Novel SLC12A1 (NKCC2) Mutations in Two Families with Bartter Syndrome Type 1

Adachi, Masanori; Asakura, Yumi; Sato, Yoshiaki; Tajima, Toshihiro; Nakajima, Takeo; Yamamoto, Toshiyuki; Fukui, Kenji

doi:10.1507/endocrj.k06-204

Cited by 35 publications

(20 citation statements)

References 13 publications

(11 reference statements)

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“…The significance of our findings is that they indicate that naturally occurring BS1 mutations, such as Y998X and N984fs (32,33), which deprive NKCC2 of its distal COOHterminal tail and thus interfere with the previously reported 1081 LLV 1083 motif (20), and the two additional 1038 LL 1039 and 1048 LI 1049 motifs identified in the present report, result in defects in the ER exit of the co-transporter. Altogether, our findings suggest that the last 101 AA of COOH terminus is an essential determinant of the ER export of NKCC2.…”

Section: Effect Of Mutation Of Kkxx Motifs Present In Nkcc2 C Terminus-supporting

confidence: 61%

Multiple Evolutionarily Conserved Di-leucine Like Motifs in the Carboxyl Terminus Control the Anterograde Trafficking of NKCC2

Zaarour

Demaretz

Defontaine

et al. 2012

Journal of Biological Chemistry

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Background: Despite the pivotal role of NKCC2 in blood pressure homeostasis, the mechanisms underlying its membrane trafficking remain poor. Results: Substitutions of 1038 LL 1039 and 1048 LI 1049 motifs result in ER retention of NKCC2. Conclusion: NKCC2 surface expression is controlled by multiple di-leucine like motifs. Significance: Elucidating the molecular mechanisms of the motif-facilitated ER export may help to develop therapeutic strategies targeting NKCC2 transport from the ER to the cell surface.

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Section: Effect Of Mutation Of Kkxx Motifs Present In Nkcc2 C Terminus-supporting

confidence: 61%

Multiple Evolutionarily Conserved Di-leucine Like Motifs in the Carboxyl Terminus Control the Anterograde Trafficking of NKCC2

Zaarour

Demaretz

Defontaine

et al. 2012

Journal of Biological Chemistry

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“…1A). A large number of mutations leading to BS1 are located at the COOH terminus of NKCC2, which is an indication of the importance of this domain in NKCC2 protein expression and function (5,6,26,27). In the present work, we studied the effect of the most distal mutation described to date in the NKCC2 COOH terminus (26), Y998X, on NKCC2 expression and function.…”

Section: Nkcc2 Cooh-terminal Removal Disrupts Glycosylation and Plasmmentioning

confidence: 99%

“…Thus, naturally occurring mutations, depriving NKCC2 of this motif, would lead to inappropriate sorting, which not only would disrupt vectorial solute transport in TAL cells but also could form the molecular basis of BS1. To further support this hypothesis, we also tested the effect of two other different mutations previously identified in the NKCC2 COOH terminus of BS1 patients, N984fs and D918fs (6,27), and studied their effect on NKCC2 expression. N984fs and D918fs mutations produce COOH-terminally truncated NKCC2 proteins missing the last 108 and 198 aa of NKCC2, respectively, and thus lacking the 1081 LLV 1083 motif.…”

Section: The Impact Of the Llv Motif Upon Maturation Of Nkcc2 Ismentioning

confidence: 99%

A Highly Conserved Motif at the COOH Terminus Dictates Endoplasmic Reticulum Exit and Cell Surface Expression of NKCC2

Zaarour

Demaretz

Defontaine

et al. 2009

Journal of Biological Chemistry

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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 cotransporter. 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 complexglycosylated 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.The Na-K-2Cl co-transporter, NKCC2, provides the major route for sodium/chloride transport across the apical plasma membrane of the thick ascending limb (TAL) 3 of the kidney (1). This co-transporter is critical for salt reabsorption, acid-base regulation, and divalent mineral cation metabolism (2). The prominent importance of NKCC2 in renal functions is evidenced by the effect of loop diuretics, which as pharmacologic inhibitors of NKCC2, are extensively used in the treatment of edematous states (2). Even more impressive, inactivating mutations of the NKCC2 gene in humans causes Bartter syndrome type 1 (BS1), a life-threatening renal tubular disorder for which the diagnosis is usually made in the antenatal-neonatal period, due to the presence of polyhydramnios, premature delivery, salt loss, hypokalemia, metabolic alkalosis, hypercalciuria, and nephrocalcinosis (3). Without appropriate treatment, patients with BS1 will not survive the early neonatal period (4). In congruen...

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“…Therefore, although not widely available in Brazil, molecular tools have become increasingly important for the diagnosis of BS. 2,5,6,9 The rational use of molecular biology techniques will certainly improve our understanding of the genetic aspects of BS, allowing an individualized approach to our patients.…”

Section: Discussionmentioning

confidence: 99%

“…2,5,6,9,12 The recent study by Brochard et al,2 who investigated mutations in 42 children with BS, is worth mentioning. Most of those children had heterozygous mutations of the KCNJ1 gene (45%).…”

Section: Discussionmentioning

confidence: 99%

Aplicação da biologia molecular na abordagem da síndrome de Bartter: relato de caso

Reis¹,

Miranda²,

Pereira³

et al. 2012

J. Bras. Nefrol.

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Novel SLC12A1 (NKCC2) Mutations in Two Families with Bartter Syndrome Type 1

Cited by 35 publications

References 13 publications

Multiple Evolutionarily Conserved Di-leucine Like Motifs in the Carboxyl Terminus Control the Anterograde Trafficking of NKCC2

Multiple Evolutionarily Conserved Di-leucine Like Motifs in the Carboxyl Terminus Control the Anterograde Trafficking of NKCC2

A Highly Conserved Motif at the COOH Terminus Dictates Endoplasmic Reticulum Exit and Cell Surface Expression of NKCC2

Aplicação da biologia molecular na abordagem da síndrome de Bartter: relato de caso

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