Identification of residues within GABA(A) receptor alpha subunits that mediate specific assembly with receptor beta subunits.

GABA(A) receptors can be constructed from a range of differing subunit isoforms: alpha, beta, gamma, delta, and epsilon. Expression studies have revealed that production of GABA-gated channels is achieved after coexpression of alpha and beta subunits. The expression of a gamma subunit isoform is essential to confer benzodiazepine sensitivity on the expressed receptor. However, how the specificity of subunit interactions is controlled during receptor assembly remains unknown. Here we demonstrate that residues 58-67 within alpha subunit isoforms are important in the assembly of receptors comprised of alphabeta and alphabetagamma subunits. Deletion of these residues from the alpha1 or alpha6 subunits results in retention of either alpha subunit isoform in the endoplasmic reticulum on coexpression with the beta3, or beta3 and gamma2 subunits. Immunoprecipitation revealed that residues 58-67 mediated oligomerization of the alpha1 and beta3 subunits, but were without affect on the production of alpha/gamma complexes. Within this domain, glutamine 67 was of central importance in mediating the production of functional alpha1beta3 receptors. Mutation of this residue resulted in a drastic decrease in the cell surface expression of alpha1beta3 receptors and the resulting expression of beta3 homomers. Sucrose density gradient centrifugation revealed that this residue was important for the production of a 9S alpha1beta3 complex representing functional GABA(A) receptors. Therefore, our studies detail residues that specify GABA(A) receptor alphabeta subunit interactions. This domain, which is conserved in all alpha subunit isoforms, will therefore play a critical role in the assembly of GABA(A) receptors composed of alphabeta and alphabetagamma subunits.     

Assembly of functional alpha6beta3gamma2delta GABA(A) receptors in vitro

Transgenic mice deficient in the alpha6 subunit of the GABA(A) receptor show reduced levels of the delta subunit protein and an altered GABA(A) receptor pharmacology, suggesting selective assembly mechanisms. Delta reduced the binding of [3H]Ro15-4513 or t-butylbicyclophosphoro[35S]thionate and, to a lesser extent, [3H]muscimol to recombinant alpha1beta1gamma2(delta), alpha4beta1gamma2(delta) and alpha6beta1gamma2(delta) receptors, paralleled by diminished GABA-evoked maximal currents in electrophysiological recordings for the latter one. The delta subunit gave rise to a lower EC50 for GABA and a slowed desensitization indicating its assembly in alpha6beta2delta, alpha6beta1gamma2delta and alpha6beta2gamma2delta receptors. The data show that the delta subunits assemble in various functional GABA(A) receptor subtypes in vitro to reduce GABA-evoked maximal currents and ligand binding, but increase the potency for GABA.     

A-kinase anchoring protein 79/150 facilitates the phosphorylation of GABA(A) receptors by cAMP-dependent protein kinase via selective interaction with receptor beta subunits

GABA(A) receptors, the key mediators of fast synaptic inhibition in the brain, are predominantly constructed from alpha(1-6), beta(1-3), gamma(1-3), and delta subunit classes. Phosphorylation by cAMP-dependent protein kinase (PKA) differentially regulates receptor function dependent upon beta subunit identity, but how this kinase is selectively targeted to GABA(A) receptor subtypes remains unresolved. Here we establish that the A-kinase anchoring protein 150 (AKAP150), directly binds to the receptor beta1 and beta3, but not to alpha1, alpha2, alpha3, alpha6, beta2, gamma2, or delta subunits. Furthermore, AKAP79/150 is critical for PKA-mediated phosphorylation of the receptor beta3 subunit. Together, our observations suggest a mechanism for the selective targeting of PKA to GABA(A) receptor subtypes containing the beta1 or beta3 subunits dependent upon AKAP150. Therefore, the selective interaction of beta subunits with AKAP150 may facilitate GABA(A) receptor subtype-specific functional modulation by PKA activity which may have profound local effects on neuronal excitation.     

Identification of amino acid residues within GABA(A) receptor beta subunits that mediate both homomeric and heteromeric receptor expression.

