PROJECT SUMMARY Ligand-gated ion channels (LGICs) enable cells to fine-tune their intracellular space based on environmental cues and cell-cell communication. Because of their role in regulating cells, dysregulation of LGICs is a major contributor to human diseases. LGICs function through binding chemicals in their extracellular domain, which opens their ion channel, enabling ion flux into cells. The mechanisms of how this occurs is referred to as gating. Because LGIC function enables our cells to react to environmental cues, they must gate on a rapid, millisecond timescale for proper function. However, it is unknown how LGIC gating occurs on this timescale because technological limitations have precluded the physiological mechanisms of gating from being studied. In addition, the function of LGICs is tightly regulated by auxiliary proteins in cells, which localize the function of LGICs in cells and provide a mechanism for cells to further control gating. Despite the importance of auxiliary subunits to fine-tune LGIC function, the mechanisms of how auxiliary subunits regulate LGICs are unclear. Furthermore, how therapeutics regulate LGIC function is unknown. Our research addresses these major knowledge gaps and will uncover the mechanisms of how LGICs gate, how function is regulated, and provide vital foundations for targeting LGICs with therapeutics to treat human diseases. To initiate our research, we are focusing on AMPA receptors (AMPARs), which are a subtype of LGIC that mediate neuronal communication, and dysregulation of AMPARs is a major driver of neurological diseases. To understand how AMPARs function on a physiological timescale, we developed a time-resolved cryo-electron microscopy (cryo-EM) approach that enables us to study AMPAR and LGIC function with millisecond resolution in this proposed research. Our research here will form the molecular and structural basis of how AMPARs gate. We will also elucidate how AMPAR gating and localization is regulated by auxiliary subunit proteins, which is a major regulation mechanism of AMPAR function, but the details are unknown. We will uncover these mechanisms with cryo-EM, biophysics, and electrophysiology. We will also use these approaches to understand the mechanisms of how therapeutic molecules alter AMPAR function. Collectively, our findings will uncover the precise molecular details into how AMPARs function and how AMPARs are regulated in health and disease, as well as provide us with critical avenues for studying LGICs in our future research.