Role of the store-operated calcium entry proteins Stim1 and Orai1 in muscarinic cholinergic receptor-stimulated calcium oscillations in human embryonic kidney cells
Role of the store-operated calcium entry proteins Stim1 and Orai1 in muscarinic cholinergic receptor-stimulated calcium oscillations in human embryonic kidney cells
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DOI：
10.1113/jphysiol.2006.125641
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发表时间：
2007-03-15
影响因子：
5.5
通讯作者：
Bird, Gary S.
中科院分区：
文献类型：
--
作者：
Wedel, Barbara;Boyles, Rebecca R.;Bird, Gary S.
我们通过检查最近描述的钙库操纵的 Ca2+ 进入蛋白 Stim1 和 Orai1 的作用，研究了人胚胎肾 (HEK293) 细胞中支持 [Ca2+](i) 振荡的 Ca2+ 进入的性质。通过 RNA 干扰 (RNAi) 敲除 Stim1，可将低浓度乙酰甲胆碱响应的 [Ca2+](i) 振荡频率降低至缺乏外部 Ca2+ 时的水平。然而，敲除 Stim1 并不会阻止表达标准瞬时受体电位 3 通道 (TRPC3) 的细胞中的振荡，也不会影响响应花生四烯酸的 Ca2+ 进入。通过用高浓度的 Gd3+ 抑制 Ca2+ 排出，或者通过 Stim1 的过表达来挽救敲低，可以逆转 Stim1 敲低的影响。类似地，Orai1 的敲低消除了 [Ca2+](i) 振荡，并且通过使用高浓度的 Gd3+ 可以逆转这一现象；然而，Orai1 的敲除并不影响花生四烯酸激活的进入。针对瞬时受体电位 (TRP) 通道超家族 34 个成员的 RNAi 并未揭示任何这些通道蛋白在 HEK293 细胞中钙池操纵的 Ca2+ 进入中的作用。这些发现表明，HEK293 细胞中支持 [Ca2+](i) 振荡的 Ca2+ 进入依赖于 Ca2+ 传感器 Stim1 和钙释放激活的 Ca2+ 通道蛋白 Orai1，并为我们的结论提供了进一步的支持，即钙池操纵机制在该途径中起主要作用。
We have investigated the nature of the Ca2+ entry supporting [Ca2+](i) oscillations in human embryonic kidney (HEK293) cells by examining the roles of recently described store-operated Ca2+ entry proteins, Stim1 and Orai1. Knockdown of Stim1 by RNA interference (RNAi) reduced the frequency of [Ca2+](i) oscillations in response to a low concentration of methacholine to the level seen in the absence of external Ca2+. However, knockdown of Stim1 did not block oscillations in canomical transient receptor potential 3 channel (TRPC3)-expressing cells and did not affect Ca2+ entry in response to arachidonic acid. The effects of knockdown of Stim1 could be reversed by inhibiting Ca2+ extrusion with a high concentration of Gd3+, or by rescuing the knockdown by overexpression of Stim1. Similarly, knockdown of Orai1 abrogated [Ca2+](i) oscillations, and this was reversed by use of high concentrations of Gd3+; however, knockdown of Orai1 did not affect arachidonic acid-activated entry. RNAi targeting 34 members of the transient receptor potential (TRP) channel superfamily did not reveal a role for any of these channel proteins in store-operated Ca2+ entry in HEK293 cells. These findings indicate that the Ca2+ entry supporting [Ca2+](i) oscillations in HEK293 cells depends upon the Ca2+ sensor, Stim1, and calcium release-activated Ca2+ channel protein, Orai1, and provide further support for our conclusion that it is the store-operated mechanism that plays the major role in this pathway.