Molecular mechanisms of the partial allosteric modulatory effects of bretazenil at gamma-aminobutyric acid type A receptor.

In central nervous system gamma-aminobutyric acid (GABA) inhibits neuronal activity by acting on GABA type A (GABAA) receptors. These heterooligomeric integral membrane proteins include a GABA-gated Cl- channel and various allosteric modulatory sites where endogenous modulators and anxiolytic drugs act to regulate GABA action. In vivo, various anxiolytic drugs exhibit a wide range of variability in their modulatory efficacy and potency of GABA action. For instance, bretazenil modulatory efficacy is much lower than that of diazepam. Such low efficacy could be due either to a preferential modulation of specific GABAA receptor subtypes or to a low modulatory efficacy at every GABAA receptor subtype. To address these questions we studied drug-induced modifications of GABA-activated Cl- currents in native GABAA receptors of cortical neurons in primary cultures and in recombinant GABAA receptors transiently expressed in transformed human embryonic kidney cells (293) after transfection with cDNAs encoding different molecular forms of alpha, beta, and gamma subunits of GABAA receptors. In cortical neurons the efficacy of bretazenil was lower than that of diazepam, whereas the potency of the two drugs was similar. In cells transfected with gamma 2 subunits and various molecular forms of alpha and beta subunits bretazenil efficacy was always lower than that of diazepam. However, in cells transfected with gamma 1 or gamma 3 subunits and various forms of alpha and beta subunits the efficacy of both diazepam and bretazenil was lower and always of similar magnitude. When bretazenil and diazepam were applied together to GABAA receptors including a gamma 2 subunit, the action of diazepam was curtailed in a manner related to the dose of bretazenil.     

Expression patterns of gamma-aminobutyric acid type A receptor subunit mRNAs in primary cultures of granule neurons and astrocytes from neonatal rat cerebella.

Using a competitive polymerase chain reaction assay, we have quantitated the absolute amounts of mRNA encoding 14 distinct subunits of the gamma-aminobutyric acid type A (GABAA) receptor in primary cultures of rat cerebellar granule neurons and cerebellar astrocytes. We found that the total amount of GABAA receptor subunit mRNA in astrocytes was 2 orders of magnitude lower than in neuronal cells. Furthermore, granule cell cultures expressed all 14 different GABAA subunit mRNAs, while the astroglial cultures contained detectable amounts of all the subunits expressed by granule cells except the alpha 6 and the gamma 2L subunits. Of the alpha subunit family members, the alpha 1, alpha 5, and alpha 6 mRNAs were prominent in granule cells, while the alpha 1 and alpha 2 mRNAs were abundant in astrocytes. Of the beta receptor subunit mRNAs, the beta 1 and beta 3 mRNAs were abundantly expressed in both cultures. The gamma 2S and gamma 2L mRNAs constituted the great majority of gamma subunit mRNAs in neurons, while the gamma 1 subunit mRNA was the most abundant gamma subunit mRNA in astrocytes. When various allosteric modulators of GABAA receptors were tested electrophysiologically, methyl 6,7-dimethoxy-4-ethyl-beta-carboline- 3-carboxylate (DMCM) was the only one to modulate chloride currents elicited by GABA in a significantly different manner in granule cells (negative modulation) compared with astrocytes (positive modulation). The latter effect was previously observed in transiently expressed recombinant GABAA receptors containing a gamma 1 instead of a gamma 2 subunit. Our quantitative mRNA results suggest that an important molecular determinant responsible for the DMCM-positive modulatory effect on astroglial native GABAA receptors is the presence of the gamma 1 subunit in the receptor assembly.     

