Rodriguez-Menchaca Aldo A, Ferrer Tania, Navarro-Polanco Ricardo A, Sanchez-Chapula Jose A, Moreno-Galindo Eloy G
Departamento de Fisiología y Biofísica, Facultad de Medicina, Universidad Autónoma de San Luis Potosí, San Luis Potosí, Mexico.
Centro Universitario de Investigaciones Biomédicas, Universidad de Colima, Colima, Col., Mexico.
J Pharmacol Toxicol Methods. 2014 May-Jun;69(3):237-44. doi: 10.1016/j.vascn.2013.12.007. Epub 2014 Jan 9.
Voltage- and state-dependent blocks are important mechanisms by which drugs affect voltage-gated ionic channels. However, spontaneous (i.e. drug-free) time-dependent changes in the activation and inactivation of hERG and Na(+) channels have been reported when using conventional whole-cell patch-clamp in HEK-293 cells.
hERG channels were heterologously expressed in HEK-293 cells and in Xenopus laevis oocytes. hERG current (IhERG) was recorded using both conventional and perforated whole-cell patch-clamp (HEK-293 cells), and two microelectrode voltage-clamp (Xenopus oocytes) in drug-free solution, and in the presence of the drug trazodone.
In conventional whole-cell setup, we observed a spontaneous time-dependent hyperpolarizing shift in the activation curve of IhERG. Conversely, in perforated patch whole-cell (HEK-293 cells) or in two microelectrode voltage-clamp (Xenopus oocytes) activation curves of IhERG were very stable for periods ~50min. Voltage-dependent inactivation of IhERG was not significantly altered in the three voltage clamp configurations tested. When comparing voltage- and state-dependent effects of the antidepressant drug trazodone on IhERG, similar changes between the three voltage clamp configurations were observed as under drug-free conditions.
The comparative analysis performed in this work showed that only under conventional whole-cell voltage-clamp conditions, a leftward shift in the activation curve of IhERG occurred, both in the presence and absence of drugs. These spontaneous time-dependent changes in the voltage activation gate of IhERG are a potential confounder in pharmacological studies on hERG channels expressed in HEK-293 cells.
电压依赖性和状态依赖性阻滞是药物影响电压门控离子通道的重要机制。然而，在HEK-293细胞中使用传统的全细胞膜片钳技术时，已报道人乙醚-a- go-相关基因（hERG）通道和钠通道的激活和失活存在自发的（即无药物的）时间依赖性变化。
hERG通道在HEK-293细胞和非洲爪蟾卵母细胞中进行异源表达。在无药物溶液以及存在药物曲唑酮的情况下，使用传统全细胞膜片钳和穿孔全细胞膜片钳（用于HEK-293细胞）以及双电极电压钳（用于非洲爪蟾卵母细胞）记录hERG电流（IhERG）。
在传统全细胞模式下，我们观察到IhERG激活曲线存在自发的时间依赖性超极化偏移。相反，在穿孔膜片全细胞模式（用于HEK-293细胞）或双电极电压钳模式（用于非洲爪蟾卵母细胞）下，IhERG的激活曲线在约50分钟内非常稳定。在所测试的三种电压钳配置中，IhERG的电压依赖性失活没有显著改变。在比较抗抑郁药曲唑酮对IhERG的电压依赖性和状态依赖性作用时，观察到三种电压钳配置之间的变化与无药物条件下相似。
本研究进行的比较分析表明，只有在传统全细胞电压钳条件下且无论有无药物时，IhERG的激活曲线才会向左偏移。IhERG电压激活门的这些自发时间依赖性变化是对HEK-293细胞中表达的hERG通道进行药理研究时的一个潜在干扰因素。
