Nedd4 mediates control of an epithelial Na+ channel in salivary duct cells by cytosolic Na+

Abstract

Epithelial Na+ channels are expressed widely in absorptive epithelia such as the renal collecting duct and the colon and play a critical role in fluid and electrolyte homeostasis. Recent studies have shown that these channels interact via PY motifs in the C terminals of their alpha, beta, and gamma subunits with the WW domains of the ubiquitin-protein ligase Nedd4. Mutation or deletion of these PY motifs (as occurs, for example, in the heritable form of hypertension known as Liddle's syndrome) leads to increased Na+ channel activity. Thus, binding of Nedd4 by the PY motifs would appear to be part of a physiological control system for down-regulation of Na+ channel activity. The nature of this control system is, however, unknown. In the present paper, we show that Nedd4 mediates the ubiquitin-dependent down-regulation of Na+ channel activity in response to increased intracellular Na+. We further show that Nedd4 operates downstream of Go in this feedback pathway. We find, however, that Nedd4 is not involved in the feedback control of Na+ channels by intracellular anions. Finally, we show that Nedd4 has no influence on Na+ channel activity when the Na+ and anion feedback systems are inactive. We conclude that Nedd4 normally mediates feedback control of epithelial Na+ channels by intracellular Na+, and we suggest that the increased Na+ channel activity observed in Liddle's syndrome is attributable to the loss of this regulatory feedback system.

Figure 1

Expression of Nedd4 protein in the mouse mandibular gland. Sections of the formalin-fixed, paraffin-embedded mandibular gland tissue were subjected to immunohistochemical analysis by using either the pre-immune serum (A and C), or a 1:200 dilution of anti-Nedd4 serum (B and D). Note the strong expression of Nedd4 (brown) in all granular duct cells. [Bars = 160 μm (A and B), or 80 μm (C and D).]

Figure 2

(A) The effects of the inclusion in NMDG-glutamate pipette solution of the GST–WW fusion protein (G-W), GST control (G), anti-Nedd4 IgG (A-Nd4), or pre-immune IgG on the chord conductance measured at −80 mV of the amiloride-sensitive Na⁺ conductance. (B) The effects of the inclusion in 72 mmol/l Na⁺ pipette solution of the GST control, the GST–WW fusion protein, pre-immune IgG, or anti-Nedd4 IgG on the chord conductance measured at −80 mV of the amiloride-sensitive Na⁺ conductance. (C) The effects of the inclusion in NMDG–NO₃ pipette solution of the GST–WW fusion protein or anti-Nedd4 antibody on the chord conductance measured at −80 mV of the amiloride-sensitive Na⁺ conductance. (D) The effects of the inclusion of activated α subunit of G_o(αo), activated α subunit of G_o plus pre-immune IgG, activated α subunit of G_o plus anti-Nedd4 IgG, or activated α subunit of G_i2 plus anti-Nedd4 IgG in the NMDG-glutamate pipette solution on the chord conductance measured at −80 mV of the amiloride-sensitive Na⁺ conductance. The chord conductance observed by using the NMDG-glutamate pipette solution is shown in each panel as a dotted line.

Figure 3

The effects of the inclusion in the 72 mmol/l Na⁺ pipette solution or in the NMDG-glutamate pipette solution of the GST–dn–ubiquitin (K48R) fusion protein (dn) and the GST–wt–ubiquitin fusion protein (wt) on the chord conductance measured at −80 mV of the amiloride-sensitive Na⁺ conductance.

Figure 4

Proposed model for feedback regulation of Na⁺ channels in salivary duct cells by cytosolic Na⁺ and Cl⁻ acting through G proteins and Nedd4.