Subcellular localization of gamma-aminobutyric acid type A receptors is determined by receptor beta subunits.

gamma-aminobutyric acid type A (GABAA) receptors are the major sites of fast synaptic inhibition in the brain. They are constructed from four subunit classes with multiple members: alpha (1-6), beta (1-4), gamma (1-4), and delta (1). The contribution of subunit diversity in determining receptor subcellular targeting was examined in polarized Madin-Darby canine kidney (MDCK) cells. Significant detection of cell surface homomeric receptor expression by a combination of both immunological and electrophysiological methodologies was only found for the beta 3 subunit. Expression of alpha/beta binary combinations resulted in a nonpolarized distribution for alpha 1 beta 1 complexes, but specific basolateral targeting of both alpha 1 beta 2 and alpha 1 beta 3 complexes. The polarized distribution of these alpha/beta complexes was unaffected by the presence of the gamma 2S subunit. Interestingly, delivery of receptors containing the beta 3 subunit to the basolateral domain occurs via the apical surface. These results show that beta subunits can selectively target GABAA receptors to distinct cellular locations. Changes in the spatial and temporal expression of beta-subunit isoforms may therefore provide a mechanism for relocating GABAA receptor function between distinct neuronal domains. Given the critical role of these receptors in mediating synaptic inhibition, the contribution of different beta subunits in GABAA receptor function, may have implications in neuronal development and for receptor localization/clustering.     

gamma-Aminobutyric acid type A receptor point mutation increases the affinity of compounds for the benzodiazepine site.

Recombinantly expressed gamma-aminobutyric acid type A (GABAA) receptors consisting of alpha 1, beta 2, and gamma 2 subunits contain a binding site for benzodiazepines that differs in its properties from that of alpha 3 beta 2 gamma 2 receptors. Amino acid substitutions between the GABAA receptor alpha subunits were analyzed for their effect on the binding of compounds to the benzodiazepine site. By converting ever smaller regions of the alpha 3 subunit sequence to that of the alpha 1 subunit, we show that a single substitution (glycine for glutamic acid) increases the affinity for several compounds approximately 10-fold without changing the affinity for nonselective compounds. Hence, the identified amino acids may interact directly with the ligand and define part of the benzodiazepine binding sites in these receptors.     

Changes in gamma-aminobutyrate type A receptor subunit mRNAs, translation product expression, and receptor function during neuronal maturation in vitro.

The amounts of mRNAs encoding alpha 1, alpha 6, beta 2, beta 3, gamma 2, and delta subunits of gamma-aminobutyrate type A (GABAA) receptors and the gold immunolabeling density of their translation products were monitored during the growth of neonatal rat granule cells in primary culture. We investigated possible correlations (i) between temporal changes in mRNA content and expression density of their respective translation products and (ii) between the quantitative changes of receptor subunit expression, the GABA EC50 for Cl- channel activation, and diazepam efficacy in modulating GABA action on the Cl- channels. At 3 days in vitro, the amount of GABAA receptor subunit mRNAs and the expression of their respective translation products were very low. During the next 2 weeks both parameters for every subunit studied increased asynchronously; moreover, at 14 days in vitro the sum of gamma 2 and delta subunit expression was smaller than the expression of the alpha 1 or alpha 6 or beta 2/beta 3 subunits. This suggests that during in vitro maturation each subunit may be regulated independently and invites speculation as to possible changes in specific GABAA receptor subtype abundance during development in vitro. The maximal current intensity elicited by GABA failed to increase from 5 to 14 days in vitro, though the amount of mRNA encoding various subunits and the expression density of their respective translation products increased. Thus, qualitative changes in the GABAA receptor subtypes expressed and/or abnormalities in the subunit assembly very likely account for the uniformity of the maximal current intensity elicited by GABA during in vitro development. Also, during maturation of neuronal cultures from 5 to 20 days in vitro the extent of the positive modulation of GABA action by diazepam decreased dramatically. This finding might be related to an increase in the abundance of GABAA receptors including the alpha 6 subunit and/or to the expression, during granule cell maturation in vitro, of GABAA receptors devoid of gamma 2 subunits.     

Five subtypes of type A gamma-aminobutyric acid receptors identified in neurons by double and triple immunofluorescence staining with subunit-specific antibodies.

