Manipulation of cellular excitability by cell fusion: effects of rapid introduction of transient outward K+ current on the guinea pig action potential.
Manipulation of cellular excitability by cell fusion: effects of rapid introduction of transient outward K+ current on the guinea pig action potential.
复制标题
通过细胞融合操纵细胞兴奋性：快速引入瞬时外向 K 电流对豚鼠动作电位的影响。
DOI：
10.1161/01.res.84.8.964
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发表时间：
1999
影响因子：
20.1
通讯作者：
O'Rourke,B
中科院分区：
文献类型：
--
作者：
Hoppe,UC;Johns,DC;Marbán,E;O'Rourke,B
— 为了研究不依赖 Ca2+ 的瞬时外向电流 (Ito1) 对心脏动作电位复极的作用，我们使用细胞融合将 Ito1 引入通常缺乏这种电流的豚鼠心肌细胞中。该技术能够在分离后数小时内将预制的功能离子通道快速递送至心肌细胞，从而消除使长期细胞培养复杂化的动作电位变化。稳定表达 Kv4.3 (CHO-Kv4.3) 的中国仓鼠卵巢 (CHO) 细胞负载荧光染料，并使用聚乙二醇与豚鼠心肌细胞融合。作为对照，未转染的 CHO 细胞使用相同的方案进行融合。与 CHO-Kv4.3 细胞融合的肌细胞表现出强大的 Ito1（+40 mV 时为 16.5±2.6 pA/pF；37°C；n=19），而对照组则没有。随着 Ito1 密度的增加，Ito1 加速了早期复极速度（r=-0.68；超调后 3 ms），并逐渐抑制平台期电压（r=-0.90）。动作电位持续时间减少至 50% 复极 (r=-0.76) 和 90% 复极 (r=-0.65) 也与 Ito1 密度密切相关。因此，Ito1对早期复极相和动作电位持续时间缩短有显着影响。细胞融合是将预先形成的膜蛋白引入天然细胞的一种有价值且通用的技术。
—To investigate the still-undetermined role of the Ca2+-independent transient outward current (Ito1) on repolarization of the cardiac action potential, we used cell fusion to introduceIto1into guinea pig cardiomyocytes, which normally lack this current. This technique enables the rapid delivery of premade functional ion channels to cardiomyocytes within hours of isolation, thus eliminating the action potential alterations that complicate prolonged cell culture. Chinese hamster ovary (CHO) cells stably expressing Kv4.3 (CHO-Kv4.3) were loaded with a fluorescent dye and fused to guinea pig cardiomyocytes using polyethylene glycol. As controls, nontransfected CHO cells were fused using the same protocol. Myocytes fused with CHO-Kv4.3 cells exhibited a robustIto1(16.5±2.6 pA/pF at +40 mV; 37°C; n=19), whereas controls had none.Ito1accelerated the early repolarization velocity (r=−0.68; 3 ms after the overshoot) and progressively suppressed the voltage of the plateau phase (r=−0.90) with increasingIto1density. Reduction of the action potential duration to 50% repolarization (r=−0.76) and to 90% repolarization (r=−0.65) also correlated well withIto1density. Thus,Ito1exerted a significant effect on the early repolarization phase and abbreviated action potential duration. Cell fusion is a valuable and generalizable technique to introduce preformed membrane proteins into native cells.