ABSTRACT: GABAA receptors are pentameric GABA-gated chloride channels. The existence of 19 different subunits (six ?, three ?, three ?, ?, ?, ?, ?, and three ?) 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, ?6-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 ?6-containing GABAA receptors. They are mostly expressed in the cerebellar granule cell layer, where they form the following subtypes: ?6?x?2, ?1?6?x?2, ?6?x?, and ?1?6?x?. In former studies, ?1?6?x?2-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 ?1 neighbors ?2 (forming a "diazepam-sensitive" receptor), or if ?6 neighbors ?2 (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 ?1- or ?6 subunit-specific antibodies followed by radioligand binding assays with either 3H-flunitrazepam or 3H-Ro 15-4513. Indeed, we were able to prove the existence of two distinct populations of ?1?6-containing GABAA-receptors and could quantify the different receptor populations: ?1?x?2 receptors constitute approximately 60% of all ?2-containing receptors in the rat cerebellum, ?6?x?2 about 20%, and both isoforms of ?1?6?x?2 9-15% each. The simple classification of GABAA-receptors into ?x-containing subtypes seems not to reflect the complexity of nature; those receptors are more diverse than previously thought.