The extraordinary structural diversity of subunits forming type A gamma-aminobutyric acid (GABAA) receptors in the brain is expected to give rise to different modes of GABAergic synaptic inhibition and different profiles of modulatory drugs effective in anxiolytic, hypnotic, and antiepileptic therapy. To identify receptor subtypes in situ, the most prevalent subunits were visualized by double and triple immunofluorescence staining in rat brain, using polyclonal antibodies to the alpha 1, alpha 3, and gamma 2 subunits and a monoclonal antibody to locate both the beta 2 and the beta 3 subunit. At both cellular and subcellular levels five distinct patterns of subunit colocalization were identified: I, alpha 1 beta 2,3 gamma 2; II, alpha 3 beta 2,3 gamma 2; III, alpha 1 alpha 3 beta 2,3 gamma 2; IV, alpha 3 gamma 2; and V, alpha 1 alpha 3 gamma 2. As analyzed by confocal laser microscopy, different subunits displayed the same local variations of staining intensity ("hot spots") along the plasma membrane. The covisualized subunits appear therefore to be coassembled in receptor subtypes. Most neurons expressed only a single major receptor subtype with no apparent distinction between synaptic and extrasynaptic sites. However, in some neurons, most notably in Purkinje cells, the subunit composition varied between the soma and the dendrites, pointing to the existence of receptor heterogeneity within single neurons. Furthermore, different populations of neurons may be characterized by particular receptor subtypes. Cells displaying alpha 1-subunit immunoreactivity were mostly identified as GABAergic, whereas monoaminergic neurons displayed intense alpha 3-subunit immunoreactivity but virtually no alpha 1-subunit immunoreactivity. The allocation of defined GABAA receptor subtypes to identified neurons opens the way for a functional analysis of receptor heterogeneity.     

Benzodiazepine-insensitive mice generated by targeted disruption of the gamma 2 subunit gene of gamma-aminobutyric acid type A receptors.

Vigilance, anxiety, epileptic activity, and muscle tone can be modulated by drugs acting at the benzodiazepine (BZ) site of gamma-aminobutyric acid type A (GABAA) receptors. In vivo, BZ sites are potential targets for endogenous ligands regulating the corresponding central nervous system states. To assess the physiological relevance of BZ sites, mice were generated containing GABAA receptors devoid of BZ sites. Following targeted disruption of the gamma 2 subunit gene, 94% of the BZ sites were absent in brain of neonatal mice, while the number of GABA sites was only slightly reduced. Except for the gamma 2 subunit, the level of expression and the regional and cellular distribution of the major GABAA receptor subunits were unaltered. The single channel main conductance level and the Hill coefficient were reduced to values consistent with recombinant GABAA receptors composed of alpha and beta subunits. The GABA response was potentiated by pentobarbital but not by flunitrazepam. Diazepam was inactive behaviorally. Thus, the gamma 2 subunit is dispensable for the assembly of functional GABAA receptors but is required for normal channel conductance and the formation of BZ sites in vivo. BZ sites are not essential for embryonic development, as suggested by the normal body weight and histology of newborn mice. Postnatally, however, the reduced GABAA receptor function is associated with retarded growth, sensorimotor dysfunction, and drastically reduced life-span. The lack of postnatal GABAA receptor regulation by endogenous ligands of BZ sites might contribute to this phenotype.     

Positioning of the alpha-subunit isoforms confers a functional signature to gamma-aminobutyric acid type A receptors

Fast synaptic inhibitory transmission in the CNS is mediated by gamma-aminobutyric acid type A (GABA(A)) receptors. They belong to the ligand-gated ion channel receptor superfamily, and are constituted of five subunits surrounding a chloride channel. Their clinical interest is highlighted by the number of therapeutic drugs that act on them. It is well established that the subunit composition of a receptor subtype determines its pharmacological properties. We have investigated positional effects of two different alpha-subunit isoforms, alpha(1) and alpha(6), in a single pentamer. For this purpose, we used concatenated subunit receptors in which subunit arrangement is predefined. The resulting receptors were expressed in Xenopus oocytes and analyzed by using the two-electrode voltage-clamp technique. Thus, we have characterized gamma(2)beta(2)alpha(1)beta(2)alpha(1), gamma(2)beta(2)alpha(6)beta(2)alpha(6), gamma(2)beta(2)alpha(1)beta(2)alpha(6), and gamma(2)beta(2)alpha(6)beta(2)alpha(1) GABA(A) receptors. We investigated their response to the agonist GABA, to the partial agonist piperidine-4-sulfonic acid, to the noncompetitive inhibitor furosemide and to the positive allosteric modulator diazepam. Each receptor isoform is characterized by a specific set of properties. In this case, subunit positioning provides a functional signature to the receptor. We furthermore show that a single alpha(6)-subunit is sufficient to confer high furosemide sensitivity, and that the diazepam efficacy is determined exclusively by the alpha-subunit neighboring the gamma(2)-subunit. By using this diagnostic tool, it should become possible to determine the subunit arrangement of receptors expressed in vivo that contain alpha(1)- and alpha(6)-subunits. This method may also be applied to the study of other ion channels.     