GABA(A) receptors are believed to be heteropentamers that can be constructed from six subunit classes: alpha(1-6), beta(1-4), gamma(1-3), delta, epsilon, and pi. Given that individual neurons often express multiple receptor subunits, it is important to understand how these receptors assemble. To determine which domains of receptor subunits control assembly, we have exploited the differing capabilities of the beta2 and beta3 subunits to form functional cell surface homomeric receptors. Using a chimeric approach, we have identified four amino acids in the N-terminal domain of the beta3 subunit that mediate functional cell surface expression of this subunit compared with beta2, which is retained within the endoplasmic reticulum. Substitution of these four amino acids-glycine 171, lysine 173, glutamate 179, and arginine 180-into the beta2 subunit was sufficient to enable the beta2 subunit to homo-oligomerize. The effect of this putative "assembly signal" on the production of heteromeric receptors composed of alphabeta and betagamma subunits was also analyzed. This signal was not critical for the formation of receptors composed of either alpha1beta2 or alpha1beta3 subunits, suggesting that mutation of these residues did not disrupt subunit folding. However, this signal was important in the formation of betagamma2 receptors. These residues did not seem to affect the initial association of beta2 and gamma2 subunits but appeared to be important for the subsequent production of functional receptors. Our studies identify, for the first time, key residues within the N-terminal domains of receptor beta subunits that mediate the selective assembly of GABA(A) receptors.     

Expression profiling of genes encoding glutamate and GABA receptor subunits in three immortalized GnRH cell lines

Gonadotropin-releasing hormone (GnRH) plays a central role in regulating development and function of the reproductive axis, and its secretion is known to be influenced by glutamate and GABA. In the present study, we used gene microarrays and RT-PCR to compare the expression profiles of glutamate and GABA receptor subunits in three immortalized GnRH cell lines: GT1-1, GT1-7, and Gn10. All of these cell lines expressed the AMPA glutamate receptor subunit genes GluR2 and GluR4, but only the GT1-1 and GT1-7 cells expressed the kainate glutamate receptor subunit gene KA2. Additionally, GluRdelta2, a subunit that can form heteromeric receptors with kainate and AMPA subunits, was present in GT1-1 and Gn10 cells but not in GT1-7 cells. Genes encoding the GABA(A) receptor alpha3, beta2, beta3, epsilon, and pi subunits, as well as the GABA(B) receptor 1 subunit, were evident in all three cell lines. However, the gene encoding the expression of GABA(A) receptor gamma subunit was noticeably absent. Taken together, these data demonstrate comprehensive screening of neurotransmitter receptor genes in a controlled neuronal culture system, and reveal novel features.     

Distribution of the major gamma-aminobutyric acid(A) receptor subunits in the basal ganglia and associated limbic brain areas of the adult rat

Within the basal ganglia, gamma-aminobutyric acid (GABA) exerts a fundamental role as neurotransmitter of local circuit and projection neurons. Its fast hyperpolarizing action is mediated through GABA(A) receptors. These ligand-gated chloride channels are assembled from five subunits, which derive from multiple genes. Using immunocytochemistry, we investigated the distribution of 12 major GABA(A) receptor subunits (alpha1-5, beta1-3, gamma1-3, and delta) in the basal ganglia and associated limbic brain areas of the rat. Immunoreactivity for an additional subunit (subunit alpha6) was not observed. The striatum, the nucleus accumbens, and the olfactory tubercle displayed strong, diffuse staining for the subunits alpha2, alpha4, beta3, and delta presumably located on dendrites of the principal medium spiny neurons. Subunit alpha1-, beta2-, and gamma2-immunoreactivities were apparently mostly restricted to interneurons of these areas. In contrast, the globus pallidus, the entopeduncular nucleus, the ventral pallidum, the subthalamic nucleus, and the substantia nigra pars reticulata revealed dense networks of presumable dendrites of resident projection neurons, which were darkly labeled for subunit alpha1-, beta2-, and gamma2-immunoreactivities. The globus pallidus, ventral pallidum, entopeduncular nucleus, and substantia nigra pars reticulata, all areas receiving innervations from the striatum, displayed strong subunit gamma1-immunoreactivity compared to other brain areas. In the substantia nigra pars compacta and in the ventral tegmental area, numerous presumptive dopaminergic neurons were labeled for subunits alpha3, gamma3, and/or delta. This highly heterogeneous distribution of individual GABA(A) receptor subunits suggests the existence of differently assembled, and presumably also functionally different, GABA(A) receptors within individual nuclei of the basal ganglia and associated limbic brain areas.     

