Institute of Cytology, Russian Academy of Sciences, Tikhoretsky Avenue 4, 194064 St. Petersburg, Russia.
Biochimie. 2013 Feb;95(2):347-53. doi: 10.1016/j.biochi.2012.10.004. Epub 2012 Oct 16.
TRPC1 is a major component of store-operated calcium entry in many cell types. In our previous studies, three types of endogenous store-operated calcium channels have been described in HEK293 cells, but it remained unknown which of these channels are composed of TRPC1 proteins. Here, this issue has been addressed by performing single-channel analysis in HEK293 cells transfected with anti-TRPC1 siRNA (siTPRC1) or a TPRC1-encoding plasmid. The results show that thapsigargin-or agonist-induced calcium influx is significantly attenuated in siTRPC1-transfected HEK293 cells. TRPC1 knockdown by siRNA results in the disappearance of store-operated I(max) channels, while the properties of I(min) and I(NS) channels are unaffected. In HEK293 cells with overexpressed TRPC1 protein, the unitary current-voltage relationship of exogenous TRPC1 channels is almost linear, with a slope conductance of about 17 pS. The extrapolated reversal potential of expressed TRPC1 channels is +30 mV. Therefore, the main electrophysiological and regulatory properties of expressed TRPC1 and native I(max) channels are identical. Moreover, TRPC1 overexpression in HEK293 cells results in an increased number of store-operated I(max) channels. All these data allow us to conclude that TRPC1 protein forms native store-operated I(max) channels but is not an essential subunit for other store-operated channel types in HEK293 cells.
TRPC1 是许多细胞类型中储存操纵性钙内流的主要组成部分。在我们之前的研究中，已经描述了 HEK293 细胞中存在三种内源性储存操纵性钙通道，但尚不清楚这些通道中的哪一种由 TRPC1 蛋白组成。在这里，通过在转染抗 TRPC1 siRNA（siTRPC1）或 TRPC1 编码质粒的 HEK293 细胞中进行单通道分析来解决这个问题。结果表明，在转染 siTRPC1 的 HEK293 细胞中，毒胡萝卜素或激动剂诱导的钙内流明显减弱。TRPC1 通过 siRNA 的敲低导致储存操纵性 I(max)通道的消失，而 I(min)和 I(NS)通道的特性不受影响。在过表达 TRPC1 蛋白的 HEK293 细胞中，外源性 TRPC1 通道的单位电流-电压关系几乎呈线性，斜率电导约为 17 pS。表达的 TRPC1 通道的外推反转电位为+30 mV。因此，表达的 TRPC1 和天然 I(max)通道的主要电生理和调节特性是相同的。此外，TRPC1 在 HEK293 细胞中的过表达导致储存操纵性 I(max)通道数量增加。所有这些数据使我们得出结论，TRPC1 蛋白形成天然储存操纵性 I(max)通道，但不是 HEK293 细胞中其他储存操纵性通道类型的必需亚基。
