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- 2016
HEK293细胞中杂合状态下无义突变体L539fs/47对野生型HERG电流的作用
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Abstract:
摘要：目的 研究单独表达无功能的无义突变体L539fs/47在HEK293细胞中杂合状态下对野生型HERG电流的作用。方法 用脂质体转染法将野生型HERG及突变体HERG-L539fs/47分别转染HEK293细胞36h后，RT-PCR检测HERG mRNA表达，免疫荧光及免疫印迹检测HERG蛋白定位及表达量，全细胞膜片钳检测IHERG电流密度。结果 野生组HERG的mRNA表达量高于L539fs/47突变组(1.066±0.612 vs. 0.254±0.397, P<0.01)。免疫荧光检测发现野生型HERG蛋白多于突变体，野生型HERG蛋白主要分布在细胞膜上；而HERG突变体蛋白大部分滞留胞质。免疫印迹显示：不同于野生型HERG135ku及155ku 2个条带，突变体仅60ku 1个条带。全细胞膜片钳检测WT+MT组IHERG较WT组下降55.23%(22.03±2.62 vs. 49.20±2.31 pApF, P<0.01)。结论 HEK293细胞中杂合状态下截断突变体L539fs/47对野生型HERG电流的不完全负显性抑制作用。
ABSTRACT: Objective To study the effect of nonsense mutation of L539fs/47, when coexpressed with wild type human ether-a-go-go-related gene (HERG) current in HEK293 cells in the heterozygous state. Methods Thirty-six hours after transfection by liposome, wild-type HERG and its mutant HERG-L539fs/47 were transfected into HEK293 cells. HERG mRNA expressions were detected by real-time PCR; its protein localization and quantitative expression were detected by immunofluorescence and Western blotting, respectively. Current densities of HERG were detected by whole cell patch clamp. Results The expression of HERG mRNA in wild-type group was higher than that in L539fs/47 mutation group (1.066±0.612 vs. 0.254±0.397, P<0.01). Immunofluorescence results showed that HERG protein was more than that in mutant group. The wild-type HERG protein was mainly distributed in the cell membrane; but retention of most mutant HERG protein L539fs/47 cytoplasm was detected. Western blotting showed that, different from the wild type HERG 135ku- and 155ku-bands, only one 60ku- band was detected in mutant group. Through the whole cell patch clamp test, the current density of HERG in heterozygous group (WT+MT group) decreased by 55.23% compared with WT group (22.03±2.62 vs. 49.20±2.31 pApF, P<0.01). Conclusion In the heterozygous state, the truncation mutant L539fs/47 has the effect of incomplete dominant negative inhibition on wild type HERG current in HEK293 cells
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