The Activity of the Epithelial Sodium Channels Is Regulated by Caveolin-1 via a Nedd4-2-dependent Mechanism

Abstract

It has recently been shown that the epithelial Na؉ channel (ENaC) is compartmentalized in caveolin-rich lipid rafts and that pharmacological depletion of membrane cholesterol, which disrupts lipid raft formation, decreases the activity of ENaC. Here we show, for the first time, that a signature protein of caveolae, caveolin-1 (Cav-1), down-regulates the activity and membrane surface expression of ENaC. Physical interaction between ENaC and Cav-1 was also confirmed in a coimmunoprecipitation assay. We found that the effect of Cav-1 on ENaC requires the activity of Nedd4-2, a ubiquitin protein ligase of the Nedd4 family, which is known to induce ubiquitination and internalization of ENaC. The effect of Cav-1 on ENaC requires the proline-rich motifs at the C termini of the ␤-and ␥-subunits of ENaC, the binding motifs that mediate interaction with Nedd4-2. Taken together, our data suggest that Cav-1 inhibits the activity of ENaC by decreasing expression of ENaC at the cell membrane via a mechanism that involves the promotion of Nedd4-2-dependent internalization of the channel.Amiloride-sensitive epithelial Na ϩ channels (ENaC) 3 are membrane proteins that are expressed in salt-absorptive epithelia, including the distal collecting tubules of the kidney, the mucosa of the distal colon, the respiratory epithelium, and the excretory ducts of sweat and salivary glands (1-4). Na ϩ absorption via ENaC is critical to the normal regulation of Na ϩ and fluid homeostasis and is important for maintaining blood pressure (5) and the volume of fluid in the respiratory passages (6). Increased ENaC activity has been implicated in the salt-sensitive inherited form of hypertension, Liddle's syndrome (7), and dehydration of the surface of the airway epithelium in the pathology associated with cystic fibrosis lung disease (8).Expression of ENaC at the cell membrane surface is regulated by the E3 ubiquitin protein ligase, Nedd4-2 (neural precursor cell expressed developmentally down-regulated protein 4) (9).Interaction between the WW domains of Nedd4-2 and the proline-rich PY motifs (PPPXY) on ENaC is essential for Nedd4-2 to exert a negative effect on the channel (10, 11). This interaction leads to ubiquitination-dependent internalization of ENaC (12, 13). Several regulators of ENaC exert their effects on the channel by modulating the action of Nedd4-2. For instance, serum and glucocorticoid-dependent protein kinase (14), protein kinase B (15), and G protein-coupled receptor kinase (16) up-regulate activity of ENaC by inhibiting Nedd4-2. Although the details of cellular mechanisms that underlie internalization of ENaC remain to be elucidated, the physiological significance of Nedd4-dependent internalization of the channel has been well established. For instance, heritable mutations that delete the cytosolic termini of the ␤-or ␥-subunit of ENaC, which contain the proline-rich motifs, are known to cause hyperactivity of ENaC in the kidney (17) and increase cell surface expression of the channel (7, 18).The plasma membranes of most cell t...

FGF23 regulates renal sodium handling and blood pressure

“…ENaC is a heteromultimeric membrane protein consisting of a-, b-, and c-subunits. The abundance of the aldosterone-induced a-subunit is the rate-limiting factor in the assembly of the ENaC complex (May et al, 1997), whereas the b-and c-subunits are involved in ubiquitination and degradation of ENaC (Lee et al, 2009). WNK4 is an important regulator of distal tubular membrane abundance of the Na + :Cl À co-transporter NCC and physically interacts with the NCC protein to regulate its membrane trafficking (Cai et al, 2006).…”

Regulation of the Epithelial Na+ Channel by the RH Domain of G Protein-coupled Receptor Kinase, GRK2, and Gαq/11

“…The chamber was connected to a VCC MC6 multichannel voltage/current clamp amplifier, controlled and monitored using the Acquire & Analyze software (V2.3.177, Physiologic Instruments, San Diego, CA). Experiments were carried out under current clamp (open circuit) conditions as previously described (24). Transepithelial potential was measured with reference to the baso-lateral side of the epithelium and current pulses were injected to assess the transepithelial resistance.…”

“…Activity of the Na ϩ /K ϩ -ATPase was determined as previously described (24). In short, amiloride (10 M) was added to the apical bathing solution to inhibit activity of ENaC.…”

“…We reconstituted ENaC in Fischer Rat Thyroid (FRT) cells by transiently expressing the ␣-, ␤-, and ␥-ENaC subunits. A similar approach has previously been used successfully to investigate regulation of ENaC (24,27). The cells were co-transfected with the wtGRK2 construct or with siRNA directed against GRK2 (Fig.…”

Plasticity in Membrane Cholesterol Contributes toward Electrical Maturation of Hearing

“…These cholesterol/ sphingolipid-rich domains are thought to confer spatial segregation of signaling pathways in cells (13). Indeed, several ion channels gravitate toward these cholesterol-rich "icebergs" in the sea of lipids, which regulate channel protein functions or their cell-surface expression (18,19). Moreover, the composition of the lipid microdomains undergoes drastic and consistent plasticity during neurodevelopment, serving as stagespecific markers for certain lineages of cells (20 -22).…”