Long-range interactions in neuronal gene expression: evidence from gene targeting in the GABA(A) receptor beta2-alpha6-alpha1-gamma2 subunit gene cluster

Clustering of GABA(A) receptor alpha1, alpha6, beta2, and gamma2 subunit genes on mouse chromosome 11/human chromosome 5 may have functional significance for coordinating expression patterns, but until now there has been no evidence for cross-talk between the genes. However, altering the structure of the alpha6 gene, specifically expressed in the cerebellum, with neomycin gene insertions in two different experiments unexpectedly reduced the expression of the widespread alpha1 and beta2 genes in the forebrain. There were corresponding reductions in the levels of alpha1 and beta2 subunit proteins and in autoradiographic ligand binding densities to GABA(A) receptors in the forebrain of alpha6-/- mice. The gamma2 mRNA level was not changed, nor were beta3 and delta mRNAs. The data suggest that elements in the neo gene may have an influence over long distances in the GABA(A) subunit gene complex on as yet undefined structures coordinating the expression of the alpha1 and beta2 genes.     

GABA(A) receptor changes in delta subunit-deficient mice: altered expression of alpha4 and gamma2 subunits in the forebrain

The delta subunit is a novel subunit of the pentameric gamma-aminobutyric acid (GABA)(A) receptor that conveys special pharmacological and functional properties to recombinant receptors and may be particularly important in mediating tonic inhibition. Mice that lack the delta subunit have been produced by gene-targeting technology, and these mice were studied with immunohistochemical and immunoblot methods to determine whether changes in GABA(A) receptors were limited to deletion of the delta subunit or whether alterations in other GABA(A) receptor subunits were also present in the delta subunit knockout (delta-/-) mice. Immunohistochemical studies of wild-type mice confirmed the restricted distribution of the delta subunit in the forebrain. Regions with moderate to high levels of delta subunit expression included thalamic relay nuclei, caudate-putamen, molecular layer of the dentate gyrus, and outer layers of the cerebral cortex. Virtually no delta subunit labeling was evident in adjacent regions, such as the thalamic reticular nucleus, hypothalamus, and globus pallidus. Comparisons of the expression of other subunits in delta-/- and wild-type mice demonstrated substantial changes in the alpha4 and gamma2 subunits of the GABA(A) receptor in the delta-/- mice. gamma2 Subunit expression was increased, whereas alpha4 subunit expression was decreased in delta-/- mice. Importantly, alterations of both the alpha4 and the gamma2 subunits were confined primarily to brain regions that normally expressed the delta subunit. This suggests that the additional subunit changes are directly linked to loss of the delta subunit and could reflect local changes in subunit composition and function of GABA(A) receptors in delta-/- mice.     

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

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

Regional and cellular localisation of GABA(A) receptor subunits in the human basal ganglia: An autoradiographic and immunohistochemical study.

