CAJ | 학술논문

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ether-a-go-go相关基因(human 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.同野生犁HERG钾通道的阻断相比,Y652A突变使阻断的IC50提高72倍,而Y652R突变使阻断的IC50提高53倍.Y652A和Y652R的阴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.
채용쌍전겁전압겸기술,연구동색림대표체재비주조섬란모세포상적야생형화Y652돌변형인류ether-a-go-go상관기인(human ether-a-go-go-related gene,HERG)갑통도적조단효응,관측HERG통도적분자위점특성개변대기조단효응적영향.결과현시,동색림이전압의뢰성화농도의뢰성적방식조단야생형적HERG갑통도전류.미전류포과정서기록전류현시동색림대HERG갑통도미소적장력성조단.조단특정부합대개방상태통도적조단특정.동색림야능조절실활상태적HERG갑통도.위우공도S6구적안기산위점돌변Y652A화Y652R가현저감약동색림대HERG통도적조단작용.동야생리HERG갑통도적조단상비,Y652A돌변사조단적IC50제고72배,이Y652R돌변사조단적IC50제고53배.Y652A화Y652R적음단효응지간몰유명현적차별.이상결과제시,동색림우선조단개방상태적HERG갑통도,이Y652시동색림여통도결합적관건위점지일.
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 voltageclamp 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 voltageand concentration-dependent manner and showed minimal tonic blockade of HERG current evaluated by the envelope of tails test. The IC50 value was (0.38±0.04) μmol/L for WT HERG potassium channel. The peaks of the Ⅰ-Ⅴ relationship for HERG channel suggesteda 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 μmol/L, (-22.39±0.94) mV at 1 μmol/L, (-23.51±0.91) mV at 10 μmol/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 thevoltage-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 S6mutation, Y652A and Y652R, significantly attenuated the blockade by ketansefin. The IC50 value were (27.13±9.40) μmol/L and (20.203±2.80) μmol/L, respectively, increased by approximately 72-fold for Y652A and 53-fold for Y652R compared to that of WT HERGchannel blockade [(0.38±0.04) μmol/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.