Summary Type-A γ-aminobutyric receptors (GABA A Rs) are ligand-gated chloride channels with a very rich pharmacology. Some of their modulators, including benzodiazepines and general anaesthetics, are among the most successful drugs in clinical use and common substances of abuse. Without reliable structural data, the mechanistic basis for pharmacological modulation of GABA A Rs remains largely unknown. Here we report high-resolution cryoEM structures of the full-length human α1β3γ2L GABA A R in lipid nanodiscs, bound to the channel blocker picrotoxin, the competitive antagonist bicuculline, the agonist GABA and the classical benzodiazepines alprazolam (Xanax) and diazepam (Valium), respectively. We describe the binding modes and mechanistic impacts of these ligands, the closed and desensitised states of the GABA A R gating cycle, and the basis for allosteric coupling between the extracellular, agonist-binding, and the transmembrane, pore-forming, regions. This work provides a structural framework to integrate decades of physiology and pharmacology research and a rational basis for development of novel GABA A R modulators.

The three-dimensional positions of atoms in protein molecules define their structure and provide mechanistic insights into the roles they perform in complex biological processes. The more precisely atomic coordinates are determined, the more chemical information can be derived and the more knowledge about protein function may be inferred. With breakthroughs in electron detection and image processing technology, electron cryomicroscopy (cryo-EM) single-particle analysis has yielded protein structures with increasing levels of detail in recent years 1,2 . However, obtaining cryo-EM reconstructions with sufficient resolution to visualise individual atoms in proteins has thus far been elusive. Here, we show that using a new electron source, energy filter and camera, we obtained a 1.7 Å resolution cryo-EM reconstruction for a prototypical human membrane protein, the β3 GABAA receptor homopentamer 3 . Such maps allow a detailed understanding of small molecule coordination, visualisation of solvent molecules and alternative conformations for multiple amino acids, as well as unambiguous building of ordered acidic side chains and glycans. Applied to mouse apo-ferritin, our strategy led to a 1.2 Å resolution .

Type A γ-aminobutyric acid receptors (GABA A Rs) are pentameric ligand-gated ion channels (pLGICs) and the main drivers of fast inhibitory neurotransmission in the vertebrate nervous system 1 , 2 . Their dysfunction is implicated in a range of neurological disorders, including depression, epilepsy and schizophrenia 3 , 4 . Amongst the numerous assemblies theoretically possible, α1β2/3γ2 GABA A Rs are most prevalent in the brain 5 . The β3 subunit plays an important role in maintaining inhibitory tone and expression of this subunit alone is sufficient to rescue inhibitory synaptic transmission in a CRISPR/Cas9 derived β1-3 triple knockout 6 . To date, efforts to generate accurate structural models for heteromeric GABA A Rs have been hampered by the use of engineered receptors and the presence of detergents 7 – 9 . Significantly, some recent cryo-EM reconstructions report “collapsed” conformations 8 , 9 which disagree with the prototypical pLGIC, the Torpedo nicotinic acetylcholine receptor 10 , 11 , the large body of structural work on homologous homopentameric receptor variants 12 , and the logic of a ion channel architecture. To address this problem, here we present a high-resolution cryo-EM structure of the full-length human α1β3γ2L, a major synaptic GABA A R isoform, functionally reconstituted in lipid nanodiscs. The receptor is bound to a positive allosteric modulator megabody and in a desensitised conformation. Unexpectedly, each GABA A R pentamer harbours two phosphatidylinositol 4,5-bisphosphate (PIP2) molecules, whose head groups occupy positively-charged pockets in the intracellular juxtamembrane regions of α1-subunits. Beyond this level, the intracellular M3-M4 loops are largely disordered, possibly because interacting post-synaptic proteins were not included. This structure illustrates the molecular principles of heteromeric GABA A receptor organization and provides a reference framework for future mechanistic investigations of GABA-ergic signalling and pharmacology.