Camacho Juan A, Hensellek Sabine, Rougier Jean-Sébastien, Blechschmidt Steve, Abriel Hugues, Benndorf Klaus, Zimmer Thomas
Institute of Physiology II, Friedrich Schiller University, 07740 Jena, Germany.
J Biol Chem. 2006 Apr 7;281(14):9498-506. doi: 10.1074/jbc.M509716200. Epub 2006 Feb 8.
In the present study, we identified a novel splice variant of the human cardiac Na(+) channel Na(v)1.5 (Na(v)1.5d), in which a 40-amino acid sequence of the DII/DIII intracellular linker is missing due to a partial deletion of exon 17. Expression of Na(v)1.5d occurred in embryonic and adult hearts of either sex, indicating that the respective alternative splicing is neither age-dependent nor gender-specific. In contrast, Na(v)1.5d was not detected in the mouse heart, indicating that alternative splicing of Na(v)1.5 is species-dependent. In HEK293 cells, splice variant Na(v)1.5d generated voltage-dependent Na(+) currents that were markedly reduced compared with wild-type Na(v)1.5. Experiments with mexiletine and 8-bromo-cyclic AMP suggested that the trafficking of Na(v)1.5d channels was not impaired. However, single-channel recordings showed that the whole-cell current reduction was largely due to a significantly reduced open probability. Additionally, steady-state activation and inactivation were shifted to depolarized potentials by 15.9 and 5.1 mV, respectively. Systematic mutagenesis analysis of the spliced region provided evidence that a short amphiphilic region in the DII/DIII linker resembling an S4 voltage sensor of voltage-gated ion channels is an important determinant of Na(v)1.5 channel gating. Moreover, the present study identified novel short sequence motifs within this amphiphilic region that specifically affect the voltage dependence of steady-state activation and inactivation and current amplitude of human Na(v)1.5.
在本研究中，我们鉴定出一种人类心脏钠通道Na(v)1.5的新型剪接变体（Na(v)1.5d），由于外显子17的部分缺失，其二/三细胞内连接子的40个氨基酸序列缺失。Na(v)1.5d在雌雄两性的胚胎和成年心脏中均有表达，表明各自的可变剪接既不依赖年龄也不具有性别特异性。相比之下，在小鼠心脏中未检测到Na(v)1.5d，表明Na(v)1.5的可变剪接具有物种依赖性。在HEK293细胞中，剪接变体Na(v)1.5d产生的电压依赖性钠电流与野生型Na(v)1.5相比明显降低。用美西律和8-溴环磷酸腺苷进行的实验表明，Na(v)1.5d通道的转运未受损。然而，单通道记录显示，全细胞电流降低主要是由于开放概率显著降低。此外，稳态激活和失活分别向去极化电位偏移了15.9和5.1 mV。对剪接区域的系统诱变分析提供了证据，表明二/三连接子中一个类似于电压门控离子通道S4电压传感器的短两亲区域是Na(v)1.5通道门控的重要决定因素。此外，本研究在该两亲区域内鉴定出了新的短序列基序，这些基序特异性地影响人类Na(v)1.5的稳态激活和失活的电压依赖性以及电流幅度。
