Dorovykh, Valentina; (2023) Exploring alpha-subunit heterogeneity in GABAA receptors. Doctoral thesis (Ph.D), UCL (University College London).

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Abstract

GABA type-A receptors (GABAARs) are the most abundant proteins responsible for inhibitory signalling in the brain. These proteins assemble into a hetero-pentameric structure, constructed from a selection of α1-6, β1-3 and γ1-3, δ, ε, ρ, π and θ subunits. Although prototypic GABAARs are generally considered to be composed of αβγ subunits, the precise composition and arrangement of native GABAARs remains poorly defined. It is commonly accepted that distinct GABA receptors comprise only a single type of α subunit, which then dictates their pharmacological profiles influencing neural circuit activity and behaviour. The aim of this study was to investigate the likelihood of hetero-α subunit assembly in the same receptor pentamer, dissecting the complexity associated with subunit assembly for GABAARs and providing novel insight into their structures and functional footprints. To address this, we first electrophysiologically analysed α1 and α2 with β2/3 γ2L wild-type and reporter-mutant subunit mixtures in HEK293 cells to establish the presence of hetero-α-GABAAR isoforms. We also examined the stoichiometry of hetero-α-receptors and whether there is preferential positioning of α subunit isoforms at α-γ benzodiazepine binding interfaces. We then demonstrate that hetero-α-GABAARs are expressed in vivo and measure their abundance and subcellular localisation in hippocampal neurons using proximity ligation assays (PLA). In addition, we assess dynamic regulation of these receptors during long term potentiation (LTP). There is a significant increase in α1α2-containing receptors upon LTP induction, and these receptors are localised preferentially at inhibitory synapses. Finally, homo- and hetero-α1/2 GABAAR numbers at synapses were estimated using Spatial Intensity Distribution Analysis (SPiDA). In conclusion, this study demonstrates that hetero-α GABAARs do assemble in the brain and proposes their physiological importance for brain inhibition. This study also describes a multidisciplinary approach that is applicable for investigating other GABAAR hetero-α-subunit assemblies, including the assessment of their pharmacological profiles.

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