Chemical signaling in the brain relies on rapid opening and closing of ligand-gated ion channels (LGICs) in the membranes of nerve cells. Members of the pentameric LGIC superfamily include nicotinic acetylcholine receptors (nAChR), serotonin-type-3 receptors (5HT3R), gamma-amino butyric acid type A receptors (GABAAR) and glycine receptors. Defects in these channels lead to a variety of neurological diseases and psychiatric disorders and a number of therapeutic drugs, including muscle relaxants, sedative-hypnotics, anti- convulsants, anxiolytics, intravenous and volatile anesthetics, anti-emetics, drugs for nicotine addiction and drugs to treat Alzheimer's disease target these channels. For these receptors, binding of neurotransmitter in the extracellular ligand-binding domain triggers opening of an intrinsic ion channel more than 50 away in the transmembrane domain of the receptor. Although we know a fair amount about the structure of these receptors, the mechanisms by which the binding of neurotransmitter triggers channel opening are still under debate and our understanding of the protein motions underlying this process limited. The general plan of this proposal is to investigate the binding-to-gating motions in the prokaryotic pLGIC homologs from Gloeobacter violaceus (GLIC) using site-directed spin labels and electron paramagnetic resonance spectroscopy (SDSL- EPR) and to test these motions in the GABAAR using an array of biochemical and electrophysiological approaches including voltage clamping, mutant cycle analysis, cysteine cross-linking, disulfide trapping and structural modeling. We will focus on three key regions: the extracellular binding domain (EBD), the gating interface and the transmembrane channel domain (TCD). These studies will build on our previous work and will provide new insights into how neurotransmitters activate pLGICs and how allosteric drugs modulate their activity. A deeper understanding of how these channels work at a molecular level will improve our ability to predict the actions of drugs and ligands that act on these channels, design safer and more effective drugs, develop better therapeutic strategies, and understand the etiology of disease-causing mutations.