Specific Palmitoyltransferases Associate with and Activate the Epithelial Sodium Channel.
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 (βCys-43 and βCys-557) and γ (γCys-33 and γCys-41) 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 inXenopusoocytes 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, whereas 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 mCCDcl1cells, and DHHC 3 was expressed in aquaporin 2-positive principal cells of mouse aldosterone-sensitive distal nephron where ENaC is localized. Treatment of polarized mCCDcl1cells 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.
From the Departments of Medicine.
Anindit
A
Mukherjee
From the Departments of Medicine.
Zhijian
Z
Wang
From the Departments of Medicine.
Carol L
CL
Kinlough
From the Departments of Medicine.
Paul A
PA
Poland
From the Departments of Medicine.
Allison L
AL
Marciszyn
From the Departments of Medicine.
Nicolas
N
Montalbetti
From the Departments of Medicine.|Cell Biology, and.
Marcelo D
MD
Carattino
Cell Biology, and.
Michael B
MB
Butterworth
From the Departments of Medicine, kleyman@pitt.edu.|Cell Biology, and.|Pharmacology and Chemical Biology, University of Pittsburgh School of Medicine, Pittsburgh, Pennsylvania 15261.
Thomas R
TR
Kleyman
From the Departments of Medicine.|Cell Biology, and.
Rebecca P
RP
Hughey
D000217:Acyltransferases / Q000235:genetics / Q000378:metabolism*; D000818:Animals; D002478:Cells, Cultured; D003593:Cytoplasm / Q000378:metabolism; D053503:Epithelial Sodium Channels / Q000235:genetics / Q000378:metabolism*; D005260:Female; D057809:HEK293 Cells; D006801:Humans; D047468:Immunoprecipitation; D015640:Ion Channel Gating / Q000502:physiology*; D017136:Ion Transport; D007668:Kidney / Q000166:cytology / Q000378:metabolism*; D054878:Lipoylation; D051379:Mice; D008810:Mice, Inbred C57BL; D009865:Oocytes / Q000166:cytology / Q000378:metabolism; D011499:Protein Processing, Post-Translational; D021122:Protein Subunits; D051102:Serine C-Palmitoyltransferase / Q000378:metabolism; D012964:Sodium / Q000378:metabolism; D014982:Xenopus laevis
D053503:Epithelial Sodium Channels; D021122:Protein Subunits; D012964:Sodium; D000217:Acyltransferases; D051102:Serine C-Palmitoyltransferase
Cys palmitoylation; DHHC; epithelial sodium channel (ENaC); ion channel; post-translational modification (PTM); protein acylation; protein palmitoylation