Abidi Affef, Devaux Jérôme J, Molinari Florence, Alcaraz Gisèle, Michon François-Xavier, Sutera-Sardo Julie, Becq Hélène, Lacoste Caroline, Altuzarra Cécilia, Afenjar Alexandra, Mignot Cyril, Doummar Diane, Isidor Bertrand, Guyen Sylvie N, Colin Estelle, De La Vaissière Sabine, Haye Damien, Trauffler Adeline, Badens Catherine, Prieur Fabienne, Lesca Gaetan, Villard Laurent, Milh Mathieu, Aniksztejn Laurent
Aix-Marseille Université, GMGF, Marseille, France; INSERM, UMR_S 910, Marseille, France.
Aix-Marseille Université, CNRS, CRN2M-UMR7286, Marseille, France.
Neurobiol Dis. 2015 Aug;80:80-92. doi: 10.1016/j.nbd.2015.04.017. Epub 2015 May 22.
Mutations in the KCNQ2 gene encoding the voltage-dependent potassium M channel Kv7.2 subunit cause either benign epilepsy or early onset epileptic encephalopathy (EOEE). It has been proposed that the disease severity rests on the inhibitory impact of mutations on M current density. Here, we have analyzed the phenotype of 7 patients carrying the p.A294V mutation located on the S6 segment of the Kv7.2 pore domain (Kv7.2(A294V)). We investigated the functional and subcellular consequences of this mutation and compared it to another mutation (Kv7.2(A294G)) associated with a benign epilepsy and affecting the same residue. We report that all the patients carrying the p.A294V mutation presented the clinical and EEG characteristics of EOEE. In CHO cells, the total expression of Kv7.2(A294V) alone, assessed by western blotting, was only 20% compared to wild-type. No measurable current was recorded in CHO cells expressing Kv7.2(A294V) channel alone. Although the total Kv7.2(A294V) expression was rescued to wild-type levels in cells co-expressing the Kv7.3 subunit, the global current density was still reduced by 83% compared to wild-type heteromeric channel. In a configuration mimicking the patients' heterozygous genotype i.e., Kv7.2(A294V)/Kv7.2/Kv7.3, the global current density was reduced by 30%. In contrast to Kv7.2(A294V), the current density of homomeric Kv7.2(A294G) was not significantly changed compared to wild-type Kv7.2. However, the current density of Kv7.2(A294G)/Kv7.2/Kv7.3 and Kv7.2(A294G)/Kv7.3 channels were reduced by 30% and 50% respectively, compared to wild-type Kv7.2/Kv7.3. In neurons, the p.A294V mutation induced a mislocalization of heteromeric mutant channels to the somato-dendritic compartment, while the p.A294G mutation did not affect the localization of the heteromeric channels to the axon initial segment. We conclude that this position is a hotspot of mutation that can give rise to a severe or a benign epilepsy. The p.A294V mutation does not exert a dominant-negative effect on wild-type subunits but alters the preferential axonal targeting of heteromeric Kv7 channels. Our data suggest that the disease severity is not necessarily a consequence of a strong inhibition of M current and that additional mechanisms such as abnormal subcellular distribution of Kv7 channels could be determinant.
编码电压依赖性钾离子M通道Kv7.2亚基的KCNQ2基因突变可导致良性癫痫或早发性癫痫性脑病（EOEE）。有人提出，疾病的严重程度取决于突变对M电流密度的抑制作用。在此，我们分析了7例携带位于Kv7.2孔结构域S6段的p.A294V突变（Kv7.2(A294V)）患者的表型。我们研究了该突变的功能和亚细胞后果，并将其与另一个与良性癫痫相关且影响相同残基的突变（Kv7.2(A294G)）进行比较。我们报告称，所有携带p.A294V突变的患者均表现出EOEE的临床和脑电图特征。在CHO细胞中，通过蛋白质印迹法评估，单独的Kv7.2(A294V)的总表达量仅为野生型的20%。在单独表达Kv7.2(A294V)通道的CHO细胞中未记录到可测量的电流。尽管在共表达Kv7.3亚基的细胞中Kv7.2(A294V)的总表达量恢复到了野生型水平，但与野生型异源通道相比，整体电流密度仍降低了83%。在模拟患者杂合基因型即Kv7.2(A294V)/Kv7.2/Kv7.3的配置中，整体电流密度降低了30%。与Kv7.2(A294V)相反，同型Kv7.2(A294G)的电流密度与野生型Kv7.2相比无显著变化。然而，与野生型Kv7.2/Kv7.3相比，Kv7.2(A294G)/Kv7.2/Kv7.3和Kv7.2(A294G)/Kv7.3通道的电流密度分别降低了30%和5%。在神经元中，p.A294V突变导致异源突变通道错误定位于体树突区室，而p.A294G突变不影响异源通道向轴突起始段的定位。我们得出结论，该位置是一个突变热点，可导致严重或良性癫痫。p.A294V突变对野生型亚基不发挥显性负效应，但改变了异源Kv7通道的优先轴突靶向。我们的数据表明，疾病的严重程度不一定是M电流受到强烈抑制的结果，其他机制如Kv7通道的异常亚细胞分布可能起决定性作用。
