The δ-Subunit of the Epithelial Sodium Channel (ENaC) Enhances Channel Activity and Alters Proteolytic ENaC Activation

Abstract

The epithelial sodium channel (ENaC) is probably a heterotrimer with three well characterized subunits (␣␤␥). In humans an additional ␦-subunit (␦-hENaC) exists but little is known about its function. Using the Xenopus laevis oocyte expression system, we compared the functional properties of ␣␤␥-and ␦␤␥-hENaC and investigated whether ␦␤␥-hENaC can be proteolytically activated. The amiloride-sensitive ENaC whole-cell current (⌬I ami ) was about 11-fold larger in oocytes expressing ␦␤␥-hENaC than in oocytes expressing ␣␤␥-hENaC. The 2-fold larger single-channel Na ؉ conductance of ␦␤␥-hENaC cannot explain this difference. Using a chemiluminescence assay, we demonstrated that an increased channel surface expression is also not the cause. Thus, overall channel activity of ␦␤␥-hENaC must be higher than that of ␣␤␥-hENaC. Experiments exploiting the properties of the known ␤S520C mutant ENaC confirmed this conclusion. Moreover, chymotrypsin had a reduced stimulatory effect on ␦␤␥-hENaC whole-cell currents compared with its effect on ␣␤␥-hENaC whole-cell currents (2-fold versus 5-fold). This suggests that the cell surface pool of so-called near-silent channels that can be proteolytically activated is smaller for ␦␤␥-hENaC than for ␣␤␥-hENaC. Proteolytic activation of ␦␤␥-hENaC was associated with the appearance of a ␦-hENaC cleavage product at the cell surface. Finally, we demonstrated that a short inhibitory 13-mer peptide corresponding to a region of the extracellular loop of human ␣-ENaC inhibited ⌬I ami in oocytes expressing ␣␤␥-hENaC but not in those expressing ␦␤␥-hENaC. We conclude that the ␦-subunit of ENaC alters proteolytic channel activation and enhances base-line channel activity.

Expression and function of the epithelial sodium channel δ-subunit in human respiratory epithelial cells in vitro

“…The specific δ-ENaC protein band at 75 kDa was of a significantly higher intensity in the transfected cells, and was demonstrated to be an N-glycosylated form of a 70 kDa protein, which was consistent with the previous reports of δ-ENaC protein being N-glycosylated [12,23]. Moreover, elevated protein levels led to an increased insertion into the apical cell membrane, as confirmed by IFM.…”

Atomic Force Microscopy Reveals the Architecture of the Epithelial Sodium Channel (ENaC)

“…A fourth ENaC subunit, ␦-ENaC, has been cloned from a human kidney library (5). In heterologous expression systems, ␦-ENaC has functional similarities with ␣-ENaC, but its physiological role is still unclear (6). Each ENaC subunit contains two transmembrane domains, a large extracellular loop, and short intracellular N and C termini.…”

“…following C-terminal tags: ␣-ENaC -V5 or FLAG; ␤-ENaC -V5 or His 6 ; and ␥-ENaC -V5. All tags were added directly to the ENaC sequence, except for ␣HA/FLAG-ENaC, which had a short linker (AGGLAS) inserted between the ENaC sequence and the FLAG epitope.…”

“…Expression and Functional Analysis of ENaCs in Xenopus laevis Oocytes-Oocyte experiments were performed essentially as described previously (6,17). Oocytes were obtained from adult female X. laevis in accordance with the principles of German legislation, with approval by the animal welfare officer for the University of Erlangen-Nürnberg, and under the governance of the state veterinary health inspectorate (permit no.…”

“…The two-electrode voltage-clamp technique used was essentially as described previously (6,17). Individual oocytes were placed in a small experimental chamber and constantly superfused with high sodium solution ND96 (96 mM NaCl, 2 mM KCl, 1.8 mM CaCl 2 , 1 mM MgCl 2 , and 5 mM HEPES, pH 7.4) supplemented with amiloride (2 M).…”