人类结肠中布美他尼德敏感的Na-K-Cl共转运体的主要结构、功能表达和染色体定位。
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Payne JA, Xu JC, Haas M, Lytle CY, Ward D, Forbush B 3
人类结肠中布美他尼德敏感的Na-K-Cl共转运体的主要结构、功能表达和染色体定位。
生物化学杂志，1995 7月28日;270(30):17977-85。
- PubMed ID
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7629105 (PubMed视图］
- 摘要
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通过将氯离子移动到上皮细胞，Na-K-Cl共转运体有助于氯离子在分泌上皮和吸收上皮的跨细胞移动。利用最近发现的弹性支分泌Na-K-Cl共转运体(sNKCC1)的cDNA探针(Xu, J. C.， Lytle, C. Zhu, T. T.， Payne, J. A.， Benz, E.， and Forbush, B.， III (1994)学术科学91,2201-2205)，我们已经确定了人类同源。通过对人结肠癌细胞系T84的cDNA文库进行筛选，从重叠克隆中鉴定出4115个碱基序列。推导出的蛋白长度为1212个氨基酸，一级结构分析显示有12个跨膜片段。其主要结构与sNKCC1的74%相同，与小鼠Na-K-Cl共转运体(mNKCC1)的91%相同，与兔和大鼠肾脏Na-K-Cl共转运体(NKCC2)的58%相同，与来自比齿鱼膀胱和大鼠肾脏的噻唑类敏感Na-Cl共转运体的43%相同。与sNKCC1和mNKCC1相似，人类结肠共转运体的5'端富含G + C。有趣的是，一个三重复(GCG)7出现在5'编码区，并有助于一个大的丙氨酸重复(Ala15)。在假定的跨膜段7和8之间的细胞外环上预测了n -链糖基化的两个位点。蛋白激酶A磷酸化的单一潜在位点存在于预测的细胞质c端结构域。 Northern blot analysis revealed a 7.4-7.5-kilobase transcript in T84 cells and shark rectal gland and a approximately 7.2-kilobase transcript in mammalian colon, kidney, lung, and stomach. Metaphase spreads from lymphocytes were probed with biotin-labeled cDNA and avidin fluorescein (the cotransporter gene was localized to human chromosome 5 at position 5q23.3). Human embryonic kidney cells stably transfected with the full-length cDNA expressed a approximately 170-kDa protein recognized by anti-cotransporter antibodies. Following treatment with N-glycosidase F, the molecular mass of the expressed protein was similar to that predicted for the core protein from the cDNA sequence (132-kDa) and identical to that of deglycosylated T84 cotransporter (approximately 135-kDa). The stably transfected cells exhibited a approximately 15-fold greater bumetanide-sensitive 86Rb influx than control cells, and this flux required external sodium and chloride. Flux kinetics were consistent with an electroneutral cotransport of 1Na:1K:2Cl. Preincubation in chloride-free media was necessary to activate fully the expressed cotransporter, suggesting a [Cl]-dependent regulatory mechanism.