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Gamma-ainobutyric acid type A (GABA(A)) receptor ionophore ligand t-[35S]butylbicyclophosphorothionate ([35S]TBPS) was used in an autoradiographic assay on brain cryostat sections to visualize and characterize atypical GABA-insensitive [35S]TBPS binding previously described in certain recombinant GABA(A) receptors and the cerebellar granule cell layer. Picrotoxinin-sensitive but 1-mM GABA-insensitive [35S]TBPS binding was present in the rat cerebellar granule cell layer, many thalamic nuclei, subiculum and the internal rim of the cerebral cortex, amounting in these regions up to 6% of the basal binding determined in the absence of exogenous GABA. Similar binding properties were detected also in human and chicken brain sections. Like the GABA-sensitive [35S]TBPS binding, GABA-insensitive binding was profoundly decreased by pentobarbital, pregnanolone, loreclezole and Mg2+. The binding was reversible and apparently dependent on Cl- ions. Localization of the GABA-insensitive [35S]TBPS binding was not identical to that of high-affinity [3H]muscimol binding and diazepam-insensitive [3H]Ro 15-4513 binding, two previously established receptor subtype-dependent binding heterogeneities in the rat brain. The present study reveals a component of the GABA-ionophore enriched in the thalamus and cerebellar granule cells, possibly representing poorly desensitized or desensitizing receptors.

We searched for subunit correlations for GABA(A) receptor-associated atypically GABA-insensitive [35S]TBPS binding. The homomeric beta3 subunit receptors could be excluded, as GABA-insensitive [35S]TBPS binding was present in beta3-/- mice. Localization of GABA-insensitive [35S]TBPS binding correlated best with those of delta, alpha4 and alpha6 subunit mRNAs. The amounts of GABA-insensitive [35S]TBPS binding components were increased in delta-/- mice, but dramatically reduced in alpha6-/- mice, suggesting a role for alpha6 but excluding delta subunits.

Eight gamma-aminobutyric acid (GABA) mimetics were tested on their ability to differentiate native GABA(A) receptor subtypes present in various rat brain regions. In rat brain cryostat sections, little regional variations by the agonistic actions of muscimol, thiomuscimol, 4,5,6,7-tetrahydroisoazolo(5,4-c)pyridin-3-ol, piperidine-4-sulphonic acid, taurine and beta-alanine on [35S]t-butylbicyclophosphorothionate ([35S]TBPS) binding to GABA(A) receptor channels were found. They were very similar to those found for GABA itself and indicated no direct correlation with single subunit distributions for any of these compounds. Only the low-efficacy GABA mimetic 5-(4-piperidyl)isoxazol-3-ol (4-PIOL) acted like a weak partial agonist or antagonist depending on the brain area. As the cerebellar granule cell layer was relatively insensitive to both modes of action, we tested 4-PIOL in recombinant alpha1beta2gamma2 (widespread major subtype) and alpha6beta2gamma2 (cerebellar granule cell restricted) receptors where it had different effects on GABA-modulated [35S]TBPS binding and on electrophysiological responses. 4-PIOL may thus serve as a potential lead for receptor subtype selective compounds.

The multisubunit gamma-aminobutyric acid type A (GABAA) receptor is heterogeneous in molecular and pharmacological aspects. We used quantitative autoradiographic techniques to generate detailed pharmacological profiles for the binding of the GABAA-receptor ionophore ligand tert-[35S]butylbicyclophosphorothionate ([35S]TBPS) and its modulation by GABA and the GABAA antagonists bicuculline and 2'-(3'-carboxy-2',3'-propyl)-3-amino-6-p-methoxyphenylpyrazinium bromide (SR 95531). Regional differences in the actions of bicuculline and SR 95531 were correlated with the expression of 13 GABAA subunits in brain as reported previously. In some brain regions SR 95531 reduced [35S]TBPS binding much more than bicuculline, as illustrated by high ratios of bicuculline- to SR 95531-modulated [35S]TBPS binding. This ratio correlated positively with alpha 2-subunit mRNA levels. Binding that was equally affected by SR 95531 and bicuculline occurred prominently in regions with abundant alpha 1 mRNA expression. The present findings thus reveal a novel pharmacological heterogeneity based on differences between alpha 1 and alpha 2 subunit-containing GABAA receptors. The data aid in developing GABAA-receptor subtype-specific antagonists and in establishing receptor domains critical for the actions of GABAA antagonists.

To determine the roles of the alternatively spliced short and long forms of the gamma 2 subunit in the effect of ethanol on the GABAA receptor function, picrotoxin-sensitive [35S]t-butylbicyclophosphorothionate ([35S]TBPS) binding was studied in recombinant rat alpha 1 beta 2 gamma 2 and alpha 6 beta 2 gamma 2 receptors expressed in human embryonic kidney 293 cells. Ethanol (10-500 mM) in the absence of added GABA had only minor effects on [35S]TBPS binding irrespective of the gamma 2 splice variant, its effects being greater in alpha 6 beta 2 gamma 2 than in alpha 1 beta 2 gamma 2 receptors. Ethanol (100 mM) decreased the binding in all four subunit combinations at various concentrations of GABA, again an effect independent of the gamma 2 variant. The two gamma 2 variants had different effects on GABA modulation of the binding, with the long gamma 2 variant decreasing the efficiency of GABA inhibition in alpha 6 beta 2 gamma 2 receptors and enhancing the biphasic GABA stimulation and inhibition in alpha 1 beta 2 gamma 2 receptors. The findings confirm the importance of the alpha subunits in the allosteric interactions between the convulsant binding site and other effector sites, which can be modified only to a minor extent by the type of the gamma 2 splice variant.

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.

Autoradiography revealed picrotoxin-sensitive [35S]TBPS ([35S]t-butylbicyclophosphorothionate) binding sites in the cerebellar granule cell layer only in the presence of GABAA receptor antagonists. However, under the same conditions, the binding was decreased in other brain regions, including the cerebellar molecular cell layer. Thus, the convulsant binding sites at GABAA receptors in the granule cell layer are highly sensitive to down-regulation by GABA, which may be due to a unique combination of receptor subunits.