Improved functional expression of human cardiac kv1.5 channels and trafficking-defective mutants by low temperature treatment.
Improved functional expression of human cardiac kv1.5 channels and trafficking-defective mutants by low temperature treatment.
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DOI：
10.1371/journal.pone.0092923
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发表时间：
2014
期刊：
影响因子：
3.7
通讯作者：
Matsuura H
中科院分区：
文献类型：
--
作者：
Ding WG;Xie Y;Toyoda F;Matsuura H
本文研究了低温暴露对人Kv1.5（hKv1.5）表达、降解、定位和活性的影响。在表达hKv1.5的CHO细胞中，与在37°C下观察到的电流相比，在降低的温度（28°C）下培养时电流显著增加。Westernblot分析表明，在低温条件下，hKv1.5的蛋白质水平（未成熟蛋白和成熟蛋白）均显著升高。用蛋白酶体抑制剂MG 132处理，在37°C下显著增加了未成熟的hKv1.5蛋白，但不增加成熟的hKv1.5蛋白，然而，在MG 132暴露后，在低温下未成熟或成熟的hKv1.5蛋白没有变化。这些观察结果表明，成熟的hKv1.5蛋白在降低温度下的增强可能不是由于蛋白水解的抑制。此外，与37°C培养的细胞相比，28°C培养的细胞中细胞表面上的hKv1.5荧光信号显著增加。重要的是，低温处理显着转移的亚细胞分布的成熟hKv1.5，这表明相当大的重叠与trans-Golgi组件。使用衣霉素（一种N-糖基化抑制剂）的实验表明，hKv1.5的N-糖基化在28°C比在37°C更有效。最后，低温处理也挽救了蛋白质表达和电流的运输缺陷hKv1.5突变体。这些结果表明，低温暴露稳定的蛋白质在细胞器或质膜上，并调节其成熟和运输，从而增强电流的hKv1.5及其运输缺陷突变体。
We herein investigated the effect of low temperature exposure on the expression, degradation, localization and activity of human Kv1.5 (hKv1.5). In hKv1.5-expressing CHO cells, the currents were significantly increased when cultured at a reduced temperature (28°C) compared to those observed at 37°C. Western blot analysis indicated that the protein levels (both immature and mature proteins) of hKv1.5 were significantly elevated under the hypothermic condition. Treatment with a proteasome inhibitor, MG132, significantly increased the immature, but not the mature, hKv1.5 protein at 37°C, however, there were no changes in either the immature or mature hKv1.5 proteins at low temperature following MG132 exposure. These observations suggest that the enhancement of the mature hKv1.5 protein at reduced temperature may not result from the inhibition of proteolysis. Moreover, the hKv1.5 fluorescence signal in the cells increased significantly on the cell surface at 28°C versus those cultured at 37°C. Importantly, the low temperature treatment markedly shifted the subcellular distribution of the mature hKv1.5, which showed considerable overlap with the trans-Golgi component. Experiments using tunicamycin, an inhibitor of N-glycosylation, indicated that the N-glycosylation of hKv1.5 is more effective at 28°C than at 37°C. Finally, the hypothermic treatment also rescued the protein expression and currents of trafficking-defective hKv1.5 mutants. These results indicate that low temperature exposure stabilizes the protein in the cellular organelles or on the plasma membrane, and modulates its maturation and trafficking, thus enhancing the currents of hKv1.5 and its trafficking defect mutants.
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影响因子：
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通讯作者：
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