Receptors belonging to the Cys-loop gene superfamily, also called pentameric ligand-gated ion channels (pLGIC), include acetylcholine, serotonin (5-HT3), GABAA, GABAr and glycine receptors. pLGIC mediate fast synaptic transmission in the nervous system. These receptors are targeted by current clinically used drugs that include antidepressants, antiepileptics, antiemetics, antipsychotics, anesthetics, muscle relaxants, spasmolytics, tranquillizers, and drugs for the treatment of substance abuse. Some family members are also found in non-excitable cells where they represent novel therapeutic targets to treat inflammatory diseases as diverse as atherosclerosis, Alzheimer's, diabetes, inflammatory bowel disease, and sepsis. All Cys-loop receptor family members in metazoans contain three domains: an extracellular domain, a transmembrane domain, and an intracellular domain (ICD). The extracellular and transmembrane domains have been functionally studied in great detail and their three-dimensional structures have been determined. The recent identification of Cys-loop receptor homologues in bacteria has propelled the structural knowledge into atomic resolution. However, the prokaryotic members lack the intracellular domain. Interestingly, the intracellular domain of Cys-loop receptors from the animal kingdom is the most diverse domain with respect to both length and amino-acid composition. The intracellular domain therefore represents an attractive target for developing subtype-selective drugs with the promise of fewer side effects than current drugs, which all target the highly- conserved extracellular or transmembrane domains. This project will focus on probing the structure and function of the so-far neglected intracellular domain. We will use functional chimeras that we have generated by inserting the ICD from select anionic and cationic pLGIC into the prokaryotic pLGIC from Gloeobacter violaceus (GLIC), a well-studied homologue that consists of ECD and TMD and lacks an ICD, and that has been established as a tool to study pLGIC. In Aim 1 we will characterize in detail the functional contributions of ICDs from specific anionic and cationic pLGIC using voltage-clamp, patch-clamp and bilayer recordings. In Aim 2 we will investigate the molecular determinants present in pLGIC ICDs that mediate interaction with the chaperone protein resistance to inhibitors of choline esterase (RIC-3). In Aim 3 we will study and compare the interactomes of wild-type and chimeric receptors and probe the overall three-dimensional structure of the ICD in the chimeras with FT-IR and CD spectroscopy, and the by-residue analysis with SDSL-EPR. Our study will be of significance because it will complete the understanding of the structure-function-interplay of all three domains of Cys-loop receptors. It will also evaluate the intracellular domain as a new drug target that can then be utilized to design innovative drugs that are not based on the conventional agonist / antagonist approach.