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目的HERG基因突变可引起遗传性QT延长综合征(longQTsyndroms,LQTS)。研究探讨HERG通道孔区的错义突变导致LQTS的分子机制。方法采用Megaprimer方法制备HERGV630A、N633S突变体,并亚克隆到pSP64和pcDNA3.1表达载体。用T7RNA聚合酶体外合成cRNA,经Ribogreen荧光定量后,将相应的cRNA注入卵母细胞,在18℃培养箱中培养3d后,用标准双电极电压钳技术测量卵母细胞的表达电流。所有数据均用pCLAMP软件采集,并应用Kaleidagraph3.5和Igor软件进行数据处理。结果当V630A、N633S分别同HERG野生型在卵母细胞共同表达时,这两种突变体均具有明显的负显性抑制效应,其抑制效应达到50%~70%。同野生型HERG通道相比,这两种异源多聚体的失活显著加快,半失活电压负向偏移。结论该研究首次阐明了两个位于HERG通道孔区外口的错义突变V630A、N633S所引起的通道电生理学功能改变,从分子水平证明HERG通道孔区的错义突变与致命性心律失常的关系。
The purpose of HERG gene mutations can cause hereditary QT prolonged syndrome (longQTsyndroms, LQTS). To investigate the molecular mechanism of missense mutations in the HERG channel pore region leading to LQTS. Methods The HERGV630A and N633S mutants were prepared by Megaprimer method and subcloned into pSP64 and pcDNA3.1 expression vector. The cRNA was synthesized in vitro by T7 RNA polymerase. After quantified by Ribogreen fluorescence, the corresponding cRNA was injected into oocytes and cultured in 18 ℃ incubator for 3 days. The oocyte expression current was measured by standard two-electrode voltage-clamp technique. All data were collected using pCLAMP software and processed using Kaleidagraph 3.5 and Igor software. Results When both V630A and N633S were overexpressed in the oocyte with HERG wild type, the two mutants showed a significant negative dominant inhibitory effect and the inhibitory effect reached 50% -70%. Inactivation of these two heteromultimers was significantly accelerated compared to the wild-type HERG channels with a negative shift of the half-inactivation voltage. Conclusions This study, for the first time, elucidated the changes in the electrophysiological function of the channels caused by the missense mutations V630A and N633S located at the mouth of the HERG channel. It was demonstrated molecularly that the missense mutation in the HERG channel was associated with fatal arrhythmia .