Different Subtypes of GABA-A Receptors Are Expressed in Human, Mouse and Rat T Lymphocytes

γ-aminobutyric acid (GABA) is the most prominent neuroinhibitory transmitter in the brain, where it activates neuronal GABA-A receptors (GABA-A channels) located at synapses and outside of synapses. The GABA-A receptors are primary targets of many clinically useful drugs. In recent years, GABA has been shown to act as an immunomodulatory molecule. We have examined in human, mouse and rat CD4(+) and CD8(+) T cells which subunit isoforms of the GABA-A channels are expressed. The channel physiology and drug specificity is dictated by the GABA-A receptor subtype, which in turn is determined by the subunit isoforms that make the channel. There were 5, 8 and 13 different GABA-A subunit isoforms identified in human, mouse and rat CD4(+) and CD8(+) T cells, respectively. Importantly, the γ2 subunit that imposes benzodiazepine sensitivity on the GABA-A receptors, was only detected in the mouse T cells. Immunoblots and immunocytochemistry showed abundant GABA-A channel proteins in the T cells from all three species. GABA-activated whole-cell transient and tonic currents were recorded. The currents were inhibited by picrotoxin, SR95531 and bicuculline, antagonists of GABA-A channels. Clearly, in both humans and rodents T cells, functional GABA-A channels are expressed but the subtypes vary. It is important to bear in mind the interspecies difference when selecting the appropriate animal models to study the physiological role and pharmacological properties of GABA-A channels in CD4(+) and CD8(+) T cells and when selecting drugs aimed at modulating the human T cells function.     

Cyclooxygenase-2 expression in astrocytomas. Relationship with microvascular parameters, angiogenic factors expression and survival

OBJECTIVE: Cyclooxygenase-2 (COX-2) is the enzyme isoform involved in the synthesis of prostaglandins (PGs) and thromboxane from arachidonic acid. The role of the up-regulation of COX-2 in the formation and progression of gliomas has been dealt with in earlier reports, which describe increased levels of PGs within gliomas. In the present study, we examined the expression of COX-2 in diffuse gliomas of astrocytic origin in relation to microvascular parameters, angiogenic factors and survival. MATERIALS AND METHODS: A total of 83 cases of diffuse astrocytomas (grade II-IV) were analyzed by immunohistochemistry for the presence of COX-2. RESULTS: COX-2 expression was detected in 79 cases (95%) with an increased expression in grade IV as compared to grades II/III (p=0.024). A positive correlation occurred between COX-2 and angiogenic factors such as vascular endothelial growth factor (VEGF) (p<0.0001) and hypoxia inducible factor (HIF)-1alpha (p=0.005), as well as the tumours' proliferative activity (expressed as the percentage of Ki-67 positive cells) (p=0.032), and total vascular area (TVA) (p=0.040). In univariate analysis, COX-2 was associated with shortened survival (p = 0.050). Multivariate survival analysis showed that the interaction model of COX-2 with grade along with age were the only significant prognostic indicators. CONCLUSION: These results implicate COX-2 in the angiogenesis and biological aggressiveness of diffuse astrocytomas, and suggest that it would be worthwhile to examine how the inhibition of COX-2 expression may influence astrocytoma patients' survival.     

Pharmacological analysis of the activation and receptor properties of the tonic GABA(C)R current in retinal bipolar cell terminals

