Su Zhi, Limberis James, Martin Ruth L, Xu Rong, Kolbe Katrin, Heinemann Stefan H, Hoshi Toshinori, Cox Bryan F, Gintant Gary A
Department of Integrative Pharmacology, Abbott Laboratories, 100 Abbott Park Road, Abbott Park, IL 60064, USA.
Biochem Pharmacol. 2007 Sep 1;74(5):702-11. doi: 10.1016/j.bcp.2007.06.002. Epub 2007 Jun 7.
Reactive species oxidatively modify numerous proteins including ion channels. Oxidative sensitivity of ion channels is often conferred by amino acids containing sulfur atoms, such as cysteine and methionine. Functional consequences of oxidative modification of methionine in human ether à go-go related gene 1 (hERG1), which encodes cardiac I(Kr) channels, are unknown. Here we used chloramine-T (ChT), which preferentially oxidizes methionine, to examine the functional consequences of methionine oxidation of hERG channels stably expressed in a human embryonic kidney cell line (HEK 293) and native hERG channels in a human neuroblastoma cell line (SH-SY5Y). ChT (300 microM) significantly decreased whole-cell hERG current in both HEK 293 and SH-SY5Y cells. In HEK 293 cells, the effects of ChT on hERG current were time- and concentration-dependent, and were markedly attenuated in the presence of enzyme methionine sulfoxide reductase A that specifically repairs oxidized methionine. After treatment with ChT, the channel deactivation upon repolarization to -60 or -100 mV was significantly accelerated. The effect of ChT on channel activation kinetics was voltage-dependent; activation slowed during depolarization to +30 mV but accelerated during depolarization to 0 or -10mV. In contrast, the reversal potential, inactivation kinetics, and voltage-dependence of steady-state inactivation remained unaltered. Our results demonstrate that the redox status of methionine is an important modulator of hERG channel.
活性物质可氧化修饰包括离子通道在内的多种蛋白质。离子通道的氧化敏感性通常由含硫原子的氨基酸赋予，如半胱氨酸和甲硫氨酸。编码心脏I(Kr)通道的人类ether à go-go相关基因1（hERG1）中甲硫氨酸氧化修饰的功能后果尚不清楚。在此，我们使用优先氧化甲硫氨酸的氯胺-T（ChT），来研究在人胚肾细胞系（HEK 293）中稳定表达的hERG通道以及人神经母细胞瘤细胞系（SH-SY5Y）中的天然hERG通道甲硫氨酸氧化的功能后果。ChT（300微摩尔）显著降低了HEK 293和SH-SY5Y细胞中的全细胞hERG电流。在HEK 293细胞中，ChT对hERG电流的影响具有时间和浓度依赖性，并且在特异性修复氧化甲硫氨酸的甲硫氨酸亚砜还原酶A存在时明显减弱。用ChT处理后，复极化至-60或-100 mV时通道的失活明显加速。ChT对通道激活动力学的影响具有电压依赖性；去极化至+30 mV时激活减慢，但去极化至0或-10 mV时激活加速。相比之下，反转电位、失活动力学以及稳态失活的电压依赖性保持不变。我们的结果表明，甲硫氨酸的氧化还原状态是hERG通道的重要调节因子。
