The epithelial Na+ channel (ENaC) has a key role in the regulation of extracellular fluid volume and blood pressure. ENaC belongs to a family of ion channels that sense the external environment and it responds to several environmental cues including Na+, Cl-, protons, proteases and shear stress. How these channels respond to these extracellular cues is poorly understood. The goal of this proposal is to determine the molecular mechanisms of ENaC regulation by external Na+ and proteases. They combine to create specific pools of channels that are constitutively active, and others that are recruited only when extracellular Na+ concentrations are low. The proposed studies based on structural models will determine molecular mechanisms of regulation by both factors. Binding sites for Na+ and an inhibitory peptide will be defined, as well as dynamic changes induced upon binding of Na+ or an inhibitory peptide. Experiments will test whether an acidic pocket hosts a Na+ inhibitory binding site, and a region adjacent to the acidic pocket hosts the binding site for an inhibitory peptide. For either effector to induce changes in channel activity, they must induce local conformational changes upon binding that propagate to the channel pore. Experiments are proposed to trap functional conformations of the channel using chemical crosslinks and electrostatic effects. A mechanistic understanding of how extracellular factors regulate ENaC will inform our understanding of ENaC functional regulation and how related channels may sense their environments.

Public Health Relevance

Epithelial sodium channels (ENaCs) are intimately involved in sodium regulation, and play parts ranging from sodium retention in the kidney to salt taste on the tongue. Many factors regulate the activity of the channel through structures present on the outside surface of cells, and contribute to blood pressure control and other human health conditions. This study will determine the mechanism of action for two of these regulators, sodium itself and proteases that remove small parts of the channel.

Agency
National Institute of Health (NIH)
Institute
National Institute of Diabetes and Digestive and Kidney Diseases (NIDDK)
Type
Research Project (R01)
Project #
1R01DK098204-01A1
Application #
8628315
Study Section
Special Emphasis Panel (KMBD)
Program Officer
Ketchum, Christian J
Project Start
2014-02-10
Project End
2019-01-31
Budget Start
2014-02-10
Budget End
2015-01-31
Support Year
1
Fiscal Year
2014
Total Cost
$333,319
Indirect Cost
$115,819
Name
University of Pittsburgh
Department
Internal Medicine/Medicine
Type
Schools of Medicine
DUNS #
004514360
City
Pittsburgh
State
PA
Country
United States
Zip Code
15213
Kashlan, Ossama B; Blobner, Brandon M; Zuzek, Zachary et al. (2015) Na+ inhibits the epithelial Na+ channel by binding to a site in an extracellular acidic cleft. J Biol Chem 290:568-76