Molecular assemblies and pharmacology of cerebellar GABA(A) receptors.

GABA(A) receptors (GABA(A)Rs) mediate fast inhibitory neurotransmission in the brain and are assembled from 19 subunit isoforms into multiple pentameric assemblies. Although α1-containing GABA(A)Rs are broadly expressed and are pharmacologically important, the molecular diversity of native α1-based assemblies in specific brain regions remains incompletely understood. Here, we use immunofluorescence, mass spectrometry, and cryogenic electron microscopy (cryo-EM) to characterize the spatial distribution, subunit composition, and structural architecture of native α1-containing GABA(A)Rs in the rat cerebellum. Confocal microscopy reveals robust colocalization of α1 and γ2 subunits across cerebellar layers, including prominent labeling at glomerular synapses. Biochemical purification and proteomic analysis identify a range of α, β, and γ subunits, along with abundant α6 and δ subunits. Using cryo-EM and automated subunit identification, we resolve eight α1-containing receptor assemblies, including the first structure of α6-containing receptors. We further determine the binding mode of the α6-selective pyrazoloquinolinone modulator PZ-II-029 at the α(+)/γ(-) interface, showing ligand-induced expansion of the entire extracellular domain (ECD). Together, our study defines the structure and subunit composition of the α1-containing cerebellar GABA(A)Rs and elaborates the molecular interactions between native receptors and pyrazoloquinolinone, thereby laying the groundwork for brain region and subunit-specific pharmacology.