Caveolin 3蛋白与心脏Kv1.5钾通道的相互作用及鉴定

Interaction of caveolin 3 protein with cardiac Kv1.5 potassium channel

目的探索caveolin 3蛋白与Kv1.5钾通道是否存在相互作用,并进一步明确caveolin3对心脏Kv1.5钾通道功能的调控作用。方法 (1)将携带caveolin 3的N端片段的pGBKT7载体转染Y187,与含人类心脏cDNA文库进行酵母双杂交,筛选与caveolin 3存在相互作用的心肌蛋白;(2)免疫荧光细胞化学分析:pEGFP-N3-KCNA5和质粒pDsRed2-N1-cav3共转染HEK293细胞,应用荧光显微镜观察caveolin 3蛋白和KCNA5蛋白的细胞亚定位情况;(3)细胞电生理检查:应用膜片钳技术记录表达caveolin 3蛋白时,观察Kv1.5钾通道电流幅度和电流密度比的变化。结果 (1)经过严格筛选,人类心脏cDNA文库中发现Kv1.5钾通道与caveolin 3-NT存在相互作用。(2)荧光免疫细胞化学分析发现在哺乳细胞中Kv1.5与caveolin 3存在细胞共定位,并主要在细胞膜上表达。(3)膜片钳技术检测发现,在电压分别为-10、0、10、20、30、40、50、60 mV时,caveolin 3蛋白的表达明显减小了Kv1.5钾通道电流幅度和电流密度比,具有统计学意义(P<0.05)。结论 Caveolin 3蛋白对心脏Kv1.5钾通道的功能具有负性调控作用。

Objectives To confirm the interaction and the regulation between caveolin 3 and cardiac Kv1.5 potassium channel.Methods(1)The AH109 transformed with the pGBKT7-caveolin 3-NT mated with Y187 pre-transformed with human heart cDNA library to screened out the caveolin 3 interacting protein.(2)Immunofluorescence cytochemistry assay:Using immune fluorescent confocal microscope to confirm the co-localization of the caveolin 3 and KCNA5.(3)Cell electrophysiological examination:Patch-clamp technique was used to study the effection of the caveolin3 protein on the Kv1.5 potassium channel.Results(1)After screening,Kv1.5 potassium channels were identified as caveolin 3 interacting proteins.(2)The co-localization of the caveolin 3 and KCNA5 occurred mostly in the membrane surface compartment,where the majority of KCNA5 presented.(3)A significant decrease in Kv1.5 current amplitude and density was observed in HEK293 cells which co-transfected with pEGFP-N3-KCNA5 plasmid and pDsRed2-N1-caveolin 3 plasmid at a test potential of about-10,0,10,20,30,40,50,60 mV(P<0.05).Conclusions Caveolin 3 negatively regulates the function of cardiac Kv1.5 channel.