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.     

Expression of functional receptors by the human gamma-aminobutyric acid A gamma 2 subunit

gamma-Aminobutyric acid A (GABA(A)) receptors are heteromeric membrane proteins formed mainly by various combinations of alpha, beta, and gamma subunits; and it is commonly thought that the gamma 2 subunit alone does not form functional receptors. In contrast, we found that cDNA encoding the gamma 2L subunit of the human GABA(A) receptor, injected alone into Xenopus oocytes, expressed functional GABA receptors whose properties were investigated by using the two-microelectrode voltage-clamp technique. GABA elicited desensitizing membrane currents that recovered after a few minutes' wash. Repetitive applications of GABA induced a "run-up" of GABA currents that nearly doubled the amplitude of the first response. The GABA currents inverted direction at about -30 mV, indicating that they are carried mainly by Cl(-) ions. The homomeric gamma 2L receptors were also activated by beta-alanine > taurine > glycine, and, like some types of heteromeric GABA(A) receptors, the gamma 2L receptors were blocked by bicuculline and were potentiated by pentobarbital and flunitrazepam. These results indicate that the human gamma 2L subunit is capable of forming fully functional GABA receptors by itself in Xenopus oocytes and suggest that the roles proposed for the various subunits that make up the heteromeric GABA(A) receptors in situ require further clarification.     

Differences in the negative allosteric modulation of gamma-aminobutyric acid receptors elicited by 4''-chlorodiazepam and by a beta-carboline-3-carboxylate ester: a study with natural and reconstituted receptors.

Cl- currents elicited by gamma-aminobutyric acid (GABA) application were recorded with the whole-cell tight-seal technique from voltage-clamped cortical neurons of neonatal rats in primary culture. The peripheral benzodiazepine recognition site ligand 4'-chlorodiazepam [Ro 5-4864; 7-chloro-1,3-dihydro-1-methyl-5-(4-chlorophenyl)-2H-[1,4]-benzodiazep in-2- one] inhibited the GABA-generated currents in a dose-dependent manner. Also, a beta-carboline (DMCM; 6,7-dimethoxy-4-ethyl-beta-carboline-3-carboxylate methyl ester), acting as a negative allosteric modulator of GABAA receptors, reduced the intensity of GABA-generated currents with similar efficacy but greater potency. Flumazenil (Ro 15-1788; 8-fluro-5,6-dihydro-5-methyl-6-oxo-4H-imidazo-[1,5-a] [1,4]-benzodiazepine-3-carboxylate ethyl ester) antagonized DMCM inhibition but not that elicited by 4'-chlorodiazepam. The isoquinoline carboxamide PK 11195, an antagonist of 4'-chlorodiazepam effects in other systems, failed to antagonize the action of 4'-chlorodiazepam. The transient expression of various molecular forms of GABAA receptors in the human embryonic kidney cell line 293 allowed a study of the minimal structural requirements for the inhibition of GABA-induced Cl- currents by bicuculline, picrotoxin, 4'-chlorodiazepam, and DMCM. GABA-elicited Cl- currents in cells coexpressing alpha 1 and beta 1 subunits of GABAA receptors were inhibited by bicuculline and picrotoxin, but not by DMCM or 4'-chlorodiazepam. Conversely, the GABA currents in cells coexpressing alpha 1 beta 1 and gamma 2 subunits were inhibited by bicuculline, picrotoxin, 4'-chlorodiazepam, and DMCM. Since the Cl- currents generated by GABA in some molecular forms of GABAA receptors are inhibited by bicuculline and picrotoxin only, 4'-chlorodiazepam cannot be acting isosterically with picrotoxin.     

