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 beta- and gamma-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.
Highlights
Amiloride-sensitive epithelial Naϩ 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 [1,2,3,4]
Biochemical and functional evidence indicates that, at the cell membrane, many receptors and ion channels localize to lipid raft microdomains [60]
It has been suggested that a proportion of ENaC is localized into cholesterol-rich lipid raft submembrane domains (34 –36) and that ENaC are associated with a protein marker of caveolae, Cav-1 [35]
Summary
DNA Constructs—Mouse ␣-, -, and ␥-ENaC (in pBluescript) were a gift from Thomas R. Cell Culture and Transfection and Reagents—Fischer rat thyroid (FRT) cells were a gift from Lucio Nitsch (University of Naples, Naples, Italy), and M1 mouse collecting duct cells, originally generated by Stoos et al [44], were a gift from Christoph Korbmacher (Universitat Erlangen, Nurnberg, Germany). Both cell types were cultured in Dulbecco’s modified Eagle’s medium/F-12 medium with 100 units/ml penicillin and 100 g/ml streptomycin at 37 °C. Short circuit current (Isc) following the addition of ouabain was used to estimate the activity of the Naϩ/Kϩ-ATPase
Sign-in/Register to view highlights & summary Sign-in/Register to access full text options 
Talk to us
Join us for a 30 min session where you can share your feedback and ask us any queries you have
Schedule a callDisclaimer: All third-party content on this website/platform is and will remain the property of their respective owners and is provided on "as is" basis without any warranties, express or implied. Use of third-party content does not indicate any affiliation, sponsorship with or endorsement by them. Any references to third-party content is to identify the corresponding services and shall be considered fair use under The CopyrightLaw.
