中山大学学报（医学科学版）2026，Vol.47Issue(2)：251-258,8.DOI:10.11714/jsysu.med.YX20250191
慢性吗啡诱导HEK293细胞GIRK1和GIRK2膜表达下调
Chronic Morphine Induces Downregulation of GIRK1 and GIRK2 Membrane Expression in HEK293 Cells
摘要
Abstract
[Objective]To investigate the expression and distribution changes of G protein-gated inwardly rectifying potassium(GIRK)channel subunits GIRK1 and GIRK2 following morphine tolerance using human embryonic kidney 293(HEK293)cells.[Methods]HEK293 cells were transfected with the lentiviral vector pLV-CMV-GIRK1-T2A-GIRK2-P2A-MOR to establish a stable cell line overexpressing GIRK1,GIRK2,and the μ-opioid receptor(MOR).Morphine(1 μmol/L,24 h)was applied to these stable cells to construct a morphine-tolerant HEK293 cell model.Cellular immunofluorescence was used to examine the distribution and co-localization of MOR with GIRK1 and GIRK2.ELISA was performed to measure cAMP levels to confirm the establishment of the morphine-tolerant model.Cellular immunofluorescence and Western blot were employed to analyze the distribution and protein expression changes of GIRK1 and GIRK2 after morphine tolerance.A multimode microplate reader was used to measure fluorescence intensity for detecting changes in cellular membrane potential following morphine tolerance.[Results]In the stable HEK293 cells,immunohistochemistry showed that GIRK1 and GIRK2 were primarily expressed on the plasma membrane,with minimal presence in the cytoplasm.Both GIRK1 and GIRK2 co-localized with MOR and with each other.Compared with the control group,cAMP levels significantly decreased after 1 h of morphine treatment(1.42±0.07 vs.0.72±0.12,P=0.001 0),while they significantly increased after 24 h of treatment(0.72±0.12 vs.1.98±0.17,P=0.000 5).Fluorescence double staining revealed that after 24 h of morphine treatment,the morphine-tolerant group showed a significant increase in cytoplasmic GIRK1(13.76±7.67 vs.63.72±16.02,P＜0.000 1)and GIRK2(7.16±2.61 vs.32.92±7.67,P=0.002 9).Western blot analysis indicated that the expression of membrane proteins GIRK1(1.11±0.14 vs.0.85±0.01,P=0.004 5)and GIRK2(1.32±0.02 vs.0.86±0.08,P=0.000 1)was significantly downregulated after morphine tolerance.Membrane potential measurements showed that the hyperpolarization response in morphine-tolerant cells was significantly attenuated(-15.53±0.12)%vs.(-8.17±0.11)%,P＜0.000 1.[Conclusion]Chronic morphine treatment can induce downregulation of GIRK1 and GIRK2 expression on the plasma membrane in stable HEK293 cells.关键词
吗啡/耐受/人胚胎肾细胞293/G蛋白门控内向整流钾离子通道亚单位1/G蛋白门控内向整流钾离子通道亚单位2Key words
morphine/tolerance/HEK293 cells/GIRK1/GIRK2分类
医药卫生引用本文复制引用
刘冰柯,崔宇,韩雪,肖力..慢性吗啡诱导HEK293细胞GIRK1和GIRK2膜表达下调[J].中山大学学报（医学科学版）,2026,47(2):251-258,8.基金项目
国家自然科学基金(82101344,82501738) （82101344,82501738）
