Xia Menghang, Imredy John P, Santarelli Vincent P, Liang Hongyu Annie, Condra Cindra L, Bennett Paul, Koblan Kenneth S, Connolly Thomas M
Department of Molecular Pharmacology, Merck Research Laboratories, West Point, PA 19486, USA.
Assay Drug Dev Technol. 2003 Oct;1(5):637-45. doi: 10.1089/154065803770380995.
Establishment of stable cell lines that constitutively express Ca(2+) channels at high density and that are useful for in vitro studies may be complicated by problems with seal quality and duration during whole-cell patch-clamp electrophysiology. The current studies describe the generation and characterization of cells that express the human alpha1H T-type Ca(2+) channel under the control of a tetracycline-inducible expression system. Western blot and immunostaining studies revealed that expression of the alpha1H protein occurred only in the presence of tetracycline. Using the whole-cell patch-clamp method, the cells displayed peak inward currents of 1.15 +/- 0.14 nA in response to voltage-clamp steps. The T-type Ca(2+) current was inhibited by the T-type Ca(2+) channel antagonist, mibefradil, with an IC(50) of 160 nM. This cell line, with inducible channel expression, sealed with longer duration during whole-cell patch-clamp recording when compared with a cell line that constitutively expresses the alpha1H Ca(2+) channel. Ca(2+) influx through this channel could also be detected after the addition of extracellular Ca(2+). The amount of Ca(2+) influx was dependent on the Ca with an EC(50) of 4 mM. The Ca(2+) influx was also inhibited by mibefradil with a potency (IC(50) = 183 nM) similar to that observed in the voltage-clamp studies. Overall, this inducible alpha1H Ca(2+) channel-expressing cell line is useful for the study of human T-type Ca(2+) channel function, and offers advantages over a similar cell line that constitutively expresses the channel.
建立在高密度下持续表达钙离子通道且适用于体外研究的稳定细胞系，可能会因全细胞膜片钳电生理过程中封接质量和持续时间的问题而变得复杂。当前的研究描述了在四环素诱导表达系统控制下表达人α1H T型钙离子通道的细胞的产生和特性。蛋白质免疫印迹和免疫染色研究表明，α1H蛋白仅在四环素存在时表达。使用全细胞膜片钳方法，细胞在电压钳阶跃刺激下显示出1.15±0.14 nA的内向峰值电流。T型钙离子电流被T型钙离子通道拮抗剂米贝地尔抑制，其半数抑制浓度（IC50）为160 nM。与持续表达α1H钙离子通道的细胞系相比，这种具有诱导性通道表达的细胞系在全细胞膜片钳记录过程中封接持续时间更长。加入细胞外钙离子后，也能检测到通过该通道的钙离子内流。钙离子内流的量取决于细胞外钙离子浓度（Ca），其半数有效浓度（EC50）为4 mM。米贝地尔也抑制钙离子内流，其效力（IC50 = 183 nM）与电压钳研究中观察到的相似。总体而言，这种诱导性表达α1H钙离子通道的细胞系可用于研究人T型钙离子通道功能，并且比持续表达该通道的类似细胞系具有优势。
