Tetrodotoxin-resistant Na+ channels in human neuroblastoma cells are encoded by new variants of Nav1.5/SCN5A
Tetrodotoxin-resistant Na+ channels in human neuroblastoma cells are encoded by new variants of Nav1.5/SCN5A
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DOI：
10.1111/j.1460-9568.2005.04280.x
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发表时间：
2005-08-01
影响因子：
3.4
通讯作者：
Kameyama, M
中科院分区：
文献类型：
--
作者：
Ou, SW;Kameyama, A;Kameyama, M
河豚毒素敏感 (TTX-S) 和 TTX 抗性 (TTX-R) 电压依赖性 Na+ 通道均在人神经母细胞瘤细胞系 NB-1 中表达，但编码 TTX-R Na+ 通道的基因尚未鉴定。在这项研究中，我们克隆了NB-1细胞中编码TTX-R Na+通道a亚基的cDNA，并将其命名为hNbR1。 hNbR1（登录号AB158469）最长的开放阅读框编码2016个氨基酸残基。序列分析表明hNbR1与人心脏Nav1.5/SCN5A高度同源，氨基酸一致性> 99%。结构域 I 的孔环区域中半胱氨酸残基 (Cys373) 的存在与 hNbR1 对 TTX 具有抗性的假设一致。对 SCN5A 基因组序列的分析揭示了编码结构域 I 的 S3 和 S4 的新外显子（外显子 6A）。此外，还发现了另一种剪接变体，缺乏外显子 18，编码结构域 II 和 III 之间的细胞内环中的 54 个氨基酸（hNbR1-2；登录号 AB158470）。转染 hNbR1 或 hNbR1-2 的人胚胎肾细胞 (HEK293) 中的 Na+ 电流显示出与 NB-1 细胞中 TTX-R I-Na 相似的电生理特性。在两种变体中，TTX 块的 IC50 大约为 8 μM。这些结果表明，SCN5A 有一个新鉴定的用于选择性剪接的外显子，并且表达范围比之前想象的更广泛。
Both tetrodotoxin-sensitive (TTX-S) and TTX-resistant (TTX-R) voltage-dependent Na+ channels are expressed in the human neuroblastoma cell line NB-1, but a gene encoding the TTX-R Na+ channel has not been identified. In this study, we have cloned cDNA encoding the a subunit of the TTX-R Na+ channel in NB-1 cells and designated it hNbR1. The longest open reading frame of hNbR1 ( accession no. AB158469) encodes 2016 amino acid residues. Sequence analysis has indicated that hNbR1 is highly homologous with human cardiac Nav1.5/SCN5A with > 99% amino acid identity. The presence of a cysteine residue (Cys373) in the pore-loop region of domain I is consistent with the supposition that hNbR1 is resistant to TTX. Analysis of the genomic sequence of SCN5A revealed a new exon encoding S3 and S4 of domain I ( exon 6A). In addition, an alternative splicing variant, lacking exon 18, that encodes 54 amino acids in the intracellular loop between domains II and III was found (hNbR1-2; accession no. AB158470). Na+ currents in human embryonic kidney cells (HEK293) transfected with hNbR1 or hNbR1-2 showed electrophysiological properties similar to those for TTX-R I-Na in NB-1 cells. The IC50 for the TTX block was approximate to 8 mu M in both variants. These results suggest that SCN5A has a newly identified exon for alternative splicing and is more widely expressed than previously thought.