Specific Palmitoyltransferases Associate With and Activate the Epithelial Sodium Channel [Membrane Biology]

The epithelial sodium channel (ENaC) has an important role in regulating extracellular fluid volume and blood pressure, as well as airway surface liquid volume and mucociliary clearance. ENaC is a trimer of three homologous subunits (α, β and γ). We previously reported that cytoplasmic residues on the β (βCys43 and βCys557) and γ (γCys33 and γCys41) subunits are palmitoylated. Mutation of Cys that blocked ENaC palmitoylation also reduced channel open probability. Furthermore, γ subunit palmitoylation had a dominant role over β subunit palmitoylation in regulating ENaC. To determine which palmitoyltransferases (termed DHHCs) regulate the channel, mouse ENaCs were co-expressed in Xenopus oocytes with each of the 23 mouse DHHCs. ENaC activity was significantly increased by DHHCs 1, 2, 3, 7 and 14. ENaC activation by DHHCs was lost when γ subunit palmitoylation sites were mutated, while DHHCs 1, 2 and 14 still activated ENaC lacking β subunit palmitoylation sites. β subunit palmitoylation was increased by ENaC co-expression with DHHC 7. Both wild type ENaC and channels lacking β and γ palmitoylation sites co-immunoprecipitated with the five activating DHHCs, suggesting that ENaC forms a complex with multiple DHHCs. RT-PCR revealed that transcripts for the five activating DHHCs were present in cultured mCCDcl1 cells, and DHHC 3 was expressed in aquaporin 2-positive principal cells of mouse aldosterone-sensitive distal nephron where ENaC is localized. Treatment of polarized mCCDcl1 cells with a general inhibitor of palmitoylation reduced ENaC-mediated Na+ currents within minutes. Our results indicate that specific DHHCs have a role in regulating ENaC.

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