Regulation of expression and function of epithelial sodium channel by lectin-like domain of TNF alpha

Alveolar liquid clearance (ALC) is crucial in order to assure a proper gas exchange between the alveoli and the lung capillaries. ALC is mediated by the transport of Na+ in type II alveolar epithelial cells, by means of the apically expressed amiloride-sensitive epithelial sodium channel (ENaC) and the basolaterally expressed Na+-K+ ATPase. These ion transporters can be impaired during mountain sickness, acute respiratory distress syndrome and pneumonia, which can contribute to the development of potentially lethal lung edema. As such, the search for novel treatments able to stimulate Na+ transport, by means of activating Na+ transporters is of the utmost clinical importance. We have previously demonstrated that the lectin-like domain of TNF, mimicked by the 17 amino acid TIP peptide, efficiently activates ENaC, in both models of cardiogenic and permeability pulmonary edema. ENaC function is controlled both at the level of expression, regulated by the interaction with Nedd4-2, as well as by the open probability of the channel. Our preliminary data indicate that the TIP peptide can increase the expression of the crucial alpha subunit of the channel in the presence of bacterial toxins and moreover increases the open probability of the channel, as measured in single channel patch clamp. The latter activity possibly occurs by means of the demonstrated direct binding of the peptide to the intracellular carboxy-terminal domain of ENaC-alpha, involved in gating of the channel. Using state-of-the-art protein chemistry and molecular biology techniques, this application aims at unraveling the mechanisms by which the TIP peptide activates ENaC. We will visualize and characterize the localization and internalization of the TIP peptide in H441 cells. We will map the binding sites between the TIP peptide and the alpha subunit and we will test the hypothesis that the TIP peptide can increase ENaC function, upon strengthening the association between the channel with PIP2 and the protein MARCKS. The results from these studies can thus contribute to the development of novel therapeutic approaches for the treatment of pulmonary edema, which represents a potentially lethal complication of both cardiogenic edema, associated with cardiac dysfunction and permeability edema, associated with pneumonia and ARDS. (AHA Program: Postdoctoral Fellowship)