Project Summary/Abstract The overarching goal of the proposal is to determine the structural basis for allosteric mechanisms governing gating and modulation in pentameric ligand-gated ion channels (pLGIC). The pLGIC superfamily governs crucial physiological processes such as gastrointestinal functions, motor functions, and pain transmission. Aberrant channel functions are implicated in mood disorders, addiction, chronic pain, and cancer. Currently used therapeutic strategies suffer from our limited knowledge of the molecular details of pLGIC function, the origin of their functional diversity, and the downstream signaling events. Using single-particle cryo-EM, we recently solved structures of the full-length serotonin receptor (5HT3AR), a cationic pLGIC, in the resting state and two serotonin-activated conformations. Building on this technical advancement and further biochemical optimization, we aim to determine the conformational changes underlying gating and lipid modulation in the full- length homomeric and heteromeric receptors within the cationic 5-HT3R and anionic glycine receptor (GlyR) subfamilies. To achieve these goals we will use an approach that combines multidisciplinary techniques, including cryo-EM, pulsed-EPR, and electrophysiology. Specifically, we will determine high-resolution snapshots of pLGIC in multiple functional states, in modulator-bound conformations, in the presence of membrane lipid constituents, and in complex with intracellular-binding proteins. These structures will be validated and complemented with protein dynamic studies in a membrane environment and extensive functional analysis. Taken together, our proposed work is expected to provide molecular blueprints of the channel in physiologically relevant conformations for therapeutic targeting and unravel the molecular mechanisms underlying channel function. These findings will, in turn, pave the way for design of novel therapeutic agents that are safer and more effective.

Project Narrative The pentameric ligand-gated ion channels (pLGIC) mediate fast synaptic transmission in the central and peripheral nervous systems, and thereby critically govern numerous cellular processes such as pain perception, motor coordination, and gastrointestinal functions. Dysfunctions in pLGIC are associated with several psychiatric, neurological, and gastrointestinal disorders, and therefore these channels are important drug targets. The goal of the proposed studies is to elucidate fundamental mechanisms governing channel function and modulation at a molecular level. Findings from this work will lead to a better understanding of the role of pLGIC in various physiological and pathophysiological processes, and hence aid strategies for development of new and safer therapeutics.