The GABA-A receptor is the most abundant inhibitory neurotransmitter receptor in the central nervous system and is the target of myriad therapeutic compounds and drugs of abuse. Function of the nervous system is governed by a balance of excitatory and inhibitory signaling; GABA-A receptor dysfunction results in disorders of anxiety and excitotoxicity including epilepsy. The principal isoform of this pentameric ligand-gated chloride channel is found on post-synaptic membranes in the brain. GABA (γ-aminobutyric acid) is the endogenous neurotransmitter and agonist of this receptor. Benzodiazepines, like diazepam (Valium) and midazolam (Versed), are positive allosteric modulators taken by 5% of the US for anxiety and insomnia. Barbiturates like pentobarbital, anesthetics like isoflurane and propofol, neurosteroids, and ethanol are all positive modulators acting through non-overlapping sites. This rich pharmacology derives from the complex subunit assembly of the synaptic GABA-A receptor. The predominant synaptic isoform consists of two α1 subunits, two β2 subunits and one γ2 subunit. Here we propose to address a lack of structural information on physiological GABA-A receptors using a direct approach. In three Specific Aims, we propose to elucidate the structural mechanism of benzodiazepine potentiation of these receptors, perform complementary electrophysiological experiments on the recombinant receptor, and in parallel characterize the structural principles underlying modulation by barbiturates, anesthetics and neurosteroids. The two structural Aims are independent and will yield fundamentally new and distinct structural information for the principal GABA-A receptor type in the central nervous system in complex with extracellular and transmembrane-site ligands. The functional Aim complements the structural work to define determinants for benzodiazepine binding, efficacy and allosteric signaling. The sum of structures and function will illuminate principles defining heteromer assembly and ligand recognition and will elucidate how drug binding changes molecular behavior, with broad relevance across the Cys-loop receptor superfamily.