A Single Nucleotide Polymorphism Alters the Activity of the Renal Na+:Cl- Cotransporter and Reveals a Role for Transmembrane Segment 4 in Chloride and Thiazide Affinity

Moreno, Erika; Tovar‐Palacio, Claudia; Heros, Paola de los; Guzmán, Blanca; Bobadilla, Norma A; Vázquez, Norma; Riccardi, Daniela; Poch, Esteban; Gamba, Gerardo

doi:10.1074/jbc.m400602200

Cited by 36 publications

(28 citation statements)

References 47 publications

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“…Here we have shown, for the thiazide-sensitive Na ϩ -Cl Ϫ cotransporter, that Cl Ϫ affinity-modifying residues reside only in the TM 1-7 region, whereas Na ϩ affinitymodifying residues reside in both the TM 1-7 and 8 -12 regions. Supporting this conclusion, we have previously shown that a single nucleotide polymorphism changing the glycine residue 264 within the fourth TM segment for alanine affected the affinity of the cotransporter for Cl Ϫ but not for Na ϩ transport (32). Thus, it is likely that, among NCC, NKCC1, and NKCC2, the affinity-modifying domains are different.…”

Section: Structure-functional Analysis Of Renal Nccsupporting

confidence: 57%

Affinity-defining Domains in the Na-Cl Cotransporter

Moreno

Cristóbal

Rivera

et al. 2006

Journal of Biological Chemistry

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The thiazide-sensitive Na ؉ -Cl ؊ cotransporter (NCC) is the major pathway for salt reabsorption in the distal convoluted tubule, serves as a receptor for thiazide-type diuretics, and is involved in inherited diseases associated with abnormal blood pressure. Little is known regarding the structure-function relationship in this cotransporter. Previous studies from our group reveal that mammalian NCC exhibits higher affinity for ions and thiazides than teleost NCC and suggest a role for glycosylation upon thiazide affinity. Here we have constructed a series of chimeric and mutant cDNAs between rat and flounder NCC to define the role of glycosylation status, the aminoterminal domain, the carboxyl-terminal domain, the extracellular glycosylated loop, and the transmembrane segments upon affinity for Na ؉ , Cl ؊ , and metolazone. Xenopus laevis oocytes were used as the heterologous expression system. We observed that elimination of glycosylation sites in flounder NCC did not affect the affinity of the cotransporter for metolazone. Also, swapping the amino-terminal domain, the carboxyl-terminal domain, the glycosylation sites, or the entire extracellular glycosylation loop between rat and flounder NCC had no effect upon ions or metolazone affinity. In contrast, interchanging transmembrane regions between rat and flounder NCC revealed that affinity-modifying residues for chloride are located within the transmembrane 1-7 region and for thiazides are located within the transmembrane 8 -12 region, whereas both segments seem to be implicated in defining sodium affinity. These observations strongly suggest that binding sites for chloride and thiazide in NCC are different.In the mammalian kidney, the apical thiazide-sensitive Na ϩ -Cl Ϫ cotransporter (NCC) 3 is the major pathway for salt reabsorption in the luminal membrane of the distal convoluted tubule (1, 2). NCC also serves as the target for the thiazide-type diuretics that are currently recommended by the Joint National Committee for the detection, evaluation, and treatment of high blood pressure as the first line pharmacological treatment of hypertension, either as the unique drug in patients with stage I hypertension or in combination with other anti-hypertensive agents for patients with stage II hypertension (3). The fundamental role for NCC in preserving the extracellular fluid volume and blood pressure homeostasis has been firmly established by the identification that Gitelman disease (4 -6) (an inherited disorder featuring arterial hypotension, renal salt wasting, hypokalemic metabolic alkalosis, hypocalcinuria, and hypomagnesemia) is caused by inactivating mutations of the SLC12A3 gene that encodes NCC. In addition, a loss of the negative effect of the serine/threonine kinases WNK1 and WNK4 upon NCC activity has been implicated in the pathogenesis of a salt-dependent form of human hypertension known as pseudohypoaldosteronism type II (7, 8), which features a clinical picture that is a mirror image of Gitelman disease (9), with striking sensitivity to hydrochlorot...

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Section: Structure-functional Analysis Of Renal Nccsupporting

confidence: 57%

Affinity-defining Domains in the Na-Cl Cotransporter

Moreno

Cristóbal

Rivera

et al. 2006

Journal of Biological Chemistry

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“…In contrast, we considered the amino acid changes p.Arg209Trp (SNP rs28936388), p.Gly264Ala (SNP rs1529927), and p.Arg928Cys (SNP rs12708965) as loss of function mutations. Indeed, in vitro expression of p.Arg209Trp and p.Gly264Ala has been shown to produce a significant reduction in NCC activity, 18,27 and the p.Arg928Cys change is predicted in silico to be deleterious and is also considered diseasecausing. 6 Eight novel missense changes detected in this study (p.Ala13Pro, p.Arg83Gln p.Val404Ile, p.Thr428Pro, p.Ser546Gly, pSer833Leu, p.Glu915Ala, and p.Gln1021Lys) were predicted in silico as nondeleterious (Supplementary Table 2) and were characterized as variants of unknown significance.…”

