Characterization of the Rapidly Activating Delayed Rectifier Potassium Current, I Kr, in HL-1 Mouse Atrial Myocytes
Characterization of the Rapidly Activating Delayed Rectifier Potassium Current, I Kr, in HL-1 Mouse Atrial Myocytes
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DOI：
10.1007/s00232-010-9257-2
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发表时间：
2010-06-01
影响因子：
2.4
通讯作者：
Matsuura, Hiroshi
中科院分区：
文献类型：
--
作者：
Toyoda, Futoshi;Ding, Wei-Guang;Matsuura, Hiroshi
HL-1是成年小鼠心肌细胞系，可在体外反复传代而不丧失分化表型。本研究旨在利用全细胞膜片钳技术研究HL-1细胞内源性表达的快速激活延迟整流钾电流I（Kr）。在尼索地平的存在下，从-50 mV的保持电位施加去极化电压阶跃诱发时间依赖性外向电流，然后在返回到保持电位时缓慢衰减外向尾电流。电流的幅度随着去极化增加至0 mV，但随后随着进一步去极化逐渐降低。时间依赖性外向电流以及尾电流对E-4031和多非利特的阻断高度敏感（IC 50分别为21.1和15.1 nM），并且几乎完全被每种药物的微摩尔浓度所消除，这表明HL-1细胞中的大部分外向电流可归因于I（Kr）。从HL-1细胞中获得的I（Kr）的大小（18.1 +/- A 1.5 pA pF（-1））足以可靠地测量各种门控参数。RT-PCR和Western blot分析揭示了小鼠ether-a-go-go相关基因（mERG 1）的可变剪接形式、全长mERG 1a和N端截短mERG 1b亚型的表达。用小干扰RNA（siRNA）敲低mERG 1转录物显著降低了I（Kr）幅度，证实了HL-1细胞中mERG 1和I（Kr）的分子联系。这些结果表明，HL-1细胞具有I（Kr）的性质与那些在天然心脏I（Kr），并提供了一个实验模型，适合于研究I（Kr）通道。
HL-1 is the adult murine cardiac cell line that can be passaged repeatedly in vitro without losing differentiated phenotype. The present study was designed to characterize the rapidly activating delayed rectifier potassium current, I (Kr), endogenously expressed in HL-1 cells using the whole-cell patch-clamp technique. In the presence of nisoldipine, depolarizing voltage steps applied from a holding potential of -50 mV evoked the time-dependent outward current, followed by slowly decaying outward tail current upon return to the holding potential. The amplitude of the current increased with depolarizations up to 0 mV but then progressively decreased with further depolarizations. The time-dependent outward current as well as the tail current were highly sensitive to block by E-4031 and dofetilide (IC50 of 21.1 and 15.1 nM, respectively) and almost totally abolished by micromolar concentrations of each drug, suggesting that most of the outward current in HL-1 cells was attributable to I (Kr). The magnitude of I (Kr) available from HL-1 cells (18.1 +/- A 1.5 pA pF(-1)) was sufficient for reliable measurements of various gating parameters. RT-PCR and Western blot analysis revealed the expression of alternatively spliced forms of mouse ether-a-go-go-related genes (mERG1), the full-length mERG1a and the N-terminally truncated mERG1b isoforms. Knockdown of mERG1 transcripts with small interfering RNA (siRNA) dramatically reduced I (Kr) amplitude, confirming the molecular link of mERG1 and I (Kr) in HL-1 cells. These findings demonstrate that HL-1 cells possess I (Kr) with properties comparable to those in native cardiac I (Kr) and provide an experimental model suitable for studies of I (Kr) channels.