreased, whereas the ability of the currents to be stimulated by low concentrations of flunitrazepam was abolished. Receptors containing a point mutant of another amino acid residue, alphaT206A, surprisingly showed an increase in affinity of 5- and 16-fold, for the negative allosteric modulator methyl-6,7-dimethoxy-4-ethyl-beta-carboline-3-carboxylate and the partial positive allosteric modulator Cl 218872, respectively, whereas there was only a small decrease in affinity for Ro 15-1788, diazepam, and zolpidem, amounting to 2-, 4-, and 5-fold. Both alpha206 and alpha209 are thus both important in determining the binding affinities for ligands of the benzodiazepine binding site. The residues are spaced at an interval of three amino acids and may be part of an alpha helix.     

Pharmacological modulation of the diazepam-insensitive recombinant gamma-aminobutyric acidA receptors alpha 4 beta 2 gamma 2 and alpha 6 beta 2 gamma 2

We characterized modulation of the gamma-aminobutyric acid (GABA)-evoked responses of the diazepam-insensitive alpha 4 beta 2 gamma2 and alpha 6 beta 2 gamma 2 recombinant GABAA receptors. The partial agonist bretazenil potentiated the responses of both receptors with similar dose dependence but with a higher maximal enhancement at the alpha 4 beta 2 gamma 2 receptor. The bretazenil-induced potentiation was reduced by the benzodiazepine antagonist flumazenil. At a high concentration (10 microM), flumazenil was a weak potentiator of the GABA response. The partial agonist imidazenil was inactive. The imidazobenzodiazepine inverse agonist Ro 15-4513, which is known to bind with high affinity to the alpha 6 beta 2 gamma 2 receptor, potentiated the GABA responses of the alpha 4 beta 2 gamma 2 and alpha 6 beta 2 gamma 2 receptor subtypes with similar dose dependence over the concentration range of 0.1-10 microM. Methyl-6, 7-dimethoxy-4-ethyl-beta-carboline, a beta-carboline inverse agonist, had a similar potentiating effect when tested at a concentration of 10 microM. The alpha 4 beta 2 gamma 2 and alpha 6 beta 2 gamma 2 receptor-mediated currents had equal sensitivities to furosemide and Zn2+ ions, both of which reduced the GABA-evoked responses. The alpha 6 beta 2 gamma 2 receptor but not the alpha 4 beta 2 gamma 2 receptor exhibited a low level of spontaneous activity in the absence of GABA; this resting current could be directly potentiated by Ro 15-4513, methyl-6,7-dimethoxy-4-ethyl-beta-carboline, bretazenil and flumazenil and was blocked by picrotoxin. Thus, although the alpha 4 beta 2 gamma 2 receptors are insensitive to benzodiazepine binding site full agonists, such as diazepam, they can be modulated by certain ligands acting as partial and inverse agonists at diazepam-sensitive receptors and thereby contribute to the respective pharmacological profiles.     

Key amino acids in the gamma subunit of the gamma-aminobutyric acidA receptor that determine ligand binding and modulation at the benzodiazepine site

Pharmacological analyses of gamma-aminobutyric acidA (GABAA) receptor subtypes have suggested that both the alpha and gamma subunits, but not the beta subunit, contribute to the benzodiazepine binding site. We took advantage of the different pharmacological properties conferred by the inclusion of different gamma subunits in the receptor macromolecule to identify amino acids gamma2Phe77 and gamma2Met130 as key determinants of the benzodiazepine binding site. gamma2Phe77 was required for high affinity binding of the benzodiazepine site ligands flumazenil, CL218,872, and methyl-beta-carboline-3-carboxylate but not flunitrazepam. This amino acid was, however, required for allosteric modulation by flunitrazepam, as well as other benzodiazepine site ligands. In contrast, gamma2Met130 was required for high affinity binding of flunitrazepam, clonazepam, and triazolam but not flumazenil, CL218, 872, or methyl-beta-carboline-3-carboxylate and did not affect benzodiazepine efficacy. Introduction of the phenylalanine and methionine into the appropriate positions of gamma1 was not sufficient to confer high affinity for the benzodiazepine site ligand zolpidem. These data show that gamma2Phe77 and gamma2Met130 are necessary for high affinity binding of a number of benzodiazepine site ligands. Although most previous studies have focused on the contribution of the alpha subunit, we demonstrated a critical role for the gamma subunit at the benzodiazepine binding site, indicating that this modulatory site is located at the interface of these two subunits. Furthermore, gamma2Phe77 is homologous to alpha1Phe64, which has been previously shown to be a key determinant of the GABA binding site, suggesting a conservation of motifs between different ligand binding sites on the GABAA receptor.     

