Project Summary/Abstract This project seeks to determine the mechanisms, structures, and structure change of integral transmembrane proteins that govern critical transmembrane processes, at the level that can lead to improved therapeutics for human disease. The premise is that alterations in molecular structures are necessary for the function of transmembrane transporters and gated channels, and are coordinated by regulatory functions. The hypothesis is that understanding the linkage between structure change and function provides a roadmap for therapeutic intervention by organic compounds or Fab fragments generated to stabilize conformational states. A major innovation is the technology and ability to determine atomic structures of membrane proteins and eukaryotic, or human membrane proteins at a resolution sufficient to instruct in the development of therapeutic development of compounds. Principal technologies include X-ray diffraction, electron cryomicroscopy, transport assays, electrophysiology. Three aims focus on different classes of transmembrane proteins. Aim 1 focuses on elaborating the mechanisms of a recently discovered class of intracellular channels that govern the release of ions and nutrients from the vacuole in plants or fungi, or the endolysosome in animals. One aim is to build on our atomic structure determination of a two-pore channel TPC1 from plants, and to determine how regulation of ion transport by voltage, by calcium ions, and by phosphorylation is brought about. The aim moves toward human TPC1 where an inhibitor seen in our structure can cure mice of Ebola virus that enters the cell through the endolysosome, and to another intracellular channel human TRPML where mutations cause a lysosomal storage disease. Aim 2 seeks to determine the mechanisms that govern secondary transmembrane transporters and their sister uniporters. The aim focuses first on a high affinity phosphate transporter where we obtained high resolution structure, made 22 mutations and recorded transport properties Vmax and Km, and effects on growth of yeast deleted of its own phosphate transporters, expressing the mutants in the plasma membrane. We also focus on mutants in the lactose transporter that for the first time converted the structure between states in the biological transport cycle. The goal is to understand how the binding and release of substrates is coupled to the transport of a driving ion, protons, and to see if this surprising mechanism is common throughout secondary transporters. This aim also addresses a human glucose transporter where we showed how drug leads block the uniporter. This transporter is relevant to many cancers. Aim 3 aims to leverage our atomic structure of human brain aquaporin 4, to understand the binding by patient antibodies with the autoimmune, sometimes lethal disease neuromyelitis optica. This will open the way to ask how we may alter this interaction to therapeutic benefit.