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The influence of neonatal administration of 6-hydroxydopamine (6-OHDA) on the maturation of GABAA receptors in the frontal cortex and hippocampus was studied using 5- to 40-day-old rats. In situ hybridization with antisense oligonucleotide probes was performed for α1, α2, α5, β2, β3 and γ2 subunit mRNAs of the GABAA receptor. We demonstrated that neonatal treatment with 6-OHDA temporarily delays the postnatal transcription of the α1 and γ2 subunits in the rat prefrontal cortex, as assessed by in situ hybridization histochemistry. The effect was selective for these subunits (the α2, α5, β2, and β3 subunit mRNAs remained unchanged) and for this region (the mRNA levels in the hippocampus were not changed). The reduction in mRNA levels at early postnatal stages (postnatal day 5, PD5, and PD10) also affected the subunit protein levels, as shown by immunohistochemistry for the α1 subunit, and the formation of GABAA receptor-associated picrotoxinin-insensitive TBPS binding sites, as shown by autoradiography. Our findings indicate that without a noradrenergic influence, the maturation of GABAergic interneurons in the frontal cortex is transiently delayed (from PD5 to PD40). However, it is possible that this transient reduction of the expression of certain GABA subunits – caused by depletion of noradrenergic innervation – cannot cause a lasting alteration to the GABAergic function in the prefrontal cortex.

Furosemide specifically reverses the inhibition by γ-aminobutyric acid (GABA) of t-[35S]-butylbicyclophosphorothionate ([35S]TBPS) binding and increases the basal [35S]TBPS binding to the cerebellar granule cell layer GABAAreceptors. For the selectivity of furosemide, an interplay between GABAAreceptor α6 and β2 subunits is needed. We have now investigated the furosemide sensitivity of cerebellar [35S]TBPS binding in the alcohol-sensitive (ANT) rat line that harbors a pharmacologically critical point mutation in the α6 subunit [α6(Q100)], increasing benzodiazepine affinity of the normally insensitive α6-containing receptors. ANT receptors were less efficiently affected by furosemide, while a normal GABAAreceptor antagonism was observed with a specific GABAAreceptor antagonist SR 95531. Reduced [3H]muscimol binding in ANT samples and small alterations in in situhybridization signals for α1, α6, β2, β3, γ2 and δ subunit mRNAs failed to correlate with impaired cerebellar furosemide efficacy in individual animals. The α6(Q100) ANT mutation was not responsible for the reduced efficacy of furosemide in the ANT rat line, as judged from the potent furosemide antagonism in recombinant ANT-type α6(Q100)β3γ2 receptors. This data suggest the presence of a novel abnormality in the structure and/or expression of α6 subunit-containing GABAAreceptors in the ANT rat line. Copyright © 1996 Elsevier Science Ltd

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

Numerous ligands affect inhibitory gamma-aminobutyric acid (GABA)A receptors, none of them showing strict receptor subtype specificity. We report here that a cerebellar GABAA receptor subtype can be uniquely modulated by furosemide but not by bumetanide, another Cl-/cation transport blocker. Furosemide specifically reversed the inhibition by GABA of t-[35S]butylbicyclophosphorothionate ([35S]TBPS) binding in the cerebellar granule cell layer, as detected by autoradiography of rat brain sections. With recombinant receptors expressed in Xenopus oocytes, furosemide antagonized potently (IC50, about 10 microM), rapidly, and reversibly GABA-evoked currents of cerebellar granule cell-specific alpha 6 beta 2 gamma 2 receptors but not alpha 1 beta 2 gamma 2 receptors (IC50, > 3 mM). Furosemide reversed GABA inhibition of [35S]TBPS binding and elevated basal [35S]TBPS binding only with alpha 6 beta 2 gamma 2 and alpha 6 beta 3 gamma 2 receptors and not with alpha 6 beta 1 gamma 2 or alpha 1 beta 1/2/3 gamma 2 receptors. It appeared to interact with the receptor complex via a novel recognition site that allosterically regulates the Cl- ionophore. Furosemide is the first subtype-selective GABAA receptor (alpha 6 beta 2/3 gamma 2) antagonist and should facilitate studies on cerebellar physiology. It might serve as a prototypic structure for the development of additional subtype-selective GABAA ligands.