Direct activation of GABAA receptors by loreclezole, an anticonvulsant drug with selectivity for the beta-subunit

Loreclezole, an anticonvulsant and antiepileptic compound, potentiates gamma-aminobutyric acid (GABA) type A receptor function, by interacting with a specific allosteric modulatory site on receptor beta-subunits. A similar selectivity for GABAA receptor beta-subunits is apparent for the direct activation of receptor-operated Cl- channels, by the general anesthetics propofol and pentobarbital. The ability of loreclezole to activate GABAA receptors directly has now been compared, biochemically and electrophysiologically, with that of propofol. In well-washed rat cortical membranes (devoid of endogenous GABA), loreclezole and propofol increased t-[35S]butylbicyclophosphorothionate ([35S]TBPS) binding by up to 28% (at 5 microM) and 80% (at 10 microM), respectively. Higher concentrations (50-100 microM) of both compounds inhibited [35S]TBPS binding with great efficacy, an effect mimicked by GABA. In contrast, the benzodiazepine diazepam increased [35S]TBPS binding, but failed to inhibit this parameter, even at high concentrations. At concentrations of 50-100 microM, loreclezole induced inward Cl- currents in the absence of GABA, in Xenopus oocytes expressing human recombinant GABAA receptors, comprised of alpha 1-, beta 2- and gamma 2S-subunits. At 100 microM, the current evoked by loreclezole was 26% of that induced by 5 microM GABA. The current evoked by 100 microM propofol was 98% of that induced by 5 microM GABA. Currents induced by loreclezole, like those evoked by propofol, were potentiated by diazepam in a flumazenil-sensitive manner and blocked by either bicuculline or picrotoxin. These data suggest that loreclezole shares, with propofol, an agonistic action at GABAA receptors containing the beta 2-subunit and that the different efficacies of the two compounds in this regard, may underlie the difference in their pharmacological profiles. The failure of loreclezole to activate GABAA receptors containing the beta 1-subunit may be responsible for its lack of hypnotic effect.     

Amino acid residue 200 on the alpha1 subunit of GABA(A) receptors affects the interaction with selected benzodiazepine binding site ligands

Mutant alph1 subunits of the GABA(A) receptor were coexpressed in combination with the wild-type beta2 and gamma2 subunits in human embryonic kidney (HEK) 293 cells. The binding properties of various benzodiazepine site ligands were determined by displacement of ethyl-8-fluoro-5,6-dihydro-5-methyl-6-oxo-4H-imidazo[1,5a]-[1,4]benzodia zepine-3-carboxylate ([3H]Ro 15-1788). The mutation G200E led to a decrease in zolpidem and 3-methyl-6-[3-(trifluoromethyl)phenyl]-1,2,4-triazolo[4,3-b]pyridazine (CL 218872) affinity amounting to 16- and 8-fold. Receptors containing a conservative T206V substitution showed a 41- and 38-fold increase in methyl-6,7-dimethoxy-4-ethyl-beta-carboline-3-carboxylate (DMCM) and CL 218872 affinity combined with a decrease in diazepam and zolpidem affinity, amounting to 7- and 10-fold. Two mutations, Q203A and Q203S showed almost no effects on the binding of benzodiazepine site ligands, indicating that this residue is not involved in the binding of benzodiazepines and related compounds.     

