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首页> 外文期刊>American Journal of Physiology >Tetramerization domain mutations in KCNA5 affect channel kinetics and cause abnormal trafficking patterns.
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Tetramerization domain mutations in KCNA5 affect channel kinetics and cause abnormal trafficking patterns.

机译：KCNA5中的四聚体结构域突变影响通道动力学并导致异常的运输模式。

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摘要

The activity of voltage-gated K(+) (K(V)) channels plays an important role in regulating pulmonary artery smooth muscle cell (PASMC) contraction, proliferation, and apoptosis. The highly conserved NH(2)-terminal tetramerization domain (T1) of K(V) channels is important for proper channel assembly, association with regulatory K(V) beta-subunits, and localization of the channel to the plasma membrane. We recently reported two nonsynonymous mutations (G182R and E211D) in the KCNA5 gene of patients with idiopathic pulmonary arterial hypertension, which localize to the T1 domain of KCNA5. To study the electrophysiological properties and expression patterns of the mutants compared with the wild-type (WT) channel in vitro, we transfected HEK-293 cells with WT KCNA5, G182R, E211D, or the double mutant G182R/E211D channel. The mutants form functional channels; however, whole cell current kinetic differences between WT and mutant channels exist. Steady-state inactivation curves of the G182R and G182R/E211D channels reveal accelerated inactivation; the mutant channels inactivated at more hyperpolarized potentials compared with the WT channel. Channel protein expression was also decreased by the mutations. Compared with the WT channel, which was present in its mature glycosylated form, the mutant channels are present in greater proportion in their immature form in HEK-293 cells. Furthermore, G182R protein level is greatly reduced in COS-1 cells compared with WT. Immunostaining data support the hypothesis that, while WT protein localizes to the plasma membrane, mutant protein is mainly retained in intracellular packets. Overall, these data support a role for the T1 domain in channel kinetics as well as in KCNA5 channel subcellular localization.
机译：电压门控K（+）（K（V））通道的活动在调节肺动脉平滑肌细胞（PASMC）的收缩，增殖和凋亡中起重要作用。 K（V）通道的高度保守的NH（2）-末端四聚结构域（T1）对于适当的通道组装，与调节性K（V）β亚基的关联以及通道向质膜的定位非常重要。我们最近报道了特发性肺动脉高压患者的KCNA5基因中的两个非同义突变（G182R和E211D），它们位于KCNA5的T1域。为了研究突变体与野生型（WT）通道的体外电生理特性和表达模式，我们用WT KCNA5，G182R，E211D或双突变体G182R / E211D通道转染了HEK-293细胞。突变体形成功能通道。然而，野生型和突变型通道之间存在全细胞电流动力学差异。 G182R和G182R / E211D通道的稳态失活曲线显示加速失活;与WT通道相比，突变型通道在更多的超极化电位下失活。突变也降低了通道蛋白的表达。与以成熟糖基化形式存在的WT通道相比，突变体通道在HEK-293细胞中以未成熟形式存在的比例更高。此外，与WT相比，COS-1细胞中的G182R蛋白水平大大降低。免疫染色数据支持以下假设：虽然WT蛋白位于质膜上，但突变蛋白主要保留在细胞内小包中。总体而言，这些数据支持T1域在通道动力学以及KCNA5通道亚细胞定位中的作用。

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