Clenbuterol Attenuates hERG Channel by Promoting the Mature Channel Degradation
Clenbuterol Attenuates hERG Channel by Promoting the Mature Channel Degradation
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DOI：
10.1177/1091581817710786
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发表时间：
2017-05
影响因子：
2.2
通讯作者：
L. Luo;Peijing Hu;C. Miao;A. Ma;Tingzhong Wang
中科院分区：
文献类型：
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作者：
L. Luo;Peijing Hu;C. Miao;A. Ma;Tingzhong Wang
盐酸克伦特罗是一种β2-选择性肾上腺素能受体激动剂，在动物生产中被广泛应用于减肥、提高生产性能等方面。越来越多的证据表明，克伦特罗可引起多种心律失常和QTc间期延长。然而，人们对潜在的机制知之甚少。大多数药物通过干扰人类ether-a-go-go-related gene（hERG）K+通道与QTc延长相关。本研究旨在探讨克伦特罗对hERG通道的作用及其机制。HEK 293细胞用hERG通道的野生型和Y 652 A或F656 A突变体转染，并用克伦特罗处理。采用全细胞膜片钳技术记录hERG电流，Western blot检测hERG蛋白水平。我们发现克伦特罗以浓度和时间依赖性方式降低细胞膜上hERG蛋白的成熟形式，而不影响未成熟形式。相应地，与急性治疗相比，克伦特罗慢性治疗在更大程度上降低了hERG电流。在Brefeldin A（BFA），这是用来阻止hERG通道运输到细胞膜的存在下，克伦特罗降低hERG在质膜上的程度比单独的BFA。此外，hERG通道的药物结合位点突变体Y 652 A和F656 A消除了克伦特罗介导的hERG降低和电流阻断。总之，克伦特罗通过促进通道降解来降低hERG通道表达和电流。克伦特罗对hERG通道的作用与位于S6结构域上的药物结合位点Tyr-652和Phe-656有关。这种生物物理机制可能是克伦特罗诱导的QTc延长或心律失常的基础。
Clenbuterol, a β2-selective adrenergic receptor agonist, is illicitly used in weight loss and performance enhancement and animal production. Increasing evidence demonstrates that clenbuterol induces various kinds of arrhythmias and QTc interval prolongation. However, little is known about the underlying mechanism. Most drugs are associated with QTc prolongation through interfering with human ether-a-go-go-related gene (hERG) K+ channels. The present study aims to investigate the effects and underlying mechanisms of clenbuterol on the hERG channel. HEK 293 cells were transfected with wild type and Y652A or F656A mutants of the hERG channel and treated with clenbuterol. The hERG current was recorded using whole-cell patch-clamp technique, and protein level was evaluated by Western blot. We found that clenbuterol decreases the mature form of the hERG protein at the cell membrane in a concentration- and time-dependent manner, without affecting the immature form. Correspondingly, clenbuterol chronic treatment reduced hERG current to a greater extent compared to acute treatment. In the presence of Brefeldin A (BFA), which was used to block hERG channel trafficking to cell membrane, clenbuterol reduced hERG on plasma membrane to a greater extent than BFA alone. In addition, the hERG channel’s drug binding sites mutant Y652A and F656A abolished clenbuterol-mediated hERG reduction and current blockade. In conclusion, clenbuterol reduces hERG channel expression and current by promoting the channel degradation. The effect of clenbuterol on the hERG channel is related to the drug-binding sites, Tyr-652 and Phe-656, located on the S6 domain. This biophysical mechanism may underlie clenbuterol-induced QTc prolongation or arrhythmia.