Molecular cloning and functional expression of the K-Cl cotransporter from rabbit, rat, and human - A new member of the cation-chloride cotransporter family
Molecular cloning and functional expression of the K-Cl cotransporter from rabbit, rat, and human - A new member of the cation-chloride cotransporter family
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DOI：
10.1074/jbc.271.27.16237
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发表时间：
1996-07-05
影响因子：
4.8
通讯作者：
Forbush, B
中科院分区：
文献类型：
--
作者：
Gillen, CM;Brill, S;Forbush, B
我们报告了K-Cl协同转运蛋白KCC1的克隆、序列分析、组织分布和功能表达，KCC1是通过搜索人类表达序列标签数据库来鉴定的，基于预期它与Na-K-Cl协同转运蛋白有远亲关系。使用同源 cDNA 探针筛选兔肾和大鼠脑 cDNA 文库克隆兔 KCC1 (rbKCC1) 和大鼠 KCC1 (rtKCC1)，人 KCC1 (hKCC1) 从 I.M.A.G.E. 获得。克隆并部分通过逆转录聚合酶链反应进行；它与 rbKCC1 具有 97% 的同一性，KCC1 编码 1085 个残基的多肽，与布美他尼敏感的 Na-K-Cl 协同转运蛋白（NKCC 或 BSC）和噻嗪类敏感的 Na-Cl 协同转运蛋白（NCC 或 TSC）具有显着的序列同源性（24-25% 同一性），KCC1 的水疗分析表明与 NKCC 的结构同源性，包括 12跨膜结构域、具有潜在 N 连接糖基化位点的大细胞外环以及细胞质 N 和 C 末端区域，Northern 印迹分析揭示了普遍表达的 3,8-kilobase 转录物。 hKCC1 的大部分基因组序列都在数据库中，并且该基因先前已定位于 16q22.1 (Larsen, F., Solhein, J., Kristensen, T., Kolsto, A. B., and Prydz, H. (1993) Hum. Mel. Genet. 2, 1589-1595)，表位标记的 rbKCC1 稳定表达在人胚胎肾 (HEK 293) 细胞中，产生类似于 150 kDa 的糖蛋白。表达 rbKCC1 的细胞中 Rb-86 的初始流出速率比对照细胞的流出速率高 7 倍以上，并被 2 mM 呋塞米抑制； Rb-86 外流受到细胞肿胀的刺激，用 1 mM N-乙基马来酰亚胺预处理 15 分钟后，rbKCC1 细胞对 Rb-86 的摄取依赖于外部氯化物，但不依赖于外部钠，并且被 K-i 类似于 40 μM 的呋塞米和 K-i 类似于 60 μM 的布美他尼抑制。这些数据表明，KCC1 cDNA 编码广泛的表达的 K-Cl 协同转运蛋白具有红细胞中已表征的 K-Cl 转运蛋白的特征。
We report the cloning, sequence analysis, tissue distribution, and functional expression of the K-Cl cotransport protein, KCC1, KCC1 was identified by searching the human expressed sequence tag data base, based on the expectation that it would be distantly related to the Na-K-Cl cotransporter. Rabbit KCC1 (rbKCC1) and rat KCC1 (rtKCC1) were cloned by screening rabbit kidney and rat brain cDNA libraries using homologous cDNA probes, Human KCC1 (hKCC1) was obtained from I.M.A.G.E. clones and in part by reverse transcription-polymerase chain reaction; it exhibits 97% identity with rbKCC1, KCC1 encodes a 1085-residue polypeptide with substantial sequence homology (24-25% identity) to the bumetanide-sensitive Na-K-Cl cotransporter (NKCC or BSC) and the thiazide-sensitive Na-Cl cotransporter (NCC or TSC), Hydropathy analysis of KCC1 indicates structural homology to NKCC, including 12 transmembrane domains, a large extracellular loop with potential N linked glycosylation sites, and cytoplasmic N- and C-terminal regions, Northern blot analysis revealed a ubiquitously expressed 3,8-kilobase transcript. Much of the genomic sequence of hKCC1 is in the data base, and the gene has been previously localized to 16q22.1 (Larsen, F., Solhein, J., Kristensen, T., Kolsto, A. B., and Prydz, H. (1993) Hum. Mel. Genet. 2, 1589-1595), Epitope-tagged rbKCC1 was stably expressed in human embryonic kidney (HEK 293) cells, resulting in production of a similar to 150-kDa glycoprotein. The initial rate of Rb-86 efflux from cells expressing rbKCC1 was more than 7 times greater than efflux from control cells and was inhibited by 2 mM furosemide; Rb-86 efflux was stimulated by cell swelling, Uptake of Rb-86 into rbKCC1 cells after a 15-min pretreatment with 1 mM N-ethylmaleimide was dependent on external chloride but not on external sodium, and was inhibited by furosemide with a K-i of similar to 40 mu M and by bumetanide with a K-i of similar to 60 mu M. These data demonstrate that the KCC1 cDNAs encode a widely expressed K-Cl cotransporter with the characteristics of the K-Cl transporter that has been characterized in red cells.