Trafficking and Regulation of the Epithelial Na+ Channel

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DESCRIPTION (provided by applicant): Maintenance of.extracellular fluid volume homeostasis is essential for hemodynamic stability, and abnormalities of renal sodium handling have been linked to cardiovascular disease and hypertension, Ultimate regulation of sodium excretion in the kidney occurs in the distal nephron via conductive transport through the amiloride sensitive epithelial Na+channel (ENaC). ENaC expression and activity in the apical membrane of epithelial cells is the rate limiting step in Na+reabsorption not only in the kidney collecting duce, but in airway epithelia and colon as well. Abnormalities of ENaC function have been demonstrated in hereditary forms of salt-sensitive hypertension, renal salt wasting, and cystic Fibrosis. The long term goal of this research is to understand the factors that regulate ENaC expression in the apical membrane of epithelial cells and the mechanisms by which hormones, physiologic conditions and other channels (such as the cystic fibrosis trahsmembrane regulator CFTR) control ENaC function. Major control of ENaC function is exerted by regulation of the number of active channels in the membrane of Na+ reabsorbing cells. Channels are activated by proteolytic cleavage. Control of apical expression of the channel is a function of delivery of the channel to the membrane, retrieval through endocytosis, and the balance between recycling and degradation of the channel. Multiple observations in responsive cell lines and native tissues including kidney and lung have demonstrated that modulation of channel activity is associated with non-coordinate regulation of the 3 separate subunits which make up the fully active channel. The current experiments will define the binding partners and regulatory sites of clathrin-mediated endocytosis of the channel, the fate of the individual subunits within the endocytic pathways and the regulation of channel recycling to the apical membrane. Experiments are designed to determine if non-coordinate regulation of channel subunits takes place in the endocytic pathway and is related to differential handling of wild type and cleaved channels which have.been activated by proteolysis. The research aims to identify regulatory mechanisms which may be subject to abnormal regulation in disease states such as hypertension.