The Ubiquitin Ligase Nedd4L Regulates the Na/K/2Cl Co-transporter NKCC1/SLC12A2 in the Colon

J Biol Chem. 2017 Feb 24;292(8):3137-3145. doi: 10.1074/jbc.M116.770065. Epub 2017 Jan 13.

Abstract

The ubiquitin ligase Nedd4-like (Nedd4L, or Nedd4-2) binds to and regulates stability of the epithelial Na+ channel (ENaC) in salt-absorbing epithelia in the kidney, lung, and other tissues. Its role in the distal colon, which also absorbs salt and fluid and expresses ENaC, is unknown. Using a conditional knock-out approach to knock out Nedd4L in mice intestinal epithelium (Nedd4Lf/f ;Vil-CreERT2 ) we show here that Nedd4L depletion leads to a higher steady-state short circuit current (Isc) in mouse distal colon tissue relative to controls. This higher Isc was partially reduced by the addition of apical amiloride and strongly reduced by basolateral bumetanide as well as by depletion of basolateral Cl-, suggesting that Na+/K+/2Cl- (NKCC1/SLC12A2) co-transporter and ENaC are targets of Nedd4L in the colon. In accordance, NKCC1 (and γENaC) protein abundance in the colon of the Nedd4L knock-out animals was increased, indicating that Nedd4L normally suppresses these proteins. However, we did not observe co-immunoprecipitation between Nedd4L and NKCC1, suggesting that Nedd4L indirectly suppresses NKCC1 expression. Low salt diet resulted in a strong increase in β and γ (but not α) ENaC mRNA and protein expression and ENaC activity. Although salt restriction also increased NKCC1 protein and mRNA abundance, it did not lead to its elevated activity (Isc). These results identify NKCC1 as a novel target for Nedd4L-mediated down-regulation in vivo, which modulates ion and fluid transport in the distal colon together with ENaC.

Keywords: E3 ubiquitin ligase; cell biology; epithelial sodium channel (ENaC); gene knockout; intestine; membrane transport; ubiquitin.

Publication types

  • Research Support, Non-U.S. Gov't

MeSH terms

  • Animals
  • Chlorides / metabolism
  • Colon / metabolism*
  • Endosomal Sorting Complexes Required for Transport / genetics
  • Endosomal Sorting Complexes Required for Transport / metabolism*
  • Epithelial Sodium Channels / genetics
  • Epithelial Sodium Channels / metabolism*
  • HEK293 Cells
  • Humans
  • Mice
  • Mice, Inbred C57BL
  • Mice, Knockout
  • Nedd4 Ubiquitin Protein Ligases
  • Potassium / metabolism
  • Salts / metabolism
  • Signal Transduction
  • Sodium / metabolism
  • Solute Carrier Family 12, Member 2 / genetics
  • Solute Carrier Family 12, Member 2 / metabolism*
  • Ubiquitin-Protein Ligases / genetics
  • Ubiquitin-Protein Ligases / metabolism*
  • Up-Regulation

Substances

  • Chlorides
  • Endosomal Sorting Complexes Required for Transport
  • Epithelial Sodium Channels
  • Salts
  • Slc12a2 protein, mouse
  • Solute Carrier Family 12, Member 2
  • Sodium
  • Nedd4 Ubiquitin Protein Ligases
  • Nedd4 protein, human
  • Nedd4L protein, human
  • Nedd4l protein, mouse
  • Ubiquitin-Protein Ligases
  • Potassium