CAJ | 학술논문

目的研究WNK4激酶对BK通道的调节作用及机制.方法将BK和WNK4野生型(WNK4-WT)或CD4(对照)质粒DNA共同转染进Cos-7细胞中,采用免疫染色-共聚焦激光显微镜、化学发光法、Western印迹法检测BK在细胞上的分布、细胞膜表面蛋白和总蛋白的表达；并使用质子泵抑制剂bafilomycin A1( Baf A1)阻断溶酶体降解检测BK蛋白表达水平的减少是否由于其蛋白降解增多所致.结果免疫染色-共聚焦激光显微镜发现,与对照组相比,WNK4-WT组BK在细胞膜表面的分布明显减少.化学发光法检测结果显示,对照组BK的细胞膜表面蛋白表达水平为299.9±18.6,WNK4-WT组中其细胞膜表面蛋白表达水平为148.4±13.7,比对照组显著下降(P＜0.01).Western印迹结果提示,WNK4-WT组BK的总蛋白表达水平比对照组明显减少.和对照组(100％)相比,WNK4-WT显著减少BK的总蛋白水平(42.3％±15.2％,P＜0.01),而Baf A1则逆转WNK4-WT对BK蛋白的抑制作用(82.2％±12.1％,P＜0.05).结论 WNK4激酶能同时抑制BK在Cos-7细胞膜表面蛋白和总蛋白的表达水平；WNK4激酶抑制BK通道蛋白的表达是通过增加其在溶酶体内的降解所致的.
목적 연구WNK4격매대BK통도적조절작용급궤제.방법 장BK화WNK4야생형(WNK4-WT)혹CD4(대조)질립DNA공동전염진Cos-7세포중,채용면역염색-공취초격광현미경、화학발광법、Western인적법검측BK재세포상적분포、세포막표면단백화총단백적표체；병사용질자빙억제제bafilomycin A1( Baf A1)조단용매체강해검측BK단백표체수평적감소시부유우기단백강해증다소치.결과 면역염색-공취초격광현미경발현,여대조조상비,WNK4-WT조BK재세포막표면적분포명현감소.화학발광법검측결과현시,대조조BK적세포막표면단백표체수평위299.9±18.6,WNK4-WT조중기세포막표면단백표체수평위148.4±13.7,비대조조현저하강(P＜0.01).Western인적결과제시,WNK4-WT조BK적총단백표체수평비대조조명현감소.화대조조(100％)상비,WNK4-WT현저감소BK적총단백수평(42.3％±15.2％,P＜0.01),이Baf A1칙역전WNK4-WT대BK단백적억제작용(82.2％±12.1％,P＜0.05).결론 WNK4격매능동시억제BK재Cos-7세포막표면단백화총단백적표체수평；WNK4격매억제BK통도단백적표체시통과증가기재용매체내적강해소치적.
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.