Dept. of Medicine, Univ. of California, San Diego, 9500 Gilman Dr., MC 0725, La Jolla, CA 92093-0725, USA.
Am J Physiol Cell Physiol. 2010 Mar;298(3):C496-509. doi: 10.1152/ajpcell.00464.2009. Epub 2009 Dec 16.
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.
电压门控钾 (K(V)) 通道的活性在调节肺动脉平滑肌细胞 (PASMC) 收缩、增殖和凋亡方面起着重要作用。K(V) 通道高度保守的 NH 2 -末端四聚化结构域 (T1) 对于正确的通道组装、与调节性 K(V)β亚基的结合以及通道在质膜上的定位是重要的。我们最近报道了特发性肺动脉高压患者 KCNA5 基因中的两个非同义突变 (G182R 和 E211D)，它们定位于 KCNA5 的 T1 结构域。为了研究突变体与野生型 (WT) 通道在体外的电生理特性和表达模式，我们用 WT KCNA5、G182R、E211D 或双突变体 G182R/E211D 转染 HEK-293 细胞。这些突变体形成功能性通道；然而，WT 和突变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出失活加速；与 WT 通道相比，突变通道在更超极化的电位下失活。突变还降低了通道蛋白的表达。与存在于成熟糖基化形式的 WT 通道相比，突变通道以其未成熟形式存在的比例更大。此外，与 WT 相比，COS-1 细胞中的 G182R 蛋白水平大大降低。免疫染色数据支持这样的假设，即尽管 WT 蛋白定位于质膜，但突变蛋白主要保留在细胞内的包囊中。总的来说，这些数据支持 T1 结构域在通道动力学以及 KCNA5 通道亚细胞定位中的作用。
