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目的:研究钾离子通道β亚单位KCNE4对KCNQ1及HERG通道电流的影响。方法:构建离子通道细胞模型,单独表达基因KCNQ1、HERG,以及联合表达基因KCNQ1+KCNE4、HERG+KCNE4。采用全细胞膜片钳方法记录通道电流曲线,比较组间电流的大小及通道的动力学特征。采用免疫荧光细胞定位方法,检测KCNE4对通道蛋白表达的影响。结果:KCNE4与KCNQ1共表达显著减低KCNQ1通道电流。单独表达KCNQ1通道的细胞,膜电位+60 mV时,平均电流密度为(24±2.9)pA/pF,共表达KCNQ1+KCNE4时,细胞电流密度降至(7.3±1.1)pA/pF。与KCNQ1电流相比,+40 mV时KCNQ1/KCNE4激活时间常数的快成分(τfast)增加,但慢成分(τslow)减慢,KC-NQ1/KCNE4无尾电流产生,表明KCNE4参与了KCNQ1通道动力学的调整。单独表达KCNE4亚单位的细胞没有产生任何电流。当KCNE4与HERG共表达时,电流的大小、电流电压关系曲线及通道稳态激活曲线都与HERG通道无明显差异,电流密度为(26.7±3.9)pA/pF,半数激活电压(V1/2)为(-3.10±0.68)mV,斜率因子为8.89±0.33。从通道的失活时间常数以及失活后恢复时间常数与测试电压的关系曲线可见,KCNE4对HERG通道的动力学特征均无影响。免疫荧光染色结果显示,KCNE4没有影响KCNQ1蛋白的表达。结论:KCNE4亚单位可显著改变KCNQ1通道的电压敏感性,表现出抑制作用,KCNE4对KCNQ1蛋白在细胞膜上的表达及HERG通道电流均无影响。
Objective: To investigate the effect of potassium channel β subunit KCNE4 on KCNQ1 and HERG channel currents. Methods: The ion channel cell model was constructed and the genes KCNQ1 and HERG were separately expressed and the genes of KCNQ1 + KCNE4 and HERG + KCNE4 were co-expressed. The whole cell patch clamp method was used to record the channel current curve, comparing the current size and the channel dynamics. Immunofluorescence cell localization method was used to detect the effect of KCNE4 on the channel protein expression. Results: Co-expression of KCNE4 with KCNQ1 significantly reduced KCNQ1 channel currents. The cells with KCNQ1 channel alone expressed an average current density of (24 ± 2.9) pA / pF at membrane potential of +60 mV and reduced the cell current density to (7.3 ± 1.1) pA / pF when KCNQ1 + KCNE4 was co-expressed. The fast component (τfast) of KCNQ1 / KCNE4 activation time constant increased at +40 mV compared to KCNQ1 current, but the slow component (τslow) slowed down and KC-NQ1 / KCNE4 produced no tail current, indicating that KCNE4 is involved in KCNQ1 channel motility Academic adjustment. Cells expressing KCNE4 subunit alone did not produce any current. When KCNE4 and HERG were co-expressed, the current density, current-voltage curve and channel steady-state activation curve had no significant difference with HERG channel. The current density was (26.7 ± 3.9) pA / pF, half activation voltage (V1 / 2) (-3.10 ± 0.68) mV, the slope factor is 8.89 ± 0.33. KCNE4 has no effect on the kinetic characteristics of HERG channel, as shown in the relationship between the inactivation time constant of the channel and the recovery time constant after inactivation and the test voltage. Immunofluorescence staining showed that KCNE4 did not affect the expression of KCNQ1 protein. Conclusion: KCNE4 subunit can significantly change the voltage sensitivity of KCNQ1 channel, showing inhibition, KCNE4 on KCNQ1 protein expression in the cell membrane and HERG channel current had no effect.