title: Multiple evolutionarily conserved Di-leucine like motifs in the carboxyl terminus control the anterograde trafficking of NKCC2.
abstract: Mutations in the apical Na-K-2Cl co-transporter, NKCC2, cause type I Bartter syndrome, a life-threatening kidney disease. Yet the mechanisms underlying the regulation of NKCC2 trafficking in renal cells are scarcely known. We previously showed that naturally occurring mutations depriving NKCC2 of its distal COOH-terminal tail and interfering with the (1081)LLV(1083) motif result in defects in the ER exit of the co-transporter. Here we show that this motif is necessary but not sufficient for anterograde trafficking of NKCC2. Indeed, we have identified two additional hydrophobic motifs, (1038)LL(1039) and (1048)LI(1049), that are required for ER exit and surface expression of the co-transporter. Double mutations of (1038)LL(1039) or (1048)LI(1049) to di-alanines disrupted glycosylation and cell surface expression of NKCC2, independently of the expression system. Pulse-chase analysis demonstrated that the absence of the terminally glycosylated form of NKCC2 was not due to reduced synthesis or increased rates of degradation of mutant co-transporters, but was instead caused by defects in maturation. Co-immunolocalization experiments revealed that (1038)AA(1039) and (1048)AA(1049) were trapped mainly in the ER as indicated by extensive co-localization with the ER marker calnexin. Remarkably, among several analyzed motifs present in the NKCC2 COOH terminus, only those required for ER exit and surface expression of NKCC2 are evolutionarily conserved in all members of the SLC12A family, a group of cation-chloride co-transporters that are targets of therapeutic drugs and mutated in several human diseases. Based upon these data, we propose abnormal anterograde trafficking as a common mechanism associated with mutations depriving NKCC2, and also all other members of the SLC12A family, of their COOH terminus.
authors.affiliation: INSERM, Centre de Recherche des Cordeliers, UMRS872, 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: Yingying
authors.initials: Y
authors.lastname: Zhu
authors.forename: Kamel
authors.initials: K
authors.lastname: Laghmani
mesh_terms: D020816:Amino Acid Motifs; D000595:Amino Acid Sequence; D019943:Amino Acid Substitution / Q000235:genetics; D000818:Animals; D002460:Cell Line; D002462:Cell Membrane / Q000378:metabolism; D017124:Conserved Sequence; D004721:Endoplasmic Reticulum / Q000378:metabolism; D019143:Evolution, Molecular; D006801:Humans; D057927:Hydrophobic and Hydrophilic Interactions; D051379:Mice; D008969:Molecular Sequence Data; D050505:Mutant Proteins / Q000737:chemistry / Q000378:metabolism; D009154:Mutation / Q000235:genetics; D009893:Opossums; D017434:Protein Structure, Tertiary; D021381:Protein Transport; D049992:Sodium Chloride Symporters / Q000378:metabolism; D028021:Sodium-Potassium-Chloride Symporters / Q000737:chemistry* / Q000235:genetics / Q000378:metabolism*; D064466:Solute Carrier Family 12, Member 1; D013329:Structure-Activity Relationship
chemical_list: D050505:Mutant Proteins; C578714:SLC12A1 protein, human; C578715:Slc12a1 protein, mouse; D049992:Sodium Chloride Symporters; D028021:Sodium-Potassium-Chloride Symporters; D064466:Solute Carrier Family 12, Member 1