# Molecular assemblies and pharmacology of cerebellar GABAA receptors

## TL;DR

This study reveals the structure and subunit composition of GABA receptors in the cerebellum, providing insights into their function and drug targeting.

## Contribution

The study identifies novel α1-containing GABAAR assemblies and their subunit arrangements in the cerebellum using cryo-EM and biochemical methods.

## Key findings

- Cerebellar GABAARs include α1 and α6 subunits arranged in a conserved pentameric scaffold.
- Cryo-EM resolved eight α1-containing receptor structures, including those with α6 subunits.
- The binding mode of a drug at the α+/γ– interface was determined, showing ligand-induced structural changes.

## Abstract

Hetero-pentameric GABAA receptors (GABAARs) are critical for inhibitory signaling in the brain, yet their molecular structure, subunit composition, and subunit arrangement remain incompletely defined, especially as a function of specific brain regions. Here, we reveal the molecular diversity of α1-containing receptors of the cerebellum using an integrated approach combining imaging, biochemistry, and cryo-EM. Our findings uncover cerebellar receptor assemblies composed of α1 and α6 subunits and define their nonrandom subunit arrangement within a conserved pentameric scaffold. This work advances our understanding of inhibitory receptor architecture and provides a structural framework for investigating region-specific receptor function and drug targeting.

GABAA receptors (GABAARs) mediate fast inhibitory neurotransmission in the brain and are assembled from 19 subunit isoforms into multiple pentameric assemblies. Although α1-containing GABAARs 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 GABAARs 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 GABAARs and elaborates the molecular interactions between native receptors and pyrazoloquinolinone, thereby laying the groundwork for brain region and subunit-specific pharmacology.