Functional Expression of Mutations in the Human NaCl Cotransporter

Jong, Joke C. de; Vliet, Walter A van der; Heuvel, L.P.W.J. van den; Willems, Peter H.G.M.; Knoers, Nine V A M; Bindels, René J. M.

doi:10.1097/01.asn.0000017904.77985.03

Cited by 137 publications

(107 citation statements)

References 28 publications

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“…We first identified well-documented disease causing mutations which had been shown biochemically to cause loss of function of the encoded cotransporter/ channel [18][19][20][21][22] . This identified 10 different biochemically proven loss of function mutations found in 23 subjects (Table 1).…”

Section: Mutations In Slc12a1 Slc12a3 and Kcnj1 In Fhsmentioning

confidence: 99%

Rare independent mutations in renal salt handling genes contribute to blood pressure variation

et al. 2008

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The effects of alleles in many genes are believed to contribute to common complex diseases such as hypertension. Whether risk alleles comprise a small number of common variants or many rare independent mutations at trait loci is largely unknown. We screened members of the Framingham Heart Study (FHS) for variation in three genes -SLC12A3 (NCCT), SLC12A1 (NKCC2) and KCNJ1 (ROMK)-causing rare recessive diseases featuring large reductions in blood pressure. Using comparative genomics, genetics, and biochemistry, we identified subjects with mutations proven or inferred to be functional. These mutations, all heterozygous and rare, produce clinically significant blood pressure reduction and protect from development of hypertension. Our findings implicate many rare alleles that alter renal salt handling in blood pressure variation in the general population, and identify alleles with health benefit that are nonetheless under purifying selection. These findings have implications for the genetic architecture of hypertension and other common complex traits.Correspondence to: Richard Lifton richard.lifton@yale.edu 203-737-4420 Howard Hughes Medical Institute, Yale University School of Medicine, TAC Room S341D, 1 Gilbert Street, New Haven, CT 06510, USA. Accession Numbers. GenBank mRNA sequences are as follows: NCCT/SLC12A3 (NM_000339); NKCC2/SLC12A1 (NM_000338); ROMK/KCNJ1 (NM_000220, NM_153764-7). GenBank protein sequences are as follows: SLC12A3 orthologs: Human (NP_000330), mouse (NP_062288), rat (NP_062218), rabbit (AAC33139), dog (XP_535292), cow (XP_871112), chicken (XP_414059), zebrafish (NP_001038545) and winter flounder (AAL26926); SLC12A1orthologs: Human (NP_000329), mouse (NP_899197), rat (NP_062007), rabbit (AAB03494), dog (XP_850426), chicken (XP_413814), zebrafish (NP_001002080) and Tetraodon (CAF99849); SLC12A1-3 invertebrate orthologs: S. purpuratus (XP_783014), C. elegans (NP_502704) and D. melanogaster (NP_648572); KCNJ1 orthologs: Human (NP_72245), mouse (NP_062633), rat (NP_058719), dog (XP_546403), chicken (XP_425795), cow (XP_585917), frog (AAH79788), zebrafish (XP_684541), fugu (ABB87033), C.elegans (NP_509138), S. purpuratus (XP_789112) and D. melanogaster (NP_651131); Other human paralogs: SLC12A2 (NP_001037), SLC12A4 (NP_005063), SLC12A5 (NP_065759), SLC12A6 (NP_005126) and SLC12A7 (NP_006589), KCNJ2 (NP_000882), KCNJ3 (NP_002230) KCNJ4 (NP_004972), KCNJ5 (NP_000881), KCNJ6 (NP_002231), KCNJ8 (NP_004973), KCNJ9 (NP_004974), KCNJ10 (NP_002232), KCNJ11 (NP_000516), KCNJ12 (NP_066292), KCNJ13 (NP_002233), KCNJ14 (NP_733838), KCNJ15 (NP_733933) and KCNJ16 (NP_733938). NIH Public AccessAuthor Manuscript Nat Genet. Author manuscript; available in PMC 2013 September 08. NIH-PA Author ManuscriptNIH-PA Author Manuscript NIH-PA Author ManuscriptHypertension affects 1 billion people world-wide and is a major contributor to death from stroke, myocardial infarction, end-stage renal disease and congestive heart failure. Although epidemiologic studies have demonstrated high heritability of blood pressure...

