Project Summary Ischemic strokes can cause long-term cognitive damage, leading to reduced mobility in nearly half of stroke survivors. Brain tissue damage that occurs during and preceding an ischemic event is often largely in part due to severe local tissue acidosis. While the molecular mechanism of how acidosis leads to tissue damage is largely unknown, proton-gated ion channels are thought to play a role. A novel proton-gated chloride channel has been recently identified as the previously uncharacterized gene, TMEM206, now commonly referred to as the Proton-Activated Chloride (PAC) Channel. While recent studies have implicated PAC in acid-induced cell death, there exists no molecular justification of the channel’s proton-activated chloride currents. This proposal will integrate electrophysiological, biochemical, and high-resolution structural experiments to elucidate the structure-based mechanisms that govern the function of PAC. In support of this goal, I will first obtain the high- resolution structures of PAC’s resting and active functional state using single particle cryo-electron microscopy (Cryo-EM). These structures will provide fundamental insights into the architecture, stoichiometry, and unique protein folding of PAC. This information will also expand our knowledge surrounding the physical and chemical properties of proton-gated ion channels, as well as broadly inform the structure/function relationship of ion channels. I will then ascertain the molecular underpinnings of PAC’s pH-dependent mechanism and pore properties by probing PAC’s function using structure-directed mutagenesis and electrophysiological experiments. These experiments will establish a link between the molecular architecture and physiology of PAC. Ultimately, this proposal will define structure-based, biochemical mechanisms for PAC’s function, which will lay the foundation for future studies and may inform the development of therapeutic agents to mitigate neuronal damage in ischemic events. As a neuroscience graduate student whose goal is to become an independent academic scientist that will study the structure/function of ligand-gated ion channels of the nervous system, this project will directly expand my knowledge base and technical skillset in ion channels, membrane protein biochemistry, electrophysiology, and cryo-EM. The study into the molecular architecture and mechanism of PAC will be pursued under the mentorship of Dr. Eric Gouaux, an expert in ligand-gated ion channels and leader in membrane protein structural biology. Dr. Gouaux is a senior scientist at the Vollum Institute at OHSU, an electrophysiology powerhouse that is home to one of three national centers for Cryo-EM, Pacific Northwest Center for Cryo-EM (PNCC). Taken together, the project will not only illuminate critical insights into a novel proton-activated channel, but will also provide me with the necessary knowledge, biophysical tool-kit, and professional skills I need to achieve my long-term ...