SB-205384: a GABA(A) receptor modulator with novel mechanism of action that shows subunit selectivity

1. SB-205384, and its (+) enantiomer (+)-SB-205384 were tested for their modulatory effects on human GABA(A) receptor subunit combinations expressed in Xenopus oocytes by electrophysiological methods. 2. The slowing of the decay rate induced by SB-205384 on native GABA-activated currents in rat neurones was also seen on GABA(A) currents in oocytes expressing human GABA(A) subunits. This temporal effect was observed for the alpha3beta2gamma2 subunit combination with little effect in subunit combinations containing either alpha1 or alpha2. 3. Potentiation of the peak amplitude of the GABA-activated currents by SB-205384 or (+)-SB-205384 was less specific for a particular subunit combination, although the greatest effect at 10 microM drug was seen on the alpha3beta2gamma2 subunit combination. 4. In contrast, zolpidem, a benzodiazepine site modulator, did not significantly slow decay rates of GABA(A) currents in oocytes expressing the alpha3beta2gamma2 subunit combination. Zolpidem, as expected, did selectively potentiate GABA-activated currents on oocytes expressing the gamma2 subunit compared to those containing the gamma1. 5. The results show that the novel kinetic modulatory profile of SB-205384 is selective for the alpha3beta2gamma2 subunit combination. This suggests that the compound is binding to a novel regulatory site on the subunit complex.     

L-lysine is a barbiturate-like anticonvulsant and modulator of the benzodiazepine receptor

Our earlier observations showed that L-lysine enhanced the activity of diazepam against seizures induced by pentylenetetrazol (PTZ), and increased the affinity of benzodiazepine receptor binding in a manner additive to that caused by gamma-aminobutyric acid (GABA). The present paper provides additional evidence to show that L-lysine has central nervous system depressant-like characteristics. L-lysine enhanced [3H]flunitrazepam (FTZ) binding in brain membranes was dose-dependent and stimulated by chloride, bromide and iodide, but not fluoride. Enhancement of [3H]FTZ binding by L-lysine at a fixed concentration was increased by GABA but inhibited by pentobarbital between 10(-7) to 10(-3)M. While GABA enhancement of [3H]FTZ binding was inhibited by the GABA mimetics imidazole acetic acid and tetrahydroisoxazol pyridinol, the enhancement by pentobarbital and L-lysine of [3H]FTZ binding was dose-dependently increased by these two GABA mimetics. The above results suggest that L-lysine and pentobarbital acted at the same site of the GABA/benzodiazepine receptor complex which was different from the GABA binding site. The benzodiazepine receptor antagonist imidazodiazepine Ro15-1788 blocked the antiseizure activity of diazepam against PTZ. Similar to pentobarbital, the anti-PTZ effect of L-lysine was not blocked by Ro15-1788. Picrotoxinin and the GABA, receptor antagonist bicuculline partially inhibited L-lysine's enhancement of [3H]FTZ binding with the IC50s of 2 microM and 0.1 microM, respectively. The convulsant benzodiazepine Ro5-3663 dose-dependently inhibited the enhancement of [3H]FTZ binding by L-lysine. This article shows the basic amino acid L-lysine to have a central nervous system depressant characteristics with an anti-PTZ seizure activity and an enhancement of [3H]FTZ binding similar to that of barbiturates but different from GABA.     

Comparative autoradiographic distribution of central omega (benzodiazepine) modulatory site subtypes with high, intermediate and low affinity for zolpidem and alpidem

Pharmacological characterization of [3H]benzodiazepine binding to membrane preparations of adult rat hippocampus and neonatal rat brain have demonstrated, in addition to the omega 1 and omega 2 populations of central omega benzodiazepine binding sites associated with GABAA receptors, the existence of binding sites with microM affinity for the imidazopyridines zolpidem and alpidem. In the present study we have investigated their comparative autoradiographic distribution using [3H]flumazenil as a ligand. In the neonatal rat CNS, the imidazopyridine derivatives zolpidem and alpidem were found to discriminate two [3H]flumazenil binding site populations with an IC50 value ratio of more than 200-fold. In the different regions investigated (spinal cord, striatum, neocortex and inferior colliculus) the low affinity component had IC50 values of 20-40 microM (zolpidem) and 5-15 microM (alpidem) and accounted for ca. 50% of the total binding site population. In the adult rat, these imidazopyridine derivatives displayed a greater displacing potency in the cerebellum (IC50 = 6 and 36 nM, respectively) than in the hippocampus (IC50 = 37 and 403 nM, respectively). In the cerebellum, [3H]flumazenil binding was fully displaced by 1 microM of either compound and Hill coefficients of displacement curves were close to unity. In the hippocampus, 25% of [3H]flumazenil binding were resistant to 3 microM zolpidem or 1 microM alpidem, but were displaced by 100 microM of either compound. CL 218,872 also displayed a greater displacing potency in the cerebellum (IC50 = 83 nM) than in the hippocampus (IC50 = 711 nM) but [3H]flumazenil binding in the hippocampus was fully displaced by 10 microM of this compound. In adult rat hippocampus, zolpidem and alpidem were found to discriminate between three central omega site subtypes which display high (IC50 = 31 and 6.1 nM, for these imidazopyridine derivatives. In contrast, CL 218,872 discriminated between omega 1 and omega 2 sites but not between two omega 2 receptor subpopulations. omega 1 sites were mainly localized in layer IV of the sensorimotor cortex, cerebellum, substantia nigra, olfactory bulb and inferior colliculus. omega 2I sites were present in the cortical mantle (with higher levels in the cingulate and olfactory than in the sensorimotor cortex) and in subcortical (hippocampus, hypothalamus and nucleus accumbens) limbic structures. In the hippocampus, hypothalamus, spinal cord and nucleus accumbens, omega 2L sites accounted for more than 25% of the specific [3H]flumazenil binding; the density of these sites was minor in the cortex and in most pyramidal and extrapyramidal system structures.(ABSTRACT TRUNCATED AT 400 WORDS)     

