ISSN 0371-0874, CN 31-1352/Q


酮色林对人类{sl ether--a--go--go}相关基因钾通道的阻断作用

涂丹娜, 邹安若, 廖玉华, 杜以梅, 王宪沛, 李璐

华中科技大学同济医学院附属协和医院心血管病研究所,心内科,离子通道病研究中心.湖北,武汉 430022


采用双电极电压钳技术,研究酮色林对表达在非洲爪蟾卵母细胞上的野生型和Y652突变型人类{sl ether--a--go--go}相关基因(human {sl ether--a--go--go}--related gene,HERG)钾通道的阻断效应,观测HERG通道的分子位点特性改变对其阻断效应的影响。结果显示,酮色林以电压依赖性和浓度依赖性的方式阻断野生型的HERG钾通道电流。尾电流包裹程序记录电流显示酮色林对HERG钾通道微小的张力性阻断。阻断特征符合对开放状态通道的阻断特征。酮色林也能调节失活状态的HERG钾通道。位于孔道S6区的氨基酸位点突变Y652A和Y652R可显著减弱酮色林对HERG通道的阻断作用。同野生型HERG钾通道的阻断相比,Y652A突变使阻断的IC_(50)提高72倍,而Y652R突变使阻断的IC_(50)提高53倍。Y652A和Y652R的阻断效应之间没有明显的差别。以上结果提示,酮色林优先阻断开放状态的HERG钾通道,而Y652是酮色林与通道结合的关键位点之一。

关键词: 人类ether-a-go-go相关基因; 钾通道; 酮色林; 电压钳技术; 突变

Blockade of the human {sl ether--a--go--go}--related gene potassium channel by ketanserin

Tu Danna, Zou Anruo, Liao Yuhua, Du Yimei, Wang Xianpei, Li Lu

Research Center of Ion Channelopathy,Department of Cardiology,Institute of Cardiovascular Diseases,Union Hospital,Tongji Medical College,Huazhong University of Science and Technology.Wuhan 430022,Hubei


In the present study, we investigated the inhibitory action of ketanserin on wild-type (WT) and Y652 mutant human ether-a-go-go-related gene (HERG) potassium channels expressed in Xenopus oocytes and the effects of changing the channel molecular determinants characteristics on the blockade with and without ketanserin intervention using standard two-microelectrode voltage- clamp techniques. Point mutations were introduced into HERG gene (Y652A and Y652R) and subcloned into the pSP64 plasmid expression vector. Complementary RNAs for injection into oocytes were prepared with SP6 Cap-Scribe after linearization of the expression construct with EcoR I. Clampfit 9.2 software was employed for data collection and analysis. Origin 6.0 software was used to fit the data, calculate time constants and plot histograms. The results showed that ketanserin blocked WT HERG currents in voltage- and concentration-dependent manner and showed minimal tonic blockade of HERG current evaluated by the envelope of tails test. The IC_(50) value was (0.38±0.04) #mu#mol/L for WT HERG potassium channel. The peaks of the I-V relationship for HERG channel suggested a negative shift in the voltage-dependence of activation after using ketanserin, whose midpoint of activation values (V1/2) were (-16.59± 1.01) mV (control) vs (-20.59±0.87) mV (ketanserin) at 0.1 #mu#mol/L, (-22.39±0.94) mV at 1 #mu#mol/L, (-23.51±0.91) mV at 10 #mu#mol/L, respectively (P<0.05, n=6). Characteristics of blockade were consistent with an open-state channel blockade, because the extent and rate of onset of blockade was voltage-dependent, increasing at more potentials even in the condition of leftward shift of activation curve. Meanwhile, in the different depolarization duration, the fractional blockade of end-pulse step current and peak tail current at 100 ms duration was significantly lower than that at 400 ms and 700 ms, which indicated that following the channel activation fractional blockade was enhanced by the activated channels. Ketanserin could also modulate the inactivation of HERG channel, which shifted the voltage-dependence of WT HERG channel inactivation curve from (-51.71±2.15) mV to (-80.76±14.98) mV (P<0.05, n=4). The S6 mutation, Y652A and Y652R, significantly attenuated the blockade by ketanserin. The IC_(50) value were (27.13±9.40) #mu#mol/L and (20.20± 2.80) #mu#mol/L, respectively, increased by approximately 72-fold for Y652A and 53-fold for Y652R compared to that of WT HERG channel blockade [(0.38±0.04) #mu#mol/L]. However, between the inhibitory effects of Y652A and Y652R, there was no significant difference. In conclusion, ketanserin blocks WT HERG currents in voltage- and concentration-dependent manner and preferentially blocks open-state HERG channels. Tyr-652 is one of the critical residues in the ketanserin-binding sites.

Key words: human ether-a-go-go-related gene;Potassium channels;ketanserin;voltage-clamp techniques;mutation

收稿日期:  录用日期:

通讯作者:  E-mail:


涂丹娜, 邹安若, 廖玉华, 杜以梅, 王宪沛, 李璐. 酮色林对人类{sl ether--a--go--go}相关基因钾通道的阻断作用[J]. 生理学报 2008; 60 (4): 525-534.

Tu Danna, Zou Anruo, Liao Yuhua, Du Yimei, Wang Xianpei, Li Lu. Blockade of the human {sl ether--a--go--go}--related gene potassium channel by ketanserin. Acta Physiol Sin 2008; 60 (4): 525-534 (in Chinese with English abstract).