Defective regulation of the epithelial Na+ channel by Nedd4 in Liddle's syndrome

Abstract

Liddle's syndrome is an inherited form of hypertension linked to mutations in the epithelial Na+ channel (ENaC). ENaC is composed of three subunits (alpha, beta, gamma), each containing a COOH-terminal PY motif (xPPxY). Mutations causing Liddle's syndrome alter or delete the PY motifs of beta- or gamma-ENaC. We recently demonstrated that the ubiquitin-protein ligase Nedd4 binds these PY motifs and that ENaC is regulated by ubiquitination. Here, we investigate, using the Xenopus oocyte system, whether Nedd4 affects ENaC function. Overexpression of wild-type Nedd4, together with ENaC, inhibited channel activity, whereas a catalytically inactive Nedd4 stimulated it, likely by acting as a competitive antagonist to endogenous Nedd4. These effects were dependant on the PY motifs, because no Nedd4-mediated changes in channel activity were observed in ENaC lacking them. The effect of Nedd4 on ENaC missing only one PY motif (of beta-ENaC), as originally described in patients with Liddle's syndrome, was intermediate. Changes were due entirely to alterations in ENaC numbers at the plasma membrane, as determined by surface binding and immunofluorescence. Our results demonstrate that Nedd4 is a negative regulator of ENaC and suggest that the loss of Nedd4 binding sites in ENaC observed in Liddle's syndrome may explain the increase in channel number at the cell surface, increased Na+ reabsorption by the distal nephron, and hence the hypertension.

Figure 1

Regulation of ENaC by Nedd4 overexpressed in Xenopus oocytes. (a) Immunoblot analysis showing expression of xNedd4 in oocytes injected with cRNA of ENaC alone (ENaC/H₂O), or ENaC coinjected with either 25 ng of xNedd4-wt cRNA (ENaC/Nedd4-wt) or the same amount of mutant xNedd4 lacking ubiquitin–protein ligase activity (ENaC/Nedd4-CS). Proteins from oocyte lysates were separated on SDS-PAGE and immunoblotted with anti-xNedd4 antibodies. The left lane shows the level of endogenous xNedd4. Faster-migrating protein bands in Nedd4-injected oocytes likely represent degradation products. (b) I_Na were measured in oocytes expressing either ENaC alone (ENaC/H₂O), ENaC plus xNedd4-wt (ENaC/Nedd4-wt), or ENaC plus xNedd4C938S (ENaC/Nedd4-CS). The measured currents were normalized to control oocytes (4.2 ± 0.6 μA/oocyte). n = 36 oocytes from six different batches. **P < 0.01 vs. ENaC/H₂O–injected oocytes. ENaC, epithelial sodium channel; I_Na, amiloride-sensitive Na⁺ current; wt, wild-type.

Figure 2

Dose–response relationship of the effect of Nedd4 on ENaC activity. (a) Immunoblot analysis showing expression of xNedd4 with increasing amounts of xNedd4 cRNA (ng/oocyte). (b) Corresponding normalized I_Na. The measured currents were normalized to control oocytes (6.1 ± 2.3 μA/oocyte). n = 16–18 oocytes from three different batches. **P < 0.01 vs. ENaC/H₂O–injected oocytes (i.e., 0 ng of xNedd4 cRNA). The difference in I_Na in oocytes injected with 0 and 0.2 ng cRNA/oocyte was not statistically significant.

Figure 3

ENaC PY motifs are necessary for the effect of Nedd4 on ENaC activity. Oocytes were either injected with ENaC-wt (left three bars), ENaC bearing mutations in all three PY motifs (ENaC-ΔPY₃; middle three bars), or a Liddle's ENaC channel missing the COOH terminus of β-ENaC (βR564stop), including its PY motif (right three bars), together with either H₂O (closed bars), Nedd4-wt (open bars), or xNedd4C938S (hatched bars). Currents were normalized to control mean values (5.5 ± 1.2 μA/oocyte). Twenty-four oocytes from four different batches were measured per condition. *P < 0.05 and **P < 0.01 represent levels of significance relative to conditions indicated by the brackets.

Figure 4

Nedd4 regulates the number of ENaC channels at the cell surface. Oocytes were coinjected with ENaC^F and either H₂O (left two bars), 25 ng xNedd4-wt cRNA (middle two bars), or xNedd4C938S (Nedd4-CS; right two bars). To quantitate the number of channels a t the cell surface, I_Na (closed bars) and binding of iodinated anti-FLAG antibodies (open bars) were measured in the same oocytes, as described previously (25). Current and binding values were normalized to control values (5.5 ± 1.2 μA/oocyte and 0.10 ± 0.01 fmol/oocyte, respectively). **P < 0.01 vs. ENaC^F/H₂O–injected oocytes.

Figure 5

Immunostaining of ENaC expressed in Xenopus oocytes coexpressing Nedd4-wt or -CS. ENaC^F-related membrane immunostainings were carried out with anti-FLAG antibodies in oocytes expressing ENaC alone (a), ENaC plus Nedd4-wt (b), or ENaC plus xNedd4C938S (Nedd4-CS; c) as detailed in Methods. Scale bar: 20 μm.