Absence of association between delta and gamma2 subunits in native GABA(A) receptors from rat brain

We investigated the possible association between delta and gamma2 subunits in native GABA(A) receptors, from different rat brain regions, using subunit-specific anti-delta and anti-gamma2 antibodies. Previous reports have provided somewhat controversial results, indicating both the presence and the absence of association between these two subunits in native receptors. Our results indicate the absence of co-localization between delta and gamma2 subunits. In immunoprecipitation experiments, anti-delta antibody consistently immunoprecipitated [3H]muscimol binding activity (GABA binding sites) from all brain areas tested (10-20% of the total binding). However, under the same conditions, no significant [3H]flumazenil or [3H]ethyl 8-azido-6-dihydro-5-methyl-6-oxo-4H-imidazol[1,5-a]-[1,4]benzodiazepine- 3-carboxylate (Ro15-4513) binding (benzodiazepine binding sites) activity was detected in the immunopellets. These results indicate the absence of association between delta and gamma2 subunits. This question was directly addressed by immunopurification and Western blot experiments. As expected, no gamma2 subunits were detected in anti-delta immunoaffinity purified receptors. Conversely, no delta subunits were identified in anti-gamma2 immunopurified receptors. Thus, these results demonstrate the absence of association between delta and gamma2 subunits in native GABA(A) receptors. Finally, our results also indicate the relevance of the solubilization conditions on the apparent association between different subunits of the native GABA(A) receptor complex.     

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1. The responses of the electrically-driven right ventricle strip of the guinea-pig heart to diazepam were recorded in the absence and in the presence of different selective cyclic nucleotide phosphodiesterase (PDE) inhibitors. 2. Diazepam, at concentrations ranging from 1 microM to 100 microM, was devoid of effect on the contractile force in this preparation. 3. Conversely, diazepam (5 microM-100 microM) produced a consistent positive inotropic response in the presence of a concentration (1 microM), that was without effect in the absence of diazepam, of either of the selective PDE 3 inhibitors milrinone or SK&F 94120, but not in the presence of the selective PDE 4 inhibitor rolipram. 4. This effect of diazepam was not gamma-aminobutyric acid (GABA)-dependent, since it was neither mimicked nor potentiated by GABA, and was not affected by either a high concentration (5 microM) of the antagonists of the benzodiazepine/GABA/channel chloride receptor complex, picrotoxin, flumazenil and beta-carboline-3-carboxylic acid methyl ester (betaCCMe), or by the inverse agonists, beta-carboline-3-carboxylic acid N-methylamide (betaCCMa) and methyl 6,7-dimethoxy-4-ethyl-beta-carboline-3-carboxylate (DMCM, 0.1 microM). Furthermore, a specific antagonist of the peripheral benzodiazepine receptors, PK 11195 (5 microM), did not influence the effect of diazepam. 5. Biochemical studies with isolated PDEs, confirmed that diazepam selectively inhibits type 4 PDE from guinea-pig right ventricle rather than the other PDEs present in that tissue. The compound inhibited this enzyme in a non-competitive manner. Diazepam was also able to inhibit PDE 5, the cyclic GMP specific PDE absent from cardiac muscle, with a potency close to that shown for PDE 4. 6. Diazepam displaced the selective type 4 PDE inhibitor, rolipram from its high affinity binding site in rat brain cortex membranes, and also potentiated the rise in cyclic AMP levels induced by isoprenaline in guinea-pig eosinophils, where only type 4 PDE is present. 7. The PDE inhibitory properties of diazepam were shared, although with lower potency, by other structurally-related benzodiazepines, that also displaced [3H]-rolipram from its high affinity binding site. The order of potency found for these compounds in these assays was not related to their potencies as modulators of the GABA receptor through its benzodiazepine binding site. 8. The pharmacological and biochemical data presented in this study indicate that diazepam behaves as a selective type 4 PDE inhibitor in cardiac tissue and this effect seems neither to be mediated by the benzodiazepine/GABA/channel chloride receptor complex nor by peripheral type benzodiazepine receptors.     

