Publications

• Primary structure and functional expression of a cDNA encoding the thiazide-sensitive, electroneutral sodium-chloride cotransporter.

  Gamba G., Saltzberg S.N., Lombardi M., Miyanoshita A., Lytton J., Hediger M.A., Brenner B.M., Hebert S.C.

  Electroneutral Na+:Cl-cotransport systems are involved in a number of important physiological processes including salt absorption and secretion by epithelia and cell volume regulation. One group of Na+:Cl-cotransporters is specifically inhibited by the benzothiadiazine (thiazide) class of diuretic ... >> More

  Electroneutral Na+:Cl-cotransport systems are involved in a number of important physiological processes including salt absorption and secretion by epithelia and cell volume regulation. One group of Na+:Cl-cotransporters is specifically inhibited by the benzothiadiazine (thiazide) class of diuretic agents and can be distinguished from Na+:K+:2Cl-cotransporters based on a lack of K+ requirement and insensitivity to sulfamoylbenzoic acid diruetics like bumetanide. We report here the isolation of a cDNA encoding a thiazide-sensitive, electroneutral sodium-chloride cotransporter from the winter flounder urinary bladder using an expression cloning strategy. The pharmacological and kinetic characteristics of the cloned cotransporter are consistent with the properties of native thiazide-sensitive sodium-chloride cotransporters in teleost urinary bladder and mammalian renal distal tubule epithelia. The nucleotide sequence predicts a protein of 1023 amino acids (112 kDa) with 12 putative membrane-spanning regions, which is not related to other previously cloned sodium or chloride transporters. Northern hybridization shows two different gene products: a 3.7-kb mRNA localized only to the urinary bladder and a 3.0-kb mRNA present in several non-bladder/kidney tissues. << Less

  Proc. Natl. Acad. Sci. U.S.A. 90:2749-2753(1993) [PubMed] [EuropePMC]

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

  Glaudemans B., Yntema H.G., San-Cristobal P., Schoots J., Pfundt R., Kamsteeg E.J., Bindels R.J., Knoers N.V., Hoenderop J.G., Hoefsloot L.H.

  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-se ... >> More

  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. << Less

  Eur. J. Hum. Genet. 20:263-270(2012) [PubMed] [EuropePMC]

• Pathophysiology of functional mutations of the thiazide-sensitive Na-Cl cotransporter in Gitelman disease.

  Sabath E., Meade P., Berkman J., de los Heros P., Moreno E., Bobadilla N.A., Vazquez N., Ellison D.H., Gamba G.

  Most of the missense mutations that have been described in the human SLC12A3 gene encoding the thiazide-sensitive Na(+)-Cl(-) cotransporter (TSC, NCC, or NCCT), as the cause of Gitelman disease, block TSC function by interfering with normal protein processing and glycosylation. However, some mutat ... >> More

  Most of the missense mutations that have been described in the human SLC12A3 gene encoding the thiazide-sensitive Na(+)-Cl(-) cotransporter (TSC, NCC, or NCCT), as the cause of Gitelman disease, block TSC function by interfering with normal protein processing and glycosylation. However, some mutations exhibit considerable activity. To investigate the pathogenesis of Gitelman disease mediated by such mutations and to gain insights into structure-function relationships on the cotransporter, five functional disease mutations were introduced into mouse TSC cDNA, and their expression was determined in Xenopus laevis oocytes. Western blot analysis revealed immunoreactive bands in all mutant TSCs that were undistinguishable from wild-type TSC. The activity profile was: wild-type TSC (100%) > G627V (66%) > R935Q (36%) = V995M (32%) > G610S (12%) > A585V (6%). Ion transport kinetics in all mutant clones were similar to wild-type TSC, except in G627V, in which a small but significant increase in affinity for extracellular Cl(-) was observed. In addition, G627V and G610S exhibited a small increase in metolazone affinity. The surface expression of wild-type and mutant TSCs was performed by laser-scanning confocal microscopy. All mutants exhibited a significant reduction in surface expression compared with wild-type TSC, with a profile similar to that observed in functional expression analysis. Our data show that biochemical and functional properties of the mutant TSCs are similar to wild-type TSC but that the surface expression is reduced, suggesting that these mutations impair the insertion of a functional protein into the plasma membrane. The small increase in Cl(-) and thiazide affinity in G610S and G627V suggests that the beginning of the COOH-terminal domain could be implicated in defining kinetic properties. << Less

  Am. J. Physiol. 287:F195-F203(2004) [PubMed] [EuropePMC]

• Characterization of the thiazide-sensitive Na(+)-Cl(-) cotransporter: a new model for ions and diuretics interaction.

  Monroy A., Plata C., Hebert S.C., Gamba G.

  The thiazide-sensitive Na(+)-Cl(-) cotransporter (TSC) is the major pathway for salt reabsorption in the apical membrane of the mammalian distal convoluted tubule. When expressed in Xenopus laevis oocytes, rat TSC exhibits high affinity for both cotransported ions, with the Michaelis-Menten consta ... >> More

  The thiazide-sensitive Na(+)-Cl(-) cotransporter (TSC) is the major pathway for salt reabsorption in the apical membrane of the mammalian distal convoluted tubule. When expressed in Xenopus laevis oocytes, rat TSC exhibits high affinity for both cotransported ions, with the Michaelis-Menten constant (K(m)) for Na(+) of 7.6 +/- 1.6 mM and for Cl(-) of 6.3 +/-1.1 mM, and Hill coefficients for Na(+) and Cl(-) consistent with electroneutrality. The affinities of both Na(+) and Cl(-) were increased by increasing concentration of the counterion. The IC(50) values for thiazides were affected by both extracellular Na(+) and Cl(-). The higher the Na(+) or Cl(-) concentration, the lower the inhibitory effect of thiazides. Finally, rTSC function is affected by extracellular osmolarity. We propose a transport model featuring a random order of binding in which the binding of each ion facilitates the binding of the counterion. Both ion binding sites alter thiazide-mediated inhibition of transport, indicating that the thiazide-binding site is either shared or modified by both Na(+) and Cl(-). << Less

  Am. J. Physiol. 279:F161-F169(2000) [PubMed] [EuropePMC]