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本文旨在研究大蒜素(allitridum,All)对SCN5A-F1473S突变的HEK293细胞钠电流降低的逆转作用,为筛选治疗Brugada综合征的新药提供理论依据。采用瞬时转染的方法,将SCN5A-F1473S通道质粒转入HEK293细胞,采用细胞外灌流和共培养模式的方法将All急性和慢性给药,使其作用浓度为30μmol·L~(-1)。采用全细胞膜片钳技术在电压钳模式下记录电流和门控动力学,采用共聚焦显微镜技术和蛋白质免疫印迹技术检测通道蛋白在细胞膜表达,探讨All对SCN5A-F1473S峰钠电流降低的逆转作用。发现All 30μmol·L~(-1)组的HEK293细胞峰钠电流(269.8±16.6 p A/p F)显著增加(P<0.01),几乎接近对照组电流密度(298.2±17.5 p A/p F,P<0.01)。All可使通道稳态失活向更正的方向移动(V_(1/2,inact)恢复至-79.5±2.4 m V,P<0.01),导致失活减慢,并且使通道中间态失活减缓(延长至598.1±22.6 ms,P<0.01)。同时,All增加通道蛋白在细胞膜的分布和表达(与F1473S相比,P<0.01)。All使SCN5A-F1473S突变的细胞电流增加,其主要机制可能与此药物能减慢通道失活及改善突变通道迁移障碍有关。
The aim of this study was to investigate the reversal effect of allitridum (All) on the reduction of sodium currents in SCN5A-F1473S mutated HEK293 cells, and to provide a theoretical basis for screening new drugs for treating Brugada syndrome. The transient transfection method was used to transfer the SCN5A-F1473S channel plasmid into HEK293 cells. All the cells were treated acutely and chronically with the method of extracellular perfusion and co-culture to make the concentration of 30μmol·L -1. The whole cell patch clamp technique was used to record the currents and gating kinetics in voltage clamp mode. The confocal microscopy and Western blotting were used to detect the expression of the channel proteins in the cell membrane, and the reversal effect of All on the reduction of peak sodium currents of SCN5A-F1473S was also investigated. It was found that the peak sodium currents (269.8 ± 16.6 p A / p F) of HEK293 cells in all 30μmol·L -1 group were significantly increased (P <0.01), almost close to those of the control group (298.2 ± 17.5 p A / p F , P <0.01). All allowed the steady-state inactivation of the channel to move in a more positive direction (V_ (1/2, inact) to -79.5 ± 2.4 mV, P <0.01), resulting in a slower inactivation and a slower inactivation of the channel intermediate state (Extended to 598.1 ± 22.6 ms, P <0.01). At the same time, All increased the distribution and expression of channel proteins in the cell membrane (P <0.01 compared to F1473S). All the SCN5A-F1473S mutation in the cell current increased, the main mechanism may be related to this drug can slow down the channel inactivation and improve the migration of mutant channels.