GABAergic inhibition in the central nervous system (CNS) can occur via rapid, transient postsynaptic currents and via a tonic increase in membrane conductance, mediated by synaptic and extrasynaptic GABA(A) receptors (GABA(A)Rs) respectively. Retinal bipolar cells (BCs) exhibit a tonic current mediated by GABA(C)Rs in their axon terminal, in addition to synaptic GABA(A)R and GABA(C)R currents, which strongly regulate BC output. The tonic GABA(C)R current in BC terminals (BCTs) is not dependent on vesicular GABA release, but properties such as the alternative source of GABA and the identity of the GABA(C)Rs remain unknown. Following a recent report that tonic GABA release from cerebellar glial cells is mediated by Bestrophin 1 anion channels, we have investigated their role in non-vesicular GABA release in the retina. Using patch-clamp recordings from BCTs in goldfish retinal slices, we find that the tonic GABA(C)R current is not reduced by the anion channel inhibitors NPPB or flufenamic acid but is reduced by DIDS, which decreases the tonic current without directly affecting GABA(C)Rs. All three drugs also exhibit non-specific effects including inhibition of GABA transporters. GABA(C)R ρ subunits can form homomeric and heteromeric receptors that differ in their properties, but BC GABA(C)Rs are thought to be ρ1-ρ2 heteromers. To investigate whether GABA(C)Rs mediating tonic and synaptic currents may differ in their subunit composition, as is the case for GABA(A)Rs, we have examined the effects of two antagonists that show partial ρ subunit selectivity: picrotoxin and cyclothiazide. Tonic and synaptic GABA(C)R currents were differentially affected by both drugs, suggesting that a population of homomeric ρ1 receptors contributes to the tonic current. These results extend our understanding of the multiple forms of GABAergic inhibition that exist in the CNS and contribute to visual signal processing in the retina.     

5-(N,N-Hexamethylene) amiloride is a GABA-A ρ1 receptor positive allosteric modulator

Guanidine compounds act as ion channel modulators. In the case of Cys-loop receptors, the guanidine compound amiloride antagonized the heteromeric GABA-A, glycine, and nicotinic acetylcholine receptors. However, amiloride exhibits characteristics consistent with a positive allosteric modulator for the human GABA-A (hGABA-A) ρ1 receptor. Site-directed mutagenesis revealed that the positive allosteric modulation was influenced by the GABA-A ρ1 second transmembrane domain 15′ position, a site implicated in ligand allosteric modulation of Cys-loop receptors. There are a variety of amiloride derivatives that provide opportunities to assess the significance of amiloride functional groups (e.g., the guanidine group, the pyrazine ring, etc.) in the modulation of the GABA-A ρ1 receptor activity. We utilized 3 amiloride derivatives (benzamil, phenamil, and 5-(N, N-Hexamethylene) amiloride) to assess the contribution of these groups toward the potentiation of the GABA-A ρ1 receptor. Benzamil and phenamil failed to potentiate on the wild type GABA-A ρ1 GABA-mediated current while HMA demonstrated efficacy only at the highest concentration studied. The hGABA-A ρ1 (I15'N) mutant receptor activity was potentiated by lower HMA concentrations compared to the wild type receptor. Our findings suggest that an exposed guanidine group on amiloride and amiloride derivatives is critical for modulating the GABA-A ρ1 receptor. The present study provides a conceptual framework for predicting which amiloride derivatives will demonstrate positive allosteric modulation of the GABA-A ρ1 receptor.     

Effects of treatment with GABA(A) receptor subunit antisense oligodeoxynucleotides on GABA-stimulated 36Cl- influx in the rat cerebral cortex

