摘要
目的本研究旨在探讨氨基糖甙类药物对纯合和杂合的HERG无义突变体的作用效应。方法采用聚合酶链反应法制备HERGR1014X突变体,并克隆至真核细胞表达载体中。将突变体的cDNA瞬时转染至HEK293细胞,应用全细胞膜片钳技术记录通道电流。药物拯救采用将转染24h后的HEK293细胞在含G-418或庆大霉素(终浓度为400μg/mL)的培养液中孵育24h。结果 R1014X能表达典型的HERG样电流,尽管与野生型(wildtype,WT)HERG相比,最大尾电流幅值明显下降(3.9±1.4pA/pF,n=8,vs.47.8±6.3pA/pF,n=12,P<0.01)。经过G-418和庆大霉素干预后,R1014X的最大尾电流密度分别增加至12.7±3.3pA/pF(n=7,P<0.05)和18.3±3.7pA/pF(n=8,P<0.05)。药物干预使R1014X的激活动力学特性亦得到恢复。Westernblot和共聚焦检测进一步证实,氨基糖甙类能促进全长的HERG通道蛋白的表达。G-418和庆大霉素对杂合的WT/R1014X通道作用效应不同:庆大霉素能使WT/R1014X的电流增加2.2倍(n=12,P<0.05),G-418却无明显效应。结论氨基糖甙类药物能恢复无义突变的HERG通道的功能性表达,且对纯合和杂合通道均有效,这为遗传性心律失常的治疗提供了新的思路。
Objective The study was designed to investigate the effect of aminoglycosides on homozygous and heterozygous HERG channels with nonsense mutations.Methods The R1014X mutant was constructed by polymerase chain reaction based mutagenesis strategy and then subcloned into pcDNA3.1 vector.The channel current was recorded by patch clamping whole cell recording technique in HEK293 cells transiently transfected with wild type or the mutant cDNA.Pharmacological rescue was applied by culturing the HEK293 cells transfected for 24 hours in medium solution containing G-418 or gentamicin at a concentration of 400μg/mL for 24 hours.Results R1014X displayed wild type HERG-like currents,despite of a significant reduction in current amplitude compared to that in wild type channel(3.9±1.4 pA/pF,n=8,vs.47.8±6.3 pA/pF,n=12,P<0.01).The current density increased to 12.7±3.3 pA/pF(n=7,P<0.05)and 18.3±3.7 pA/pF(n=8,P<0.05)after G-418 and gentamicin treatment.And the voltage dependence of activation of R1014X was also restored by drug treatment.Furthermore,the expression of full-length proteins for R1014X induced by drugs was detected by western blot and confocal imaging.In the cells co-transfected with WT/R1014X,gentamicin and G-418 demonstrated different results:gentamicin,but not G-418,increased the current density by 2.2-fold(n=12,P<0.05).Conclusions Aminoglycosides may restore the functional expression of homozygous and heterozygous HERG channels produced by nonsense mutants,which may have clinical implications in the treatment of inherited arrhythmia syndromes.
出处
《中国分子心脏病学杂志》
CAS
2010年第6期339-343,共5页
Molecular Cardiology of China
基金
国家973重点基础研究项目(No.2007CB512008)
国家自然科学基金项目(No.30571040)