不同浓度药物PTC124和G418对HERG通道无义突变体的作用  

The Pharmaceutical Effects of PTC124 and G418 on Rescuing Nonsense Mutations of HERG Gene Channels

作　　者：于海云[1] 姚艳[1] 张银辉[1] 龚菁[1] 黄健[1] 浦介麟[1] 

机构地区：[1]北京协和医学院中国医学科学院阜外心血管病医院心血管疾病国家重点实验室,北京市100037

出　　处：《中国循环杂志》2014年第6期461-465,共5页Chinese Circulation Journal

基　　金：国家自然科学基金资助项目(No.81070150)

摘　　要：目的:通过对HERG基因无义突变体应用不同剂量PTC124和G418来观察其通道功能的恢复情况,并探讨药物的作用机制。方法:制备HERG基因R1014X突变体,将野生型(WT)HERG及R1014X分别瞬时转染至HEK293细胞,转染24 h后将其置入含不同浓度的PTC124和G418的培养液中孵育24 h。逆转录聚合酶链反应法检测各组细胞HERG信使核糖核酸(mRNA)的表达,蛋白印迹法检测野生型和R1014X给药前后通道蛋白的表达,全细胞膜片钳技术记录通道电流以评价HERG基因编码通道的功能性表达。结果:与野生型相比,R1014X突变体编码的通道蛋白的蛋白条带变短,蛋白量及mRNA水平无明显差异(P>0.05)。药物PTC124和G418均能恢复R1014X完整蛋白的表达,并呈剂量依赖性变化(P<0.05),PTC124的作用弱于G418(P<0.05)。药物干预后,R1014X突变体HERG通道的功能得到不同程度的恢复,其最大尾电流密度由(4.75±0.88)pA/pF分别增加至(9.62±0.73)pA/pF(PTC124,P<0.05)和(22.57±2.26)pA/pF(G418,P<0.05),G418作用使通道的半激活电压得到恢复(P<0.05),差异均有统计学意义。结论:药物PTC124和G418能不同程度恢复HERG基因无义突变的结构和功能性表达,其恢复HERG基因的蛋白表达呈剂量依赖性变化。Objective： To study the pharmaceutical effects of PTC124 and G418 at different doses on rescuing the nonsense mutation of HERG gene channels with its mechanism. Methods： The wide type （WT） and R1014X mutant of HERG gene were transiently transfected into HEK293 cells for 24 hours, and the cells were cultured with pharmaceuticals of PTC124 and G418 at different concentrations respectively for 24 hours. HERG mRNA expression was examined by RT-PCR, HERG WT and R1014X mutant protein expressions were detected by Western blot analysis, HERG channel function was evaluated by patch clamping recorded current flow. Results： Compared with WT HERG, the R1014X mutant showed shorter protein zone in HERG channel protein, while the expressions of mRNA and protein were similar between WT and mutant, P〉0.05. Both PTC 124 and G418 may recover the entire protein expression of R1014X mutant in a dose-dependent model, P〈0.05, and the effect of PTC124 was weaken than that of G418, P〈0.05. With pharmaceutical intervention, the R1014X mutant of HERG channel function may recover at different degrees, the maximal tail current densities of RI014X channels increased from （4.75±0.88） pA/pF to （9.62±0.73） pA/pF by PTC124, P〈0.05 and to （22.57±2.26） pA/pF by G418, P〈0.05. The G418 recovered the semi-activation voltage of HERG channels, P〈0.05. Conclusion： Both pharmaceuticals of PTC124 and G418 could recover the structure and function of HERG channel in nonsense mutation at different degree; they recover HERG channel protein expression in a dose-dependent model.

关 键 词：HERG 无义突变 PTC124 遗传性心律失常 

分 类 号：R541[医药卫生—心血管疾病]