A highly conserved motif at the COOH terminus dictates endoplasmic reticulum exit and cell surface expression of NKCC2

J Biol Chem. 2009 Aug 7;284(32):21752-64. doi: 10.1074/jbc.M109.000679. Epub 2009 Jun 17.


Mutations in the apically located Na(+)-K(+)-2Cl(-) co-transporter, NKCC2, lead to type I Bartter syndrome, a life-threatening kidney disorder, yet the mechanisms underlying the regulation of mutated NKCC2 proteins in renal cells have not been investigated. Here, we identified a trihydrophobic motif in the distal COOH terminus of NKCC2 that was required for endoplasmic reticulum (ER) exit and surface expression of the co-transporter. Indeed, microscopic confocal imaging showed that a naturally occurring mutation depriving NKCC2 of its distal COOH-terminal region results in the absence of cell surface expression. Biotinylation assays revealed that lack of cell surface expression was associated with abolition of mature complex-glycosylated NKCC2. Pulse-chase analysis demonstrated that the absence of mature protein was not caused by reduced synthesis or increased rates of degradation of mutant co-transporters. Co-immunolocalization experiments revealed that these mutants co-localized with the ER marker protein-disulfide isomerase, demonstrating that they are retained in the ER. Cell treatment with proteasome or lysosome inhibitors failed to restore the loss of complex-glycosylated NKCC2, further eliminating the possibility that mutant co-transporters were processed by the Golgi apparatus. Serial truncation of the NKCC2 COOH terminus, followed by site-directed mutagenesis, identified hydrophobic residues (1081)LLV(1083) as an ER exit signal necessary for maturation of NKCC2. Mutation of (1081)LLV(1083) to AAA within the context of the full-length protein prevented NKCC2 ER exit independently of the expression system. This trihydrophobic motif is highly conserved in the COOH-terminal tails of all members of the cation-chloride co-transporter family, and thus may function as a common motif mediating their transport from the ER to the cell surface. Taken together, these data are consistent with a model whereby naturally occurring premature terminations that interfere with the LLV motif compromise co-transporter surface delivery through defective trafficking.

Publication types

  • Research Support, Non-U.S. Gov't

MeSH terms

  • Amino Acid Motifs
  • Amino Acid Sequence
  • Animals
  • Chlorides / chemistry
  • Endoplasmic Reticulum / metabolism*
  • Gene Expression Regulation*
  • Glycosylation
  • Mice
  • Models, Biological
  • Molecular Sequence Data
  • Mutation
  • Opossums
  • Protein Structure, Tertiary
  • Protein Transport
  • Sodium-Potassium-Chloride Symporters / metabolism
  • Sodium-Potassium-Chloride Symporters / physiology*
  • Solute Carrier Family 12, Member 1


  • Chlorides
  • Slc12a1 protein, mouse
  • Sodium-Potassium-Chloride Symporters
  • Solute Carrier Family 12, Member 1