Ji H L, DuVall M D, Patton H K, Satterfield C L, Fuller C M, Benos D J
Department of Physiology and Biophysics, University of Alabama at Birmingham 35294, USA.
Am J Physiol. 1998 Feb;274(2):C455-64. doi: 10.1152/ajpcell.1998.274.2.C455.
We have isolated a niflumic acid-insensitive, Ca(2+)-activated Cl- channel (CaCC) from bovine trachea that migrates at 38 kDa (p38) on reducing sodium dodecyl sulfate-polyacrylamide gel electrophoresis. However, a cloned CaCC isolated from a tracheal cDNA expression library by screening with an antibody raised against p38 has a primary cDNA transcript of 2712 base pairs that codes for a 100-kDa protein and is not susceptible to dithiothreitol reduction. To test the hypothesis that the functional channel may be a much smaller posttranslationally processed form of the 100-kDa protein, we generated a mutant construct (CaCCX, 42.5-kDa protein) truncated at the NH2 and COOH termini. The whole cell currents of wild-type (wt) CaCC and CaCCX expressed in Xenopus oocytes were 10-fold higher than those of water-injected oocytes and were further increased by ionomycin or A-23187 and inhibited by 4,4'-diisothiocyanostilbene-2,2'-disulfonic acid and dithiothreitol. Whole cell currents in wtCaCC- and CaCCX-expressing oocytes could also be activated by phorbol 12-myristate 13-acetate and could be inhibited by chelerythrine chloride, suggesting that the cloned CaCC is regulated by protein kinase C. These results suggest that a smaller form of the full-length CaCC can form a functional channel.
我们从牛气管中分离出一种对氟尼缩酸不敏感的Ca(2+)激活的Cl-通道（CaCC），该通道在还原型十二烷基硫酸钠-聚丙烯酰胺凝胶电泳上的迁移分子量为38 kDa（p38）。然而，通过用针对p38的抗体筛选从气管cDNA表达文库中分离出的克隆CaCC，其初级cDNA转录本为2712个碱基对，编码一种100 kDa的蛋白质，且不易被二硫苏糖醇还原。为了验证功能性通道可能是100 kDa蛋白质经翻译后加工形成的小得多的形式这一假设，我们构建了一个在NH2和COOH末端截短的突变体构建体（CaCCX，42.5 kDa蛋白质）。非洲爪蟾卵母细胞中表达的野生型（wt）CaCC和CaCCX的全细胞电流比注射水的卵母细胞高10倍，并且离子霉素或A-23187可使其进一步增加，4,4'-二异硫氰基芪-2,2'-二磺酸和二硫苏糖醇可抑制该电流。表达wtCaCC和CaCCX的卵母细胞中的全细胞电流也可被佛波醇12-肉豆蔻酸酯13-乙酸酯激活，并可被氯化白屈菜红碱抑制，这表明克隆的CaCC受蛋白激酶C调节。这些结果表明，全长CaCC的较小形式可以形成功能性通道。
