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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。
OBJECTIVE: To construct hKCNQ1 / hKCNQ2 chimera and to study the electrophysiological characteristics of chimeric channels, which laid the foundation for the analysis of the regulation mechanism of hKcnq1 channel by PIP2. Methods: hKCNQ1 / hKCNQ2 chimera plasmid (Q1ctQ2-pCI, Q1ctQ2-pcDNA3.1) was constructed by over-lap PCR and transfected into HEK293 cells. Whole cell patch clamp technique was used to record the chimeric currents in HEK293 cells. Results: In the whole-cell patch-clamp recording mode, the current density I60 of HEK293 cells transfected with chimera Q1ctQ2-pCI was 5.2 ± 0.87pA / pf and the half activation voltage V0.5 was 23.3 ± 8.9mV (n = 6) ; The HEK293 cell current density I60 transfected with the chimeric Q2ctQ1-pcDNA3.1 was 21.7 ± 4.2pA / pf, and its half activation voltage V0.5 was -37.5 ± 3.6mV (n = 9). CONCLUSION: The hKCNQ1 / hKCNQ2 chimera was successfully constructed and the heterologous expression of chimeric recombinant plasmid and patch clamp recording were completed.