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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

Moreno, E., Tovar-Palacio, C., de los Heros, P., Guzman, B., Bobadilla, N. A., Vazquez, N., Riccardi, Daniela, Poch, E. and Gamba, G. 2004. 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. Journal of Biological Chemistry 279 (16) , pp. 16553-16560. 10.1074/jbc.M400602200

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Abstract

The thiazide-sensitive Na+:Cl- cotransporter is the major salt transport pathway in the distal convoluted tubule of the kidney, and a role of this cotransporter in blood pressure homeostasis has been defined by physiological studies on pressure natriuresis and by its involvement in monogenic diseases that feature arterial hypotension or hypertension. Data base analysis revealed that 135 single nucleotide polymorphisms along the human SLC12A3 gene that encodes the Na+:Cl- cotransporter have been reported. Eight are located within the coding region, and one results in a single amino acid change; the residue glycine at the position 264 is changed to alanine (G264A). This residue is located within the fourth transmembrane domain of the predicted structure. Because Gly-264 is a highly conserved residue, we studied the functional properties of this polymorphism by using in vitro mutagenesis and the heterologous expression system in Xenopus laevis oocytes. G264A resulted in a significant and reproducible reduction (∼50%) in 22Na+ uptake when compared with the wild type cotransporter. The affinity for extracellular Cl- and for thiazide diuretics was increased in G264A. Western blot analysis showed similar immunoreactive bands between the wild type and the G264A cotransporters, and confocal images of oocytes injected with enhanced green fluorescent protein-tagged wild type and G264A cotransporter showed no differences in the protein surface expression level. These observations suggest that the G264A polymorphism is associated with reduction in the substrate translocation rate of the cotransporter, due to a decrease in the intrinsic activity. Our study also reveals a role of the transmembrane segment 4 in defining the affinity for extracellular Cl- and thiazide diuretics.

Item Type: Article
Date Type: Publication
Status: Published
Schools: Biosciences
Publisher: American Society for Biochemistry and Molecular Biology
ISSN: 0021-9258
Last Modified: 04 Jun 2017 06:40
URI: http://orca-mwe.cf.ac.uk/id/eprint/63463

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