The chemical synapse plays a central role in communication between neurons. Ligand-gated ion channels responsible for synaptic transmission were discovered more than four decades ago and have attracted much attention in neuroscience. Devastating neurological diseases arise when these protein receptors go awry. Most of the prior research has focused on understanding molecular architecture, conformational changes, interactions, and trafficking of individual receptor molecules separately. There has been a recent trend in investigating complex neuronal activities at the systems level. Since receptors are considered to be the “switches” of the neuronal networks, it is essential to understand them under a system context with unified perspective. The unification proposed in this project is anchored on a recent biotechnological innovation in which I (the main coordinator) possess an expertise: the unnatural amino acid (UAA) site-directed mutagenesis. The project will focus on two critical aspects of the technology : 1) to demonstrate the feasibility to incorporate various UAAs in two types of ligand-gated ion-channels and 2) to identify new structural and dynamic properties of the receptors. The development will bring together experts from chemistry, electrophysiology, and high-resolution fluorescent imaging. The combination of several techniques involved will help understand the mechanisms of receptor functions from multiple aspects. We have designed the project into a series of well-defined tasks which focus on the incorporation of two kinds of UAAs in ligand gated ion channels: UAAs that are photoreactive ; and UAAs that serve as chemical handles that can be conjugated with spectroscopic probes. We aim at 1) identifying key structural elements regulating channel activities through a novel photocrosslinking approach; 2) engineering ligand gated ion channels that can be activated by light stimuli; and 3) imaging receptors in their native environment using high resolution fluorescent imaging techniques (stochastic optical reconstruction microscopy and fluorescence correlation spectroscopy) to understand trafficking and diffusion properties. The establishment of those approaches may not only lead to discoveries, but also can be translated to studies of many other receptors. In addition, the combination of these approaches in the studies of a specific receptor will facilitate the emergence of coherent understanding of receptors and their physiological functions. To further demonstrate the power of the UAA mutagensis technology, we will integrated a task to be conducted with an external collaborator in the University Paris-Sud 11 (Institut de Génétique et Microbiologie) centered on the identification of novel ribozymes (catalytic RNAs). The emerging team will perform key functions in the field of chemical biology: advancing the UAA site-directed mutagenesis by developing novel methods to address questions related to ligand-gated ion channels and synaptic transmissions; therefore, importing important technology into the field of neuroscience. By collaborating with experts in fields other than neuroscience, the project will help promote the application of the technology in a variety of problems.