Effect of subunit composition and Liddle's syndrome mutations on biosynthesis of ENaC

Abstract

The epithelial Na+ channel (ENaC) is comprised of three homologous subunits: alpha, beta, and gamma, all of which are required for formation of the fully functional channel. This channel is responsible for salt reabsorption in the kidney, the airway, and the large bowel. Mutations in ENaC can cause human disease by increasing channel function in Liddle's syndrome, a form of hereditary hypertension, or by decreasing channel function in pseudohypoaldosteronism type I, a salt-wasting disease of infancy. We previously showed that ENaC is expressed on the cell surface as a minimally glycosylated, Triton-insoluble protein. In the present study we found that ENaC existed initially as a Triton-soluble protein that contained high-mannose glycosylation, presumably in the endoplasmic reticulum. This form of the protein disappeared as the Triton-insoluble, minimally glycosylated form became the more prevalent species. In pulse-chase studies of individually expressed subunits, we found that the Triton-soluble form of beta-ENaC accumulated initially, whereas the Triton-soluble form of alpha-ENaC decreased throughout the time course. However, when all three subunits were coexpressed, the alpha- and beta-subunits showed a similar pattern. The complex became Triton insoluble at some point after the endoplasmic reticulum, as incubation at 15 degrees C blocked the conversion to the insoluble form. Deletion of the carboxy-terminal tail of beta-ENaC causes Liddle's syndrome. This mutation increased the amount of newly synthesized Triton-insoluble ENaC heteromultimers but did not affect the half-life of insoluble protein. Therefore, subunit composition and mutations in individual subunits can influence biosynthesis of the ENaC complex.