Person:

Miller, Keith

Type A γ-aminobutyric acid receptors (GABAARs) are pentameric ligand-gated ion channels (pLGICs) and the main drivers of fast inhibitory neurotransmission in the vertebrate nervous system. Their dysfunction is implicated in a range of neurological disorders, including depression, epilepsy and schizophrenia. Amongst the numerous assemblies theoretically possible, α1β2/3γ2 GABAARs are most prevalent in the brain. 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. To date, efforts to generate accurate structural models for heteromeric GABAARs have been hampered by the use of engineered receptors and the presence of detergents. Significantly, some recent cryo-EM reconstructions report collapsed conformations which disagree with the prototypical pLGIC, the Torpedo nicotinic acetylcholine receptor, the large body of structural work on homologous homopentameric receptor variants, and the logic of an 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 GABAAR isoform, functionally reconstituted in lipid nanodiscs. The receptor is bound to a positive allosteric modulator megabody and in a desensitized conformation. Unexpectedly, each GABAAR 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 GABAA receptor organization and provides the reference framework for future mechanistic investigations of GABA-ergic signaling and pharmacology.

Type-A -aminobutyric receptors (GABAARs) 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 GABAARs remains largely unknown. Here we report high-resolution cryoEM structures of the full-length human 132L GABAAR 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 GABAAR 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 GABAAR modulators.