Ethanol, flunitrazepam, and pentobarbital modulation of GABAA receptors expressed in mammalian cells and Xenopus oocytes

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

The gamma-aminobutyric acid receptor gamma 3 subunit gene (GABRG3) is tightly linked to the alpha 5 subunit gene (GABRA5) on human chromosome 15q11-q13 and is transcribed in the same orientation

GABAA receptors are heterooligomeric ligand-gated ion channels that mediate the effect of the inhibitory neurotransmitter gamma-aminobutyric acid. The GABAA receptors consist of at least 15 different receptor subunits that can be classified into 5 subfamilies (alpha, beta, gamma, delta, rho) on the basis of sequence similarity. Chromosomal mapping studies have revealed that several of the GABAA receptor subunit genes appear to be organized as clusters. One such cluster, which consists of the GABAA receptor beta 3 (GABRB3) and alpha 5 (GABRA5) subunit genes, is located in chromosome 15q11-q13. It is shown here that the GABAA receptor gamma 3 subunit gene (GABRG3) also maps to this region. Lambda and P1 phage clones surrounding both ends of GABRG3 were isolated; the clones derived from the 5' end of GABRG3 were linked to an existing phage contig spanning the 3' end of GABRA5. The two genes are located within 35 kb of each other and are transcribed in the same orientation.     

Distinct deactivation and desensitization kinetics of recombinant GABAA receptors

The functional role of the large heterogeneity in GABAA receptor subunit genes and its role in setting the properties of inhibitory synapses in the CNS is poorly understood. A kinetic comparison between currents elicited by ultra-rapid application with a piezoelectric translator of 1 mM GABA to mammalian cells transfected with cDNAs encoding distinct GABAA receptor subunits revealed that the intrinsic deactivation and desensitization properties depend on subunit combination. In particular, receptors containing alpha 6 with beta 2 gamma 2 subunits were endowed with a significantly slower deactivation as compared to those receptors containing alpha 1 with beta 2 gamma 2 subunits. While desensitization produced by prolonged GABA applications on alpha 1 beta 2 gamma 2 receptors was characterized by a rapid exponential decay followed by a slower decay and a steady state response, alpha 6 beta 2 gamma 2 receptors lacked desensitization. Furthermore, GABAA receptors lacking the gamma 2 subunit were characterized by a much larger non-desensitization component and a very rapid deactivation. Lastly, analysis of GABA-activated currents in cells cotransfected with alpha 1 and alpha 6 together with beta 2 gamma 2 subunit revealed unique kinetic properties. Our results suggest that distinct subunit composition confers specific deactivation and desensitization properties that may profoundly affect synaptic decay kinetics and the capability to sustain high frequency synaptic inputs.     

Modulation of GABAA receptor function by alcohols: effects of subunit composition and differential effects of ethanol

We sought to test the hypotheses that closely related alcohols would have effects on GABAA receptor function that were not predicted by differences in lipid solubility, and that the subunit structure of the GABAA receptor would significantly affect the actions of different alcohols. Cloned subunits of human GABAA receptors were expressed in Xenopus oocytes, and two-electrode voltage-clamp recording was used to quantify the membrane current response to GABA in the presence and absence of different alcohols. 1-Butanol and 2-butanol differentially potentiated the response to 20 microM GABA in oocytes expressing the alpha 1 beta 2 gamma 2L and alpha 2 beta 2 gamma 2L receptor isoforms. In the alpha 1 beta 2 gamma 2L receptor construct, 1-butanol was more potent than 2-butanol to potentiate GABAA receptor function, but 2-butanol had a greater efficacy. In the alpha 2 beta 2 gamma 2L receptor construct, 1-butanol and 2-butanol were equipotent, but 2-butanol again had a greater efficacy. In the alpha 2 beta 2 receptor construct, both 1-butanol and 2-butanol produced large potentiations of the current response to 3 microM GABA. The efficacy for butanol potentiation of GABA responses in the absence of a gamma 2L subunit was greater, but the potency was greatly reduced. Low concentrations (20 mM) of ethanol potentiated GABA responses in the alpha 1 beta 2 gamma 2L receptor construct. Ethanol potentiation of GABAA receptor function was completely blocked by the benzodiazepine receptor partial inverse agonist RO15-4513 at a concentration (0.5 microM) that did not alter the control GABA response. In contrast, RO15-4513 did not block potentiation of GABAA receptor activity induced by n-propanol, 1-butanol, 2-butanol, 1-heptanol, or propofol (2,6-diisopropylphenol). These results suggest that alcohols have specific interactions with GABAA receptors, and that ethanol may have unique effects not shared by other longer chain alcohols.     

