Overview. Electrical signal transmission between cells underlies almost all physiological processes in human, from heartbeat, to learning and memory. Neurotransmitter receptors are organized in clusters at micron-sized inter-cellular machineries, namely synapses, throughout our nervous system to enable signal transmission. Decades of intense research have characterized the working mechanism of most individual receptors. However, due to technical difficulties, the clusters of receptors remain enigmatic – neither the structure, nor the functional significance is known. In this proposal, my lab will use state-of-the-art methods and a reconstitution system to systematically characterize the architecture of synaptic glycine receptors, which is the last major neurotransmitter receptors whose architecture remained elusive before our work. We will also develop novel technologies for quantitative characterization of higher-order assemblies of glycine receptors, and other proteins in the 2D setting of lipid membranes. Following is a brief description of the proposed work. Glycine receptors and its clustering with gephyrin scaffold. Glycine receptor (GlyR) belongs to the Cys-loop family of pentameric ligand-gated ion channels. GlyRs in adult tissue are heteromeric receptors composed of both the α and β subunits, which form clusters with scaffold protein gephyrin at synapses through a specific interaction between the β subunit and gephyrin. Disfunction of GlyR signaling is the major cause of the rare congenital disease hyperekplexia and related to chronical neurological pain and autism spectrum disorders. The architecture of heteromeric GlyRs and how they form clusters with gephyrin is very limited – even the α:β subunit stoichiometry has been under debate for decades. We have discovered an unexpected subunit composition of GlyR, explaining the unique function of heteromeric GlyRs and clearing long-lasting confusion. We will use this established platform to characterize all major types of GlyRs and how they interact with gephyrin. Develop novel technologies for characterizing protein clusters in lipid membrane. My lab will develop a novel correlated raised-total internal refection fluorescence microscopy (TIRF) and electrophysiology reconstitution system to characterize ion channel/receptor clusters. This system has the high signal/noise ratio and single- molecule sensitivity as traditional TIRF, and allow complex electrophysiological experiments to be performed simultaneously with imaging. We will learn how clusters form, how they are regulated, and whether clustering gives rise to functional effects and regulate physiological activities. The knowledge gained here will guide the reconstitution of functional clusters for structural characterization using cryo-EM single particle and/or tomography methods. These new methods will allow quantitative characterization of the spatial organization and functional significance of clustering, as well as the mechanisms ...