【摘要】 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。更多还原

【Abstract】 Objective To identify the electrophysiological properties of long-QT syndrome (LQTS) associated missense mutations in the outer mouth of the HERG potassium channel in vitro.Methods Mutations V630A and N633S were constructed by Megaprimer PCR method and cRNA were produced by T7 RNA polymerase. The electrophysiological properties of the mutation were investigated in the Xenopus oocyte heterologous expression system.Results Coexpression of mutant and wild-type HERG subunits caused a dominant-negative effect,and the currents were significantly decreased. Compared with wild-type HERG channels,V630A and N633S mutations were related to decreased time constants for inactivation for V630A/WT and N633S/WT at all potentials,reduced slope conductance and the voltage dependence of steady-state inactivation was shifted to negative potentials for V630A/WT and N633S/WT.Conclusion Present study shows that LQTS associated missense mutations located in the outer mouth of HERG cause a dominant-negative effect and alterations in steady-state voltage dependence of channel gating of heteromultimeric channels suggesting a reduction in expressional current might be one of the pathophysiologic mechanisms of LQTS.更多还原