# Modular Structure and Polymerization Status of GABAA Receptors Illustrated with EM Analysis and AlphaFold2 Prediction

## TL;DR

This study explores the structure of GABAA receptors using EM and AlphaFold2, revealing how subunits form pentamers and offering insights for drug development.

## Contribution

The study provides new insights into the modular structure and polymerization of GABAA receptor subunits using experimental and computational methods.

## Key findings

- α1 and β2 subunit fragments formed homopentamers and heteropentamers with γ2.
- γ2 subunit proteins did not form homopentamers.
- AlphaFold2 predictions aligned with EM results, revealing subunit structure and polymerization.

## Abstract

Type-A γ-aminobutyric acid (GABAA) receptors are channel proteins crucial to mediating neuronal balance in the central nervous system (CNS). The structure of GABAA receptors allows for multiple binding sites and is key to drug development. Yet the formation mechanism of the receptor’s distinctive pentameric structure is still unknown. This study aims to investigate the role of three predominant subunits of the human GABAA receptor in the formation of protein pentamers. Through purifying and refolding the protein fragments of the GABAA receptor α1, β2, and γ2 subunits, the particle structures were visualised with negative staining electron microscopy (EM). To aid the analysis, AlphaFold2 was used to compare the structures. Results show that α1 and β2 subunit fragments successfully formed homo-oligomers, particularly homopentameric structures, while the predominant heteropentameric GABAA receptor was also replicated through the combination of the three subunits. However, homopentameric structures were not observed with the γ2 subunit proteins. A comparison of the AlphaFold2 predictions and the previously obtained cryo-EM structures presents new insights into the subunits’ modular structure and polymerization status. By performing experimental and computational studies, a deeper understanding of the complex structure of GABAA receptors is provided. Hopefully, this study can pave the way to developing novel therapeutics for neuropsychiatric diseases.