高糖对肾小球系膜细胞BK_(Ca)-Orai1信号复合物表达与功能的影响

Expression and function of Orai1-BK_(Ca) complex in high glucose treatment rat glomerular mesangial cells

目的 探讨在肾小球系膜细胞(GMCs)中,钙库操纵钙内流(SOCE)的主要通道蛋白Orai1与其下游信号分子大电导钙激活钾通道(BK_Ca)的物理性与功能性相互作用,及其在高糖环境下的改变。方法 采用免疫共沉淀法(co-IP)检测Orai1与BKCa相互作用;利用钙成像检测SOCE引起的细胞内游离Ca^2+浓度变化;利用DiBAC_4(3)作为荧光指示剂,检测SOCE引起细胞膜电位的改变;免疫蛋白电泳法检测Orai1与BK_Ca的表达水平。结果 在GMCs,SOCE的主要通道蛋白Orai1可与其下游信号分子BK_Ca通道蛋白形成信号复合物,引起GMCs膜电位超极化。高糖培养可以明显上调Orai1与BK_Ca蛋白的表达水平,并增强SOCE引起的膜电位超极化水平。结论 GMCs的Orai1可与BK_Ca形成信号复合物,参与调节细胞膜超极化过程,并可能参与高糖培养对细胞膜超极化的影响。

Aim To investigate whether Orai1 and BK（Ca） form a physical and functional signal complex in glomerular mesangial cells（GMCs） and reveal the role of high glucose treatment on Orai1-BK（Ca） complex and SOCE-induced hyperpolarization. Methods The interaction of Orai1 and BK（Ca） was evaluated by co-immunoprecipitation（co-IP）. Western blot method was used to detect the expression levels of Orai1 and BK（Ca）.SOCE was activated by Ca（2＋） depletion evoked by TG in normal glucose and high glucose. The Di BAC4（3）was employed as fluorescence indicator to measure the potential change of membrane.. Results The co-IP experiment results showed that Orai1 interacted with BK（Ca） in GMCs. SOCE induced the hyperpolarization of GMCs. SOCE and SOCE-induced membrane hyperpolorizaiton were enhanced by high glucose treatment for three days. In addition,the expression levels of Orai1 and BK（Ca）were enhanced significantly in the high glucose-cultured cells. Conclusions Orai1 can form a signaling complex with BK（Ca）,which participates in the regulation of hyperpolarization in GMCs and may be involved in the hyperpolarization in high glucose cultured GMCs.