The regional and cellular localisation of gamma-aminobutyric acid(A) (GABA(A)) receptors was investigated in the human basal ganglia using receptor autoradiography and immunohistochemical staining for five GABA(A) receptor subunits (alpha(1), alpha(2), alpha(3), beta(2, 3), and gamma(2)) and other neurochemical markers. The results demonstrated that GABA(A) receptors in the striatum showed considerable subunit heterogeneity in their regional distribution and cellular localisation. High densities of GABA(A) receptors in the striosome compartment contained the alpha(2), alpha(3), beta(2, 3), and gamma(2) subunits, and lower densities of receptors in the matrix compartment contained the alpha(1), alpha(2), alpha(3), beta(2,3), and gamma(2) subunits. Also, six different types of neurons were identified in the striatum on the basis of GABA(A) receptor subunit configuration, cellular and dendritic morphology, and chemical neuroanatomy. Three types of alpha(1) subunit immunoreactive neurons were identified: type 1, the most numerous (60%), were medium-sized aspiny neurons that were immunoreactive for parvalbumin and alpha(1), beta(2,3), and gamma(2) subunits; type 2 (38%) were medium-sized to large aspiny neurons immunoreactive for calretinin and alpha(1), alpha(3), beta(2,3), and gamma(2) subunits; and type 3 (2%) were large sparsely spiny neurons immunoreactive for alpha(1), alpha(3), beta(2,3), and gamma(2) subunits. Type 4 neurons were calbindin-positive and immunoreactive for alpha(2), alpha(3), beta(2,3), and gamma(2) subunits. The remaining neurons were immunoreactive for choline acetyltransferase (ChAT) and alpha(3) subunit (type 5) or were neuropeptide Y-positive with no GABA(A) receptor subunit immunoreactivity (type 6). The globus pallidus contained three types of neurons: types 1 and 2 were large neurons and were immunoreactive for alpha(1), alpha(3), beta(2,3), and gamma(2) subunits and for parvalbumin alone (type 1) or for both parvalbumin and calretinin (type 2); type 3 neurons were medium-sized and immunoreactive for calretinin and alpha(1), beta(2, 3), and gamma(2) subunits. These results show that the subunit composition of GABA(A) receptors displays considerable regional and cellular variation in the human striatum but are more homogeneous in the globus pallidus.     

Decreased expression of the GABAA receptor in fragile X syndrome

After our initial discovery of under expression of the GABA(A) receptor delta subunit in a genome wide screening for differentially expressed mRNAs in brain of fragile X mice, a validated model for fragile X mental retardation syndrome, we analyzed expression of the 17 remaining subunits of the GABA(A) receptor using real-time PCR. We confirmed nearly 50% under expression of the delta subunit and found a significant 35%-50% reduction in expression of 7 additional subunit mRNAs, namely alpha(1), alpha(3), and alpha(4), beta(1) and beta(2) and gamma(1) and gamma(2), in fragile X mice compared to wild-type littermates. In concordance with previous results, under expression was found in cortex, but not in cerebellum. Moreover, decreased expression of specific GABA(A) receptor subunits in fragile X syndrome seems to be an evolutionary conserved hallmark since in the fragile X fly (Drosophila melanogaster) model we also found almost 50% under expression of all 3 subunits which make up the invertebrate GABA receptor, namely Grd, Rdl and Lcch3. In addition, we demonstrated a direct correlation between the amount of dFmrp and the expression of the GABA receptor subunits Rdl and Grd. Our results add evidence to previous observations of an altered GABAergic system in fragile X syndrome. Because GABA(A) receptors are the major inhibitory receptors in brain, involved in anxiety, depression, insomnia, learning and memory and epilepsy, processes also disturbed in fragile X patients, the well described GABA(A) receptor pharmacology might open new powerful opportunities for treatment of the behavioral and epileptic phenotype associated with fragile X syndrome.     

CaMK-II modulation of GABA(A) receptors expressed in HEK293, NG108-15 and rat cerebellar granule neurons

The gamma-aminobutyric acid type A (GABA(A)) receptor is a pentameric ligand-gated ion channel responsible for fast synaptic inhibition in the brain. Phosphorylation of the GABA(A) receptor by serine/threonine protein kinases, at residues located in the intracellular loop between the third and fourth transmembrane domains of each subunit, can dynamically modulate receptor trafficking and function. In this study, we have assessed the effect that Ca(2+)-calmodulin-dependent protein kinase-II (CaMK-II) has on GABA(A) receptors. The intracellular application of preactivated CaMK-II failed to modulate the function of alphabeta and alphabetagamma subunit GABA(A) receptors heterologously expressed in human embryonic kidney (HEK)293 cells. However, application of similarly preactivated alpha-CaMK-II significantly potentiated the amplitudes of whole-cell GABA currents recorded from rat cultured cerebellar granule neurons and from recombinant GABA(A) receptors expressed in neuroblastoma, NG108-15, cells. The modulation by alpha-CaMK-II of current amplitude depended upon the subunit composition of GABA(A) receptors. alpha-CaMK-II potentiated GABA currents recorded from alpha1beta3 and alpha1beta3gamma2 GABA(A) receptors, but was unable to functionally modulate beta2 subunit-containing receptors. Similar results were obtained from beta2 -/- mouse cerebellar granule cell cultures and from rat granule cell cultures overexpressing recombinant alpha1beta2 or alpha1beta3 GABA(A) receptors. alpha-CaMK-II had a greater effect on the modulation of GABA responses mediated by alpha1beta3gamma2 compared with alpha1beta3 receptors, indicating a possible role for the gamma2 subunit in CaMK-II-mediated phosphorylation. In conclusion, CaMK-II can upregulate the function of GABA(A) receptors expressed in neurons or a neuronal cell line that is dependent on the beta subunit co-assembled into the receptor complex.     