We expressed in cultured cells recombinant gamma-aminobutyric acid type A (GABAA) receptors of the subunit compositions alpha 1 beta j gamma k and alpha 5 beta j gamma k (j = 1, 2, or 3 and k = 2 or 3). A comparison of ligand-binding properties revealed a functional role for individual beta variants, which depended on the alpha subunit in the GABAA receptor. Recombinant alpha 5 beta x gamma 2/3 receptors recognized the cage convulsant t-butylbicyclophosphoro[35S]thionate, as well as the benzodiazepine (BZ) receptor inverse agonist [3H]Ro 15-4513, only with the beta 3 variant. In contrast, the exchange of beta variants in alpha 1 beta x gamma 2 receptors imparted differential modulation of t-butyl-bicyclophosphoro[35S]thionate binding by BZ receptor ligands. The BZ site of gamma 3-containing receptors was partially independent of the accompanying alpha and beta variants. alpha 1/5 beta 3 gamma 3 receptors were zolpidem insensitive but distinguished from alpha 5 beta 3 gamma 2 receptors by high affinity for the partial BZ receptor agonist CI 218,872. The distinct affinities of recombinant receptors for CI 218,872 suggested that the alpha 5 beta 3 gamma 2 receptor is the dominant zolpidem-insensitive GABAA receptor in the brain. Hence, alpha 5 beta 3 gamma 3 recpetors are not a major fraction of the native zolpidem-insensitive receptors, even though their genes are colocalized on mouse chromosome 7 and on human chromosome 15.

gamma-Aminobutyric acid (GABA) modulates the convulsant binding site on GABAA receptors labeled by t-butylbicyclophosphoro[35S] thionate ([35S]TBPS). The modulation varies between different brain regions, reflecting the molecular heterogeneity of the GABAA receptors. In rat brain cryostat sections, the main sensitivity difference to GABA between brain regions was observed within the cerebellum. [35S]TBPS binding in the granule cell layer was more sensitive to GABA than was that in the molecular layer and was detected only after blockade of the GABA agonist sites by the specific GABAA antagonists SR 95531, RU 5135, and bicuculline. This indicates that the [35S]TBPS binding sites in cerebellar granule cells were blocked by endogenous GABA. In contrast, the internal rim of the granule cell layer had a small amount of binding that was largely insensitive to 50 microM GABA. The molecular basis for the sensitivity difference could be traced to the alpha subunits of the GABAA receptor. Expression in human embryonic kidney 293 cells of alpha 6 beta 2 gamma 2 receptors produced [35S] TBPS binding sites that were about 10-fold more sensitive to inhibition by GABA than were those inherent to alpha 1 beta 2 gamma 2 receptors. Coexpression of alpha 6 and beta 2 subunits produced [35S]TBPS binding sites that were largely insensitive to GABA inhibition, resembling in their pharmacological profile the sites in the internal granule cell layer. Furthermore, the differences between alpha 6 beta 2 and alpha 6 beta 2 gamma 2 receptors stress the importance of the gamma 2 subunit for the proper pharmacological fingerprint of the rest of the granule cell layer. The neurosteroid 5 alpha-pregnan-3 alpha-ol-20-one affected the binding in both alpha 1 beta 2 gamma 2 and alpha 6 beta 2 gamma 2 receptors, but inhibition was greater in alpha 6-containing than in alpha 1-containing receptors, suggesting differential coupling of both GABA and neurosteroid sites with the convulsant site. These data might serve as a platform for additional studies to assess the amino acid residues in the two alpha subunits that are critically involved in the allosteric interactions between the GABAA agonist/antagonist or neurosteroid domains and the convulsant site.