The epithelial Na+ channel (ENaC) is exposed to a variety of factors in the kidney, lung, vasculature, taste buds, and other tissues. There, proteases, changes in sodium, chloride, pH, and mechanical stress modulate channel activity. My laboratory seeks to understand the molecular mechanisms of channel regulation by these factors. One focus of our work is activation by proteases. Our research provided evidence that proteases liberate an autoinhibitory sequence that stabilizes an interaction between two hinge domains in the alpha subunit of the channel that are far from the channel pore. We are currently examining protease activation through the gamma subunit, and whether there is functional symmetry between the subunits. A second focus of our work is inhibition by extracellular Na+, and relatedly - activation by protons. We identified a key aspartate within an allosterically active acidic pocket of the channel that accounted for most of the inhibition by extracellular Na+. We also found that protons activate the channel by protonating the key aspartate, reducing Na+ affinity for the site and relieving inhibition by Na+. Our research also suggested at least one additional site for Na+ binding within the channel. We are comparing related and ancestral ENaC subunits that vary in their response to Na+ to identify the key binding and transduction steps in the molecular mechanism. A third focus of our laboratory is modularity in the ENaC family of proteins. Proteins in this family share a common fold, but vary greatly in their peripheral domains, and in their responses to extracellular cues. To gain insight into how this family of proteins evolved to perform a variety of functions, we are determining whether certain variable domains are responsible for ligand sensitivity, and whether that sensitivity can be transferred from one protein to another. Our ultimate goal is to define the molecular mechanisms of channel regulation in this family of ion channels. Our approach is multidisciplinary, using techniques from molecular biology, electrophysiology, protein biochemistry, structural biology, cell biology, and computational biology.