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.
ABSTRACT
Objective To investigate the effects of protein expressions and the urea transport activity of aldosterone on urea transporter A1 (UT-A1) and urea transporter A3 (UT-A3) in HEK293 cells and Xenopus laevis oocytes.Methods (1) Western Blot was used to investigate the protein expressions of UT-A1 and UT-A3.(2) Cell surface biotinylation was used to investigate the protein expressions of UT-A1 and UT-A3 on the cell surface of Xenopus laevis oocytes.(3) 14C-urea transport experiment was conducted to investigate the transport activity of UT-A1 and UT-A3 in Xenopus laevis oocytes.Results (1) Compared with UT-A1 or UT-A3 high expression groups,the total protein levels of UT-A 1 and UT-A3 were all significantly reduced in aldosterone treatment groups (all P ＜ 0.01).(2) Compared with UT-A1 or UT-A3 high expression groups,the levels of protein expression on cell surface were all significantly reduced in aldosterone groups (all P ＜ 0.01).(3) Compared with UT-A1 or UT-A3 high expression groups,14C-urea transport experiment results showed that aldosterone treatment groups had significantly reduced the urea transporter activity of UT-A1 (1 min:94.32±9.044vs 40.68±4.274,P＜0.01,n=6;3 min:165.0±4.7 vs 80.3±0.6,P＜0.01,n=6),and UT-A3 (1 min:204.6± 3.1 vs 176.7± 9.1,P＜0.05,n=6;3 min:371.4 ± 14.9 vs 318.8 ± 12.0,P＜0.05,n=6).Conclusion Aldosterone can directly down-regulate the protein expressions of UT-A1 and UT-A3 in both total protein and cell surface level,which reduces their urea transport activity.