摘要
目的鉴定HERG钾通道的相互作用蛋白,并进一步研究该相互作用蛋白对HERG钾通道的功能调控。方法 (1)应用酵母双杂交技术,构建含有HERG氨基末端的诱饵载体,将转染有诱饵载体的酵母菌AH109与预转染有人类cDNA文库的Y187酵母菌进行双杂交,初步筛选出HERG的相互作用蛋白;(2)应用免疫共沉淀技术进一步验证酵母双杂交所筛选蛋白与HERG之间的相互作用;(3)GSTpull-down分析:应用GST-HERGT-NT融合蛋白和谷光苷肽-琼脂糖4B小球从大鼠心肌裂解物中沉淀蛋白质,应用抗PTPN12抗体对沉淀物进行WesternBlot分析;(4)免疫荧光组织化学分析:应用抗PTPN12和抗HERG的抗体和荧光标记二抗显示PTPN12及HERG的亚细胞定位,应用激光共聚焦显微镜观察。结果 (1)酵母双杂交筛选发现蛋白酪氨酸磷酸酶非受体型12(Proteintyrosinephosphatasenonreceptortype12,PTPN12)与HERG氨基末端存在相互作用;(2)免疫共沉淀分析发现抗HERG的抗体能够沉淀HERG和PTPN12复合物;(3)GSTpull-down分析发现GST-HERG-NT能够将PTPN12沉淀,而GST蛋白则不能沉淀PTPN12;(4)免疫荧光组织化学分析发现PTPN12和HERG两个蛋白共定位的地方主要出现在细胞膜。结论 PTPN12与HERG氨基末端相互作用,这一发现将有助于更加透彻地理解HERG通道特性多样性的分子基础和LQTS的发病机理。
Objective To identify the cellular proteins which interact with HERG channel and to study the effects of these protein interaction on the HERG channel function. Methods： （1） Yeast two-hybrid screen. Construct the bait plasmid pGBKT7-herg-NT. The AH109 transformed with pGBKT7-herg-NT was mated with a pre-transformed human heart cDNA library and then the mating mixture was grown on the selected medium. The prey plasmids were isolated from the putative positive colonies, and then were sequenced. （2） To further confirm the interaction, the Co-immunoprecipitation was performed by using specifi c antibody. （3） GST pull-down assay. To study the PTPN12-HERG interaction, the GST-HERG- NT fusion protein was expressed and purifi ed, then the proteins pulled down by the GST-HERG-NT was Western blot analyzed using anti-PTPN12 antibody. （4） Immuno？ uorescence analysis. To study the co-localization between PTPN12 and HERG, the anti-PTPN12 antibody and anti-HERG antibody were used to probe the subcellular localization of these two proteins. Results： （1） In the yeast two-hybrid screening using the HERG-NT as the bait, Protein tyrosine phosphatase nonreceptor type 12 （PTPN12） was identifi ed as the potential HERG partner. （2） Co-immunoprecipitation. The anti-HERG antibody precipitated the PTPN12 and the HERG complex from the rat heart lysates. （3） GST pull-down assay. The GST-HERG-NT fusion protein, but not the GST protein, pulled the PTPN12 down from the rat heart lysates. （4） Immunofluorescence analysis. The co-localization of the PTPN12 and HERG occurred mostly in the membrane surface compartment, where the majority of HERG present. Conclusions： PTPN12 interacts with HERG potassium channel in cardiac cell. This novel fi nding may help to further understand the molecular basis of HERG channel diversity and arrhythmogenesis in the long-QT syndrome.
出处
《中国分子心脏病学杂志》
CAS
2010年第3期147-151,共5页
Molecular Cardiology of China
基金
国家自然科学基金
项目批准号:30600254
广东省自然科学基金
项目批准号:9151008901000157
首批中国博士后科学基金特别资助
批准号:200801246
