Project Summary Volume control is critical for cellular responses to osmotic stress and for modulating cell size during growth, migration, and death. Osmotic forces dictate that a cell exposed to a hypotonic solution will swell and even burst, a property routinely used for cell lysis. However, researchers noted in the 1980's that many vertebrate cell types respond to swelling by releasing chloride ions. This generates an observable current, corrects the osmotic imbalance, and leads to cell shrinking. The hypothetical channel mediating this apparently ubiquitous activity was termed the “volume-regulated anion channel” or VRAC. In addition to controlling cell volume, VRAC is implicated in membrane passage of physiologically and medically important molecules, such as the neurotransmitter glutamate and the anti-cancer drug Cisplatin. Recently, the molecular identity of VRAC activity has been determined to be heteromeric complexes of LRRC8 proteins. This knowledge opens up multiple questions about VRAC function, including precisely how the channel is composed, how these channels sense osmotic changes, how they are gated, and which features of the VRAC pore determine anion selectivity and passage of diverse small molecules. This project addresses these questions using a combined structural and electrophysiological approach. The specific aims of the study are: (1) To express and purify defined LRRC8 complexes; (2) to determine the structure of LRRC8 channels; and (3) to conduct in vivo and in vitro electrophysiological experiments on channels to determine the mechanism for their function and regulation. Completion of these aims will reveal the architecture of a novel cellular gateway and provide a molecular mechanism for cellular volume control.