Functional analysis of potassium channels in Kv7.2 G271V mutant causing early onset familial epilepsy
Functional analysis of potassium channels in Kv7.2 G271V mutant causing early onset familial epilepsy
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DOI：
10.1016/j.brainres.2015.04.060
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发表时间：
2015-08-07
期刊：
影响因子：
2.9
通讯作者：
Zhou, Xihui
中科院分区：
文献类型：
--
作者：
Wang, Juanjuan;Li, Yuan;Zhou, Xihui
KV7(KCNQ)通道位于一类电压门控K+电流之下，以调节神经元兴奋性而闻名。Kv7.2(KCNQ2)亚基的第一甘氨酸(G)残基在不同类别的KV7通道家族中高度保守。在一个大的四代家系中发现了一个错义突变，导致Kv7.2中相应的G残基被Valine(p.G271V)取代。在这里，我们开始利用膜片钳技术结合生化和免疫细胞化学技术在瞬时转染人胚胎肾(HEK)293细胞中检测G271V突变的分子发病机制。野生型(WT)和G271V的Kv7.2蛋白表达强度相同。在转基因的HEK细胞中，G271V突变导致电导-电压关系的去极化移动，与WT相比，电流激活动力学明显减慢。此外，在Kv7.2/G271V/Kv7.3异构体条件下，G271V突变体取消了同源通道的电流，使电流减少了约50%，表明存在更严重的功能缺陷。为了测试G271V突变通道在表膜上的表达，我们进行了荧光共聚焦显微镜成像，结果显示突变体和WT之间没有差异，这表明G271V通道即使存在于膜上，也不能对去极化做出反应。此外，药物干预实验显示，当表达G271V异多聚体通道的HEK 293细胞与KV7通道增强剂雷替加宾(Ezogabine)一起特异性孵育时，钾电流显著增加，提示雷替加宾作为基因特异性治疗的可能性。(C)2015爱思唯尔B.V.保留所有权利。
Kv7 (KCNQ) channels underlying a class of voltage-gated K+ current are best known for regulating neuronal excitability. The first glycine (G) residue in the pore helix of Kv7.2 (KCNQ2) subunit is highly conserved among different classes of Kv7 channel family. A missense mutation causing the replacement of the corresponding G residues with a valine (p.G271V) in Kv7.2 was found in a large, four-generation pedigree. Here, we set out to examine the molecular pathomechanism of G271V mutants using patch clamp technology combined with biochemical and immunocytochemical techniques in transiently transfected human embryonic kidney (HEK) 293 cells. The expression of Kv7.2 protein had the same intensity for both wild type (WT) and G271V. In transfected HEK cells, G271V mutants induced large depolarizing shifts of the conductance-voltage relationships and marked slowing of current activation kinetics compared to WT. In addition, G271V mutants abolished currents in homomeric channels, and resulted in about 50% reduction of current in Kv7.2/G271V/Kv7.3 heteromultimeric condition, indicating a more severe functional defect. To test for G271V mutant channel expression in surface membrane, we performed fluorescence confocal microscopy imaging, which revealed no differences between the mutant and WT, suggesting that G271V channels fail to open in response to depolarization even though they are present in the membrane. Furthermore, pharmacologic intervention experiments revealed that upon specific incubation of transfected HEK 293 cells expressing G271V heteromultimeric channels in presence of Kv7 channel enhancer retigabine (ezogabine), the potassium currents increased significantly, suggesting the potential of retigabine as gene-specific therapy. (C) 2015 Elsevier B.V. All rights reserved.