State Key Laboratory of Cardiovascular Disease, Physiology and Pathophysiology Laboratory, Fuwai Hospital, National Center for Cardiovascular Diseases, Chinese Academy of Medical Sciences and Peking Union Medical College, Beijing 100037, P.R. China.
Int J Mol Med. 2014 Mar;33(3):729-35. doi: 10.3892/ijmm.2013.1601. Epub 2013 Dec 23.
Aminoglycosides promote the readthrough of premature stop codons introduced by nonsense mutations to produce full-length proteins in genetic disease models. The read-through effects of different aminoglycosides and PTC124 on HERG gene have yet to be adequately elucidated. The wild-type (WT) or mutant genes were transiently transfected in HEK293 cells. The read-through effect was examined by adding drugs into culture medium for 24 h. Western blot analysis and patch clamping were performed to evaluate the expression and function of the genes. The mRNA levels were determined using qPCR. The results showed that G418 and PTC124 significantly increased the protein expression of R1014X mutant in a dose-dependent manner and produced a full-length protein. The maximal protein levels after G418, gentamicin or PTC124 treatment were 39.1±2.4, 18.6±0.3 or 10.3±1.0%, respectively, of the WT level. Tobramycin did not exhibit a read-through effect. The mRNA levels, however, did not differ between WT and mutant gene. The tail current densities of R1014X channels at 40 mV were 22.57±2.26 pA/pF for G418, 16.21±1.49 pA/pF for gentamicin and 9.62±0.73 pA/pF for PTC124. The leftward shift of the activation curve was corrected only by G418 and gentamicin. The read-through effects of W927X, R863X and E698X revealed that as the mutation site approached the N-terminal, the rescue efficiency was decreased. The above results suggest that aminoglycosides and PTC124 induced different effects on rescue nonsense mutations of the HERG gene. The mutation site was a significant factor in determining the pharmacological rescue efficiency.
氨基糖苷类抗生素可促进无义突变引起的提前终止密码子通读，从而在遗传疾病模型中产生全长蛋白。不同的氨基糖苷类抗生素和 PTC124 对 HERG 基因的通读效果尚未得到充分阐明。将野生型（WT）或突变基因瞬时转染至 HEK293 细胞中。通过在培养物中添加药物 24 小时来检测通读效果。通过 Western blot 分析和膜片钳技术评估基因的表达和功能。使用 qPCR 测定 mRNA 水平。结果表明，G418 和 PTC124 以剂量依赖性方式显著增加 R1014X 突变体的蛋白表达，并产生全长蛋白。G418、庆大霉素或 PTC124 处理后的最大蛋白水平分别为 WT 水平的 39.1±2.4%、18.6±0.3%和 10.3±1.0%。妥布霉素则没有通读效果。然而，WT 和突变基因之间的 mRNA 水平没有差异。在 40 mV 时，R1014X 通道的尾电流密度分别为 G418 组 22.57±2.26 pA/pF、庆大霉素组 16.21±1.49 pA/pF 和 PTC124 组 9.62±0.73 pA/pF。只有 G418 和庆大霉素能纠正激活曲线的左移。W927X、R863X 和 E698X 的通读效果表明，随着突变部位接近 N 端，挽救效率降低。上述结果表明，氨基糖苷类抗生素和 PTC124 对 HERG 基因无义突变的挽救效果不同。突变部位是决定药物挽救效率的重要因素。
