【摘要】 目的:构建hKCNQ1/hKCNQ2嵌合体,研究嵌合体通道的电生理学特征,为分析PIP2对hKcnq1通道的调节机制打下基础。方法:利用over-lap PCR方法构建hKCNQ1/hKCNQ2嵌合体质粒(Q1ctQ2-pCI、Q1ctQ2-pcDNA3.1),转染HEK293细胞,采用全细胞膜片钳技术记录HEK293细胞上嵌合体电流。结果:在全细胞膜片钳记录模式下,转染了嵌合体Q1ctQ2-pCI的HEK293细胞电流密度I60为5.2±0.87pA/pf,其半数激活电压V0.5为23.3±8.9mV(n=6);转染了嵌合体Q2ctQ1-pcDNA3.1的HEK293细胞电流密度I60为21.7±4.2pA/pf,其半数激活电压V0.5为-37.5±3.6mV(n=9)。结论:成功构建了hKCNQ1/hKCNQ2嵌合体并完成了嵌合体重组质粒的异源性表达和膜片钳记录。更多还原

【Abstract】 Objective:To construct and characterize hKCNQ1/hKCNQ2 Chimeras.Methods:Chimeras(Q1ctQ2、Q1ctQ2) were constructed by over-lap PCR.The plasmids were transfected into HEK293 cells and the current of chimeras was recorded with whole-cell mode.Results:When membrane potential holded at -80 mV and stepped from -80 to 60 mV for 4 seconds,the average current density at 60 mV(I60) and half-maximal activation(V0.5) of HEK293 cells transfected with Q1ctQ2 was 5.2±0.87 pA/pf and 23.3±8.9 mV(n=6),respectively;The average current density at 60 mV(I60) and half-maximal activation(V0.5) of HEK293 cells transfected with Q2ctQ1 were 21.7±4.2 pA/pf and -37.5 ±3.6 mV(n=9).Conclusion:The hKCNQ1/hKCNQ2 Chimeras,Q1ctQ2 and Q2ctQ1 were successfully constructed and their electrophysiologic characters were analyzed in a heterologous expression system.更多还原