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Section: Mutations In Slc12a1 Slc12a3 and Kcnj1 In Fhsmentioning

confidence: 99%

Rare independent mutations in renal salt handling genes contribute to blood pressure variation

et al. 2008

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“…So far, functional analysis has been performed for only a limited number of mutations. [12][13][14][15] To confirm disease-causing NCC mutants, Xenopus laevis oocytes were used as a model system to investigate Na + transport by NCC and cell surface localization. These mutants are categorized according to standard nomenclature as established for renal Na + transporter's NCC and NKCC2 mutations.…”

Section: Introductionmentioning

confidence: 99%

Novel NCC mutants and functional analysis in a new cohort of patients with Gitelman syndrome

et al. 2011

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Gitelman syndrome (GS) is an autosomal recessive disorder characterized by hypokalemic metabolic alkalosis in conjunction with significant hypomagnesemia and hypocalciuria. The GS phenotype is caused by mutations in the solute carrier family 12, member 3 (SLC12A3) gene that encodes the thiazide-sensitive NaCl cotransporter (NCC). We analyzed DNA samples of 163 patients with a clinical suspicion of GS by direct sequencing of all 26 exons of the SLC12A3 gene. In total, 114 different mutations were identified, 31 of which have not been reported before. These novel variants include 3 deletions, 18 missense, 6 splice site and 4 nonsense mutations. We selected seven missense mutations to investigate their effect on NCC activity and plasma membrane localization by using the Xenopus laevis oocyte expression system. The Thr392Ile mutant did not display transport activity (probably class 2 mutation), while the Asn442Ser and Gln1030Arg NCC mutants showed decreased plasma membrane localization and consequently function, likely due to impaired trafficking (class 3 mutation). Even though the NaCl uptake was hampered for NCC mutants Glu121Asp, Pro751Leu, Ser475Cys and Tyr489His, the transporters reached the plasma membrane (class 4 mutation), suggesting an effect on NCC regulation or ion affinity. The present study shows the identification of 38 novel mutations in the SLC12A3 gene and provides insight into the mechanisms that regulate NCC.

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“…Alternatively, long-term PMA treatment may lead to inhibition at some step of the sorting process. In contrast to hNCC-MDCK cells, Xenopus laevis oocytes predominantly express the approximately 110-kD protein (24,28). This glycosylation pattern is not likely to be due to excessive hNCC production because AQP2, when expressed at similar levels, was hardly detected in the high mannose glycosylated form (34).…”

Section: Discussionmentioning

confidence: 96%

“…By use of the Xenopus laevis expression system, we (24) and others (29) have previously shown that complex glycosylation is a prerequisite for functional targeting to the plasma membrane. Here, we show that MDCK cells predominantly express the approximately 120-to 140-kD form of the hNCC, with only very little expression of the 110-kD protein.…”

Section: Discussionmentioning

confidence: 96%

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Functional Expression of the Human Thiazide-Sensitive NaCl Cotransporter in Madin-Darby Canine Kidney Cells

Jong¹,

Willems²,

Heuvel³

et al. 2003

Journal of the American Society of Nephrology

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Abstract. The thiazide-sensitive Na ϩ -Cl Ϫ cotransporter (NCC), which is expressed on the apical membrane of epithelial cells lining the distal convoluted tubule, is responsible for the reabsorption of 5% to 10% of filtered Na ϩ and Cl Ϫ . To date, functional studies on the structural and regulatory requirements for localized trafficking and ion-transporting activity of NCC have been hampered by lack of a suitable cell system expressing this cotransporter. Reported here is the functional expression of human NCC (hNCC) in a polarized mammalian cell of renal origin-that is, the high-resistance Madin-Darby canine kidney (MDCK) cell. Western blot testing revealed that the cells predominantly expressed the complex glycosylated (approximately 140 kD) form of hNCC. hNCC was present primarily in the apical part of the cell. The functionality of hNCC was demonstrated by the gain of thiazide-sensitive Na ϩ uptake and transepithelial transport activity. Na ϩ uptake was significantly increased after short-term (15 min) treatment with forskolin, whereas cyclic guanosine monophosphate, wortmannin, phorbol 12-myriatate 13-acetate, and staurosporine were without effect. This indicates that hNCC activity is regulated through cyclic adenosine monophosphate, rather than via cyclic guanosine monophosphate, phosphoinositide 3-kinases or protein kinase C. Aldosterone did not alter Na ϩ uptake in the short term (15 min) but significantly increased the transport activity in the long term (16 h). The latter effect of aldosterone was due to an effect on the cytomegalovirus promoter/enhancer driving the expression of hNCC. hNCC-MDCK cells are a good model for the study of the regulation of apical trafficking and ion-transporting activity of hNCC.

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Functional Expression of Mutations in the Human NaCl Cotransporter

Cited by 137 publications

References 28 publications

Rare independent mutations in renal salt handling genes contribute to blood pressure variation

Rare independent mutations in renal salt handling genes contribute to blood pressure variation

Novel NCC mutants and functional analysis in a new cohort of patients with Gitelman syndrome

Functional Expression of the Human Thiazide-Sensitive NaCl Cotransporter in Madin-Darby Canine Kidney Cells

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