Human granulocyte-macrophage colony-stimulating factor receptor signal transduction requires the proximal cytoplasmic domains of the alpha and beta subunits

Human granulocyte-macrophage colony-stimulating factor (GM-CSF) controls the production, maturation, and function of cells in multiple hematopoietic lineages. These effects are mediated by a cell-surface receptor (GM-R) composed of alpha and beta subunits, each containing 378 and 881 amino acids, respectively. Whereas the alpha subunit exists as several isoforms that bind GM-CSF with low affinity, the beta common subunit (beta c) does not bind GM-CSF itself, but acts as a high- affinity converter for GM-CSF, interleukin-3 (IL-3), and IL-5 receptor alpha subunits. The cytoplasmic region of GM-R alpha consists of a membrane-proximal conserved region shared by the alpha 1 and alpha 2 isoforms and a C-terminal variable region that is divergent between alpha 1 and alpha 2. The cytoplasmic region of beta c contains membrane proximal serine and acidic domains. To investigate the amino acid sequences that influence signal transduction by this receptor complex, we constructed a series of cytoplasmic truncation mutants of the alpha 2 and beta subunits. To study these truncations, we stably transfected the IL-3-dependent murine cell line Ba/F3 with wild-type or mutant cDNAs. We found that the wild-type and mutant alpha subunits conferred similar low-affinity binding sites for human GM-CSF to Ba/F3, and the wild-type or mutant beta subunit converted some of these sites to high- affinity; the cytoplasmic domain of beta was unnecessary for this high- affinity conversion. Proliferation assays showed that the membrane- proximal conserved region of GM-R alpha and the serine-acidic domain of beta c are required for both cell proliferation and ligand-dependent phosphorylation of a 93-kD cytoplasmic protein. We suggest that these regions may represent an important signal transduction motif present in several cytokine receptors.     

Long-lasting reduction of inhibitory function and gamma-aminobutyric acid type A receptor subunit mRNA expression in a model of temporal lobe epilepsy.

This study evaluated hippocampal inhibitory function and the level of expression of gamma-aminobutyric acid type A (GABAA) receptor mRNA in an in vivo model of epilepsy. Chronic recurrent limbic seizures were induced in rats using injections of pilocarpine. Electrophysiological studies performed on hippocampal slices prepared from control and epileptic animals 1 to 2 months after pilocarpine injections demonstrated a significant hyperexcitability in the epileptic animals. Reduced levels of mRNA expression for the alpha 2 and alpha 5 subunits of the GABAA receptors were evident in the CA1, CA2, and CA3 regions of the hippocampus of epileptic animals. No decrease in mRNA encoding alpha 1, beta 2, or gamma 2 GABAA receptor subunits was observed. In addition, no change in the mRNA levels of alpha CaM kinase II was seen. Selective decreases in mRNA expression did not correlate with neuronal cell loss. The results indicate that selective, long-lasting reduction of GABAA subunit mRNA expression and increased excitability, possibly reflecting loss of GABAergic inhibition, occur in an in vivo model of partial complex epilepsy.     

Human chromosomal localization of genes encoding the gamma 1 and gamma 2 subunits of the gamma-aminobutyric acid receptor indicates that members of this gene family are often clustered in the genome.