Molecular and pharmacological characterization of GABAA receptors in the rat pituitary

Levels of mRNA for the major subunits of the GABAA receptor were assayed in the rat pituitary anterior and neurointermediate lobes by ribonuclease protection assay. alpha 1, beta 1, beta 2, beta 3, and gamma 2s were found to be the predominant subunits in the anterior lobe, whereas alpha 2, alpha 3, beta 1, beta 3, gamma 2s, and gamma 1 were the predominant subunits expressed in the neurointermediate lobe. alpha 5, alpha 6, and delta subunits were not detectable. Hill and Scatchard analysis of [3H] muscimol binding to anterior and neurointermediate lobe membranes showed high-affinity binding sites with dissociation constants of 5.6 and 4.5 nM, respectively, and Hill coefficients near 1. Muscimol sites were present at a maximum of 126 fmol/mg in the anterior lobe and 138 fmol/mg in the neurointermediate lobe. The central-type benzodiazepine antagonist [3H]Ro 15-1788 bound to a high-affinity site with a dissociation constant of 1.5 nM in both tissues, at a maximum of 60 fmol/mg in anterior pituitary and 72 fmol/mg in neurointermediate lobe. A Hill coefficient of 1 was measured for this site in both tissues. Assays of CL 218,872 displacement of Ro 15-1788 were consistent with a pure type I benzodiazepine site in the anterior lobe and a pure type II site in the intermediate lobe. These results are consistent with both tissue-specific expression of particular GABAA receptor subunits and receptor heterogeneity within individual cells in the pituitary.     

U-89843A is a novel allosteric modulator of gamma-aminobutyric acidA receptors

A group of pyrrolopyrimidine derivatives were examined for their interaction with rat recombinant gamma-aminobutyric acid (GABA)A receptors using the whole cell patch clamp and equilibrium binding techniques. In the alpha 1 beta 2 gamma 2 subtype of GABAA receptors expressed in human embryonic kidney cells, a prototype pyrrolopyrimidine, U-89843A (7H-pyrrol[2,3-d]pyrimidine,6,7-methyl-2,4-di- 1-pyrrolidinyl,hydrochloride), dose-dependently enhanced 5 microM GABA-induced Cl- currents with a maximal enhancement of 362 +/- 91%, a half-maximal concentration of 2 +/- 0.4 microM and a slope factor of 1.1 +/- 0.4. The drug also inhibited [35S]t-butylbicyclophosphorothionate binding in rat cerebrocortical membranes with a similar half-maximal inhibitory concentration. The enhancement of Cl- currents by U-89843A was insensitive to Ro 15-1788 (a benzodiazepine antagonist), was also observed in the alpha 3 beta 2 gamma 2 and alpha 6 beta 2 gamma 2 subtypes (no selectivity to different alpha-isoforms unlike many benzodiazepines), but was absent in the receptor subtypes consisting of two subunits (alpha 1 beta 2, alpha 1 gamma 2 and beta 2 gamma 2). It has been known that neurosteroids and barbiturates are uniformly active in both the two subunit receptors, substituted pyrazinones are only active in the alpha 1 beta 2 subtype and loreclezole is active in the subtypes containing beta 2. We propose that U-89843A interacts with an allosteric site on GABAA receptors distinct from the sites for benzodiazepines, barbiturates, neurosteroids, substituted pyrazinones or loreclezole.(ABSTRACT TRUNCATED AT 250 WORDS)     

