Eukaryotic pentameric ligand-gated ion channels (pLGIC) represent targets for a wide variety of drugs such as skeletal muscle relaxants, anti-psychotics, anti-epileptics and drugs against Parkinson's and Alzheimer's diseases. Each subunit consists of an extracellular domain (ECD), a transmembrane domain (TMD) with 4 α-helical segments, and an intracellular domain (ICD) that is 4-14 amino acids long in prokaryotes and ∼50-270 amino acids in eukaryotes. The ICD of eukaryotes has been implicated in modulating single channel conductance and kinetic properties of the channel. Additionally, it interacts with cytosolic proteins such as the resistance to inhibitors of cholinesterase protein, RIC-3, that affects plasma membrane-expression of some pLGIC. We created chimeras by introducing the ICD of cationic (5-HT3A , nACh α7) as well as anionic (Glycine α1, GABAA ρ1) eukaryotic pLGICs into the Gloeobacter violaceus pLGIC, GLIC, a well-studied prokaryotic homologue, that only contains ECD and TMD. Electrophysiological experiments after X. laevis oocyte expression (EC50 and IC50) demonstrate that for each of the four chimera sets, we have constructs that act as functional proton-gated ion channels, similar to the parent GLIC. Co-expression of cationic chimeras as well as wild-type pLGIC together with RIC-3 showed significant changes in current amplitudes, whereas RIC-3 did not affect anionic receptor chimeras or wild-type channels. We established that cationic chimeras and RIC-3, both overexpressed and purified to homogeneity from E. coli, bind to each other. Currently, we are using the chimeras to identify interacting proteins in native brain lysates. Our results clearly demonstrate a direct interaction between the ICD of cationic pLGIC and RIC-3. Specific interaction points will be identified in further studies.