致LQT2的无义突变的HERG通道的表达和药物拯救研究

Expression and pharmacological correction of nonsense mutant HERG channels in LQT2

目的本研究旨在探讨氨基糖苷类抗生素庆大霉素在异源表达系统中对导致长QT综合征2型的无义突变的HERG通道的作用。方法采用聚合酶链反应法制备相关突变体——W927X、R863X和E698X,并克隆到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细胞在含庆大霉素(400μg/mL)的培养液中孵育24h。结果W927X能表达典型的HERG电流,尽管与野生型HERG相比,电流幅值明显下降;R863X和E698X仅表达与未转染细胞上类似的内生性电流,说明未能形成功能性的HERG通道。庆大霉素能增强W927X的功能性表达,使其最大尾电流密度由11.6±2.4pA/pF(n=8)增至19.5±2.7pA/pF(n=7,P<0.05)。通道激活动力学特征在野生型HERG、W927X和W927X加庆大霉素干预组均没有明显差异。然而,庆大霉素对R863X和E698X并无明显作用。结论氨基糖苷类抗生素能部分恢复无义突变的HERG通道的功能性表达,且对不同的突变体其作用效应不同。

Objective To investigate the effect of aminoglyeoside antibiotics gentamicin on nonsense mutant HERG channels leading to LQT2 in heterologous expression system. Methods The mutations were constructed by polymerase chain reaction（PCR） 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 HERG or three nonsense mutants. Pharmacological rescue was applied by culturing the HEK293 cells transfeeted for 24 hours in medium solution containing gentamicin at a concentration of 400μg/mL for 24 hours. Results W927X displayed wild type HERG-like currents, despite of a significant reduction in current amplitude compared to that in wild type channel; while R863X and E698X generated currents that were similar to endogenous currents, indicating their incapability of forming functional channels. Gentamicin increased the functional expression of W927X, its current density was enhanced from 11. 6± 2.4 pA/pF（ n = 8） to 19.5 ± 2.7 pA/pF （ n = 7, P 〈 0.05 ）. And the voltage dependence of activation was not much different among wild type HERG, W927X alone and W927X plus gentamicin treatment. However, gentamiein did not demonstrate any effect on R863X and E698X. Conclusions Aminoglycoside antibiotics may partially restore the functional expression of HERG channels produced by nonsense mutations, and the effect is various to different mutants.