Defective processing and expression of thiazide-sensitive Na-Cl cotransporter as a cause of Gitelman's syndrome.
Gitelman's syndrome is an autosomal recessive disorder of salt wasting and hypokalemia caused by mutations in the thiazide-sensitive Na-Cl cotransporter. To investigate the pathogenesis of Gitelman's syndrome, eight disease mutations were introduced into the mouse thiazide-sensitive Na-Cl cotransporter and studied by functional expression in Xenopus oocytes. Sodium uptake into oocytes that expressed the wild-type clone was more than sevenfold greater than uptake into control oocytes. Uptake into oocytes that expressed the mutated transporters was not different from control. Hydrochlorothiazide reduced Na uptake by oocytes expressing the wild-type gene to control values but had no effect on oocytes expressing the mutant clones. Western blots of oocytes injected with the wild-type clone showed bands representing glycosylated (125 kDa) and unglycosylated (110 kDa) forms of the transport protein. Immunoblot of oocytes expressing the mutated clones showed only the unglycosylated protein, indicating that protein processing was disrupted. Immunocytochemistry with an antibody against the transport protein showed intense membrane staining of oocytes expressing the wild-type protein. Membrane staining was completely absent from oocytes expressing mNCC(R948X); instead, diffuse cytoplasmic staining was evident. In summary, the results show that several mutations that cause Gitelman's syndrome are nonfunctional because the mutant thiazide-sensitive Na-Cl cotransporter is not processed normally, probably activating the "quality control" mechanism of the endoplasmic reticulum.
Kunchaparty
S
S
Department of Internal Medicine, Yale University School of Medicine, New Haven, Connecticut 06520, USA.
Palcso
M
M
Berkman
J
J
Velázquez
H
H
Desir
G V
GV
Bernstein
P
P
Reilly
R F
RF
Ellison
D H
DH
D000471:Alkalosis / Q000235:genetics / Q000378:metabolism*; D000818:Animals; D002352:Carrier Proteins / Q000235:genetics / Q000378:metabolism*; D003001:Cloning, Molecular; D005260:Female; D007008:Hypokalemia / Q000235:genetics / Q000378:metabolism*; D007022:Hypotension / Q000235:genetics / Q000378:metabolism*; D051379:Mice; D008807:Mice, Inbred BALB C; D009154:Mutation / Q000502:physiology; D009865:Oocytes / Q000378:metabolism; D011499:Protein Processing, Post-Translational; D011955:Receptors, Drug / Q000235:genetics / Q000378:metabolism*; D049992:Sodium Chloride Symporters; D064486:Solute Carrier Family 12, Member 3; D027981:Symporters; D013577:Syndrome; D014982:Xenopus laevis
D002352:Carrier Proteins; D011955:Receptors, Drug; C578875:Slc12a3 protein, mouse; D049992:Sodium Chloride Symporters; D064486:Solute Carrier Family 12, Member 3; D027981:Symporters; C064415:thiazide receptor