Effect of peripherally and centrally administered calcitonin on gallbladder emptying in dogs

The vast molecular heterogeneity of brain gamma-aminobutyric acid type A (GABAA) receptors forms the basis for receptor subtyping. Using autoradiographic techniques, we established the characteristics of cerebellar granule cell GABAA receptors by comparing wild-type mice with those with a targeted disruption of the alpha6 subunit gene. Cerebellar granule cells of alpha6(-/-) animals have severe deficits in high affinity [3H]muscimol and [3H]SR 95531 binding to GABA sites, in agonist-insensitive [3H]Ro 15-4513 binding to benzodiazepine sites, and in furosemide-induced increases in tert-[35S]butylbicyclophosphorothionate binding to picrotoxin-sensitive convulsant sites. These observations agree with the known specific properties of these sites on recombinant alpha6beta2/3gamma2 receptors. In the presence of GABA concentrations that fail to activate alpha1 subunit-containing receptors, methyl-6,7-dimethoxy-4-ethyl-beta-carboline (30 microM), allopregnanolone (100 nM), and Zn2+ (10 microM) are less efficacious in altering tert-[35S]butylbicyclophosphorothionate binding in the granule cell layer of the alpha6(-/-) than alpha6(+/+) animals. These data concur with the deficiency of the cerebellar alpha6 and delta subunit-containing receptors in the alpha6(-/-) animals and could also account for the decreased affinity of [3H]muscimol binding to alpha6(-/-) cerebellar membranes. Predicted additional alterations in the cerebellar receptors of the mutant mice may explain a surplus of methyl-6,7-dimethoxy-4-ethyl-beta-carboline-insensitive receptors in the alpha6(-/-) granule cell layer and an increased diazepam-sensitivity in the molecular layer. These changes may be adaptive consequences of altered GABAA receptor subunit expression patterns in response to the loss of two subunits (alpha and delta) from granule cells.     

Chronic neurosteroid treatment produces functional heterologous uncoupling at the gamma-aminobutyric acid type A/benzodiazepine receptor complex in mammalian cortical neurons

We have investigated the effects of chronic treatment with the neurosteroid 5 alpha-pregnan-3 alpha-ol-20-one (5 alpha 3 alpha) on the gamma-aminobutyric acid (GABA)A receptor complex in cultured mammalian cortical neurons. Chronic 5 alpha 3 alpha treatment (up to 2 microM, 5 days) did not produce any changes in the morphological appearance or the cell protein content of cortical neurons. The basal binding of [3H]flunitrazepam, [3H]Ro15-1788, and [3H]Ro15-4513 was not altered after the chronic treatment. Chronic 5 alpha 3 alpha treatment did not alter the Kd or Bmax values of [3H]flunitrazepam binding to intact cortical neurons. However, chronic 5 alpha 3 alpha treatment produced uncoupling between GABA, barbiturate, and neurosteroid sites and the benzodiazepine site. The EC50 values of these ligands were not significantly altered; however, their Emax values were decreased after chronic 5 alpha 3 alpha treatment. The 5 alpha 3 alpha-induced uncoupling was time and concentration dependent. The binding of [3H]GABA and t-[35S]butylbicyclophosphorothionate was also decreased after chronic 5 alpha 3 alpha treatment. Chronic 5 alpha 3 alpha treatment decreased the Bmax of the low affinity GABAA receptor sites, without affecting the high affinity sites, and decreased the Bmax of t-butylbicyclophosphorothionate binding sites. The EC50 value for GABA-induced 36Cl- influx was not altered, whereas the Emax value was decreased after chronic 5 alpha 3 alpha treatment. Furthermore, the 5 alpha 3 alpha-induced uncoupling was reversed by concomitant exposure of the cortical neurons to 5 alpha-pregnan-3 beta-ol-20-one or R5135, suggesting an involvement of the neurosteroid and GABA recognition sites in the observed uncoupling. Taken together, these results suggest that chronic 5 alpha 3 alpha treatment produces heterologous uncoupling at the GABAA receptor complex.     

