Acidic extracellular solution activates transient H+-gated currents in dorsal root ganglion (DRG) neurons. The biophysical properties of three degenerin/epithelial sodium (DEG/ENaC) channel subunits (BNC1, ASIC, and DRASIC), and their expression in DRG, suggest that they might underlie these H+-gated currents and function as sensory transducers. However, it is uncertain which of these DEG/ENaC subunits generate the currents, and whether they function as homomultimers or heteromultimers. We found that the biophysical properties of transient H+-gated currents from medium to large mouse DRG neurons differed from BNC1, ASIC, or DRASIC expressed individually, but were reproduced by coexpression of the subunits together. To test the contribution of each subunit, we studied DRG from three strains of mice, each bearing a targeted disruption of BNC1, ASIC, or DRASIC. Deletion of any one subunit did not abolish H+-gated currents, but altered currents in a manner consistent with heteromultimerization of the two remaining subunits. These data indicate that combinations of two or more DEG/ENaC subunits coassemble as heteromultimers to generate transient H+-gated currents in mouse DRG neurons

The epithelial Na ؉ channel (ENaC) forms the pathway for Na ؉ absorption across epithelia, including the kidney collecting duct, where it plays a critical role in Na ؉ homeostasis and blood pressure control. Na ؉ absorption is regulated in part by mechanisms that control the expression of ENaC at the apical cell surface. Nedd4 family members (e.g. Nedd4, Nedd4-2) bind to the channel and decrease its surface expression by catalyzing its ubiquitination and degradation. Conversely, serum and glucocorticoid-regulated kinase (SGK), a downstream mediator of aldosterone, increases the expression of ENaC at the cell surface. Here we show that SGK and human Nedd4-2 (hNedd4-2) converge in a common pathway to regulate epithelial Na ؉ absorption. Consistent with this model, we found that SGK bound to hNedd4-2 and hNedd4. A PY motif in SGK mediated the interaction and was required for SGK to stimulate ENaC. SGK phosphorylated hNedd4-2 (but not hNedd4), altering hNedd4-2 function; phosphorylation reduced the binding of hNedd4-2 to ␣ENaC, and hence, the hNedd4-2-mediated inhibition of Na ؉ absorption. These data suggest that SGK regulates epithelial Na ؉ absorption in part by modulating the function of hNedd4-2.

Liddle's syndrome is an inherited form of hypertension caused by mutations that truncate the C-terminus of human epithelial Na+ channel (hENaC) subunits. Expression of truncated beta and gamma hENaC subunits increased Na+ current. However, truncation did not alter single-channel conductance or open state probability, suggesting there were more channels in the plasma membrane. Moreover, truncation of the C-terminus of the beta subunit increased apical cell-surface expression of hENaC in a renal epithelium. We identified a conserved motif in the C-terminus of all three subunits that, when mutated, reproduced the effect of Liddle's truncations. Further, both truncation of the C-terminus and mutation of the conserved C-terminal motif increased surface expression of chimeric proteins containing the C-terminus of beta hENaC. Thus, by deleting a conserved motif, Liddle's mutations increase the number of Na+ channels in the apical membrane, which increases renal Na+ absorption and creates a predisposition to hypertension.

Agonist-stimulated ␤ 2 -adrenergic receptor (␤ 2 AR) ubiquitination is a major factor that governs both lysosomal trafficking and degradation of internalized receptors, but the identity of the E3 ubiquitin ligase regulating this process was unknown. Among the various catalytically inactive E3 ubiquitin ligase mutants that we tested, a dominant negative Nedd4 specifically inhibited isoproterenol-induced ubiquitination and degradation of the ␤ 2 AR in HEK-293 cells. Moreover, siRNA that downregulates Nedd4 expression inhibited ␤ 2 AR ubiquitination and lysosomal degradation, whereas siRNA targeting the closely related E3 ligases Nedd4-2 or AIP4 did not. Interestingly, ␤ 2 AR as well as ␤-arrestin2, the endocytic and signaling adaptor for the ␤ 2 AR, interact robustly with Nedd4 upon agonist stimulation. However, ␤ 2 AR-Nedd4 interaction is ablated when ␤-arrestin2 expression is knocked down by siRNA transfection, implicating an essential E3 ubiquitin ligase adaptor role for ␤-arrestin2 in mediating ␤ 2 AR ubiquitination. Notably, ␤-arrestin2 interacts with two different E3 ubiquitin ligases, namely, Mdm2 and Nedd4 to regulate distinct steps in ␤ 2 AR trafficking. Collectively, our findings indicate that the degradative fate of the ␤ 2 AR in the lysosomal compartments is dependent upon ␤-arrestin2-mediated recruitment of Nedd4 to the activated receptor and Nedd4-catalyzed ubiquitination.