ABSTRACT
Objective To investigate the mechanism underlying the WNK4 kinasemediated inhibitory effect on BK channel. Methods Cos-7 cells were cotransfected with BK in combination with either CD4 (control group) or wild type WNK4 (WNK4-WT).Immunostaining and confocal microscopy,chemiluminescence,Western blotting analysis were then employed to determine the BK localization in cells,BK surface expression and total protein level,respectively.To further investigate whether the reduction of BK protein expression is due to an increase in degradation through a lysosomal pathway,BK protein level was determined after treated with bafilomycin A1(Baf A1),a proton pump inhibitor affecting lysosomal degradation. Results Immunostaining and confocal microscopic study showed that BK was localized both in plasma membrane and cytosol in the control group.After cells transfected with WNK4-WT,BK expression was markedly reduced.Chemiluminescent assay found that BK surface expression level was 299.9±18.6 in the control group,whereas it was significantly reduced (148.4±13.7,P＜0.01) in the WNK4-WT group.Western blotting analysis showed that total BK protein level was markedly reduced in the presence of WNK4-WT compared to the control group.WNK4-WT was shown to significantly reduce the BK total protein level (42.3％±15.2％) compared to the control group (100％) (P＜0.01).When the cells was treated with Bafilomycin A1 (Baf A1,0.5 μmol/L),WNK4-mediated reduction in BK protein was reversed (82.2％±12.1％,P＜0.05). Conclusions WNK4 inhibits total and surface protein expression of BK in Cos-7 cells whick is likely due to an increase in BK degradation through a lysosomal pathway.