Alterations in the expression of GABAA receptor subunits in cerebellar granule cells after the disruption of the alpha6 subunit gene

Any given subunit of the heteromultimeric type-A gamma-aminobutyric acid (GABA) GABAA receptor may be present in several receptor subtypes expressed by individual neurons. Changes in the expression of a subunit may result in differential changes in the expression of other subunits depending on the subunit composition of the receptor subtype, leading to alterations in neuronal responsiveness to GABA. We used the targeted disruption of the alpha6 subunit gene to test for changes in the expression of other GABAA receptor subunits. Immunoprecipitation and ligand binding experiments indicated that GABAA receptors were reduced by approximately 50% in the cerebellum of alpha6 -/- mice. Western blot experiments indicated that the alpha6 subunit protein completely disappeared from the cerebellum of alpha6 -/- mice, which resulted in the disappearance of the delta subunit from the plasma membrane of granule cells. The amount of beta2, beta3 and gamma2 subunits was reduced by approximately 50%, 20% and 40%, respectively, in the cerebella of alpha6 -/- mice. A comparison of the reduction in the level of alpha1, beta2, beta3, gamma2, or delta-subunit-containing receptors in alpha6 -/- cerebellum with those observed after removal of alpha6-subunit-containing receptors from the cerebella of alpha6 +/+ mice by immuno-affinity chromatography demonstrated the presence of a significantly higher than expected proportion of receptors containing beta3 subunits in alpha6 -/- mice. The receptors containing alpha1, beta2, beta3 and gamma2 subunits were present in the plasma membrane of granule cells of alpha6 -/- mice at both synaptic and extrasynaptic sites, as shown by electron microscopic immunocytochemistry. Despite the changes, the alpha1 subunit content of Golgi-cell-to-granule-cell synapses in alpha6 -/- animals remained unaltered, as did the frequency of alpha1 immunopositive synapses in the glomeruli. Furthermore, no change was apparent in the expression of the alpha1, beta2 and gamma2 subunits in Purkinje cells and interneurons of the molecular layer. These results demonstrate that in alpha6 -/- mice, the cerebellum expresses only half of the number of GABAA receptors present in wild-type animals. Since these animals have no gross motor deficits, synaptic integration in granule cells is apparently maintained by alpha1-subunit-containing receptors with an altered overall subunit composition, and/or by changes in the expression of other ligand and voltage gated channels.     

Increased expression of GABA(A) receptor beta-subunits in the hippocampus of patients with temporal lobe epilepsy

It has been postulated that dysfunction of the GABA-ergic transmission is causatively related to the development of epilepsy. Animal models of temporal lobe epilepsy (TLE) revealed considerable changes in the expression of GABA(A) receptor subunits in the hippocampus. Using immunocytochemistry, we investigated the expression of GABA(A) receptor subunits alpha1, alpha3, beta1-3, and gamma2 in hippocampal specimens obtained at surgery from TLE patients with and without hippocampal sclerosis and in autopsy controls. Consistent with the severe neurodegeneration in the CA1 sector, significant decreases in alpha1-, alpha3-, beta3-, and gamma2-subunit immunoreactivity (IR) were detected in sclerotic but not in nonsclerosic specimens. In contrast, pronounced increases in IR of all 3 beta-subunits were observed in most sectors of the hippocampal formation both in sclerotic and nonsclerotic specimens, being especially pronounced in the dentate molecular layer and in the subiculum where subunit alpha3- and gamma2-IR were also elevated. Using in situ hybridization for subunits beta2 and beta3, increased expression of the respective mRNAs was detected in dentate granule cells of patients with and without hippocampal sclerosis. Beta-subunits are important constituents of the GABA(A) receptor and contribute to the binding site of GABA. Our data indicate pronounced adaptive changes in the expression of these GABA(A) receptor subunits related to seizure activity and indicate altered assembly of GABA(A) receptors in TLE.     

