Cryo-EM structure of the benzodiazepine-sensitive α1β1γ2S tri-heteromeric GABAA receptor in complex with GABA.

Swastik Phulera,Hongtao Zhu,Jie Yu,Derek P Claxton,Nate Yoder,Eric Gouaux,Craig Yoshioka

doi:10.7554/elife.39383

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https://doi.org/10.7554/elife.39383

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Abstract

Fast inhibitory neurotransmission in the mammalian nervous system is largely mediated by GABAA receptors, chloride-selective members of the superfamily of pentameric Cys-loop receptors. Native GABAA receptors are heteromeric assemblies sensitive to many important drugs, from sedatives to anesthetics and anticonvulsant agents, with mutant forms of GABAA receptors implicated in multiple neurological diseases. Despite the profound importance of heteromeric GABAA receptors in neuroscience and medicine, they have proven recalcitrant to structure determination. Here we present the structure of a tri-heteromeric α1β1γ2SEM GABAA receptor in complex with GABA, determined by single particle cryo-EM at 3.1-3.8 Å resolution, elucidating molecular principles of receptor assembly and agonist binding. Remarkable N-linked glycosylation on the α1 subunit occludes the extracellular vestibule of the ion channel and is poised to modulate receptor assembly and perhaps ion channel gating. Our work provides a pathway to structural studies of heteromeric GABAA receptors and a framework for rational design of novel therapeutic agents.

Highlights

GABAA receptors are chloride permeable, g-amino butyric acid (GABA)-gated ion channels that are responsible for the majority of fast inhibitory neurotransmission in the mammalian nervous system (Sigel and Steinmann, 2012)
While previous studies have suggested that GABAA receptor assembly occurs via defined pathways that limit the receptor diversity (Sarto-Jackson and Sieghart, 2008), precise molecular mechanisms regarding assembly are unknown
If we consider how heteromeric GABAA receptors form from a, b and g subunits, we speculate that the larger contact areas in the extracellular domain (ECD) region makes formation of the b(+)/a(-) interface favorable, leading to a/b assembly intermediates, which transition to (a/b)2 intermediates

Summary

Introduction

GABAA receptors are chloride permeable, g-amino butyric acid (GABA)-gated ion channels that are responsible for the majority of fast inhibitory neurotransmission in the mammalian nervous system (Sigel and Steinmann, 2012). GABAA receptors belong to the pentameric ligand-gated ion channel (pLGIC) superfamily (Thompson et al, 2010) Other members of this family are nicotinic acetylcholine (nAChR), 5-HT3A, glycine, and the invertebrate GluCl and Zn2+-activated cation channels (Thompson et al, 2010). Recent structures of diheteromeric nAChRs further our understanding of subunit arrangement and function in heteromeric Cys-loop receptors (Walsh et al, 2018). These studies, together with a large number of biochemical and biophysical experiments, have defined the transmembrane ion channel pore, its lining by the M2 helices and likely mechanisms of ion selectivity (Cymes and Grosman, 2016; Sine et al, 2010). The structural analysis defines subunit organization, but it uncovers the mode of GABA binding to the orthosteric agonist binding site and suggests a critical role of N-linked glycosylation of the a1 subunit in governing receptor assembly and, perhaps, ion channel activity

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Discussion

Materials and methods

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