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1 Molecular Physiology Unit, Instituto Nacional de Ciencias Medicas y Nutricion Salvador Zubiran, Mexico City, Mexico; Instituto de Investigaciones Biomedicas, UNAM, Mexico City, Mexico
2 Division of Nephrology and Hypertension, Department of Mecidine, Oregon Health & Science University, Portland, Oregon, USA
* To whom correspondence should be addressed. E-mail: gamba{at}biomedicas.unam.mx.
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 get insights into structure-function relationships on the cotransporter, five functional disease mutations were introduced into the 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 a 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 when 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 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 suggest that the beginning of the carboxyl terminal domain could be implicated in defining kinetic properties.
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