gamma-Aminobutyric acid type A receptors in the rat brain can contain both gamma 2 and gamma 3 subunits, but gamma 1 does not exist in combination with another gamma subunit

Antibodies specific for the gamma 1, gamma 2, and gamma 3 subunits of the gamma-aminobutyric acid (GABA)A receptor have been used to probe the composition of naturally occurring GABAA receptors in the rat brain. Most GABAA receptors contain at least one of these three subunits. The percentage of each, determined by immunoprecipitation of [3H]muscimol binding, was 11 +/- 1%, 59 +/- 3%, and 14 +/- 2% for gamma 1, gamma 2, and gamma 3 subunits, respectively. Receptors containing gamma 2 or gamma 3 subunits were labeled by benzodiazepine site ligands with high affinity, whereas gamma 1-containing receptors could be labeled only by [3H]muscimol. Receptors immunoprecipitated by anti-gamma 2 or anti-gamma 3 antibodies were labeled with [3H]Ro 15-1788 with similar affinities (Kd for anti-gamma 2-immunoprecipitated receptors, 1.9 nM; Kd for anti-gamma 3-immunoprecipitated receptors, 1.7 nM). Immunoprecipitation or Western blot analysis of GABAA receptors solubilized from rat cerebellar or whole-brain preparations indicated that gamma 1 was not present coassembled with any other gamma subunit. Western blot analysis of receptors purified on alpha-specific immunoaffinity resins showed that gamma 1 was predominantly assembled with the alpha 2 subunit. Some GABAA receptors may contain more than one type of gamma subunit. Quantitative immunoprecipitation and Western blot analysis both indicated that gamma 2 and gamma 3 subunits can exist in the same receptor complex. A large proportion of GABAA receptors immunopurified on a gamma 3 affinity resin also appeared to contain a gamma 2 subunit. In contrast, when receptors were purified on a gamma 2 affinity resin a small proportion also appeared to contain a gamma 3 subunit. We conclude that most gamma 1-containing receptors have no other gamma subunit in the same receptor complex but some GABAA receptors contain both gamma 2 and gamma 3 subunits.     

Chronic GABA treatment downregulates the GABAA receptor alpha 2 and alpha 3 subunit mRNAS as well as polypeptide expression in primary cultured cerebral cortical neurons

Chronic GABA exposure of mammalian primary cultured cortical neurons results in a downregulation of the GABA-benzodiazepine receptor complex. In the present study, the mRNA levels, as well as polypeptide expression, for the GABAA receptor alpha 2 and alpha 3 subunits in cultured embryonic mouse cerebral cortical neurons (7 day old) were examined using northern analysis and immunoblotting techniques following chronic GABA treatment. The alpha 1 subunit mRNA or polypeptide could not be detected in these neurons. The steady state levels of mRNA for the GABAA receptor alpha 2 and alpha 3 subunits showed a decrease in comparison with untreated neurons. There was no change in the level of the beta actin or poly(A)+ RNA under the same experimental conditions. This agonist-induced reduction in the GABAA receptor alpha 2 and alpha 3 subunit mRNA was blocked by the concomitant exposure of neurons to R 5135, an antagonist of GABAA receptor. The polypeptide expression for the GABAA receptor alpha 2 and alpha 3 subunits in chronically GABA-treated neurons also showed a decline and this change was also blocked by the concomitant exposure of cells to GABA and R 5135. These results indicate that the chronic exposure of the GABAA receptor complex to agonist downregulates the expression of the alpha subunits of the receptor complex, which may be related to an observed decreases in the number of binding sites and GABA-induced 36Cl-influx in the cortical neurons.     

Positive modulation of human gamma-aminobutyric acid type A and glycine receptors by the inhalation anesthetic isoflurane

The interactions of the inhalation anesthetic agent isoflurane with ligand-gated chloride channels were studied using transient expression of recombinant human receptors in a mammalian cell line. Isoflurane enhanced gamma-aminobutyric acid (GABA)-activated chloride currents in cells that expressed heteromeric GABAA receptors consisting of combinations of alpha 1 or alpha 2, beta 1, and gamma 2 subunits and in cells that expressed receptors consisting of combinations of only alpha and beta subunits. Receptors consisting of alpha 2 and gamma 2 subunits were poorly expressed but were sensitive to isoflurane. Receptors consisting of beta 1 and gamma 2 subunits were not expressed. Isoflurane also enhanced glycine-activated chloride currents through homomeric alpha glycine receptors but did not enhance GABA currents in cells expressing homomeric rho 1 receptors. These results show that not all ligand-gated chloride channel receptors are sensitive to isoflurane and, therefore, that the anesthetic interacts with specific structural determinants of these ion channel proteins.     

