Two Distinct Populations of α1α6-Containing GABAA-Receptors in Rat Cerebellum

Abstract

GABAA receptors are pentameric GABA-gated chloride channels. The existence of 19 different subunits (six α, three β, three γ, δ, ε, θ, π, and three ρ) in mammalian systems gives rise to an enormous theoretical diversity of GABAA receptor subtypes with distinct subunit composition and unique pharmacological properties. These receptors are already now the drug targets of several clinically used compounds, such as benzodiazepines, anesthetics, and many more. There is a constant quest to identify novel molecules and possible future drug targets: Currently, α6-containing GABAA receptors are being discussed in the context of treating sensorimotor gating deficits in neuropsychiatric disorders, such as tic disorders and schizophrenia. Therefore, we aim to learn more about α6-containing GABAA receptors. They are mostly expressed in the cerebellar granule cell layer, where they form the following subtypes: α6βxγ2, α1α6βxγ2, α6βxδ, and α1α6βxδ. In former studies, α1α6βxγ2-containing GABAA receptors were considered a single receptor population. In the current study, we investigate the possibility, that this population can consist of two subgroups with alternative arrangements depending if α1 neighbors γ2 (forming a “diazepam-sensitive” receptor), or if α6 neighbors γ2 (forming a “diazepam-insensitive” receptor) and aimed to prove the existence of both subtypes in native tissue. We performed immunoprecipitation experiments on rat cerebellar lysates using α1- or α6 subunit-specific antibodies followed by radioligand binding assays with either 3 H-flunitrazepam or 3 H-Ro 15-4513. Indeed, we were able to prove the existence of two distinct populations of α1α6-containing GABAA-receptors and could quantify the different receptor populations: α1βxγ2 receptors constitute approximately 60% of all γ2-containing receptors in the rat cerebellum, α6βxγ2 about 20%, and both isoforms of α1α6βxγ2 9–15% each. The simple classification of GABAA-receptors into αx-containing subtypes seems not to reflect the complexity of nature; those receptors are more diverse than previously thought.

Probes for the heterogeneity of muscimol binding sites in rat brain

“…As a clear indication for differences not only in the number of available sites but also for somewhat different affinities, we observed non-sigmoidal displacement curves in hippocampus, striatum and thalamus, while sigmoidal curves are seen for cerebellum. This supports the notion that chiefly the diversity in α subunits drives the affinity of the sites for the test molecules, because the cerebellum contains only two α isoforms in appreciable amounts ( Pirker et al, 2000 ; Scholze et al, 2020 ). Generally, a limited subset of 6 out of 13 GABA A -receptor subunits (α1, α6, β2, β3, γ2 and δ) was found in cerebellum ( Pirker et al, 2000 ), and therefore only selected receptor subtypes are being expressed ( Jechlinger et al, 1998 ; Poltl et al, 2003 ).…”

α6-Containing GABAAReceptors: Functional Roles and Therapeutic Potentials

“…These could be pentameric a1bc2 and a6bc2 receptors sitting side by side in the same synapse or pentameric a1a6bc2 receptors containing two different a subunit types. The actual existence of a1a6bc2 or a1a6bd receptors is supported by several lines of evidence, resulting in their classification as "existing with high probability" (Olsen and Sieghart, 2008;Scholze et al, 2020) (Fig. 1).…”

“…The two a and b subunits can be identical or different. The upper left image depicts the experimentally verified subunit arrangement for the major GABA A R subtype composed of a1bc2 subunits, and the other three panels depict a6-containing arrangements that have been shown to exist in cerebellar granule cells (Scholze et al, 2020). Each subunit has a principal (1) and a complementary (À) side.…”

The delta subunit of the GABAA receptor is necessary for the GPT2-promoted breast cancer metastasis

“…GABAA receptors are heteromeric complexes composed of 2α, 2β, and either one of the γ-subunit or δ, ε, θ, π, or ρ subunit, which were encoded by GABR genes GABRA (1 to 6), GABRB (1 to 3), GABRG (1 to 3), GABRD, GABRE, GABRQ, GABRP, and GABRRs, respectively [33] [34]. In contrast, GABAB receptors are obligatory heterodimers composed of R1 and R2 subunits [35] [36].…”