WNK3激酶对肾脏大电导钙激活钾通道的调节作用及机制  

Effects of WNK3 kinase on regulation of large- conductance calcium- activated potassium channels and its mechanisms

作　　者：胡小涵 毕叶 陈新新[2] 陈丽虹 张玉桦 陈敏广[1] 蔡晖[2] 庄捷秋[1] Hu Xiaohan;Bi Ye;Chen Xinxin;Chen Lihong;Zhang Yuhua;Chen Minguang;Cai Hui;Zhuang Jieqiu(Department of Pediatric Nephrology,the Second Affiliated Hospital ＆ Yuying Children＇s Hospital of Wenzhou Medical University,Wenzhou 325027,China)

机构地区：[1]温州医科大学附属第二医院育英儿童医院儿童肾内科,325027 [2]温州医科大学附属第二医院肾内科,325027

出　　处：《中华肾脏病杂志》2018年第8期616-621,共6页Chinese Journal of Nephrology

基　　金：国家自然科学基金（81170709）;浙江省自然科学基金（Y2080291）

摘　　要：目的探讨WNK3激酶对肾脏大电导钙激活钾通道（Maxi K通道）的调节作用及其机制。方法（1）在非洲绿猴肾成纤维细胞（Cos-7细胞）中转染0．5μg Maxi K质粒的同时分别转染0、0．6、1．2、1．8μg WNK3质粒，Western印迹检测细胞总蛋白中Maxi K通道的表达及丝裂原活化蛋白激酶（MAPK）细胞外信号调节激酶1／2（ERK1／2）的磷酸化水平。（2）Cos-7细胞分为转染2．5μg Maxi K质粒组（对照组）和转染2．5μg Maxi K质粒＋2．5μg WNK3质粒组（实验组），细胞表面生物素化方法检测细胞表面膜蛋白中Maxi K通道的表达，免疫沉淀和Westem印迹检测Maxi K通道蛋白泛素化水平。（3）用WNK 3siRNA沉默WNK3激酶基因，用质子泵抑制剂（Baf—A1）阻断溶酶体降解途径，Cos-7细胞分为Maxi K＋阴性对照siRNA组、Maxi K＋WNK3 siRNA组和Maxi K＋WNK3 siRNA＋Baf-A1组。Western印迹检测细胞中Maxi K通道蛋白的表达。结果（1）与0μg WNK3质粒组比较，转染0．6、1．2、1．8μg WNK3质粒组细胞总蛋白中Maxi K通道的表达均升高，MAPKERKl／2的磷酸化水平均降低（均P〈0．01），且呈剂量依赖性。（2）与对照组比较，同时转染WNK3和Maxi K的实验组细胞总蛋白和膜蛋白中Maxi K通道的表达均增加，Maxi K通道蛋白的泛素化水平降低（均P〈0．01）。（3）与Maxi K＋阴性对照siRNA组比较，Maxi K＋WNK 3siRNA组Maxi K蛋白表达降低（P〈0．01），Maxi K＋WNK 3siRNA＋Bar-A1组的Maxi K蛋白表达无明显变化（P〉0．05）；与Maxi K＋WNK 3siRNA组比较，Maxi K＋WNK 3siRNA＋Baf—A1组的Maxi K蛋白表达升高（P〈0．01）。结论WNK3激酶通过减少Maxi K通道蛋白的泛素化来抑制Maxi K通道的溶酶体降解途径，从而促进Maxi K通道在细胞内和细胞膜上的表达量。其机制可能与MAPK ERK1／2转导通路有关。Objective To investigate the effects of WNK3 kinase on the regulation of large- conductance calcium-activated potassium channels （Maxi K channels） on African green monkey kidney fibroblast-like cells （Cos-7 cells） and its mechanisms. Methods （1） Cos-7 cells were transfected with 0, 0.6, 1.2, 1.8 μg WNK3 plasmid＋0.5 μg Maxi K plasmid. The total protein expression of Maxi K channel and the phosphorylation of mitogen- activated protein kinase （MAPK） extraeellular regulated kinase-1 and-2 （ERK1/2） were detected by Western blotting. （2） Cos-7 cells were divided into the control group （2.5 μg Maxi K plasmid） and the experimental group （2.5 μg WNK3 plasmid＋2.5 μg Maxi K plasmid）. Cell surface biotinylation was used to investigate the cell surface protein expression of Maxi K channel in Cos-7 cells. Immunoprecipitation and Western blotting were used to detect the ubiquitination of Maxi K channel protein. （3） WNK3 kinase was knocked down by WNK3 siRNA. The lysosomal degradation pathway was blocked by the proton pump inhibitor （Baf-A1）. Cos-7 cells were divided into Maxi K＋negative control siRNA group, Maxi K＋WNK3 siRNA group and Maxi K＋WNK3 siRNA＋Baf-A1 group. The protein expression of Maxi K channel protein was detected by Western blotting. Results （1） Compared with those in 0 μg WNK3 plasmid groups, in 0.6, 1.2, 1.8 μg WNK3 plasmid groups the total protein expression of the Maxi K channel increased and the phosphorylation level of MAPK ERK1/2 reduced on a dose- dependent manner （all P 〈 0.01）. （2） Compared with those in the control group, the total protein expression and cell surface membrane protein expression of the Maxi K channel increased in the experimental group （P 〈 0.01）, while the ubiquitination of the Maxi K channel protein reduced （P 〈 0.01）. （3） Compared with the Maxi K＋ negative control siRNA group, the expression of Maxi K protein reduced in the Maxi K＋WNK3 siRNA group （P〈 0.01）, but did not change in the Max

关 键 词：蛋白质丝氨酸苏氨酸激酶 大电导钙激活钾通道 泛素化 MAP激酶信号系统 溶酶体降解途径 

分 类 号：R692[医药卫生—泌尿科学]