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.     

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.     

alpha2,3-sialyltransferase mRNA and alpha2,3-linked glycoprotein sialylation are increased in malignant gliomas

Microinjection of a substance P analogue (1 mM; 7 or 10 nl) into laminae I and II of the L7 dorsal horn of decerebrate cats significantly potentiated (P < 0.05) the increase in arterial pressure evoked by microinjection of L-glutamate (109 mM; 7 or 10 nl) into these spinal sites. Microinjection of the substance P analogues (i.e., GR73638 and [Sar9,Met(O2)11]-substance P) which were selective NK-1 receptor agonists, had no impact on the cardioacceleration evoked by microinjection of L-glutamate (P > 0.05). In addition, microinjection of these analogues had no effect on the modest and non-significant increase in phrenic nerve discharge evoked by L-glutamate. We conclude that stimulation of NK-1 receptors in the superficial laminae of the dorsal horn potentiates the pressor responses to microinjection of L-glutamate.     

Intrathecal chemotherapy for treatment of overt meningeal leukemia: comparison between intraventricular and traditional intralumbar route

The developmental changes in the expression of mRNAs encoding the alpha 1 and alpha 2 subunits of inhibitory glycine receptors in the spinal cord of fetal and postnatal rats were examined by in situ hybridization. During embryonic periods (E11-18), the mantle zone was scarce in the alpha 1 mRNA, but the germinal zone (matrix layer) at E11-14 expressed higher levels of the message. At postnatal day 0 (P0), the alpha 1 signals became manifested throughout the gray matter of the spinal cord. The intensities of the signals were increased to reach a maximal level at P21. By contrast, the spinal tissues at P0 exhibited the highest levels of alpha 2 mRNA, which decreased with the postnatal development. In P50 rats, the alpha 2 mRNA was barely expressed in the ventral horn, but a significant number of grains could still be detectable in a population of cells in the dorsal horn. During postnatal development from P0 to P10, the spinal tissues were rich in the alpha 1 and alpha 2 mRNAs, both of which were detected in the presumed motoneurons. The coexistence of the two subunits in single neurons might correlate with the modification of the glycine receptor function during the development of the spinal cord.     

The time-course of abeta-evoked c-fos expression in neurons of the dorsal horn and gracile nucleus after peripheral nerve injury

We have examined the mechanisms underlying Abeta-evoked c-fos expression in the dorsal horn and gracile nucleus following either sciatic nerve section or crush injury. The results indicate that in the spinal cord Abeta-evoked c-fos does not depend on primary afferent sprouting but is associated with the disconnection from the peripheral target since its expression in the dorsal horn reverts to normal after crush injury when regeneration occurs but persists after cut and ligation where regeneration is prevented. In contrast, however, Abeta-evoked c-fos expression in the gracile nucleus may be under some other control since its expression appears independent of peripheral nerve regeneration.     

Properties and sex-specific differences of GABAA receptors in neurons expressing gamma1 subunit mRNA in the preoptic area of the rat

Gamma-aminobutyric acid type A (GABAA) receptors expressed within the medial preoptic area (mPOA) are known to play a critical role in regulating sexual and neuroendocrine functions. In the rat brain, high levels of expression of the gamma1 subunit mRNA of the GABAA receptor are restricted to a limited number of regions that mediate sexual behaviors, including the mPOA. The biophysical and pharmacological profiles of native gamma1-containing receptors in neurons are unknown. Here, we have characterized the properties of GABAA receptor-mediated spontaneous inhibitory postsynaptic currents (sIPSCs) and currents elicited by fast perfusion of GABA to isolated mPOA neurons of juvenile male and female rats. No significant sex-specific differences were evident in the mean peak amplitude, distribution of event amplitudes, kinetics of current decay, or the frequency of sIPSCs. The profile of modulation of sIPSCs by diazepam, beta-CCM and zolpidem, allosteric modulators that act at the benzodiazepine (BZ) site of the GABAA receptor, support the assertion that mPOA neurons of both sexes express functional gamma1-containing receptors. The ability of zolpidem to modulate both sIPSC amplitude and currents elicited by rapid perfusion of GABA to mPOA neurons differed significantly between the sexes. Zolpidem reversibly induced negative modulation of currents in mPOA neurons isolated from male rats, but had no effect in mPOA neurons from female rats. Concentration-response analysis of responses in neurons acutely isolated from male rats indicated an IC50 of 58 nM with maximal decreases of approximately 50% of control peak current amplitude. In situ hybridization analysis demonstrated that levels of the gamma1 subunit mRNA are significantly higher in mPOA neurons from male than female rats. No significant sex-specific differences were detected in the levels of alpha1, alpha2, or alpha5 mRNAs. These results suggest that native gamma1-containing receptors are expressed in primary neurons of the mPOA and that sex-specific differences in the expression of this subunit may contribute to sexual dimorphism in GABAA receptor modulation by compounds acting at the BZ site.     