Functional characterization of human gamma-aminobutyric acidA receptors containing the alpha 4 subunit

The alpha subunits are an important determinant of the pharmacology of gamma-aminobutyric acidA (GABAA) receptors with respect to agonists, antagonists, and modulatory compounds, particularly the benzodiazepines. The alpha 4 subunit is the least abundant subunit in the brain and the most similar in deduced primary amino acid sequence to the alpha 6 subunit. We demonstrate that the human alpha 4 subunit forms a functional receptor when expressed with beta gamma 2, demonstrating some properties similar to alpha 6 beta gamma 2 and some properties more akin to alpha 1 beta gamma 2. It also exhibited some properties that were unlike any other alpha subunit-containing receptor. GABA affinity seemed to be identical to that of the alpha 1 beta 1 gamma 2 receptor; however, the partial agonists 4,5,6,7-tetrahydroisoxazolo-[5,4-c]pyridin-3-ol and piperidine-4-sulfonic acid showed lower efficacy than at either alpha 1 beta 1 gamma 2 or alpha 6 beta 1 gamma 2. Benzodiazepine pharmacology of alpha 4-containing receptors was similar to that of alpha 6-containing receptors with the exception of dimethoxy-4-ethyl-beta-carboline-3-carboxylate, which behaved as a partial inverse agonist. Pentobarbital potentiated alpha 4 beta 1 gamma 2 receptor GABA responses to a level comparable with alpha 6 beta 1 gamma 2 (approximately 700% of EC20); however, unlike alpha 6 beta 1 gamma 2 receptors, it did not elicit any direct activation of the receptor. Propofol also potentiated alpha 4 beta 1 gamma 2 GABA responses but to a level more comparable to that of alpha 1 beta 1 gamma 2, suggesting that these compounds act via different sites. Unlike other subunit combinations, propofol did not elicit a direct activation of the receptor. These results suggest that the mechanism for direct activation of the GABAA receptor by pentobarbital and propofol is absent on alpha 4-containing receptors. Furosemide, which non-competitively inhibits the GABAA receptor, showed 700-fold selectivity for alpha 6 beta 3 gamma 2 receptors over alpha 1-, alpha 2-, alpha 3-, and alpha 5-containing receptors and exhibited selectivity for alpha 4 beta 3 gamma 2 receptors (> 50-fold). These experiments reveal a unique pharmacology for alpha 4-containing receptors with some similarities to both alpha 6- and alpha 1-containing receptors.     

Anticonvulsant activity of azirino[1,2-d][1,4]benzodiazepines and related 1,4-benzodiazepines in mice

The anticonvulsant properties of several 1,4-benzodiazepine and azirino[1,2-d][1,4]benzodiazepine (ABDZ) derivatives were studied after intraperitoneal (IP) administration in DBA/2 mice (a strain genetically susceptible to sound-induced seizures) and in Swiss mice. The anticonvulsant effects were evaluated on seizures evoked by means of auditory stimulation (109 dB, 12-16 kHz) in animals placed singly under a hemispheric Perspex dome or on seizures induced by administration of pentylenetetrazole. The 1,4-benzodiazepines were generally more potent than the related ABDZ derivatives. The rank order of potency for anticonvulsant activity was flunitrazepam > diazepam > pinazepam > ABDZ5 > ABDZ4 > prazepam > halazepam > ABDZ1 > ABDZ3 > camazepam > ABDZ6 > ABDZ2. The impairment of locomotor performance following IP administration of these derivatives was also evaluated by means of the rotarod test. The rank order of potency for impairment of coordinated motor movements was pinazepam > flunitrazepam > diazepam > ABDZ5 > prazepam > halazepam > ABDZ4 > ABDZ3 > ABDZ1 > camazepam > ABDZ2 = ABDZ6. The potency of various 1,4-benzodiazepines and ABDZs as inhibitors of specific [3H]flumazenil binding to membranes from cerebellum or cortex was evaluated. In general, ABDZs were active as anticonvulsants and inhibited [3H]flumazenil binding in the micromolar range. Radioligand binding studies carried out in stable cell lines demonstrated that none of the ABDZs tested showed a particular subtype specificity. The pharmacological actions of ABDZ4 and ABDZ5, which appeared to be the most potent ABDZs as anticonvulsants, were significantly reduced by treatment with flumazenil (8.24 mumol/kg IP), suggesting a clear involvement of benzodiazepine mechanisms in the anticonvulsant activity of these compounds or their metabolites. The anticonvulsant activity of ABDZ4 and ABDZ5 was also evaluated against seizures induced in DBA/2 mice by two beta-carbolines: methyl-beta-carboline-3-carboxylate (beta-CCM) and methyl-6,6-dimethoxy-4-ethyl-beta-carboline-3-carboxylate (DMCM). Both ABDZ4 and ABDZ5 give better protection against seizures induced by beta-CCM than DMCM, suggesting a preferential action on the benzodiazepine receptor subtype BDZ1.     