GABA(A) receptor function was studied in cerebral cortical vesicles prepared from rats after intracerebroventricular microinjections of antisense oligodeoxynucleotides (aODNs) for alpha1, gamma2, beta1, beta2 subunits. GABA(A) receptor alpha1 subunit aODNs decreased alpha1 subunit mRNA by 59+/-10%. Specific [3H]GABA binding was decreased by alpha1 or beta2 subunit aODNs (to 63+/-3% and 64+/-9%, respectively) but not changed by gamma2 subunit aODNs (94+/-5%). Specific [3H]flunitrazepam binding was increased by alpha1 or beta2 subunit aODNs (122+/-8% and 126+/-11%, respectively) and decreased by gamma2 subunit aODNs (50+/-13%). The "knockdown" of specific subunits of the GABA(A )receptor significantly influenced GABA-stimulated 36Cl- influx. Injection of alpha1 subunit aODNs decreased basal 36Cl- influx and the GABA Emax; enhanced GABA modulation by diazepam; and decreased antagonism of GABA activity by bicuculline. Injection of gamma2 subunit aODNs increased the GABA Emax; reversed the modulatory efficacy of diazepam from enhancement to inhibition of GABA-stimulation; and reduced the antagonist effect of bicuculline. Injection of beta2 subunit aODNs reduced the effect of diazepam whereas treatment with beta1 subunit aODNs had no effect on the drugs studied. Conclusions from our studies are: (1) alpha1 subunits promote, beta2 subunits maintain, and gamma2 subunits suppress GABA stimulation of 36Cl- influx; (2) alpha1 subunits suppress, whereas beta2, and gamma2 subunits promote allosteric modulation by benzodiazepines; (3) diazepam can act as an agonist or inverse agonist depending on the relative composition of the receptor subunits: and (4) the mixed competitive/non-competitive effects of bicuculline result from activity at alpha1 and gamma2 subunits and the lack of activity at beta1 and beta2 subunits.     

Interactions between rho and gamma2 subunits of the GABA receptor

The gamma-aminobutyric acid type C (GABA(C)) receptor is a ligand-gated chloride channel with distinct physiological and pharmacological properties. Although the exact subunit composition of native GABA(C) receptors has yet to be firmly established, there is general agreement that GABA rho subunits participate in their formation. Recent studies on white perch suggest that certain GABA rho subunits can co-assemble with the GABA(A) receptor gamma2 subunit to form a heteromeric receptor with electrophysiological properties that correspond more closely to the native GABA(C) receptor on retinal neurons than any of the homomeric rho receptors. In the present study we examined the interactions among various perch GABA rho and gamma2 subunits. When co-expressed in Xenopus oocytes, the gamma2 subunit co-immunoprecipitated with Flag-tagged perch rho1A, rho1B, and rho2B subunits, but not with the Flag-tagged perch rho2A subunit. Immunocytochemical studies indicated that the membrane surface expression of the gamma2 subunit was detected only when it was co-expressed with perch rho1A, rho1B, or rho2B subunit, but not with the perch rho2A subunit or when expressed alone. In addition, co-immunoprecipitation of perch rho1B and gamma2 subunits was also detected in protein samples of the teleost retina. Taken together, these findings suggest that a heteromeric rho(gamma2) receptor could represent one form of GABA(C) receptor on retinal neurons.     

An mRNA Encoding a Putative GABA-Gated Chloride Channel Is Expressed in the Human Cardiac Conduction System

Abstract: GABA-gated chloride channels are the main inhibitory neurotransmitter receptors in the CNS. Conserved domains among members of previously described GABA_A receptor subunits were used to design degenerate sense and antisense oligonucleotides. A PCR product from this amplification was used to isolate a full-length cDNA. The predicted protein has many of the features shared by other members of the ligand-gated ion channel family. This channel subunit has significant amino acid identity (25–40%) with members of GABA_A and GABA_C receptor subunits and thus may represent a new subfamily of the GABA receptor channel. Although we cannot rule out that this clone encodes a receptor for an unidentified ligand, it was termed GABA _χ. This gene is mainly expressed in placenta and in heart; however, placenta appears to express only an unspliced mRNA. In situ hybridization reveals that the GABA _χ subunit mRNA is present in the electrical conduction system of the human heart. Our results suggest that novel GABA receptors expressed outside of the CNS may regulate cardiac function.     

