Varghese Anthony, Tenbroek Erica M, Coles James, Sigg Daniel C
CS Department, University of Wisconsin, River Falls, WI 54022, USA.
Prog Biophys Mol Biol. 2006 Jan-Apr;90(1-3):26-37. doi: 10.1016/j.pbiomolbio.2005.05.002. Epub 2005 Jun 8.
A transformed line of human embryonic kidney epithelial cells (HEK 293) is commonly used as an expression system for exogenous ion channel genes. Previously, it has been shown that these cells contain mRNAs for a variety of ion channels. Expression of some of these genes has been confirmed at the protein level. Patch-clamp electrophysiology experiments confirm the presence of multiple ion channels and molecular data agree with pharmacological profiles of identified channels. In this work, we show that endogenous voltage-gated potassium channels in HEK cells are a significant source of outward current at positive potentials. We show that both non-transfected HEK cells and HEK cells transfected with hyperpolarization-activated cyclic-nucleotide gated (HCN) channels have a significant amount of voltage-gated potassium (K(V)) current when certain tail current voltage-clamp protocols are used to assay HCN current activation. Specifically, tail current protocols that use a depolarized holding potential of -40 mV followed by hyperpolarizing pulses (-80 to -140 mV) and then a tail pulse potential of +20 mV indicate K(V) channels undergo closed-state inactivation at the more depolarized holding potential of -40 mV, followed by recovery from inactivation (but no activation) at hyperpolarizing potentials and high amount of activation at the positive tail potential. Our results indicate that pulse protocols with positive tail pulses are inaccurate assays for HCN current in certain HEK cells. Surprisingly, HEK-293 cells were found to contain mRNA for HCN2 and HCN3 although we have not detected a significant and consistent endogenous I(f)-like current in these cells.
一种转化的人胚肾上皮细胞系（HEK 293）通常用作外源离子通道基因的表达系统。以前的研究表明，这些细胞含有多种离子通道的mRNA。其中一些基因的表达已在蛋白质水平得到证实。膜片钳电生理学实验证实了多种离子通道的存在，并且分子数据与已鉴定通道的药理学特征相符。在这项工作中，我们表明HEK细胞中的内源性电压门控钾通道是正电位下外向电流的重要来源。我们发现，当使用某些尾电流电压钳制方案来检测HCN电流激活时，未转染的HEK细胞和转染了超极化激活的环核苷酸门控（HCN）通道的HEK细胞都有大量的电压门控钾（K(V)）电流。具体而言，使用-40 mV的去极化钳制电位，随后进行超极化脉冲（-80至-140 mV），然后是+20 mV的尾脉冲电位的尾电流方案表明，K(V)通道在-40 mV的更去极化钳制电位下经历关闭状态失活，随后在超极化电位下从失活中恢复（但未激活），并在正尾电位下大量激活。我们的结果表明，对于某些HEK细胞中的HCN电流，具有正尾脉冲的脉冲方案是不准确的检测方法。令人惊讶的是，尽管我们在这些细胞中未检测到显著且一致的内源性I(f)样电流，但发现HEK-293细胞含有HCN2和HCN3的mRNA。
