Properties of HERG channels stably expressed in HEK 293 cells studied at physiological temperature
Properties of HERG channels stably expressed in HEK 293 cells studied at physiological temperature
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DOI：
10.1016/s0006-3495(98)77782-3
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发表时间：
1998-01-01
影响因子：
3.4
通讯作者：
January, CT
中科院分区：
文献类型：
--
作者：
Zhou, ZF;Gong, QM;January, CT
我们已经建立了稳定转染的 HEK 293 细胞系，该细胞系表达高水平的功能性人醚 - go-go 相关基因 (HERG) 通道。我们使用这些细胞来研究HERG蛋白的生化特性，并研究35℃下HERO通道电流的电生理和药理学特性。HERG转染的细胞表达4.0 kb的mRNA条带。蛋白质印迹分析显示两条蛋白质条带（155 和 135 kDa）略大于预测的分子量（127 kDa）。用 N-糖苷酶 F 处理将两条带转化为较小的分子量，表明两者都被糖基化，但水平不同。 HEG 电流在正电压至 -50 mV 时激活，通过去极化步骤至 -10 mV 达到最大电流，并且电流幅度在更正的电压下下降，类似于其他异源系统中表达的 HERG 通道电流。与 23 摄氏度相比，35 摄氏度时的电流密度增加了一倍多，达到最大值 53.4 +/- 6.5 pA/pF。激活、失活、失活恢复和失活动力学在 35°C 时快速，并且更接近于生理温度下快速激活延迟整流 K+ 电流 (I-Kr) 的报告值。 HERO 通道对 K+ 具有高度选择性。当我们使用动作电位钳技术时，HERG 电流在动作电位波形上升后不久就开始激活。 HERG 电流在复极期间增加，在心脏动作电位的第 2 相和第 3 相期间达到最大幅度。 HERG 在膜返回静息电位的整个过程中贡献电流，HERO 电流的失活可以参与第 4 相去极化，HERG 电流被低浓度的 E-4031 阻断（IC50 7.7 nM），该值接近报道的天然心肌细胞中 I-Kr 的值。我们的数据支持 HERG 编码 I-Kr 主要成分的假设，并表明在生理温度下 HERG 在大部分动作电位和复极后时期贡献电流。
We have established stably transfected HEK 293 cell lines expressing high levels of functional human ether-a go-go-related gene (HERG) channels. We used these cells to study biochemical characteristics of HERG protein, and to study electrophysiological and pharmacological properties of HERO channel current at 35 degrees C. HERG-transfected cells expressed an mRNA band at 4.0 kb. Western blot analysis showed two protein bands (155 and 135 kDa) slightly larger than the predicted molecular mass (127 kDa). Treatment with N-glycosidase F converted both bands to smaller molecular mass, suggesting that both are glycosylated, but at different levels. HERG current activated at voltages positive to -50 mV, maximum current was reached with depolarizing steps to -10 mV, and the current amplitude declined at more positive voltages, similar to HERG channel current expressed in other heterologous systems. Current density at 35 degrees C, compared with 23 degrees C, was increased by more than twofold to a maximum of 53.4 +/- 6.5 pA/pF. Activation, inactivation, recovery from inactivation, and deactivation kinetics were rapid at 35 degrees C, and more closely resemble values reported for the rapidly activating delayed rectifier K+ current (I-Kr) at physiological temperatures. HERO channels were highly selective for K+. When we used an action potential clamp technique, HERG current activation began shortly after the upstroke of the action potential waveform. HERG current increased during repolarization to reach a maximum amplitude during phases 2 and 3 of the cardiac action potential. HERG contributed current throughout the return of the membrane to the resting potential, and deactivation of HERO current could participate in phase 4 depolarization, HERG current was blocked by low concentrations of E-4031 (IC50 7.7 nM), a value close to that reported for I-Kr in native cardiac myocytes. Our data support the postulate that HERG encodes a major constituent of I-Kr and suggest that at physiological temperatures HERG contributes current throughout most of the action potential and into the postrepolarization period.