Premium
Regulation of ENaC by specific palmitoyltransferases
Author(s) -
Mukherjee Anindit,
Wong Zhijian,
Poland Paul A,
Montalbetti Nicolas,
Butterworth Michael,
Fukata Masaki,
Kleyman Thomas R,
Hughey Rebecca P
Publication year - 2016
Publication title -
the faseb journal
Language(s) - English
Resource type - Journals
SCImago Journal Rank - 1.709
H-Index - 277
eISSN - 1530-6860
pISSN - 0892-6638
DOI - 10.1096/fasebj.30.1_supplement.1223.5
Subject(s) - epithelial sodium channel , palmitoylation , xenopus , protein subunit , chemistry , microbiology and biotechnology , mutant , extracellular , hek 293 cells , phosphorylation , biochemistry , biology , enzyme , sodium , cysteine , receptor , gene , organic chemistry
The epithelial sodium channel (ENaC) is a key mediator of extracellular fluid volume homeostasis and blood pressure control. It is a member of the ENaC/degenerin family and is regulated by extracellular protons, chloride, sodium, and proteases. ENaC is also regulated by phosphorylation, ubiquitination, inositol phospholipids, and Cys‐palmitoylation. We have previously shown that mouse ENaC is palmitoylated at Cys residues in the β(C43, C557) and γ(C33, C41) subunit cytoplasmic tails. ENaCs lacking β and/or γ subunit palmitoylation had normal surface expression but reduced open probability. A family of enzymes, referred to as DHHCs based on their conserved Asp‐His‐His‐Cys (DHHC) catalytic motif, catalyzes Cys‐palmitoylation. The 23 mouse DHHCs were individually co‐expressed in Xenopus oocytes with either wild‐type ENaC, or ENaC with a mutant β and/or mutant γ subunit lacking Cys palmitoylation sites. Five of the 23 DHHCs activated ENaC. Both the β and γ subunit palmitoylation sites were required for full ENaC activation by DHHCs. As the five activating DHHCs are expressed in mouse CCD cells as evidenced by RT‐PCR, we tested whether inhibiting DHHC activity with the irreversible palmitoylation inhibitor, 2‐bromopalmitate (2‐BP) altered ENaC activity. Short circuit currents (Isc) were measured in mCCD cells. Isc began to fall within 5 min after addition of apical 2‐BP (25 μM), and reached a steady state after 30 minutes (50.1 ± 2.8 mA/cm 2 (vehicle control) vs. 11.3 ± 1.2 mA/cm 2 (2‐BP) (N=9, p<0.0001). Conserved cytoplasmic His‐Gly (HG) tracts that regulate ENaC gating are in close proximity to both βC43 and γC41. Mutations of the HG tract have been shown to decrease ENaC activity and open probability. We examined whether mutating the HG tract affects ENaC activation by Cys‐palmitoylation. While DHHC2 increased activity of wild type ENaC, channels with a βH36A mutation were not activated by DHHC2. These data suggest that an intact HG tract is required for channel activation by Cys‐palmitoylation. Support or Funding Information DK051391, P30‐DK079307