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

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+Baf-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.