细胞外钾对HERG基因无义突变L539fs/47表达的调控  

Effects of extracellular potassium on expression of HERG gene nonsense mutant L539fs/47

作　　者：吕颖[1] 张军波[2] 刘仲伟[1] 张爱峰[3] 潘军强[1] 王军奎[1] 潘硕[1] 韩稳琦[2] 孙超峰[2] 

机构地区：[1]陕西省人民医院心内一科,陕西西安710068 [2]西安交通大学第一附属医院心内科,陕西西安710061 [3]西安交通大学第二附属医院心内科,陕西西安710004

出　　处：《中国病理生理杂志》2016年第2期228-233,共6页Chinese Journal of Pathophysiology

基　　金：国家自然科学基金资助项目(No.30800473)

摘　　要：目的:研究持续细胞外钾对野生型HERG与其突变体L539fs/47蛋白表达的影响。方法:用脂质体转染法将野生型HERG(WT)及其突变体HERG-L539fs/47(MT)分别转染HEK293细胞36 h后,0.8、4.3及10mmol/L的钾干预6 h,流式细胞术检测HERG蛋白表达量;干预12 h用激光共聚焦成像和免疫印迹法进行HERG蛋白定位及表达量检测。结果:用激光共聚焦成像检测发现野生型HERG蛋白主要分布在细胞膜上;而HERG突变体L539fs/47蛋白大部分滞留胞浆;并且HERG蛋白的表达均随细胞外钾浓度升高而增多。流式细胞术结果显示细胞外高钾组荧光增多(P<0.01),WT组荧光阳性细胞百分比和荧光强度均高于MT组(P<0.05)。免疫印迹显示,不同于野生型HERG 135 kD及155 kD 2个条带,突变体仅60 kD 1个条带,3个条带均受细胞外钾影响(P<0.05)。结论:细胞外高钾增强细胞膜上野生型和突变型HERG通道蛋白的稳定性,持续低钾干预时间依赖性地减少HERG通道蛋白的表达。AIM： To study the effects of extracellular potassium on the protein expression of wild- type HERG and its mutant L539fs/47. METHODS： Wild-type HERG （WT） or its mutant HERG-L539fs/47 （MT） were transfected into HEK293 cells for 36 h. The cells were incubated in different media containing 0. 8, 4. 3 or 10 mmoL/L potassium. Af- ter 6 h of incubation, the protein expression of HERG was detected by flow cytometry. After 12 h of incubation, the locali- zation and quantity of the proteins were detected by laser confocal imaging and Western blot. RESULTS ： Different from the retention of mutant protein in cytoplasm, wild-type HERG protein was mainly distributed in the cell membrane. The 2 proteins both increased with the changes of extracellular potassium. Flow cytometry showed that the fluorescence in the 2 groups both increased with the changes of extracellular potassium （P 〈 0.01 ）. The fluorescence in WT group was signifi- cantly higher than that in MT group （ P 〈 0. 01 ）. Western blot showed that mutant HERG protein included only one 60 kD band, different from the 135 kD and 155 kD bands in wild-type HERG, which were affected by the changes of extracellular potassium （P 〈 0. 05 ）. CONCLUSION： The retention of HERG mutant L539fs/47 protein in the cytoplasm is more than wild-type HERG. Chronic high extracellular potassium keeps the stability of wild-type and mutant HERG proteins on the cell membrane. Chronic low potassium reduces the expression of HERG channel proteins in a time-dependent manner.

关 键 词：HERG基因 无义突变 细胞外钾 HEK293细胞 

分 类 号：R541.7[医药卫生—心血管疾病] R363[医药卫生—内科学]