Specific Palmitoyltransferases Associate with and Activate the Epithelial Sodium Channel

Area of Science:

• Molecular Biology
• Cell Biology
• Physiology

Background:

• The epithelial sodium channel (ENaC) is crucial for fluid balance and blood pressure regulation.
• ENaC activity is modulated by post-translational modifications, including palmitoylation.
• Previous studies identified specific cysteine residues on ENaC subunits as palmitoylation sites.

Purpose of the Study:

• To identify the specific palmitoyltransferases (DHHCs) that regulate ENaC.
• To investigate the role of specific DHHCs in ENaC subunit palmitoylation and channel activity.
• To determine the physiological relevance of DHHC-mediated ENaC regulation in kidney cells.

Main Methods:

• Co-expression of mouse ENaC subunits with 23 mouse DHHC enzymes in Xenopus oocytes.
• Site-directed mutagenesis of ENaC palmitoylation sites.
• Co-immunoprecipitation to assess ENaC-DHHC complex formation.
• RT-PCR and immunofluorescence to detect DHHC expression in mCCDcl1 cells.
• Measurement of ENaC-mediated Na+ currents in response to palmitoylation inhibition.

Main Results:

• Five DHHCs (DHHC 1, 2, 3, 7, and 14) significantly increased ENaC activity.
• DHHC-mediated activation depended on γ subunit palmitoylation sites, with DHHCs 1, 2, and 14 also activating ENaC lacking β subunit sites.
• DHHC 7 specifically increased β subunit palmitoylation.
• ENaC forms complexes with multiple DHHCs.
• DHHC transcripts are present in mCCDcl1 cells, and DHHC 3 is localized to principal cells in the distal nephron.
• Palmitoylation inhibition rapidly reduced ENaC activity in polarized mCCDcl1 cells.

Conclusions:

• Specific DHHC enzymes directly regulate ENaC activity through subunit palmitoylation.
• The γ subunit plays a dominant role in DHHC-mediated ENaC regulation.
• ENaC interacts with multiple DHHCs, forming a functional complex.
• DHHC-mediated regulation of ENaC is physiologically relevant in the kidney.