Molecular cloning and transmembrane structure of hCLCA2 from human lung, trachea, and mammary gland.
Molecular cloning and transmembrane structure of hCLCA2 from human lung, trachea, and mammary gland.
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DOI：
10.1152/ajpcell.1999.276.6.c1261
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发表时间：
1999-06
期刊：
影响因子：
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通讯作者：
A. Gruber;K. D. Schreur;H. Ji;C. Fuller;B. Pauli
中科院分区：
文献类型：
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作者：
A. Gruber;K. D. Schreur;H. Ji;C. Fuller;B. Pauli
钙激活氯离子通道的CLCA家族最近被发现，越来越多的密切相关的成员分离自不同物种。在这里，我们报告的第二个人类同源物，hCLCA 2，从人肺cDNA文库的克隆。北方印迹和RT-PCR分析显示在气管和乳腺中有额外的表达。在转染的HEK-293细胞中，120 kDa的初级翻译产物被切割成86和34 kDa的两种细胞表面相关糖蛋白。hCLCA 2是第一个跨膜结构已被系统研究的CLCA同源物。糖基化位点扫描和蛋白酶保护测定揭示了五个跨膜结构域，具有一个大的、富含半胱氨酸的氨基末端胞外域。全细胞膜片钳记录hCLCA 2转染的HEK-293细胞检测到一个轻微向外整流阴离子电导增加的Ca 2+离子载体离子霉素的存在下，抑制DIDS，二硫苏糖醇，尼氟灭酸，和他莫昔芬。在人气管和肺中的表达表明hCLCA 2可能在囊性纤维化的复杂发病机制中发挥作用。
The CLCA family of Ca2+-activated Cl- channels has recently been discovered, with an increasing number of closely related members isolated from different species. Here we report the cloning of the second human homolog, hCLCA2, from a human lung cDNA library. Northern blot and RT-PCR analyses revealed additional expression in trachea and mammary gland. A primary translation product of 120 kDa was cleaved into two cell surface-associated glycoproteins of 86 and 34 kDa in transfected HEK-293 cells. hCLCA2 is the first CLCA homolog for which the transmembrane structure has been systematically studied. Glycosylation site scanning and protease protection assays revealed five transmembrane domains with a large, cysteine-rich, amino-terminal extracellular domain. Whole cell patch-clamp recordings of hCLCA2-transfected HEK-293 cells detected a slightly outwardly rectifying anion conductance that was increased in the presence of the Ca2+ ionophore ionomycin and inhibited by DIDS, dithiothreitol, niflumic acid, and tamoxifen. Expression in human trachea and lung suggests that hCLCA2 may play a role in the complex pathogenesis of cystic fibrosis.