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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 investigate the effects of continuous extracellular potassium on the expression of wild-type HERG and its mutant L539fs / 47 protein. METHODS: HEK293 cells were transfected with wild-type HERG (WT) and its mutant HERG-L539fs / 47 (MT) by lipofectamine for 36 h, and treated with 0.8, 4.3 and 10 mmol / The expression of HERG protein was detected by flow cytometry. The expression of HERG protein was detected by confocal laser scanning microscopy and immunoblotting 12 h after intervention. Results: Confocal laser scanning confocal microscope showed that the wild-type HERG protein mainly distributed on the cell membrane, while the HERG mutant L539fs / 47 protein mostly remained in the cytoplasm. The expression of HERG protein increased with the increase of extracellular potassium concentration. The results of flow cytometry showed that the fluorescence of extracellular potassium increased (P <0.01), the percentage of fluorescent positive cells and the fluorescence intensity of WT group were higher than that of MT group (P <0.05). Immunoblotting showed that there were only 1 band of 60 kD mutant in the mutant with 135 kD of wild-type HERG and 155 kD of wild-type. The three bands were affected by extracellular potassium (P <0.05). CONCLUSION: Extracellular high potassium enhances the stability of wild-type and mutant HERG channel proteins in the plasma membrane. Continuous low potassium intervention reduces the expression of HERG channel protein in a time-dependent manner.