The promiscuous role of the epsilon subunit in GABAA receptor biogenesis

The formation of alpha1beta2gamma2epsilon receptors suggests that the epsilon subunit does not displace the single gamma2 subunit in alpha1beta2gamma2 receptors. Thus, epsilon must replace alpha and/or beta subunit(s) if the pentameric receptor structure is to be preserved. To assess the potential for which subunit is replaced in alphabetaepsilon and alphabetagammaepsilon receptors we analyzed the assembly and functional expression of the epsilon subunit with respect to alpha1, beta2 and gamma2 subunits. Using concatenated subunits, we have determined that epsilon is capable of substituting for either (but not both) of the alpha subunits, one of the beta subunits, and possibly the gamma2 subunit. However, the most likely sites at which the epsilon subunit may contribute to receptor function appears to be at position 1 (replaces alpha1) in alphabetagammaepsilon (epsilon-beta2-alpha1-beta2-gamma2) receptors, or at position 4 (replaces beta2) in alphabetaepsilon (alpha1-beta2-alpha1-epsilon-beta2) receptors. In both cases, it appears that only a single GABA binding site is present.     

Molecular basis underlying GABA(A) responses in rat mesencephalic trigeminal neurons

We studied the molecular basis of GABA(A) receptor (GABA(A)R) expressed in the rat mesencephalic trigeminal (Vmes) sensory neuron using the immunohistochemical and single-cell RT-PCR methods. Using anti-GABA(A)R alpha2 subunit antibody, abundant GABA(A)Rs were visualized in Vmes neurons. A single-cell RT-PCR clarified that GABA(A)Rs expressed in Vmes neurons were predominantly composed of alpha2, alpha5, beta1, gamma1 and gamma2 subunits. Novel splicing variants in both alpha5 and beta1 were found invariably, and they lacked multiple amino acid sequence in the extracellular N-terminal portion. Known functional roles of both beta and gamma subunits in regulating the expression at the cell surface suggest that the unique subunit composition of GABA(A)Rs may be involved in the characteristics of GABA(A) response in Vmes neurons.     

Functional analysis of GABA(A) receptors in nucleus tractus solitarius neurons from neonatal rats.

To gain insight into specific GABA(A) receptor configurations functionally expressed in the nucleus tractus solitarius (NTS), we conducted several physiological and pharmacological assessments. NTS neurons were characterized in thin brain slices from 1-14 day old rats using whole-cell patch clamp recordings. GABA(A-) receptor-mediated currents were detected in all neurons tested, with an average EC(50) of 22.2 microM. GABA currents were consistently stimulated by diazepam (EC(50)=63 nM), zolpidem (EC(50)=85 nM), loreclezole (EC(50)=10.1 microM) and the neurosteroid 5alpha-pregnan-3alpha-hydroxy-20-one (3alpha-OH-DHP). In contrast, GABA-gated currents of the NTS were inhibited by the divalent cation Zn(2+) (IC(50)=33.6 microM) picrotoxin (IC(50)=2.4 microM) and blockade of endogenous protein tyrosine kinase. GABA-activated currents were insensitive to furosemide (10-1000 microM) in all NTS neurons tested. Collectively, the data suggest that in neonatal rats, the predominant alpha subunit isoform present in GABA(A) receptors of the NTS appears to be the alpha1 and/or alpha2 subunit. beta2 and/or beta3 subunits are the major beta isoform, while the predominant gamma subunit is likely gamma2. Our data suggest the contribution to NTS GABA currents by alpha3-alpha6, beta1, gamma1 and delta subunits, if present, is minor by comparison.