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

我们已经确定Kv1.5和Kvβ1.2是新生儿动脉导管（DA）和肺动脉（PA）中表达的两种主要的氧敏感kv。为了阐明PO2变化诱导Kv开闭的调控机制，研究了Kv1.5-Kvβ1.2相互作用及其翻译后修饰。我们建立了稳定表达Kv1.5的HEK 293细胞。从转染或未转染Kvβ1.2的HEK 293-Kv1.5细胞中分离Kv复合物，使用蓝色天然聚丙烯酰胺凝胶电泳（BN-PAGE）进行浓缩和分离。Kv1.5、Kvβ1.2和Kv1.5/KvB1.2复合物在BN-PAGE上的表观分子大小分别为630、530和800 kDa。功能Kv是由Kv α亚基组成的四聚体，它们与Kv β亚基共同组装。由于Kv1.5、Kvβ1.2和Kv1.5/KvB1.2的四聚体的分子大小分别为263、182和445 kDa，因此Kv复合物的表观分子大小远远大于这些分子大小，这表明Kv复合物可能含有相互作用的蛋白质和/或翻译后修饰。Western blotting对Kv1.5复合物的分析表明，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复合物增加了这些条带的流动性，表明Kv1.5在HEK 293细胞中被糖基化。KvB1.2单体在45 kDa处呈带状。蛋白间二硫化物可能在Kv1.5/KvB1.2复合体的形成中起重要作用。利用免疫细胞化学方法验证了Kv1.5和KvB1.2在HEK 293细胞质膜上的共定位。采用免疫组化方法检测缺氧条件下胎儿DA和PA的谷胱甘肽化和硝基酪氨酸化。在谷胱甘肽化实验中没有观察到差异。DA内膜可见特异性抗硝基酪氨酸免疫染色，PA无特异性免疫染色，缺氧条件下DA有轻微程度染色。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