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 cotransport er. To investigate the pathogenesis of Gitelman's syndrome, eight disease m utations were introduced into the mouse thiazide-sensitive Na-Cl cotranspor ter and studied by functional expression in Xenopus oocytes. Sodium uptake into oocytes that expressed the wild-type clone was more than sevenfold gre ater than uptake into control oocytes. Uptake into oocytes that expressed t he mutated transporters was not different from control. Hydrochlorothiazide reduced Na uptake by oocytes expressing the wild-type gene to control valu es but had no effect on oocytes expressing the mutant clones. Western blots of oocytes injected with the wild-type clone showed bands representing gly cosylated (125 kDa) and unglycosylated (110 kDa) forms of the transport pro tein. Immunoblot of oocytes expressing the mutated clones showed only the u nglycosylated protein, indicating that protein processing was disrupted. Im munocytochemistry with an antibody against the transport protein showed int ense membrane staining of oocytes expressing the wild-type protein. Membran e staining was completely absent from oocytes expressing mNCC(R948X); inste ad, diffuse cytoplasmic staining was evident. In summary, the results show that several mutations that cause Gitelman's syndrome are nonfunctional bec ause the mutant thiazide-sensitive Na-Cl cotransporter is not processed nor mally, probably activating the "quality control" mechanism of the endoplasm ic reticulum.