Anatomical Involvement of the Subventricular Zone Predicts Poor Survival Outcome in Low-Grade Astrocytomas

The subventricular zone (SVZ) has been implicated in the origination, development, and biological behavior of gliomas. Tumor-SVZ contact is also postulated to be a poor prognostic factor in glioblastomas. We aimed to evaluate the prognostic consequence of the anatomical involvement of low-grade gliomas with the SVZ. To that end, we reviewed 143 patients with diffuse astrocytomas, and tumor lesions were manually delineated on magnetic resonance images. We initially investigated the prognostic role of SVZ contact in all patients. Additionally, we investigated the influence of the anatomical proximity of the tumor lesion centroids to the SVZ in the SVZ-involved patient cohorts, as well as location within the SVZ. We found SVZ contact with tumors to be a significant prognostic factor of overall survival in all patients with diffuse astrocytomas (p = 0.027). In the SVZ-involved cohort, a shorter distance from the tumor centroid to the SVZ (≤30 mm) correlated with shorter overall survival (p = 0.022) on univariate analysis. However, there was no significant difference in overall survival with respect to the SVZ region involved with the tumor (p = 0.930). Multivariate analysis showed that a shorter distance between the tumor centroid and the SVZ (p = 0.039) was significantly associated with poor overall survival in SVZ-involved patients. Hence, this study helps establish the prognostic role of the anatomical interaction of tumors with the SVZ in low-grade astrocytomas.     

Structure and subunit composition of GABA(A) receptors.

GABA(A) receptors are the major inhibitory neurotransmitter receptors in the brain and are the site of action of many clinically important drugs. These receptors are composed of five subunits that can belong to eight different subunit classes. If all GABA(A) receptor subunits could randomly combine with each other, an extremely large number of GABA(A) receptor subtypes with distinct subunit composition and arrangement would be formed. Depending on their subunit composition, these receptors would exhibit distinct pharmacological and electrophysiological properties. Recent evidence, however, indicates that not all subunits can assemble efficiently with each other and form functional homo- or hetero-oligomeric receptors. In addition, the efficiency of formation of hetero-oligomeric assembly intermediates determines the subunit stoichiometry and subunit arrangement for each receptor and thus further reduces the possible heterogeneity of GABA(A) receptors in the brain. Studies investigating the subunit composition of native GABA(A) receptors support this conclusion, but also indicate that receptors composed of one, two, three, four, or five different subunits might exist in the brain. Using a recently established immunodepletion technique, the subunit composition and quantitative importance of native GABA(A) receptor subtypes can be determined. This information, together with studies on the regional, cellular and subcellular distribution of these receptor subtypes, will be the basis for a rational development of drugs that specifically affect the GABAergic system.     

Interactions between GABA-B1 receptors and Kir 3 inwardly rectifying potassium channels

γ-aminobutyric acid (GABA) is the principal inhibitory neurotransmitter in the mammalian brain. It acts via both ionotropic GABA-A and metabotropic GABA-B receptors. We evaluated the interaction of receptors with members of the inwardly rectifying potassium (Kir 3) channel family, which also play an important role in neuronal transmission and membrane excitability. These channels are functionally regulated by GABA-B receptors. Possible physical interactions between GABA-B receptor and Kir 3 channels expressed in HEK cells were evaluated using Bioluminescence Resonance Energy Transfer (BRET) experiments, co-immunoprecipitation and confocal microscopy. Our data indicate that Kir 3 channels and Gβγ subunits can interact with the GABA-B₁ subunits independently of the GABA-B₂ subunit or Kir 3.4 which are ultimately responsible for their targetting to the cell surface. Thus signalling complexes containing GABA-B receptors, G proteins and Kir channels are formed shortly after biosynthesis most likely in the endoplasmic reticulum.     

Function of gamma-aminobutyric acid receptor/channel rho 1 subunits in spinal cord

gamma-Aminobutyric acid (GABA) receptor/channel rho 1 subunits are important components in inhibitory pathways in the central nervous system. However, the precise locations and roles of these receptors in the central nervous system are unknown. We studied the expression localization of GABA receptor/channel rho 1 subunit in mouse spinal cord and dorsal root ganglia (DRG). The immunohistochemistry results indicated that GABA receptor/channel rho 1 subunits were expressed in mouse spinal cord superficial dorsal horn (lamina I and lamina II) and in DRG. To understand the functions of the GABA receptor/channel rho 1 subunit in these crucial sites of sensory transmission in vivo, we generated GABA receptor/channel rho 1 subunit mutant mice (rho 1-/-). GABA receptor/channel rho 1 subunit expression in the rho 1-/- mice was eliminated completely, whereas the gross neuroanatomical structures of the rho 1-/- mice spinal cord and DRG were unchanged. Electrophysiological recording showed that GABA-mediated spinal cord response was altered in the rho 1-/- mice. A decreased threshold for mechanical pain in the rho 1-/- mice compared with control mice was observed with the von Frey filament test. These findings indicate that the GABA receptor/channel rho 1 subunit plays an important role in modulating spinal cord pain transmission functions in vivo.     

