Department of Physiology, Development, and Neuroscience, University of Cambridge, Cambridge, United Kingdom.
Am J Physiol Cell Physiol. 2013 May 1;304(9):C858-72. doi: 10.1152/ajpcell.00368.2012. Epub 2013 Feb 13.
We have isolated a K(+)-selective, Ca(2+)-dependent whole cell current and single-channel correlate in the human erythroleukemia (HEL) cell line. The whole cell current was inhibited by the intermediate-conductance KCa3.1 inhibitors clotrimazole, TRAM-34, and charybdotoxin, unaffected by the small-conductance KCa2 family inhibitor apamin and the large-conductance KCa1.1 inhibitors paxilline and iberiotoxin, and augmented by NS309. The single-channel correlate of the whole cell current was blocked by TRAM-34 and clotrimazole, insensitive to paxilline, and augmented by NS309 and had a single-channel conductance in physiological K(+) gradients of ~9 pS. RT-PCR revealed that the KCa3.1 gene, but not the KCa1.1 gene, was expressed in HEL cells. The KCa3.1 current, isolated in HEL cells under whole cell patch-clamp conditions, displayed an activated current component during depolarizing voltage steps from hyperpolarized holding potentials and tail currents upon repolarization, consistent with voltage-dependent modulation. This activated current increased with increasing voltage steps above -40 mV and was sensitive to inhibition by clotrimazole, TRAM-34, and charybdotoxin and insensitive to apamin, paxilline, and iberiotoxin. In single-channel experiments, depolarization resulted in an increase in open channel probability (Po) of KCa3.1, with no increase in channel number. The voltage modulation of Po was an increasing monotonic function of voltage. In the absence of elevated Ca(2+), voltage was ineffective at inducing channel activity in whole cell and single-channel experiments. These data indicate that KCa3.1 in HEL cells displays a unique form of voltage dependence modulating Po.
我们已经从人红白血病（HEL）细胞系中分离出一种 K(+) 选择性、Ca(2+) 依赖性的全细胞电流和单通道相关物。全细胞电流被中电导钙激活钾通道（KCa3.1）抑制剂克霉唑、TRAM-34 和蟾毒灵抑制，不受小电导钙激活钾通道（KCa2）家族抑制剂蜂斗菜碱和大电导钙激活钾通道（KCa1.1）抑制剂白屈菜碱的影响，而被 NS309 增强。全细胞电流的单通道相关物被 TRAM-34 和克霉唑阻断，对白屈菜碱不敏感，被 NS309 增强，在生理 K(+) 梯度下具有约 9 pS 的单通道电导。RT-PCR 显示 KCa3.1 基因，但不是 KCa1.1 基因，在 HEL 细胞中表达。在全细胞膜片钳条件下从超极化保持电位去极化电压步骤中分离出的 KCa3.1 电流在复极化时显示激活电流成分，与电压依赖性调制一致。这种激活电流随着超过-40 mV 的电压步骤增加而增加，对克霉唑、TRAM-34 和蟾毒灵敏感，对白屈菜碱、蜂斗菜碱和白屈菜碱不敏感。在单通道实验中，去极化导致 KCa3.1 的开放通道概率（Po）增加，而通道数没有增加。Po 的电压调制是电压的递增单调函数。在没有升高的 Ca(2+) 的情况下，电压在全细胞和单通道实验中均无法诱导通道活性。这些数据表明，HEL 细胞中的 KCa3.1 表现出独特的电压依赖性调节 Po 的形式。
