Identification of endogenous outward currents in the human embryonic kidney (HEK 293) cell line.
Identification of endogenous outward currents in the human embryonic kidney (HEK 293) cell line.
复制标题
人胚肾 (HEK 293) 细胞系内源性外向电流的鉴定。
DOI：
10.1016/s0165-0270(98)00019-3
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发表时间：
1998
影响因子：
3
通讯作者：
Jiang,C
中科院分区：
文献类型：
--
作者：
Zhu,G;Zhang,Y;Xu,H;Jiang,C
人胚胎肾细胞（HEK 293）作为离子通道的表达系统被广泛应用于离子通道的研究。然而，它们的内源性离子电流在很大程度上仍未被确定。为了表征这些电流，我们对该表达系统进行了膜片钳实验。在全细胞电压箝位模式下，HEK 293细胞主要通过生理浓度的Na+和K+以及跨质膜对称浓度的Cl - (150 mM)向外显示电流。当细胞外K+浓度从3到150 mM变化时，逆转电位只发生了~ 20 mV的变化，因此K+电流贡献了这些向外电流的一小部分。相反，当细胞外Cl−减少到50 mM时，逆转电位发生了~ 25 mV的变化，这表明大部分向外电流由Cl−携带。在从内到外的补丁中，可以识别出几种不同的Cl -电流。它们是：(1)350 pS Cl -电流，电压激活，具有适度的向外整流；(2) 240 pS Cl−电流，弱向外整流；(3) 55 pS Cl−电流，电压激活，对DIDS敏感，并表现出强烈的外整流。这些Cl -电流的激活不需要细胞质中游离Ca2+水平的升高。除了这三个电流外，我们还观察到另外两个电导小得多的Cl -电流（分别为25和16 pS）。在HEK 293细胞中观察到两种不同的K+电流，其中一种（125 pS）向内整流，另一种（70 pS）向外整流。此外，在这些细胞中记录到一个50ps的阳离子通道。HEK 293细胞中多种离子通道的存在表明，当将该表达系统用于几种类似离子通道的研究时，需要采取很大的预防措施。
Human embryonic kidney cells (HEK 293) are widely used as an expression system in studies of ion channels. However, their endogenous ionic currents remain largely unidentified. To characterize these currents, we performed patch clamp experiments on this expression system. In whole-cell voltage clamp mode, the HEK 293 cells showed mainly outward currents using physiological concentrations of Na+and K+and symmetric concentrations of Cl−(150 mM) across the plasma membranes. K+currents contributed to a small portion of these outward currents, since a shift of the reversal potentials of only ∼20 mV was seen with a change of extracellular K+concentration from 3 to 150 mM. In contrast, the reversal potential shifted ∼25 mV when extracellular Cl−was reduced to 50 mM, indicating that most of the outward currents are carried by Cl−. In inside-out patches, several distinct Cl−currents were identified. They were: (1) 350 pS Cl−current, which was voltage-activated and had a moderate outward rectification; (2) 240 pS Cl−current with a weak outward rectification; and (3) 55 pS Cl−current, which was voltage-activated, sensitive to DIDS, and showed a strong outward rectification. Activation of these Cl−currents did not require an elevation of free Ca2+level in the cytosol. Besides these three currents, we observed two other Cl−currents with much smaller conductances (25 and 16 pS, respectively). Two different K+currents were seen in the HEK 293 cells, with one of them (125 pS) showing inward rectification and the other (70 pS) outward rectification. Moreover, a 50 pS cation channel was recorded in these cells. The presence of a variety of ion channels in the HEK 293 cells suggests that a great precaution needs to be taken when this expression system is used in studies of several similar ion channels.