The gamma-aminobutyric acid (GABA) receptors are the major inhibitory neurotransmitter receptors in the brain and the site of action of a number of important pharmacological agents including barbiturates, benzodiazepines, and ethanol. The gamma 1 and gamma 2 subunits have been shown to be important in mediating responses to benzodiazepines, and a splicing variant of the gamma 2 subunit, gamma 2L, has been shown to be necessary for ethanol actions on the receptor, raising the possibility that the gamma 2 gene may be involved in human genetic predisposition to the development of alcoholism. We have assigned the human genes encoding the gamma 1 and gamma 2 subunits of the GABAA receptor to chromosomes 4 and 5, respectively, by PCR amplification of human-specific products from human-hamster somatic cell hybrid DNAs. Using panels of chromosome-specific natural deletion hybrids, we have further localized the gamma 1 gene (GABRG1) to 4p14-q21.1 and the gamma 2 gene (GABRG2) to 5q31.1-q33.2. These data indicate that the gamma 1 gene may be clustered together with the previously mapped alpha 2 and beta 1 genes on chromosome 4 and that the gamma 2 gene may be close to the previously localized alpha 1 gene on chromosome 5. To further examine the latter possibility the alpha 1 gene was mapped using the chromosome 5 deletion hybrids and shown to be within the same region as the gamma 2 gene, 5q31.1-q33.2. A PCR-based screening strategy was used to isolate a 450-kilobase human genomic yeast artificial chromosome clone containing both the alpha 1 and gamma 2 genes. Pulsed-field gel restriction mapping of the yeast artificial chromosome indicates that the two genes are within 200 kilobases of each other. The data presented here provide further evidence for the nonrandom organization of the human genome by demonstrating that members of the GABAA receptor gene family often occur in small gene clusters widely distributed in the genome.     

Expression of gamma-aminobutyric acid A receptor subunits α1，β1，γ2 mRNA in rats with hepatic encephalopathy

AIM: To investigate the mRNA expression of gammaaminobutyric acid A (GABAA) receptor subunitsα1,β1,γ2 in different parts of the brain of rats with hepatic encephalopathy.METHODS: Twelve adult male Sprague-Dawley rats were randomly divided into two groups: (1) hepatic encephalopabhy model group (n = 6), which was induced by intraperitoneal injection of thioacetamide (TAA, 350 mg/kg) for three consecutive days; (2) control group (n = 6), in which the rats were treated with same dose of normal saline solution. After the freeze slice of cerebrum was made, in situ hybridization was used to detect the mRNA of GABAA receptor subunits α1,β1,, and γ2 in rat cerebral cortex,basal nuclei, substantia nigra and hippocampi. Image data were collected and analyzed quantitatively by QWin550CW model image signal gather and analysis system.RESULTS: In rats with hepatic encephalopathy, mRNA expression levels of GABAA receptor subunits α1,β1 increased significantly in basal nuclei, substantia nigra pars compacta, substantia nigra pars reticularis and hippocampi (144.7±15.67/184.14±4.41, 60.61±33.66/113.07±32.44,87.71± 21.25/128.40±18.85, 122.34±5.56/161.60±4.56,123.29±5.21/140.65±4.15, 123.40±4.42/140.09±4.52,124.76±4.18/140.09±4.12, 141.62±15.09/182.80±5.20,69.13±30.74/134.21±43.76, 87.87±25.16/151.01±19.49,122.14±6.30/162.33±3.92, 122.81±5.09/137.19±7.12,123.00±4.63/138.11±5.92, 125.75 ±2.43/138.81±6.10,P<0.01), but did not change in the cerebral cortex compared to the control group. Similar changes were found in the mRNA expression levels of GABAA receptor subunit γ2,which increased significantly in basal nuclei, substantia nigra pars compacta, substantia nigra pars reticularis (136.81±26.41/167.97±16.23, 51.00±36.14/113.18±36.52,86.35±20.30/126.90±19.74, P<0.01), CA1 of hippocampal(162.15:1:9.05/178.62:1:6.45, P<0.05), and no changes were found in the cerebral cortex and CA2, CA3, CA4 of hippocampi.CONCLUSION: In rats with hepatic encephalopathy,mRNA expression levels of GABAA receptor subunits α1,β1,γ2 increase significantly in basal nuclei, substantia nigra and hippocampi, suggesting that the changes of mRNA expression levels in GABAA receptor subunits may contribute to the pathogenesis of hepatic encephalopathy.     

