Multiple Evolutionarily Conserved Di-leucine Like Motifs in the Carboxyl Terminus Control the Anterograde Trafficking of NKCC2 |
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Authors: | Nancy Zaarour Sylvie Demaretz Nadia Defontaine Yingying Zhu Kamel Laghmani |
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Institution: | From the ‡INSERM, Centre de Recherche des Cordeliers, UMRS872, Paris, France. CNRS, ERL7226, 75006 Paris, France.;the §Université Pierre et Marie Curie, 75006 Paris, France, and ;the ¶Faculté de Médecine, Université Paris-Descartes, 75006 Paris, France |
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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 1081LLV1083 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, 1038LL1039 and 1048LI1049, that are required for ER exit and surface expression of the co-transporter. Double mutations of 1038LL1039 or 1048LI1049 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 1038AA1039 and 1048AA1049 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. |
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Keywords: | Cell Surface ER Quality Control Membrane Sodium Transport Trafficking ER Export NKCC2 |
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