先天性长QT综合征HERG基因L539fs／47及A561V突变的功能研究

Function expression of congenital long QT syndrome related HERG mutation L539fs/47 and A561V

目的探讨HERG基因L539fs／47突变及A561V突变的功能。方法采用交叠聚合酶链式反应（PCR）及克隆载体快速PCR构建突变体，用限制性内切酶法将突变体克隆到真核表达载体pcDNA3中，用Superfect转染试剂将野生型及突变型HERG质粒与荧光载体pRK5-GFP共转染至HEK293细胞，用免疫荧光化学法及蛋白免疫印记法检测蛋白质表达，用全细胞膜片钳法检测HERG通道的电流表达。结果构建的突变体经DNA直接测序示HERG基因L539fs／47缺失突变及A561V点突变，L539fs／47突变体蛋白质位于细胞膜上，A561V突变体蛋白质位于细胞膜上及细胞浆中，L539fs／47突变体蛋白质条带小于80kDa，A561V突变蛋白仅有135kDa一条蛋白条带，野生型通道记录到尾电流而L539fs／47突变及A561V突变型通道均未检测到电流表达。结论成功构建并表达了HERG基因L539fs／47突变及A561V突变的功能，L539fs／47突变通过电压门控异常机制抑制HERG电流，而A561V突变通过滞留降解及膜通道功能异常机制抑制HERG电流。

Objective To investigate the function expression of HERG mutation L539fs/47 and A561V. Methods Mutation L539fs/47 was constructed by over-lap PCR and A561V by quick site-directed mutagenesis PCR. The mutations were cloned into eukaryotic expressive vector pcDNA3 by restriction enzymes. The wild-type HERG, HERG mutation L539fs/47 and A561V were cotransfected with pRKS-GFP into HEK293 cells by Superfect transfection regents,respectively. The protein was measured by immunofluorescence method and Western-blot. The electrophysiological experiments was carried out by whole cell patch-clamp. Results Direct sequencing analyses revealed a mutation of 19 bp bases deletion on codon 539 and a mutation of C to T transition on codon 561. Mutation were correctly combined to eukaryotic expressive vector pcDNA3 and expressed in HEK293 cells. The protein of mutation A561V was expressed in cytoplasm and cellular membrane while L539fs/47 only on cellular membrane. Below 80 kDa protein segment bands were detected for L539fs/47 and only 135kDa protein band for A561V. In electrophysiological experiments, significant HERG tail-current was recorded in wild type HERG channel but no current was found in these two mutational channels. Conclusions The protocol can be used successfully to construct and express HERG mutations. The results disclose that mutation L539fs/47 suppress HERG current by dysfunction of voltage-gate controlled channel. Mutation A561V blocks HERG current by protein retention and degrade in cytoplasm and malfunction of cellular membrane channel.