Mapping convulsants' binding to the GABA-A receptor chloride ionophore: a proposed model for channel binding sites

Gamma-aminobutyric acid (GABA) type A receptors play a key role in brain inhibitory neurotransmission, and are ligand-activated chloride channels blocked by numerous convulsant ligands. Here we summarize data on binding of picrotoxin, tetrazoles, beta-lactams, bicyclophosphates, butyrolactones and neurotoxic pesticides to GABA-A ionophore, and discuss functional and structural overlapping of their binding sites. The paper reviews data on convulsants' binding sensitivity to different point mutations in ionophore-lining second trans-membrane domains of GABA-A subunits, and maps possible location of convulsants' sites within the chloride ionophore. We also discuss data on inhibition of glycine, glutamate, serotonin (5-HT3) and N-acetylcholine receptors by GABA-A channel blockers, and examine the applicability of this model to other homologous ionotropic receptors. Positioning various convulsant-binding sites within ionophore of GABA-A receptors, this model enables a better understanding of complex architectonics of ionotropic receptors, and may be used for developing new channel-modulating drugs.     

The mRNAs encoding GABAA/benzodiazepine receptor subunits are localized in different cell populations of the bovine cerebellum

The expression of the mRNAs encoding the alpha and beta subunits of the GABAA/benzodiazepine receptor was examined in the bovine cerebellum by in situ hybridization histochemistry. The alpha subunit mRNA, which encodes the benzodiazepine binding site, was localized in all Purkinje and granule cells and in some cells of the molecular layer. The distribution of the beta subunit mRNA, which encodes the GABA binding site, only partially overlapped with that of the alpha subunit mRNA. While cells in the granule cell layer expressed the beta subunit mRNA, no message could be detected in other cell populations. These findings suggest that the subunit composition of the GABAA/benzodiazepine receptor is heterogeneous and that additional, as yet unidentified, beta subunits exist.     

Antisense suppression of GABA(A) receptor beta subunit levels in cultured cerebellar granule neurons demonstrates their importance in receptor expression

GABA(A) receptors in the CNS are pentameric molecules composed of alpha, beta, gamma, delta, epsilon and theta subunits. Studies on transfected cells have shown that GABA(A) receptor beta subunit isoforms can direct alpha1 subunit localization within the cell. To examine the role of selected subunits in governing GABA(A) receptor expression in neurons, cultures of rat cerebellar granule cells were grown with antisense or sense oligodeoxynucleotides (ODNs) specific for the alpha 1, beta 2 or gamma 2 subunits. These subunits are all expressed in granule neurons where they are thought to contribute to an abundant receptor type. Following ODN treatment, subunit expression and distribution were examined by western blotting, immunocytochemistry and RT-PCR. Treatment of the cultures with the antisense, but not the corresponding sense, ODNs reduced the levels of the targeted subunit polypeptides. In addition, the beta 2 antisense ODN reduced the level of the alpha1 subunit polypeptide without altering the level of its mRNA. In contrast, treatment with the beta 2 subunit antisense ODN did not alter gamma 2 subunit polypeptide expression, distribution or mRNA level. These findings suggest that the alpha1 subunit requires a beta subunit for assembly into GABA(A) receptors in cerebellar granule neurons.     