The role of the GABA(A) receptor alpha1 subunit N-terminal extracellular domain in propofol potentiation of chloride current

Propofol (2,6-diisopropylphenol), an intravenous general anesthetic in active clinical use today, potentiates the action of gamma-aminobutyric acid (GABA) at the type-A receptor and also directly induces current in the absence of GABA. We expressed different combinations of murine GABA(A) receptor alpha1, beta3 and gamma2 subunits in Xenopus oocytes to investigate the subunit dependence of propofol potentiation of pentobarbital-induced current. Pentobarbital induces current in all beta3-subunit-containing receptors, whereas current gating by GABA requires the presence of both alpha1 and beta3 subunits. Therefore, pentobarbital rather than GABA was used to induce current in order to separate the subunit dependence of current gating from the subunit dependence of potentiating action of propofol. alpha1beta3gamma2, alpha1beta3, beta3gamma2, or beta3 subunit combinations all responded to pentobarbital in a dose-dependent manner. True potentiation was defined as the current magnitude to simultaneous application of pentobarbital and propofol exceeding the additive responses to individual drug applications. A dose-dependent propofol potentiation of pentobarbital-induced current was observed in oocytes injected with alpha1beta3 or alpha1beta3gamma2 but not in beta3gamma2 or beta3 subunits, suggesting that the alpha1 subunit was necessary for this modulatory action of propofol. Further examination of the propofol potentiation in chimeras between the alpha1 and beta3 subunits showed that the extracellular amino-terminal half of the alpha1 subunit was sufficient to support propofol potentiation. The different requirements of the receptor structure for the agonistic (gating) and the potentiating actions suggest that these two actions of propofol are distinct processes mediated through its action at distinct sites.     

Dihydroergosine: anticonflict effect in rats and enhancing effects on [3H]muscimol binding in the human brain post mortem

The anticonflict activity of the ergot alkaloid, dihydroergosine, a drug which binds to 5-hydroxytryptamine1 (5-HT1) receptors and to gamma-aminobutyric acidA (GABAA) receptor-associated Cl- ionophore, was studied in water-deprived rats. In vitro effects of this drug on [3H]muscimol and [3H]flunitrazepam binding to the crude synaptosomal pellet of the human frontal cortex post-mortem were also investigated. Dihydroergosine, given 2 h prior to testing, enhanced drinking under punished (0.8 mA) conditions, and diminished it under unpunished conditions. The mechanism of this effect was (-)-propranolol- and pindolol-insensitive and picrotoxin-sensitive. Flumazenil either failed to affect, or at a higher dose (10 mg/kg), counteracted the dihydroergosine-induced enhancement of punished drinking. This dose of flumazenil was itself anxiogenic. Dihydroergosine had mild sedative and analgesic properties. Low concentrations of dihydroergosine (10 nM to 100 microM) enhanced the binding of [3H]muscimol but not of [3H]flunitrazepam. The results suggest that dihydroergosine may possess anxiolytic properties presumably mediated by its specific action at the GABA/benzodiazepine/chloride channel complex.     

An approach for assaying benzodiazepine receptor binding with radioiodinated ligand: 125I-labeled diazepam derivative

[125I]2'-Iododiazepam (IDZ) was prepared and its application in a benzodiazepine receptor binding assay was studied. [125I]2'-IDZ binds to the rat cortical membrane with a high affinity (Kd, 0.66 nM). Various benzodiazepines showed competition with [125I]2'-IDZ for the binding sites in the rat cortical membrane, and the specificity of its binding correlated well with that of [3H]diazepam (r = 0.992, p < 0.001). These findings suggested that [125I]2'-IDZ binds to the same sites as [3H]diazepam and indicated that [125I]2'-IDZ can be used in a benzodiazepine receptor assay.