Project Summary and Abstract: Glycine receptors (GlyR) are the primary mediators of synaptic neural inhibition in the brainstem and spinal cord. They are essential to many physiological processes and intriguing, yet untapped, therapeutic targets. The overall goal of this project is to understand the processes that control GlyR activity and provide a template for therapeutic design. GlyR is a pentameric ligand-gated ion channel expressed in homomeric and heteromeric forms. Heteromeric GlyR is essential for synaptic activity as it alone can bind the synaptic anchoring protein gephryin. This proposal uses cryogenic electron microscopy (cryo-EM), electrophysiology and electron paramagnetic resonance (EPR) to achieve mechanistic understanding of heteromeric GlyR bound to gephryin. This will build upon past cryo-EM studies of homomeric GlyR. The specific aims of this proposal are: First, to characterize subunit-specific structures in hetero-GlyR known to regulate subunit composition, ion conduction and intracellular regulation. Second, study conformational coupling between the neurotransmitter binding pocket and channel gate. Third, explore the allosteric effects of ligands that bind within the transmembrane domain of hetero-GlyR. The applicant has shown significant progress by expressing and purifying heteromeric GlyR and performing preliminary cryo-EM studies resulting in a 2.25-2.5 Å cryo-EM map. In accomplishing this project, the applicant will receive training in membrane protein biochemistry, cryo-EM, electrophysiology and EPR. This project will take place in Sudha Chakrapani’s lab at Case Western Reserve University. This lab has made significant recent contributions to the field with cryo-EM studies of homomeric GlyR and other ligand-gated ion channels. Available resources include a cryo-EM facility with a Titan Krios microscope and lab- dedicated electrophysiology rigs. Several members of the lab have extensive experience in cryo- EM, electrophysiology and/or EPR providing an excellent training environment.