Nitric oxide in the stress axis

Calcitonin gene-related peptide in sensory primary afferent neurons has an excitatory effect on postsynaptic neurons and potentiates the effect of substance P in the rat spinal dorsal horn. It has been established that calcitonin gene-related peptide expression in dorsal root ganglion neurons is depressed, and the effect of calcitonin gene-related peptide on dorsal horn neurons is attenuated, following peripheral nerve injury. We report here that a subpopulation of injured dorsal root ganglion neurons show increased expression of calcitonin gene-related peptide. Using in situ hybridization and the retrograde tracer, FluoroGold, we detected an increased number of medium- to large-sized rat dorsal root ganglion neurons projecting to the gracile nucleus that expressed alpha-calcitonin gene-related peptide messenger RNA following spinal nerve transection. Immunohistochemistry revealed a significant increase in calcitonin gene-related peptide immunoreactivity in the gracile nucleus and in laminae III-IV of the spinal dorsal horn. These results indicate that a subpopulation of dorsal root ganglion neurons express alpha-calcitonin gene-related peptide messenger RNA in response to peripheral nerve injury, and transport this peptide to the gracile nucleus and to laminae III-IV of the spinal dorsal horn. The increase of the excitatory neuropeptide, calcitonin gene-related peptide, in sites of primary afferent termination may affect the excitability of postsynaptic neurons, and have a role in neuronal plasticity following peripheral nerve injury.     

The gamma 2 subunit of the GABAA receptor is concentrated in synaptic junctions containing the alpha 1 and beta 2/3 subunits in hippocampus, cerebellum and globus pallidus

The gamma 2 subunit is necessary for the expression of the full benzodiazepine pharmacology of GABAA receptors and is one of the major subunits in the brain. In order to determine the location of channels containing the gamma 2 subunit in relation to GABA-releasing terminals on the surface of neurons, a new polyclonal antipeptide antiserum was developed to the gamma 2 subunit and used in high resolution, postembedding, immunoelectron-microscopic procedures. Dual immunogold labelling of the same section for two subunits, and up to three sections of the same synapse reacted for different subunits, were used to characterize the subunit composition of synaptic receptors. The gamma 2 subunit was present in type 2, "symmetrical" synapses in each of the brain areas studied, with the exception of the granule cell layer of the cerebellum. The gamma 2 subunit was frequently co-localized in the same synaptic junction with the alpha 1 and beta 2/3 subunits. The immunolabelling of synapses was coincident with the junctional membrane specialization of the active zone. Immunolabelling for the receptor often occurred in multiple clusters in the synapses. In the hippocampus, the gamma 2 subunit was present in basket cell synapses on the somata and proximal dendrites and in axo-axonic cell synapses on the axon initial segment of pyramidal and granule cells. Some synapses on the dendrites of GABAergic interneurones were densely labelled for the gamma 2, alpha 1 and beta 2/3 subunits. In the cerebellum, the gamma 2 subunit was present in both distal and proximal Purkinje cell dendritic synapses established by stellate and basket cell, respectively. On the soma of Purkinje cells, basket cell synapses were only weakly labelled. Synapses on interneuron dendrites were more densely labelled for the gamma 2, alpha 1 and beta 2/3 subunits than synapses on Purkinje or granule cells. Although immunoperoxidase and immunofluorescence methods show an abundance of the gamma 2 subunit in granule cells, the labelling of Golgi synapses was much weaker with the immunogold method than that of the other cell types. In the globus pallidus, many type 2 synapses were labelled for the gamma 2 subunit together with alpha 1 and beta 2/3 subunits. The results show that gamma 2 and beta 2/3 subunits receptor channels are highly concentrated in GABAergic synapses that also contain the alpha 1 and beta 2/3 subunits. Channels containing the gamma 2 subunit are expressed in synapses on functionally distinct domains of the same neuron receiving GABA from different presynaptic sources. There are quantitative differences in the density of GABAA receptors at synapses on different cell types in the same brain area.