使用PD - 118057和毒胡萝卜素对E637K - hERG突变体中蛋白质转运缺陷的药理学方法。
Pharmacologic Approach to Defective Protein Trafficking in the E637K-hERG Mutant with PD-118057 and Thapsigargin.
作者信息
Mao Haiyan, Lu Xiaoli, Karush Justin Michael, Huang Xiaoyan, Yang Xi, Ba Yanna, Wang Ying, Liu Ningsheng, Zhou Jianqing, Lian Jiangfang
机构信息
LiHuiLi Hospital, Medical School of NingBo University, NingBo, China.
出版信息
PLoS One. 2013 Jun 19;8(6):e65481. doi: 10.1371/journal.pone.0065481. Print 2013.
BACKGROUND
Treatment of LQT2 is inadequate. Many drugs which can pharmacologically rescue defective protein trafficking in LQT2 also result in potent blockade of HERG current, negating their therapeutic benefit. It is reported that PD-118057 and thapsigargin can rescue LQT2 without hERG channel blockade, but the precise mechanism of action is unknown. Furthermore, the effect of PD-118057 and thapsigargin on the dominant negative E637K-hERG mutant has not been previously investigated.
OBJECTIVE
IN THIS STUDY, WE INVESTIGATED: (a) the effect of PD-118057 and thapsigargin on the current amplitudes of WT-hERG and WT/E637K-hERG channels; (b) the effect of PD-118057 and thapsigargin on the biophysical properties of WT-hERG and WT/E637K-hERG channels; (c) whether drug treatment can rescue channel processing and trafficking defects of the WT/E637K-hERG mutant.
METHODS
The whole-cell Patch-clamp technique was used to assess the effect of PD-118057 and thapsigargin on the electrophysiological characteristics of the rapidly activating delayed rectifier K(+) current (Ikr) of the hERG protein channel. Western blot was done to investigate pharmacological rescue on hERG protein channel function.
RESULTS
In our study, PD-118057 was shown to significantly enhance both the maximum current amplitude and tail current amplitude, but did not alter the gating and kinetic properties of the WT-hERG channel, with the exception of accelerating steady-state inactivation. Additionally, thapsigargin shows a similar result as PD-118057 for the WT-hERG channel, but with the exception of attenuating steady-state inactivation. However, for the WT/E637K-hERG channel, PD-118057 had no effect on either the current or on the gating and kinetic properties. Furthermore, thapsigargin treatment did not alter the current or the gating and kinetic properties of the WT/E637K-hERG channel, with the exception of opening at more positive voltages.
CONCLUSION
Our findings illustrate that neither PD-118057 nor thapsigargin play a role in correcting the dominant-negative effect of the E637K-hERG mutant.
背景
长QT综合征2型（LQT2）的治疗并不充分。许多能够从药理学上挽救LQT2中缺陷蛋白转运的药物也会强力阻断人类ether-à-go-go相关基因（HERG）电流，从而抵消其治疗益处。据报道，PD - 118057和毒胡萝卜素可以在不阻断HERG通道的情况下挽救LQT2，但具体作用机制尚不清楚。此外，PD - 118057和毒胡萝卜素对显性负性E637K - HERG突变体的影响此前尚未被研究过。
目的
在本研究中，我们调查了：（a）PD - 118057和毒胡萝卜素对野生型HERG（WT - HERG）和野生型/ E637K - HERG通道电流幅度的影响；（b）PD - 118057和毒胡萝卜素对WT - HERG和野生型/ E637K - HERG通道生物物理特性的影响；（c）药物治疗是否能挽救野生型/ E637K - HERG突变体的通道加工和转运缺陷。
方法
采用全细胞膜片钳技术评估PD - 118057和毒胡萝卜素对HERG蛋白通道快速激活延迟整流钾电流（Ikr）电生理特性的影响。进行蛋白质免疫印迹法以研究对HERG蛋白通道功能的药理学挽救作用。
结果
在我们的研究中，PD - 118057被证明能显著提高最大电流幅度和尾电流幅度，但除了加速稳态失活外，并未改变WT - HERG通道的门控和动力学特性。此外，对于WT - HERG通道，毒胡萝卜素显示出与PD - 118057类似的结果，但除了减弱稳态失活外。然而，对于野生型/ E637K - HERG通道，PD - 118057对电流以及门控和动力学特性均无影响。此外，毒胡萝卜素处理除了使野生型/ E637K - HERG通道在更正的电压下开放外，并未改变其电流以及门控和动力学特性。
结论
我们的研究结果表明，PD - 118057和毒胡萝卜素在纠正E637K - HERG突变体的显性负性效应方面均不起作用。
Zotero 插件