This project seeks to determine how voltage sensing domains control ion channel gating, a process fundamental to all neuronal transmission, to control of endolysosomal delivery into cells, and recycling of membrane proteins out to the plasma membrane. The aim is to determine the motion that is brought about in voltage sensing domains upon activation based on the first atomic structures of a resting state and an activated state in the same channel, an endolysosomal two-pore channel. The aim is to determine how this structural change evoked upon voltage change is relayed to gate a central channel, by separating covalent attachment by means of a direct protein sequence, from non-covalent interactions between the sensor and the channel. The approach uses different two-pore channels, chemical cross linking, chemical trapping, and mutations coupled to channel recordings, followed by atomic structural definition of intermediate states in the process. With a related protein the aim is to visualize cellular complexes that associate with mucolipin in the endolysosome. These partnerships impact many lysosomal and neuronal diseases and offer a map for design of inhibitors of these processes that will lead the way to drugs that can be therapeutically advantageous. The interactome of channels in the endolysosome offer an amazing network of connections to disease that are a subject of this proposal. A second set of aims concerns how transmembrane transporters, crucial to cell viability, transport essential nutrients into the cell, and function at the molecular and structural level. By atomic structure determination of a transporter of essential phosphate, and others of essential glucose, and of uric acid these transporters provide a roadmap to targeting these processes with inhibitors and activators, that will be of therapeutic advantage in cancers, and are of use in regulating the 'master' regulators in the cell.