Molecular and pharmacological properties of GABA-ρ subunits from white perch retina

Five γ-aminobutyric acid (GABA)-ρ subunits were cloned from a white perch retinal cDNA library and expressed in Xenopus oocytes. The deduced amino acid sequences indicated that all are highly homologous to the GABA-ρ subunits cloned from mammalian retinas; two clones (perch-ρ1A and perch-ρ1B) were in the ρ1 family, two (perch-ρ2A and perch-ρ2B) were in the ρ2 family, and one clone has been tentatively identified as a perch-ρ3 subunit. When expressed in Xenopus oocytes, all but one of the subunits (ρ3) formed functional homooligomeric receptors. However, the receptors expressed by each of the GABA-ρ subunits displayed unique response properties that distinguished one from the other. For example, receptors formed by perch-ρ1B subunits were more sensitive to GABA than the receptors formed by other GABA-ρ subunits, the dose–response curves for the various receptors revealed different Hill coefficients, and there were differences in the kinetics of the GABA-induced currents. In addition, the GABA-mediated current–voltage curve for ρ2 receptors was approximately linear, whereas the responses from ρ1 receptors showed outward rectification. A further division in the properties of the GABA-ρ subunits was revealed in their responses to imidazole-4-acetic acid (I4AA); the drug behaved as an antagonist on A-type ρ receptors and a partial agonist on the B-type ρ receptors. These results suggest that there is a large diversity of GABA_c receptors in the vertebrate retina, probably formed by homooligomeric and heterooligomeric combinations of GABA ρ subunits, that exhibit different functional properties. © 1998 John Wiley & Sons, Inc. J Neurobiol 37: 305–320, 1998     

Regionally selective effects of GABA on hypothalamic GABAA receptor mRNA in vitro

We tested whether GABAA receptor (R) subunit mRNA levels are homeostatically influenced by short-term exposure to GABA in two adjacent regions of the posterior hypothalamus. mRNA levels for seven GABAAR subunits and GABA-synthesizing enzyme (GAD) were quantified in the perifornical (PF) and dorsomedial (DM) hypothalamus following superfusion of slices for 90 min with a drug-free medium, GABA uptake blocker with or without GABAAR antagonist, gabazine, or GABAAR agonist with tetrodotoxin. Increasing endogenous GABA decreased mRNAs for all seven GABAAR subunits in the PF, and for three also in the DM, region; gabazine antagonized these effects in the PF region only and increased GAD-65 mRNA. Stimulation of GABAARs in the presence of tetrodotoxin decreased mRNA for one GABAAR subunit (beta1). We conclude that, in the PF region where GABA facilitates sleep, increased GABA release may limit GABAAR-mediated inhibition, whereas in the DM region, GABA-induced changes are mainly mediated by non-GABAA receptors.     

GABAC receptor subunit mRNA expression in the rat superior colliculus is regulated by calcium channels, neurotrophins, and GABAC receptor activity

The distribution of mRNA for the rho2 subunit of the GABA(C) receptor is much broader in organotypic SC cultures than in vivo, suggesting that GABA(C) receptor expression is regulated by environmental factors. Electrophysiological recordings indicate that neurons in SC cultures have functional GABA(C) receptors, although these receptors exhibited smaller conductance than in vivo, probably due to increased rho2 subunit expression. Adding cortical input, treatment with various neuromodulators, and blocking neuronal activity with TTX failed to affect the expression of rho2 subunits. Electrophysiological recordings revealed the presence of spontaneous Ca(2+) currents in SC cultures and preventing these, by treatment with blockers of L-type Ca(2+) channels, caused rho2 mRNA expression to decline to in vivo levels. In contrast, rho1 subunit mRNA levels remained unchanged, indicating that the two subunits are independently regulated. Surprisingly, both tonic activation and blockade of GABA(C) receptors upregulated rho1/rho2 mRNA expression. Further, NGF and BDNF promoted such expression during an early postnatal time window. In vivo, expression of the rho2 mRNA in the SC, and the rho2/rho3 mRNA in the retina increased with age. Expression of the rho2 mRNA in the visual cortex, and the rho1 mRNA in the retina and SC was constant. Subunit mRNA expression was similar in dark-reared animals, indicating that visual experience has no influence. These experiments suggest that GABA(C) receptor expression in the SC is regulated during postnatal development. While visual experience seems to have no influence on GABA(C) receptor subunits, spontaneous calcium currents selectively promote rho2 expression and both rho1 and rho2 are autoregulated both by GABA(C) receptor activity and by neurotrophic factors.     

