Molecular Basis for Transmembrane Conduction & Signaling

University Of California, San Francisco, San Francisco CA

Investigators

Abstract

PROJECT SUMMARY/ABSTRACT This proposal seeks to determine fundamental mechanisms of action of integral membrane proteins at the level of atomic structure, and energy coupling. This knowledge involves defining structural transitions between multiple states that drive and regulate cyclical processes. This knowledge can be useful in targeting control over these elements in health and disease. How dynamic changes in structure are harnessed for energy coupling of directional transport, and inhibition or modulation of function are goals. How voltage sensing domains activate voltage-gated ion channels is a current theme. We and others define changes in voltage sensing domains that accompany resting to activated state transitions in terms of atomic structures. We hope to see if it is possible to determine changes using methods of triggering voltage change across the membrane in liposomes. We wish to define how these changes in voltage sensing domains are so effectively coupled to gate the central ion channel when often the 'active state' reveals an apparently closed pore, a seeming contradiction. Our hypothesis is that it is the loosening of structural elements in the channel rather than a gate opening that alters probability of opening. Hence the structural changes will be accompanied by computational simulations that help understand the dynamics that control gating. Our current approach uses carefully selected, mostly intracellular voltage-gated ion channels since they often can be tuned to obtain both electrophysiologically defined resting and activated states at zero voltage. These include two-pore channels from endolysosomes, and a hyperpolarization activated channel, AKT1 from plant vacuoles. A second theme concerns the fundamental mechanisms by which secondary transmembrane transporters, crucial to cell viability, couple an energy source such as an ion gradient, or electrostatic potential, to control import or export of specific biomolecules, and export of drugs. By measuring transport, specificity, and determination of atomic structures in multiple states throughout their cycle we seek to define mechanisms using mutational analysis, and computational simulation. The portfolio includes an essential transporter, and therefore a drug target, from Mycobacterium tuberculosis, of yet unknown function, a uric acid transporter important in human disease, and transporters of phosphate and sulfate as paradigms of organic anion import in eukaryotes. These transporters provide a roadmap to targeting these processes with inhibitors and activators, that is of therapeutic advantage in infectious diseases like tuberculosis, diseases of mis regulation, and cancers. In proposing a MIRA I seek to enhance the scope of our NIGMS funded research to include fundamental studies of ATP-driven ABC transporters. The number of target transporters and channels at any one time is chosen to balance the barriers in some research targets, against more rapid progress in more mature projects.