Effects of WNK3 kinase on regulation of large-conductance calcium-activated potassium channels and its mechanisms
10.3760/cma.j.issn.1001-7097.2018.08.009
- VernacularTitle:WNK3激酶对肾脏大电导钙激活钾通道的调节作用及机制
- Author:
Xiaohan HU
1
;
Ye BI
;
Xinxin CHEN
;
Lihong CHEN
;
Yuhua ZHANG
;
Minguang CHEN
;
Hui CAI
;
Jieqiu ZHUANG
Author Information
1. 325027,温州医科大学附属第二医院育英儿童医院儿童肾内科
- Keywords:
Protein-serine-threonine kinases;
Large-conductance calcium-activated potassium channels;
Ubiquitination;
MAP kinase signaling system;
Lysosomal degradation pathway
- From:
Chinese Journal of Nephrology
2018;34(8):616-621
- CountryChina
- Language:Chinese
-
Abstract:
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) extracellular 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 Maxi K+WNK3 siRNA + Bar-A1 group (P ＞ 0.05).The expression of Maxi K protein in Maxi K+WNK3 siRNA+Baf-A1 group was higher than that in Maxi K+WNK3 siRNA group (P ＜ 0.01).Conclusions WNK3 kinase inhibits the lysosomal degradation pathway of Maxi K channel protein by reducing the ubiquitination of Maxi K channel,and promotes the expression of Maxi K channel protein in cells and on cell membrane.These effects may be achieved by suppressing MAPK ERK1/2 signal transduction pathway.