A single amino acid in the second transmembrane domain of GABA ρ subunits is a determinant of the response kinetics of GABAC receptors

The ρ subunits that constitute the γ‐aminobutyric acid (GABA)_C receptors of retinal neurons form a unique subclass of ligand‐gated chloride channels that give rise to sustained GABA‐evoked currents that exhibit slow offset (deactivation) kinetics. We exploited this property to examine the molecular mechanisms that govern the disparate response kinetics and pharmacology of perch GABA ρ1B and ρ2A subunits expressed in Xenopus oocytes. Using a combination of domain swapping and site‐directed mutagenesis, we identified the residues at amino acid position 320 in the second transmembrane domain as an important determinant of the receptor kinetics of GABA_C receptors. When the site contains a proline residue, as in wild‐type ρ1 subunits, the receptor deactivates slowly; when serine occupies the site, as in wild‐type ρ2 subunits, the time course of deactivation is more rapid. In addition, we found that the same site also altered the pharmacology of GABA ρ receptors, e.g., when the serine residue of the ρ2A receptor was changed to proline, the response of the mutant receptor to imidazole‐4‐acetic acid (I4AA) mimicked that of the ρ1B receptor. However, despite gross changes in receptor pharmacology, the apparent binding affinity for the drug was not significantly altered. These findings provide further evidence that the second transmembrane domain is involved in the gating mechanism that governs the response properties of the various ρ receptor subunits. It is noteworthy that the proline residue in native ρ1 subunits and the serine residue of ρ2 subunits are well conserved in all species, a good indication that the presence of multiple GABA ρ subunits serves to generate GABA_C receptors that display the wide range of response kinetics observed on various types of retinal neurons. © 1999 John Wiley & Sons, Inc. J Neurobiol 40: 67–76, 1999     

Two pools of Triton X-100-insoluble GABA(A) receptors are present in the brain, one associated to lipid rafts and another one to the post-synaptic GABAergic complex

Rat forebrain synaptosomes were extracted with Triton X-100 at 4 degrees C and the insoluble material, which is enriched in post-synaptic densities (PSDs), was subjected to sedimentation on a continuous sucrose gradient. Two pools of Triton X-100-insoluble gamma-aminobutyric acid type-A receptors (GABA(A)Rs) were identified: (i) a higher-density pool (rho = 1.10-1.15 mg/mL) of GABA(A)Rs that contains the gamma2 subunit (plus alpha and beta subunits) and that is associated to gephyrin and the GABAergic post-synaptic complex and (ii) a lower-density pool (rho = 1.06-1.09 mg/mL) of GABA(A)Rs associated to detergent-resistant membranes (DRMs) that contain alpha and beta subunits but not the gamma2 subunit. Some of these GABA(A)Rs contain the delta subunit. Two pools of GABA(A)Rs insoluble in Triton X-100 at 4 degrees C were also identified in cultured hippocampal neurons: (i) a GABA(A)R pool that forms clusters that co-localize with gephyrin and remains Triton X-100-insoluble after cholesterol depletion and (ii) a GABA(A)R pool that is diffusely distributed at the neuronal surface that can be induced to form GABA(A)R clusters by capping with an anti-alpha1 GABA(A)R subunit antibody and that becomes solubilized in Triton X-100 at 4 degrees C after cholesterol depletion. Thus, there is a pool of GABA(A)Rs associated to lipid rafts that is non-synaptic and that has a subunit composition different from that of the synaptic GABA(A)Rs. Some of the lipid raft-associated GABA(A)Rs might be involved in tonic inhibition.