Analysis of oxygen sensitive voltage-gated potassium channels in ductus arteriosus

我们已经确定Kv1.5和Kvβ1.2是在新生儿动脉导管(DA)和肺动脉(PA)表达的两种主要的氧敏感KV。为了阐明氧分压变化引起Kv开放/闭合的调控机制，我们研究了Kv1.5-Kvβ1.2的相互作用及其翻译后的修饰。从HEK293-Kv1.5细胞中分离、浓缩KV复合体，并用蓝色天然聚丙烯酰胺凝胶电泳法(BN-PAGE)进行分离。Kv1.5、Kvβ1.2和Kv1.5/KvB1.2络合物在BN-PAGE上的表观分子尺寸分别为630、530和800 kDa。功能性Kv是由Kvα亚基和Kvβ亚基共同组装而成的四聚体。由于Kv1.5、KvB1.2和Kv1.5/KvB1.2四聚体的分子大小分别为263kDa、182kDa和445kDa，因此Kv络合物的表观分子尺寸比推定的β…大得多更大的分子尺寸，这表明Kv复合体可能含有相互作用蛋白(S)和/或翻译后修饰(S)。Western blotting分析表明，90-95 kDa和300-400 kDa的条带与Kv1.5单体和聚合物有关。PNGase F处理Kv1.5复合体增加了这些带的迁移率，表明Kv1.5在HEK 293细胞中是糖基化的。KvB1.2单体在45 kDa出现条带。蛋白间二硫键可能在Kv1.5/KvB1.2复合体的形成中起重要作用。用免疫细胞化学方法检测Kv1.5和KvB1.2在HEK 293细胞质膜上的共定位，并用免疫组织化学方法检测缺氧条件下胎儿DA和PA中的谷胱甘肽和亚硝酸酪氨酸化。谷胱甘肽基化实验未见差异。抗硝基酪氨酸免疫组织化学染色主要见于DA的内层，而在PA上未见，低氧处理的DA有轻微的染色。较少

We have determined Kv1.5 and Kvβ1.2 are two of major oxygen-sensitive Kvs expressed in the neonatal ductus arteriosus (DA) and pulmonary artery (PA). In order to elucidate mechanism that regulates open/closure of Kv induced by change of PO2, Kv1.5-Kvβ1.2 interaction and their post-translational modification were investigated.We have established HEK 293 cells stably expressed Kv1.5. Kv complexes from the HEK 293-Kv1.5 cells transfected with or without Kvβ1.2 were isolated, concentrated, and separated using blue native polyacrylamide gel-electrophoresis (BN-PAGE). Apparent molecular sizes of the Kv1.5, Kvβ1.2, and Kv1.5/KvB1.2 complexes on the BN-PAGE are 630, 530, and 800 kDa, respectively. Functional Kv is composed of a tetramer of Kv alpha subunits, which co-assemble with Kv beta subunits. Since deduced molecular sizes of tetramer of Kv1.5, Kvβ1.2, and Kv1.5/KvB1.2 are 263, 182, and 445 kDa, respectively, apparent molecular sizes of Kv complexes were much bigger than these deduced mol … More ecular sizes, which suggested Kv complexes might contain interacting protein(s) and/or post-translational modification(s). Analysis of the Kv1.5 complex by Western blotting showed the bands at 90-95 kDa and 300-400 kDa were relevant to Kv1.5 monomers and polymers. PNGase F treatment of the Kv1.5 complex increased mobility of these bands, suggesting Kv1.5 is glycosylated in the HEK 293 cells. The KvB1.2 monomer appeared as band at 45 kDa. Inter-protein disulphide might play an important role to form Kv1.5/KvB1.2 complex. Immunocytochemistry was used to verify the co-localization of Kv1.5 and KvB1.2 on plasma membrane in the HEK 293 cells.Glutathionylation and nitrotyrosination in the fetal DA and PA under hypoxic condition were examined by immunohistochemistry. No difference was observed in the glutathionylation experiments. Characteristic immunostaining with anti-nitrotyrosine was observed on inner media of the DA but not on the PA, and a slightly extent staining is present in the DA treated with hypoxic condition. Less