Abstract
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.
| Original language | English (US) |
|---|---|
| Journal | American Journal of Physiology - Renal Physiology |
| Volume | 287 |
| Issue number | 2 56-2 |
| DOIs | |
| State | Published - Aug 2004 |
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Keywords
- Distal tubule
- Diuretics
- Na-Cl cotransporter
- Salt reabsorption
- Structure
ASJC Scopus subject areas
- Physiology
Cite this
Pathophysiology of functional mutations of the thiazide-sensitive Na-Cl cotransporter in Gitelman disease. / Sabath, Ernesto; Meade, Patricia; Berkman, Jennifer; De Los Heros, Paola; Moreno, Erika; Bobadilla, Norma A.; Vázqaez, Norma; Ellison, David; Gamba, Gerardo.
In: American Journal of Physiology - Renal Physiology, Vol. 287, No. 2 56-2, 08.2004.Research output: Contribution to journal › Article
}
TY - JOUR
T1 - Pathophysiology of functional mutations of the thiazide-sensitive Na-Cl cotransporter in Gitelman disease
AU - Sabath, Ernesto
AU - Meade, Patricia
AU - Berkman, Jennifer
AU - De Los Heros, Paola
AU - Moreno, Erika
AU - Bobadilla, Norma A.
AU - Vázqaez, Norma
AU - Ellison, David
AU - Gamba, Gerardo
PY - 2004/8
Y1 - 2004/8
N2 - 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.
AB - 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.
KW - Distal tubule
KW - Diuretics
KW - Na-Cl cotransporter
KW - Salt reabsorption
KW - Structure
UR - http://www.scopus.com/inward/record.url?scp=3242671652&partnerID=8YFLogxK
UR - http://www.scopus.com/inward/citedby.url?scp=3242671652&partnerID=8YFLogxK
U2 - 10.1152/ajprenal.00044.2004
DO - 10.1152/ajprenal.00044.2004
M3 - Article
C2 - 15068971
AN - SCOPUS:3242671652
VL - 287
JO - American Journal of Physiology - Renal Fluid and Electrolyte Physiology
JF - American Journal of Physiology - Renal Fluid and Electrolyte Physiology
SN - 1931-857X
IS - 2 56-2
ER -