title: A highly conserved motif at the COOH terminus dictates endoplasmic reticulum exit and cell surface expression of NKCC2.
abstract: 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.
authors.affiliation: INSERM, UMRS 872-Equipe 3-ERL7226, 75006 Paris, France.
authors.forename: Nancy
authors.initials: N
authors.lastname: Zaarour
authors.forename: Sylvie
authors.initials: S
authors.lastname: Demaretz
authors.forename: Nadia
authors.initials: N
authors.lastname: Defontaine
authors.forename: David
authors.initials: D
authors.lastname: Mordasini
authors.forename: Kamel
authors.initials: K
authors.lastname: Laghmani
mesh_terms: D020816:Amino Acid Motifs; D000595:Amino Acid Sequence; D000818:Animals; D002712:Chlorides / Q000737:chemistry; D004721:Endoplasmic Reticulum / Q000378:metabolism*; D005786:Gene Expression Regulation; D006031:Glycosylation; D051379:Mice; D008954:Models, Biological; D008969:Molecular Sequence Data; D009154:Mutation; D009893:Opossums; D017434:Protein Structure, Tertiary; D021381:Protein Transport; D028021:Sodium-Potassium-Chloride Symporters / Q000378:metabolism / Q000502:physiology*; D064466:Solute Carrier Family 12, Member 1
chemical_list: D002712:Chlorides; C578715:Slc12a1 protein, mouse; D028021:Sodium-Potassium-Chloride Symporters; D064466:Solute Carrier Family 12, Member 1