Cloning of an epithelial chloride channel from bovine trachea
Cloning of an epithelial chloride channel from bovine trachea
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DOI：
10.1074/jbc.270.52.31016
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发表时间：
1995-12-29
影响因子：
4.8
通讯作者：
Fuller, CM
中科院分区：
文献类型：
--
作者：
Cunningham, SA;Awayda, MS;Fuller, CM
我们利用抗体探针从牛气管cDNA锡永文库中分离并克隆了一个新的上皮细胞Cl-通道蛋白。该抗体（α p38）提出了对一个38 kDa的组分的均聚蛋白，表现为钙/钙调蛋白激酶II-，DIDS，和二硫苏糖醇（DTT）敏感，阴离子选择性通道时，纳入平面脂质双层。全长cDNA为3001个碱基对长，编码903个氨基酸的蛋白质。克隆没有显示任何显着的同源性，任何其他先前报道的Cl-通道序列。用与克隆序列对应的cDNA探针对牛气管mRNA进行北方分析，发现3.1千碱基处有一条带，表明已克隆出接近全长的序列。全长开放阅读框架（2712个碱基对）已在非洲爪蟾卵母细胞和哺乳动物COS-7细胞中表达。在卵母细胞中，克隆的表达与一种新的DIDS-和DTT-敏感的阴离子选择性电导的出现有关，该电导向外整流并显示出接近0 mV的逆转电位。在转染克隆的COS-7细胞中进行的全细胞膜片钳研究确定了在nock转染或对照细胞中不明显的离子霉素和DTT敏感的氯离子电导。体外翻译研究表明，初级转录物编码在还原条件下以140 kDa迁移的蛋白质，显著大于α p38识别的多肽。因此，我们建议，无论是140 kDa的翻译产物是一个prepro形式的38 kDa亚基的先前确定的牛气管阴离子通道和初级转录后裂解产生的最终产品，或克隆的通道和先前确定的牛气管阴离子通道蛋白共享一个表位，被识别的α p38抗体。
We have isolated and cloned a novel epithelial Cl- channel protein from a bovine tracheal cDNA expres sion library using an antibody probe. The antibody (alpha p38) was raised against a 38-kDa component of a homopolymeric protein that behaves as a Ca2+/calmodulin kinase II-, DIDS, and dithiothreitol (DTT)-sensitive, anion-selective channel when incorporated into planar lipid bilayers. The full-length cDNA is 3001 base pairs long and codes for a 903-amino acid protein. The clone does not show any significant homology to any other previously reported Cl- channel sequence. Northern analysis of bovine tracheal mRNA with a cDNA probe corresponding to the cloned sequence revealed a band at 3.1 kilobases, suggesting that close to the full-length sequence has been cloned. The full-length open reading frame (2712 base pairs) has been expressed in Xenopus oocytes and in mammalian COS-7 cells. In oocytes, expression of the clone was associated with the appear ance of a novel DIDS-, and DTT-sensitive, anion-selective conductance that was outwardly rectified and exhibited a reversal potential close to 0 mV. Whole-cell patch clamp studies in COS-7 cells transfected with the clone identified an ionomycin-, and DTT sensitive chloride conductance that was not apparent in nock-transfected or control cells. In vitro translation studies have shown that the primary transcript codes for a protein migrating at 140 kDa under reduced conditions, significantly larger than the polypeptide recognized by alpha p38. We therefore suggest that either the 140 kDa translated product is a prepro form of the 38-kDa subunit of the previously identified bovine tracheal anion channel and that the primary transcript is post-translationally cleaved to yield the final product, or that the cloned channel and the previously identified bovine tracheal anion channel protein share an epitope that is recognized by the alpha p38 antibody.