De Jong Joke C, Van Der Vliet Walter A, Van Den Heuvel Lambertus P W J, Willems Peter H G M, Knoers Nine V A M, Bindels René J M
Department of Cell Physiology, University Medical Center Nijmegen, Nijmegen, The Netherlands.
J Am Soc Nephrol. 2002 Jun;13(6):1442-8. doi: 10.1097/01.asn.0000017904.77985.03.
Gitelman's syndrome is an autosomal recessive renal tubular disorder characterized by hypokalemic metabolic alkalosis, hypomagnesemia, and hypocalciuria. This disorder results from mutations in the thiazide-sensitive NaCl cotransporter (NCC). To elucidate the functional implications of mutations associated with this disorder, metolazone-sensitive (22)Na(+) uptake, subcellular localization, and glycosidase-sensitive glycosylation of human NCC (hNCC) were determined in Xenopus laevis oocytes expressing FLAG-tagged wild-type or mutant hNCC. Injection of 10 ng of FLAG-tagged hNCC cRNA resulted in metolazone-sensitive (22)Na(+) uptake of 3.4 +/- 0.2 nmol Na(+)/oocyte per 2 h. Immunocytochemical analysis revealed sharp immunopositive staining at the plasma membrane. In agreement with this finding, a broad endoglycosidase H-insensitive band of 130 to 140 kD was present in Western blots of total membranes. The plasma membrane localization of this complex-glycosylated protein was confirmed by immunoblotting of purified plasma membranes. The mutants could be divided into two distinct classes. Class I mutants (G439S, T649R, and G741R) exhibited no significant metolazone-sensitive (22)Na(+) uptake. Immunopositive staining was present in a diffuse band just below the plasma membrane. This endoplasmic reticulum and/or pre-Golgi complex localization was further suggested by the complete absence of the endoglycosidase H-insensitive band. Class II mutants (L215P, F536L, R955Q, G980R, and C985Y) demonstrated significant metolazone-sensitive (22)Na(+) uptake, although uptake was significantly lower than that obtained with wild-type hNCC. The latter mutants could be detected at and below the oocyte plasma membrane, and immunoblotting revealed the characteristic complex-glycosylated bands. In conclusion, this study substantiates NCC processing defects as the underlying pathogenic mechanism in Gitelman's syndrome.
吉特曼综合征是一种常染色体隐性遗传性肾小管疾病，其特征为低钾血症性代谢性碱中毒、低镁血症和低钙尿症。该疾病由噻嗪类敏感的氯化钠协同转运蛋白（NCC）突变引起。为了阐明与该疾病相关的突变的功能影响，在表达FLAG标记的野生型或突变型人NCC（hNCC）的非洲爪蟾卵母细胞中，测定了美托拉宗敏感的（22）Na（+）摄取、亚细胞定位以及糖苷酶敏感的糖基化情况。注射10 ng的FLAG标记的hNCC cRNA导致每2小时每个卵母细胞的美托拉宗敏感的（22）Na（+）摄取量为3.4±0.2 nmol Na（+）。免疫细胞化学分析显示在质膜处有明显的免疫阳性染色。与该发现一致，在总膜的蛋白质印迹中出现了一条130至140 kD的广泛的内切糖苷酶H不敏感条带。通过对纯化质膜的免疫印迹证实了这种复杂糖基化蛋白的质膜定位。突变体可分为两个不同的类别。I类突变体（G439S、T649R和G741R）未表现出明显的美托拉宗敏感的（22）Na（+）摄取。免疫阳性染色出现在质膜下方的一条弥散带中。内质网和/或高尔基体前体复合物的这种定位进一步由完全不存在内切糖苷酶H不敏感条带所提示。II类突变体（L215P、F536L、R955Q、G980R和C985Y）表现出明显的美托拉宗敏感的（22）Na（+）摄取，尽管摄取量明显低于野生型hNCC。后一类突变体可在卵母细胞质膜处及以下检测到，免疫印迹显示出特征性的复杂糖基化条带。总之，本研究证实NCC加工缺陷是吉特曼综合征潜在的致病机制。
