Informatic and functional approaches to identifying a regulatory region for the cardiac sodium channel.
Informatic and functional approaches to identifying a regulatory region for the cardiac sodium channel.
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DOI：
10.1161/circresaha.110.235630
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发表时间：
2011-06-24
影响因子：
20.1
通讯作者：
Roden DM
中科院分区：
文献类型：
--
作者：
Atack TC;Stroud DM;Watanabe H;Yang T;Hall L;Hipkens SB;Lowe JS;Leake B;Magnuson MA;Yang P;Roden DM
尽管多种证据表明心脏钠通道基因 SCN5A 的可变表达在心律失常易感性中发挥作用，但对其转录调控知之甚少。我们通过计算机和体外实验来鉴定对 SCN5A 转录调控重要的可能非编码序列。结果扩展到删除了假定的调控区域的小鼠。我们鉴定了人和小鼠 SCN5A 直向同源物之间高度保守 (>70%) 的 92 个非编码区。三个保守的非编码序列 (CNS) 在荧光素酶测定中显示出显着的（>5 倍）活性。进一步的体外研究表明，内含子 1 中的 CNS28 是潜在的调控区域。使用重组酶介导的盒交换 (RMCE)，我们生成了包含 CNS28 的 435 bp 区域被去除的小鼠。缺失纯合的动物显示 SCN5A 转录本、NaV1.5 蛋白丰度和分离心室肌细胞中测量的钠电流显着增加。心电图显示 QRS 明显较短（对照组为 10.7±0.2 毫秒，敲除组为 9.7±0.2 毫秒），表明心室传导更快。 CNS28 的体外分析发现该区域内有一个短的 3' 片段，该片段是调节活性所需的，并且包含 E-box 基序。删除该片段使 CHO 细胞中的报告活性降低至基线的 3.6±0.3%，肌细胞中的报告活性降低至基线的 16±3%（均 P<0.05），而 E-box 中各个位点的突变使 CHO 细胞和肌细胞中的报告活性分别恢复至基线的 62±4% 和 57±2%（均 P<0.05）。这些发现证实心脏钠通道表达的调节可调节体内通道功能，并确定了这种调节背后的非编码区。
Although multiple lines of evidence suggest variable expression of the cardiac sodium channel gene SCN5A plays a role in susceptibility to arrhythmia, little is known about its transcriptional regulation. We used in silico and in vitro experiments to identify possible non-coding sequences important for transcriptional regulation of SCN5A. The results were extended to mice in which a putative regulatory region was deleted. We identified 92 non-coding regions highly conserved (>70%) between human and mouse SCN5A orthologs. Three conserved non-coding sequences (CNS) showed significant (>5-fold) activity in luciferase assays. Further in vitro studies indicated one, CNS28 in intron 1, as potential regulatory region. Using Recombinase-Mediated Cassette Exchange (RMCE), we generated mice in which a 435 bp region encompassing CNS28 was removed. Animals homozygous for the deletion showed significant increases in SCN5A transcripts, NaV1.5 protein abundance, and sodium current measured in isolated ventricular myocytes. ECGs revealed a significantly shorter QRS (10.7±0.2ms in controls vs. 9.7±0.2ms in knockouts) indicating more rapid ventricular conduction. In vitro analysis of CNS28 identified a short 3′ segment within this region required for regulatory activity and including an E-box motif. Deletion of this segment reduced reporter activity to 3.6±0.3% of baseline in CHO cells and 16±3% in myocytes (both P<0.05), and mutation of individual sites in the E-box restored activity to 62±4% and 57±2% of baseline in CHO cells and myocytes, respectively (both P<0.05). These findings establish that regulation of cardiac sodium channel expression modulates channel function in vivo, and identify a non-coding region underlying this regulation.