Department of Biochemistry and Molecular Cell Biology, College of Veterinary Medicine, Konkuk University, 1 Hwayang-dong, Gwangjin-gu, Seoul, Republic of Korea.
Biochem Biophys Res Commun. 2011 May 20;408(4):559-65. doi: 10.1016/j.bbrc.2011.04.060. Epub 2011 Apr 20.
We have successfully isolated a novel anoctamin (xANO2), Ca(2+)-activated chloride channel (ANO1, TMEM16A), from Xenopus laevis. The cDNA sequence was determined to belong to the anoctamin family by comparison with the xTMEM16A sequence in a previous report. Full length cDNA synthesis was performed by repeating 5'- and 3'-rapid amplification of cDNA end (RACE). We successfully completed the entire cDNA sequence and transiently named this sequence xANO2. The xANO2 cDNA is 3884 base pair (bp) long and codes 980 amino acid (aa) proteins. According to an aa homology search using the Basic Local Alignment Search Tool (BLAST), xANO2 showed an overall identity of 92% to xTMEM16A (xANO1) independently sub-cloned in our laboratory. A primary sequence of xANO2 revealed typical characteristics of transmembrane proteins. In tissue distribution analysis, the gene products of anoctamins were ubiquitously detected by real-time PCR (RT-PCR). The expression profiles of each anoctamin were different among brain, oocytes, and digestive organs with relatively weak expression. To clarify the anoctamin activity, physiological studies were performed using the whole cell patch-clamp technique with HEK293T cells, enhanced green fluorescent protein (EGFP), and expression vectors carrying anoctamins. Characteristics typical of voltage-dependent chloride currents were detected in cells expressing both xANO2 and xTMEM16A but not with EGFP alone. Sensitive reactions to the anion channel blocker niflumic acid (NFA) were also revealed. Considering these results, xANO2 was regarded as a new TMEM16A belonging to the Xenopus anoctamin family.
我们已经成功地从非洲爪蟾中分离出一种新型的钙激活氯离子通道（ANO1，TMEM16A）。通过与之前报道的 xTMEM16A 序列进行比较，确定 cDNA 序列属于 anoctamin 家族。通过重复 5'和 3'快速扩增 cDNA 末端（RACE）进行全长 cDNA 合成。我们成功完成了整个 cDNA 序列，并将其暂时命名为 xANO2。xANO2 cDNA 长 3884 个碱基对（bp），编码 980 个氨基酸（aa）的蛋白质。根据使用基本局部比对搜索工具（BLAST）进行的 aa 同源性搜索，xANO2 与我们实验室独立克隆的 xTMEM16A（xANO1）具有 92%的整体同一性。xANO2 的一级序列显示出跨膜蛋白的典型特征。在组织分布分析中，通过实时 PCR（RT-PCR）检测到 anoctamins 的基因产物广泛存在。每个 anoctamin 的表达谱在大脑、卵母细胞和消化器官之间存在差异，表达较弱。为了阐明 anoctamin 的活性，使用全细胞膜片钳技术在表达 GFP 的 HEK293T 细胞和携带 anoctamins 的表达载体上进行了生理研究。在表达 xANO2 和 xTMEM16A 的细胞中检测到典型的电压依赖性氯离子电流特性，但在仅表达 GFP 的细胞中没有检测到。还揭示了对阴离子通道阻滞剂尼氟灭酸（NFA）的敏感反应。考虑到这些结果，xANO2 被认为是属于非洲爪蟾 anoctamin 家族的新的 TMEM16A。
