Project Summary: Type A γ-aminobutyric acid receptors (GABAA) are pentameric ligand-gated ion channels found abundantly in the central nervous system. The binding of GABA, the major brain inhibitory neurotransmitter, to GABAA receptors, accounts for ~30% of the fast synaptic signaling in the brain. These receptors are important for sensory and motor processing, central autonomic control, and cognition. While much of the research in GABAA receptor structure and function has focused on the subunit assemblies found at synapses, a tremendous diversity of pharmacologically important GABAA receptor subtypes are found outside of synapses. The goal of this proposal is to determine and validate high-resolution structures of extrasynaptic GABAA receptors bound to distinct drug classes of relevance to neurological disorders, anesthesia, addiction and drug abuse. These receptors are potentiated by mildly intoxicating concentrations of ethanol, have high affinity for neurosteroids and are targets of anesthetics, analgesics, and anticonvulsant drugs. In two Aims, I propose to optimize conditions for expression and purification of defined heteromeric GABAA receptor assemblies, refine sample preparation for cryo-electron microscopy, collect and process single particle cryo-EM datasets, and build and refine atomic models of receptor-drug complexes. High-resolution structural information for physiologically important but understudied subunit assemblies, in complex with allosteric and orthosteric-site ligands will broadly inform on principles of ligand recognition and subunit assembly. !