Subunit composition and pharmacological characterization of gamma-aminobutyric acid type A receptors in frog pituitary melanotrophs

The frog pars intermedia is composed of a single population of endocrine cells directly innervated by gamma-aminobutyric acid (GABA)ergic nerve terminals. We have previously shown that GABA, acting through GABA(A) receptors, modulates both the electrical and secretory activities of frog pituitary melanotrophs. The aim of the present study was to take advantage of the frog melanotroph model to determine the relationship between the subunit composition and the pharmacological properties of native GABA(A) receptors. Immunohistochemical labeling revealed that in situ and in cell culture, frog melanotrophs were intensely stained with alpha2-, alpha3-, gamma2-, and gamma3-subunit antisera and weakly stained with a gamma1-subunit antiserum. Melanotrophs were also immunolabeled with a monoclonal antibody to the beta2/beta3-subunit. In contrast, frog melanotrophs were not immunoreactive for the alpha1-, alpha5-, and alpha6-isoforms. The effects of allosteric modulators of the GABA(A) receptor on GABA-activated chloride current were tested using the patch-clamp technique. Among the ligands acting at the benzodiazepine-binding site, clonazepam (EC50, 5 x 10(-9) M), diazepam (EC50, 10(-8) M), zolpidem (EC50, 3 x 10(-8) M), and beta-carboline-3-carboxylic acid methyl ester (EC50, 10(-6) M) were found to potentiate the whole cell GABA-evoked current in a dose-dependent manner. Methyl-6,7-dimethoxy-4-ethyl-beta-carboline-3-carboxylate (IC50, 3 x 10(-5) M) inhibited the current, whereas Ro15-4513 had no effect. Among the ligands acting at other modulatory sites, etomidate (EC50, 2 x 10(-6) M) enhanced the GABA-evoked current, whereas 4'-chlorodiazepam (IC50, 4 x 10(-7) M), ZnCl2 (IC50, >5 x 10(-5) M), and furosemide (IC50, >3 x 10(-4) M) depressed the response to GABA. PK 11195 did not affect the GABA-evoked current or its inhibition by 4'-chlorodiazepam. The results indicate that the native GABA(A) receptors in frog melanotrophs are formed by combinations of alpha2-, alpha3-, beta2/3-, gamma1-, gamma2-, and gamma3-subunits. The data also demonstrate that clonazepam is the most potent, and zolpidem is the most efficient positive modulator of the native receptors. Among the inhibitors, 4'-chlorodiazepam is the most potent, whereas ZnCl2 is the most efficient negative modulator of the GABA(A) receptors. The present study provides the first correlation between subunit composition and the functional properties of native GABA(A) receptors in nontumoral endocrine cells.     

Specific enhancement of beta-adrenergic receptor kinase activity by defined G-protein beta and gamma subunits.

The beta and gamma subunits of heterotrimeric guanine nucleotide-binding proteins (G proteins) have recently been shown to play an active role in signal transduction. Among other effects they enable translocation of the beta-adrenergic receptor kinase (beta ARK) from the cytosol to the plasma membrane and thus permit phosphorylation and ultimately desensitization of beta-adrenergic receptors and other G-protein-coupled receptors. To investigate the specificity of this effect, we have purified various combinations of recombinant beta and gamma subunits expressed in Sf9 cells and measured their effects on beta ARK-catalyzed phosphorylation of beta 2-adrenergic receptors and of rhodopsin. The combinations tested were beta 1 gamma 2, beta 1 gamma 3, beta 2 gamma 2, beta 2 gamma 3, and transducin beta gamma (beta 1 gamma 1). There were clear differences in enhancement of rhodopsin phosphorylation, with an order of efficacy beta 2 gamma 2 > beta 1 gamma 2 >> beta 2 gamma 3 approximately beta 1 gamma 3 approximately beta 1 gamma 1. The first two combinations had larger effects than a mixed beta gamma preparation from bovine brain. In enhancing phosphorylation of beta 2-adrenergic receptors, beta 1 gamma 2 was more efficient and potent than all other combinations. These data suggest a twofold specificity of beta gamma complexes in enhancing beta ARK-catalyzed receptor phosphorylation: the gamma subunits may be important in interacting with beta ARK, with gamma 2 being more potent than gamma 3, whereas the beta subunits may determine coupling to the receptors, with beta 2 being more effective than beta 1 for rhodopsin and beta 1 being more effective than beta 2 for beta 2-adrenergic receptors.