Section: Detection Of Point Mutationsmentioning

confidence: 99%

Spectrum of Mutations in Gitelman Syndrome

Vargas‐Poussou

Dahan

Kahila

et al. 2011

Journal of the American Society of Nephrology

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Gitelman's syndrome (GS) is a rare, autosomal recessive, salt-losing tubulopathy caused by mutations in the SLC12A3 gene, which encodes the thiazide-sensitive NaCl cotransporter (NCC). Because 18 to 40% of suspected GS patients carry only one SLC12A3 mutant allele, large genomic rearrangements may account for unidentified mutations. Here, we directly sequenced genomic DNA from a large cohort of 448 unrelated patients suspected of having GS. We found 172 distinct mutations, of which 100 were unreported previously. In 315 patients (70%), we identified two mutations; in 81 patients (18%), we identified one; and in 52 patients (12%), we did not detect a mutation. In 88 patients, we performed a search for large rearrangements by multiplex ligationdependent probe amplification (MLPA) and found nine deletions and two duplications in 24 of the 51 heterozygous patients. A second technique confirmed each rearrangement. Based on the breakpoints of seven deletions, nonallelic homologous recombination by Alu sequences and nonhomologous end-joining probably favor these intragenic deletions. In summary, missense mutations account for approximately 59% of the mutations in Gitelman's syndrome, and there is a predisposition to large rearrangements (6% of our cases) caused by the presence of repeated sequences within the SLC12A3 gene.

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“…Surface expression of TSC and TTB chimera (see Nomenclature and construction of chimeric proteins) was assessed by fluorescence using enhanced green fluorescent protein (EGFP)-TSC and EGFP-TTB fusion constructs, a strategy that we have reported previously (15,27,29,45). Oocytes were microinjected with water or with EGFP-TSC or EGFP-TTB cRNA and 4 days later were monitored for EGFP fluorescence using a Zeiss laser-scanning confocal microscope (objective lens ϫ10; Nikon).…”

Section: Silent Mutagenesis and Construction Of Chimeric Cotransportersmentioning

confidence: 99%

“…A recent characterization of single nucleotide polymorphisms in TSC revealed that a glycine in the transmembrane segment 4 plays a role in defining affinity for extracellular chloride and metolazone (29). In the basolateral isoform of the Na ϩ -K ϩ -2Cl Ϫ cotransporter (BSC2), Isenring and Forbush (for a review, see Ref.…”

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confidence: 99%

Ion and diuretic specificity of chimeric proteins between apical Na⁺-K⁺-2Cl⁻and Na⁺-Cl⁻cotransporters

Tovar‐Palacio

Bobadilla

Cortés

et al. 2004

American Journal of Physiology-Renal Physiology

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The mammalian kidney bumetanide-sensitive Na+-K+-2Cl− and thiazide-sensitive Na+-Cl− cotransporters are the major pathways for salt reabsorption in the thick ascending limb of Henle's loop and distal convoluted tubule, respectively. These cotransporters serve as receptors for the loop- and thiazide-type diuretics, and inactivating mutations of corresponding genes are associated with development of Bartter's syndrome type I and Gitleman's disease, respectively. Structural requirements for ion translocation and diuretic binding specificity are unknown. As an initial approach for analyzing structural determinants conferring ion or diuretic preferences in these cotransporters, we exploited functional differences and structural similarities between Na+-K+-2Cl− and Na+-Cl− cotransporters to design and study chimeric proteins in which the NH2-terminal and/or COOH-terminal domains were switched between each other. Thus six chimeric proteins were produced. Using the heterologous expression system of Xenopus laevis oocytes, we observed that four chimeras exhibited functional activity. Our results revealed that, in the Na+-K+-2Cl− cotransporter, ion translocation and diuretic binding specificity are determined by the central hydrophobic domain. Thus NH2-terminal and COOH-terminal domains do not play a role in defining these properties. A similar conclusion can be suggested for the Na+-Cl− cotransporter.

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A Single Nucleotide Polymorphism Alters the Activity of the Renal Na+:Cl- Cotransporter and Reveals a Role for Transmembrane Segment 4 in Chloride and Thiazide Affinity

Cited by 36 publications

References 47 publications

Affinity-defining Domains in the Na-Cl Cotransporter

Affinity-defining Domains in the Na-Cl Cotransporter

Spectrum of Mutations in Gitelman Syndrome

Ion and diuretic specificity of chimeric proteins between apical Na⁺-K⁺-2Cl⁻and Na⁺-Cl⁻cotransporters

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A Single Nucleotide Polymorphism Alters the Activity of the Renal Na+:Cl- Cotransporter and Reveals a Role for Transmembrane Segment 4 in Chloride and Thiazide Affinity

Cited by 36 publications

References 47 publications

Affinity-defining Domains in the Na-Cl Cotransporter

Affinity-defining Domains in the Na-Cl Cotransporter

Spectrum of Mutations in Gitelman Syndrome

Ion and diuretic specificity of chimeric proteins between apical Na+-K+-2Cl−and Na+-Cl−cotransporters

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Ion and diuretic specificity of chimeric proteins between apical Na⁺-K⁺-2Cl⁻and Na⁺-Cl⁻cotransporters